WO2023230461A2 - Screening for inhibitors of integrin-mediated curved adhesion - Google Patents

Screening for inhibitors of integrin-mediated curved adhesion Download PDF

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WO2023230461A2
WO2023230461A2 PCT/US2023/067339 US2023067339W WO2023230461A2 WO 2023230461 A2 WO2023230461 A2 WO 2023230461A2 US 2023067339 W US2023067339 W US 2023067339W WO 2023230461 A2 WO2023230461 A2 WO 2023230461A2
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split
integrin
fcho2
protein
cell
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WO2023230461A3 (en
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Bianxiao Cui
Wei Zhang
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • a Sequence Listing is provided herewith as a Sequence Listing XML file, “STAN-1951 WO” created on May 10, 2023 and having a size of 35,554 bytes. The contents of the Sequence Listing XML file are incorporated by reference herein in their entirety.
  • Integrins have long been identified as therapeutic targets. Integrins are heterodimers composed of alpha and beta subunits forming 24 distinct integrins, which recognize a broad spectrum of ligands in the extracellular matrix (ECM).
  • ECM extracellular matrix
  • integrin inhibitors targeting four integrins have been approved by The United States Food and Drug Administration (FDA) for the treatment of thrombosis, inflammatory bowel disease, multiple sclerosis, psoriasis, and dry eye disease.
  • FDA Food and Drug Administration
  • drug developments targeting integrins that are prominently implicated in fibrosis and cancer, such as the ⁇ v subfamily including integrins ⁇ v ⁇ 1 , ⁇ v ⁇ 3 , ⁇ v ⁇ 5, and ⁇ v ⁇ 6, have faced significant challenges. These molecules often exhibited encouraging in vitro results, but failed to show significant therapeutic benefits in clinical trials.
  • Cilengitide a potent inhibitor of ⁇ v ⁇ 3 and ⁇ v ⁇ 5 at subnanomolar regime in binding assays
  • Cilengitide a potent inhibitor of ⁇ v ⁇ 3 and ⁇ v ⁇ 5 at subnanomolar regime in binding assays
  • compositions, methods, and kits are provided for screening for candidate agents that inhibit formation of integrin-mediated curved adhesions. Screening assays may further include determining the effectiveness of candidate agents in reducing cell adhesion, proliferation, cell spreading, migration, or survival of cells and screening candidate agents for efficacy in treating disorders associated with integrin-mediated formation of curved adhesions.
  • a method of screening for an inhibitor that inhibits formation of integrin- mediated curved adhesions comprising: contacting a cell, a population of cells, or a tissue with a candidate agent; and measuring proximity of integrin isoform ⁇ V ⁇ 5 to FCHo2 in the presence of the candidate agent to determine whether the inhibitor inhibits colocalization of integrin isoform ⁇ V ⁇ 5 and FCHo2 in integrin-mediated curved adhesions, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • the method further comprises providing a vertical nanostructure having an end curvature diameter ranging from 100 nm to 3 pm, wherein the cell forms the integrin-mediated curved adhesions on the surface of the nanostructure.
  • the nanostructure is a nanobar or a nanopillar. In some embodiments, the nanostructure is coated with vitronectin or fibronectin.
  • the method further comprises providing fibers comprising collagen, fibronectin, or vitronectin, or a combination thereof, wherein the cell forms the integrin- mediated curved adhesions on the surface of the fibers.
  • the method further comprises providing a curved extracellular matrix (ECM) surface, wherein the cell forms the integrin-mediated curved adhesions on the curved ECM surface.
  • ECM extracellular matrix
  • the method further comprises detecting talin or paxillin in the integrin-mediated curved adhesions.
  • measuring the proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises performing a complementation assay using a split reporter protein, a fluorescence resonance energy transfer (FRET) assay, a bioluminescence resonance energy transfer (BRET) assay, an antibody-based proximity ligation assay (PLA), or a proximity- dependent initiation of hybridization chain reaction (proxHCR) assay.
  • FRET fluorescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • PDA antibody-based proximity ligation assay
  • proxHCR proximity- dependent initiation of hybridization chain reaction
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split fluorescent protein connected to the FCHo2, wherein a reconstituted fluorescent protein forms when the first component and the second component of the split fluorescent protein are expressed and interact to produce a detectable fluorescent signal; and detecting the fluorescent signal to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 from the fluorescent signal, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
  • the fluorescent signal is measured using fluorescence microscopy two-photon excitation microscopy, fluorescence confocal laser scanning microscopy, or light sheet fluorescence microscopy.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split luminescent protein connected to the FCHo2, wherein a reconstituted luminescent protein forms when the first component and the second component of the split luminescent protein are expressed and interact to produce a detectable luminescent signal; and detecting the luminescent signal to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 from the luminescent signal, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhe
  • the split luminescent protein is a split aequorin or a split luciferase such as, but not limited to, a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
  • the luminescent signal is measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) donor connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the integrin isoform ⁇ V ⁇ 5 or FCHo2 indicates whether a cellular adhesion is an integrin- mediated curved adhesion.
  • FRET fluorescence resonance energy transfer
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) acceptor connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • FRET fluorescence resonance energy transfer
  • FRET efficiency is measured using fluorescence microscopy or fluorescence confocal laser scanning microscopy.
  • the FRET donor and the FRET acceptor are fluorescent proteins or fluorescent dyes.
  • the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein
  • the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein
  • the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5)
  • the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) donor connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin- mediated curved adhesion.
  • BRET bioluminescence resonance energy transfer
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) acceptor connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin- mediated curved adhesion.
  • BRET bioluminescence resonance energy transfer
  • the BRET efficiency is measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
  • the BRET donor or the BRET acceptor is a bioluminescent protein or a luminescent quantum dot.
  • the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises performing an antibody-based proximity ligation assay (PLA) or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay.
  • PLA antibody-based proximity ligation assay
  • proxHCR proximity-dependent initiation of hybridization chain reaction
  • performing the PLA comprises: contacting the cell, population of cells, or tissue with a first antibody that specifically binds to ⁇ V ⁇ 5; contacting the cell, population of cells, or tissue with a second antibody that specifically binds to FCHo2; contacting the first antibody with a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; contacting the second antibody with a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody; contacting the first probe and the second probe with a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with the first oligonucleotide and a second portion sufficiently complementary to hybridize with the second oligonucleotide when the first oligonucleotide and the second oligonucleot
  • amplifying comprises performing polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification. In some embodiments, amplifying comprises performing quantitative PCR. In some embodiments, the amplicon is detected with a detectably labeled probe that binds to the amplicon. In some embodiments, the probe comprises a fluorescent label, a bioluminescent label, or a chemiluminescent label.
  • PCR polymerase chain reaction
  • isothermal amplification or rolling circle amplification.
  • amplifying comprises performing quantitative PCR.
  • the amplicon is detected with a detectably labeled probe that binds to the amplicon.
  • the probe comprises a fluorescent label, a bioluminescent label, or a chemiluminescent label.
  • the first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an F v fragment, and an scF v fragment.
  • the method further comprises quantifying the number and area of the integrin-mediated curved adhesions.
  • the method further comprises contacting a population of cancer cells with the candidate agent if the candidate agent inhibits the formation of the integrin-mediated curved adhesions; and measuring cell adhesion, proliferation, cell spreading, migration, or survival of the population of cancer cells, wherein reduced proliferation, cell spreading, migration, or survival in presence of the candidate agent compared to that in a negative control population of cancer cells that are not treated with the candidate agent indicates that the candidate agent has anti-cancer activity.
  • the candidate agent is a small molecule, a peptide, a protein, an aptamer, an antibody, an antibody mimetic, an inhibitory nucleic acid, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a F v fragment, and a nanobody, wherein the antibody specifically binds to the integrin isoform ⁇ V ⁇ 5 or the FCHo2.
  • the inhibitory nucleic acid is selected from the group consisting of a small interfering RNA (siRNA), a microRNA (miRNA), a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), an antisense oligonucleotide, and a peptide nucleic acid, wherein the inhibitory nucleic acid inhibits transcription or protein translation of the integrin isoform ⁇ V ⁇ 5 or the FCHo2.
  • siRNA small interfering RNA
  • miRNA microRNA
  • piRNA Piwi-interacting RNA
  • snRNA small nuclear RNA
  • antisense oligonucleotide an antisense oligonucleotide
  • the CRISPR system targets a gene or an RNA transcript encoding the integrin isoform ⁇ V ⁇ 5 or the FCHo2, or makes epigenetic changes that reduce expression of the integrin isoform ⁇ V ⁇ 5 or the FCHo2.
  • the CRISPR system comprises Cas9, Cas12a, Cas12d, Cas13a, Cas13b, Cas13d, or a dead Cas9 (dCas9).
  • an inhibitor of integrin-mediated curved adhesion identified by a method described herein, is provided.
  • composition comprising an inhibitor of integrin-mediated curved adhesion, identified by a method described herein.
  • the composition further comprises a pharmaceutically acceptable excipient.
  • the composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment.
  • a reporter system for measuring curved adhesion comprising a cell expressing a split fluorescent or luminescent protein comprising a first component of a fluorescent or luminescent protein and a second component of the fluorescent or luminescent protein, wherein the cell comprises: a first nucleic acid comprising a coding sequence encoding a first fusion protein comprising the first component of the fluorescent or luminescent protein connected to integrin isoform ocVJ35; and a second nucleic acid comprising a coding sequence encoding a second fusion protein comprising the second component of the fluorescent or luminescent protein connected to FCHo2; wherein a reconstituted fluorescent or luminescent protein forms when the first component and the second component of the split fluorescent or luminescent protein are expressed and interact to produce a detectable fluorescent or luminescent signal indicative of the proximity of the integrin isoform ⁇ V ⁇ 5 and the FCHo2 in the cell.
  • the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split- mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
  • the split luminescent protein is a split aequorin or a split luciferase such as, but not limited to, a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
  • a split firefly luciferase such as, but not limited to, a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
  • kits comprising a reporter system described herein and instructions for detecting integrin-mediated curved adhesions is provided.
  • a system for screening a candidate agent to determine its effects on formation of integrin-mediated curved adhesions by a cell comprising a vertical nanostructure having an end curvature diameter ranging from 100 nm to 3 pm, wherein the cell forms the integrin-mediated curved adhesions on the surface of the nanostructure.
  • the nanostructure is a nanobar or a nanopillar.
  • the nanostructure is coated with vitronectin or fibronectin.
  • the system further comprises a cell or cell fragment of interest such as, but not limited to, a cancer cell, a blood cell, a platelet, an inflammatory cell, a fibroblast, or an epithelial cell.
  • a cell or cell fragment of interest such as, but not limited to, a cancer cell, a blood cell, a platelet, an inflammatory cell, a fibroblast, or an epithelial cell.
  • the system further comprises reagents for measuring the proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 with a complementation assay using a split reporter protein, a fluorescence resonance energy transfer (FRET) assay, a bioluminescence resonance energy transfer (BRET) assay, an antibody-based proximity ligation assay (PLA), or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay.
  • FRET fluorescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • PDA antibody-based proximity ligation assay
  • proxHCR proximity-dependent initiation of hybridization chain reaction
  • the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to integrin isoform ⁇ V ⁇ 5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split fluorescent protein connected to FCHo2, wherein a reconstituted fluorescent protein forms when the first component and the second component of the split fluorescent protein are expressed and interact to produce a detectable fluorescent signal.
  • the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, or a split yellow-green fluorescent protein.
  • the system further comprises a fluorescence microscope, a two-photon excitation microscope, a fluorescence confocal laser scanning microscope, or a light sheet fluorescence microscope.
  • the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to integrin isoform ⁇ V ⁇ 5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split luminescent protein connected to FCHo2, wherein a reconstituted luminescent protein forms when the first component and the second component of the split luminescent protein are expressed and interact to produce a detectable luminescent signal.
  • the split luminescent protein is a split aequorin or a split luciferase such as, but not limited to, a split firefly luciferase, a Renilla luciferase, an Elateroidea luciferase, a Metridia luciferase, a Vibrio luciferase, a dinoflagellate luciferase, or a nano-lantern luciferase.
  • the system further comprises a luminescence microscope, luminescence digital imaging microscope, time-gated luminescence microscope, or a luminometer.
  • the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) donor connected to integrin isoform ⁇ V ⁇ 5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to FCHo2.
  • FRET fluorescence resonance energy transfer
  • the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) acceptor connected to the integrin isoform ⁇ V ⁇ 5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2.
  • FRET fluorescence resonance energy transfer
  • the system further comprises a fluorescence microscope or a fluorescence confocal laser scanning microscope.
  • the FRET donor and the FRET acceptor are fluorescent proteins or fluorescent dyes.
  • the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein
  • the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein
  • the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5)
  • the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine.
  • the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) donor connected to the integrin isoform ⁇ V ⁇ 5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2.
  • BRET bioluminescence resonance energy transfer
  • the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) acceptor connected to the integrin isoform ⁇ V ⁇ 5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2.
  • BRET bioluminescence resonance energy transfer
  • the system further comprises a luminescence microscope, a luminescence digital imaging microscope, a time-gated luminescence microscope, or a luminometer.
  • the BRET donor or the BRET acceptor is a bioluminescent protein or a luminescent quantum dot.
  • the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises performing an antibody-based proximity ligation assay (PLA) or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay.
  • PLA antibody-based proximity ligation assay
  • proxHCR proximity-dependent initiation of hybridization chain reaction
  • the system further comprises reagents for performing the PLA.
  • the system comprises: a first antibody that specifically binds to ⁇ V ⁇ 5; a second antibody that specifically binds to FCHo2; a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; and a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody.
  • the system further comprises a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with the first oligonucleotide and a second portion sufficiently complementary to hybridize with the second oligonucleotide when the first oligonucleotide and the second oligonucleotide are in sufficient proximity to each other to simultaneously hybridize to the bridge oligonucleotide.
  • the system further comprises a ligase.
  • the system further comprises reagents for performing polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification.
  • the first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an F v fragment, and an scF v fragment.
  • the system further comprises a detectably labeled probe that binds to the amplicon.
  • the detectably labeled probe comprises a fluorescent label, a bioluminescent label, or a chemiluminescent label
  • kits comprising a system described herein and instructions for detecting integrin-mediated curved adhesions is provided.
  • FIGS. 1A-1 G Positive membrane curvature induces selective accumulation of integrin ⁇ V ⁇ 5.
  • FIG. 1A Scanning electron microscopy (SEM) image of nanobars viewed at 45o angle.
  • FIG. 1 B SEM images of a nanobar viewed at 45o angle (top) or from the top (bottom).
  • FIG. 1C SEM image viewed at 45° angle showing nanobars deforming the cell membrane.
  • FIG. 1 D A chart of integrin p subunits probed in this study, which form heterodimers with different a 703 subunits to bind specific ECM proteins.
  • FIG. 1 E Top: schematic illustration of integrins binding to the ECM protein-coated surface.
  • FIG. 1F Zoom-in fluorescence images of isoforms and a membrane marker FusionRed-CaaX on nanobar substrates coated with different ECM proteins.
  • the 5 shows preferential accumulation toward the nanobar ends, on both vitronectin and fibronectin coatings. Additional images of [31 ,
  • FIG. 1G Quantification of the curvature preferences of
  • 3 isoforms by measuring their nanobar end/side ratio normalized by the membrane marker. The end/side ratios of -100 nanobars per cell were individually calculated and then averaged. N 12 cells from two independent experiments were quantified for each condition. Data are mean ⁇ standard deviation (SD). Scale bars: 5 ⁇ m (FIGS. 1A, 1 C, 1 E, 1 F); 1 pm (1 B).
  • FIGS. 2A-2I ⁇ V ⁇ 5 forms curved adhesions to extracellular ligands and bear forces.
  • FIG. 2A SEM images of nanopillars.
  • FIG. 2B Left: top-view SEM image of a cell on nanopillars. Right: schematic drawing of the interface between the cell membrane and a nanopillar.
  • FIG. 2C EDTA treatment induces a dramatic reduction of [35-GFP accumulation at nanopillars in a live cell labelled with CellMask membrane dye. Bottom: the normalized [35/membrane ratio is shown in the Parula colour scale.
  • FIG. 2D Quantification of the normalized ⁇ 5/membrane ratio at nanopillars and their surrounding flat regions.
  • N 968/691 nanopillars before/after EDTA, from three independent cells.
  • FIG. 2E Schematic drawing of the centrifugation adhesion assay.
  • FIG. 2F Nucleus staining shows that more cells remain on nanopillar areas than on flat areas after spin.
  • FIG. 2H Nanopillars induce ⁇ 5 accumulation and promote early-stage (30 min after seeding) cell spreading. ⁇ 5 KD largely abolished the nanopillar-induced early cell spreading.
  • FIG. 2H Nanopillars induce ⁇ 5 accumulation and promote early-stage (30 min after seeding) cell spreading. ⁇ 5 KD largely abolished the nanopillar-induced early cell spreading.
  • FIG. 2H Nanopillars induce ⁇ 5 accumulation and promote early-stage (30 min after seeding) cell spreading. ⁇ 5 KD largely abolished the nanopillar-induced early cell spreading.
  • FIG. 2H Nanopillars induce ⁇ 5
  • Data are mean ⁇ SD.
  • FIGS. 3A-3F Curved adhesions involve a subset of focal adhesion proteins to anchor F- actin.
  • FIG. 3A At vitronectin-coated nanopillars, ⁇ 5-GFP accumulation ( ⁇ 5/membrane, green) spatially correlates with talin and paxillin, but not with vinculin or pFAK (magenta). Talin was tagged with mCherry. Endogenous paxillin, vinculin and FAK were immunolabeled.
  • FIG. 3B Both 35-GFP and LifeAct-mCherry, a marker for F-actin, strongly accumulate at nanopillars, but their intensities are not spatially correlated.
  • FIGS. 3A, 3B Pearson correlation coefficients between ⁇ 5/membrane and focal adhesion proteins at nanopillars.
  • N 12 cells with each cell interfacing with -300 nanopillars were quantified for each condition, from two independent experiments.
  • FIGS. 3D, 3E Dynamics of F- actin at nanopillars coated with vitronectin (FIG. 3D) or gelatin (FIG. 3E) imaged at 15 s/frame for 15 min.
  • 35 nanopillars (vitronectin coating), and N 153 nanopillars (gelatin coating). Data are mean ⁇ SD. Rvalues calculated using Kruskal- Wallis test with Dunn’s multiple-comparison (FIG. 3F). Scale bars, 10 pm.
  • FIGS. 4A-4H The formation of curved adhesion requires curvature-sensing proteins FCHol /2.
  • FIG.4A Mutant ⁇ 5 (Y752A Y770A) that does not bind talin still accumulates at the ends of vitronectin coated nanobars in cells co-expressing Fusion Red-CaaX.
  • FIG. 4B Quantifications of the end/side ratios show that ⁇ 5 (Y752A Y770A) mutant and WT ⁇ 5 are not statistically different in sensing membrane curvatures.
  • FIG.4A Mutant ⁇ 5 (Y752A Y770A) that does not bind talin still accumulates at the ends of vitronectin coated nanobars in cells co-expressing Fusion Red-CaaX.
  • FIG. 4B Quantifications of the end/side ratios show that ⁇ 5 (Y752A Y770A) mutant and WT ⁇ 5 are not statistically different in sensing membrane curvatures.
  • FIG. 4C Top: ⁇ 5 (green) colocalizes with clathrin plaques, marked by AP2 (magenta), on vitronectin- but not on fibronectin-coated flat surfaces. Bottom: at vitronectin coated nanopillars, 35 accumulation (35/membrane) does not spatially correlate with AP2.
  • FIG. 4D The shRNA KD of FCHo1/2 but not CHC, AP2-p, Eps15/R or intersectin 1/2 significantly reduces the nanobar end/side ratio of 35, compared with treatment with negative control shRNA.
  • FIG. 4E Top: 5 (green) does not colocalize with FCHo2 (magenta) on vitronectin-coated flat surfaces.
  • FIG. 4F Pearson correlation coefficients of AP2 and FCHo2 against 35/membrane at vitronectin-coated nanopillars.
  • FIG. 4G Dynamics of 35 and FCHo2 at vitronectin-coated nanopillars imaged at 15 s/frame for 20 min. Left: representative fluorescence images. Right: kymographs generated with nanopillars indicated by the boxes in left images.
  • FIGS. 5A-5G Cells form curved adhesions on ECM fibres.
  • FIG. 5A ⁇ 5-GFP accumulates at places where the plasma membrane is deformed along sparse ECM fibres.
  • FIG. 5B Intensity profiles of fibre (blue), (35 (green) and membrane (magenta) vertically crossing the membrane- wrapped fibre segment indicated in FIG. 5A. Lines 776 and shaded areas indicate means and SD, respectively, along the fibre segment.
  • FIG. 5A ⁇ 5-GFP accumulates at places where the plasma membrane is deformed along sparse ECM fibres.
  • FIG. 5B Intensity profiles of fibre (blue), (35 (green) and membrane (magenta) vertically crossing the membrane-
  • FIG. 5D On dense 3D ECM fibres, curved adhesions marked by the colocalizations of ⁇ 5 and FCHo2 are abundant, while focal adhesions marked by the colocalization of ⁇ 5 and vinculin are almost absent.
  • FIG. 5F Probing the range of curvatures that facilitate the formation of curved adhesions using the gradient nanobar array.
  • First row bright-field (BF) image of the gradient nanobars.
  • Second row fluorescent images of the gradient bars coated with Cy3- vitronectin (Vn).
  • FIG. 5G Quantification of the end/side ratio of ⁇ 5/membrane on gradient bars.
  • N 8 imaging fields. Data are mean ⁇ SD, from two independent experiments. P values calculated using one-way ANOVA with Tukey's (FIGS. 5C, 5E) or Bonferroni’s (FIG. 5G) multiple comparison test. Scale bars, 10 pm.
  • FIG. 6 Schematic illustrations of focal adhesion and curved adhesion structures.
  • FIGS. 7A-7C Characterizations of nanobars, nanopillars and gradient bars.
  • FIG. 7A Representative SEM images of a 200 nm-wide vertical nanobar array. The same sample was either viewed from the top (left) or tilted 45° (right). Scale bar, 5 pm. Statistical analysis of the geometrical dimensions for nanobars is shown in the table. Data are mean ⁇ SD.
  • FIG. 7B Representative SEM images of a vertical nanopillar array. The sample was tilted 45°. Zoom-in image shows one single nanopillar. The top, middle, and bottom diameters of each nanopillar were measured. Scale bars, 1 pm. Statistical analysis of the geometrical dimensions for nanopillars is shown in the table. Data are mean ⁇ SD.
  • FIG. 7A Representative SEM images of a 200 nm-wide vertical nanobar array. The same sample was either viewed from the top (left) or tilted 45° (right). Scale bar, 5 pm. Statistical analysis of the geometrical dimensions for nanobars is shown in the
  • FIGS. 8A-8B The ECM protein-coated quartz surfaces can support integrins to form adhesion structures.
  • FIG. 8A The mRNA levels of the integrin a and ⁇ subunits (presented in FIG. 1 D) in U2OS cells are listed. The mRNA levels are shown in the unit of transcripts per million (TPM) 21 .
  • FIG. 8B Fluorescence images of p subunits on flat surfaces that were coated with different ECM proteins (either gelatin, laminin, fibronectin or vitronectin). Endogenous pi (in the activated state) was immunolabelled; [33, [34, ⁇ 5, [36, and P8 were tagged with GFP and transiently expressed. Scale bar, 10 pm.
  • FIGS. 9A-9G Curvature induces the preferential accumulation of ⁇ V ⁇ 5 exclusively, related to FIG. 1 .
  • FIG. 9A Fluorescence images of integrin isoforms in U2OS cells 816 expressing a plasma membrane marker Fusion Red-CaaX on 200-nm nanobar arrays that were coated with different ECM proteins. Endogenous pi (in the activated state) was immunolabelled; [33, [36, and P8 were tagged with GFP and transiently expressed. The p subunits above show no preference for the ends of nanobars where the membranes are positively curved. Quantifications of their curvature preferences are presented in FIG. 1G. Scale bar, 5 pm. FIG.
  • FIG. 9B Fluorescence images of immunolabelled P5 (top) and ⁇ V ⁇ 5 (bottom) in U2OS cells on vitronectin-coated flat surfaces. Scale bar, 10 pm.
  • FIG. 9C Fluorescence images showing that immunolabelled P5 (top) and ⁇ V ⁇ 5 (bottom) preferentially accumulate at the ends of vitronectin-coated nanobars in U2OS cells expressing a plasma membrane marker FusionRed-CaaX. Scale bar, 5 pm.
  • FIG. 9D The curvature preferences of p5 and ⁇ V ⁇ 5 quantified, as in FIG. 1 G, by the nanobar end/side ratio of integrin/membrane. The ratios for individual nanobars were averaged to calculate the ratio for each cell.
  • FIG. 9E Fluorescence images of P5-GFP in MEFs (top) and Hela (bottom) cells on vitronectin-coated flat surfaces. Scale bar, 10 pm.
  • FIG. 9F Fluorescence images shows that P5-GFP preferentially accumulates at the ends of vitronectin-coated nanobars in MEFs (top) and Hela (bottom) cells transiently expressing a plasma membrane marker FusionRed-CaaX. Scale bar, 5 pm.
  • FIG. 9G The curvature preferences of P5 in MEFs and Hela cells quantified by the nanobar end/side ratio of integrin/membrane, as in FIG. 9D and FIG. 1 G.
  • N 12 cells for each condition, from two independent experiments. Data are mean ⁇ SD.
  • FIGS. 10A-10G ⁇ V ⁇ 5 accumulates at vitronectin-coated nanopillars to form curve adhesions, related to FIG. 2.
  • FIGS. 10A-10B Fluorescence images of ⁇ 5-GFP and plasma membrane marker Fusion Red-CaaX on flat surfaces (top) and nanopillars (bottom) coated with vitronectin (FIG. 10A) or gelatin (FIG. 10B). Ratiometric images of ⁇ 5/membrane (normalized by its mean per cell) are shown in the Parula colour scale. Scale bars, 10 pm.
  • FIG. 10C Quantification of the normalized 841 ⁇ 5/membrane ratio at individual nanopillars and flat surrounding regions coated with vitronectin or gelatin.
  • FIG. 10D Photo of a 3D-printed support for cell centrifugation assay.
  • FIG. 10F Immunofluorescence of pFAK in cells on vitronectin- coated nanopillars (left) and flat surfaces (right). Scale bar, 10 pm.
  • FIG. 10G The percentage of focal adhesion area in total cell area, for cells treated as in FIG. 10F. The focal adhesions are defined as regions with pFAK accumulation.
  • N 21 and 17 cells on nanopillars and flat surfaces, respectively, from two independent experiments. Data are mean ⁇ SD. Rvalues calculated using Kruskal-Wallis test with Dunn’s multiple-comparison (FIG. 10C) and unpaired two-tailed t test (FIGS. 10E, 10G).
  • FIGS. 11A-11 C Curved adhesions promote early-stage cell spreading.
  • FIGS. 11A-11 B Overlay of the bright-field and fluorescence 854 images showing wild type (left) and ⁇ 5-KD (right) cells stably expressing a plasma membrane marker GFP-CaaX on both nanopillars (left half) and flat surfaces (right half) in the same imaging fields. The cells were seeded on substrates coated with vitronectin (FIG. 11 A) or fibronectin (FIG. 11 B) 30 min before fixation. Scale bars, 50 pm.
  • FIG. 11 A vitronectin
  • FIG. 11 B fibronectin
  • FIGS. 12A-12H ⁇ 5 colocalizes with focal adhesion proteins on flat surfaces.
  • FIGS. 12A- 12H Fluorescence images show that ⁇ 5-GFP colocalizes with focal adhesion proteins talin (FIG. 12A), paxillin (FIG. 12B), vinculin (FIG. 12C), pFAK (FIG. 12D), LifeAct (FIG. 12E), zyxin (FIG. 12F), a-Actinin 1 (FIG. 12G), and a-Actinin 4 (FIG. 12H) at focal adhesion-like patches on vitronectin-coated flat surfaces. Endogenous paxillin, vinculin, and pFAK were immunolabelled.
  • FIGS. 13A-13H Nanopillar-induced ⁇ 5 accumulation correlate with a subset of focal adhesion proteins.
  • FIGS. 13A-13H Fluorescence images showing that at vitronectin-coated nanopillars,
  • FIGS. 14A-14E Curved adhesions are static.
  • FIG. 14A Fluorescence images of a live cell expressing ⁇ 5-GFP and a plasma membrane marker Fusion Red-CaaX on vitronectin-coated nanopillars. The ratiometric image of ⁇ 5/membrane (normalized by its mean per cell) is shown in the Parula colour scale. Scale bar, 10 pm.
  • FIG. 14B Kymograph of the normalized ⁇ 5/membrane ratio at nanopillars indicated by the white box in FIG. 14A. Images were acquired at 15 s/frame for 80 min. Scale bar, 1 pm.
  • FIGS. 14D-14E Time-lapse images of ⁇ 5-GFP and FusionRed-CaaX at nanopillar 1 (FIG. 14D) and 2 (FIG. 14E).
  • FIGS. 14D-14E Time-lapse images of ⁇ 5-GFP and FusionRed-CaaX at nanopillar 1 (FIG. 14D) and 2 (FIG. 14E).
  • the curved adhesion gradually assembled.
  • the curved adhesion gradually disassembled.
  • Scale bars 1 pm.
  • FIGS. 15A-15E The curvature preference of aV ⁇ 5 is determined by FCHo proteins, but not by talin or clathrin lattices, related to FIG. 4.
  • FIG. 15A Fluorescence images showing that mutant ⁇ 5 (Y752A Y770A) does not colocalize with talin897 mCherry at focal adhesions. Scale bar, 10 pm.
  • FIG. 15B Fluorescence images showing that both ⁇ 5-GFP and AP2-a, a marker for clathrin lattices, strongly accumulate at vitronectin-coated nanopillars, but their intensities are not spatially correlated.
  • the cell membrane was marked with transiently expressed surface SNAP- tag conjugated with Alexa Fluor 647.
  • the zoom-in images of ⁇ 5/membrane ratio and AP2-a in the white box-indicated area are shown in FIG. 4C.
  • FIG. 15C Scatter plot of the
  • N 299 pillars covered by the cell in FIG. 15B.
  • 15D Fluorescence images showing that 35-GFP accumulation spatially correlates with FCHo2-mCherry at vitronectin-coated nanopillars.
  • the cell membrane was marked with transiently expressed surface SNAP-tag conjugated with Alexa Fluor 647.
  • FIGS. 4E The zoom-in images of ⁇ 5/membrane ratio and FCHo2 in the white box-indicated area are shown in FIGS. 4E.
  • FIG. 15E Scatter plot of the 35/membrane ratio (normalized by its mean within the cell) against FCHo2 intensity at nanopillars.
  • N 305 pillars covered by the cell in FIG. 15D.
  • FIG. 16 FCHo1/2 KD reduces the curvature preference of ⁇ 5.
  • the transcription of shRNA in cells was indicated by the expression of blue fluorescent protein.
  • FIGS. 17A-17C aV918 35 ligands incorporation and super-resolution analysis of ECM fibres.
  • FIG. 17A Fluorescence images showing the colocalization of Alexa Fluor 647-collagen with fibronectin (immunolabelled with Alexa Fluor 488) and Cy3-vitronectin in sparse (left) and dense (right) ECM fibres. Scale bars, 10 pm.
  • FIG. 17B 3D super-resolution reconstruction of Alexa Fluor 647-collagen fibres. The widefield diffraction-limited image (top left) depicts collagen fibres.
  • FIG. 17C Extracting the diameter of individual fibres, (i) The image shows an example of a resolved individual fibre. Transverse line profiles (representative example shown at top) are taken across the fibre to estimate its diameter. Scale bar, 1 pm. (ii) Localizations from the line profile are binned into a histogram which is then fit to a Gaussian function (orange line). The full width at half maximum is extracted to estimate the diameter. The line profile in (i) yields the histogram and fit depicted in (ii).
  • FIGS. 18A-18C The formation of curved adhesions on ECM fibres requires ligand, curvature, and FCHol/2, related to FIG. 5.
  • FIG. 18A Top: fluorescence images showing that 35- GFP does not accumulate at places where the plasma membrane is deformed along sparse pure collagen fibres in WT cells.
  • Bottom fluorescence images showing that FCHol/2 KD reduces the 35-GFP accumulation at places where the plasma membrane is deformed along sparse ECM collagen fibres with aV ⁇ 5 ligands. Scale bar, 10 pm.
  • FIG. 18B Intensity profiles of fibre (blue), ⁇ 5 (green) and membrane (magenta) vertically crossing the membrane-wrapped fibre segments indicated in FIG.
  • FIG. 18A Lines and shaded areas indicate means and SD, respectively, along the fibre segments.
  • FIG. 18C Top: fluorescence images showing that ⁇ 5-GFP does not colocalize with FCHo2-mCherry on 2D surfaces coated with the same components of ECM fibres (i.e., collagen, fibronectin and vitronectin).
  • compositions, methods, and kits are provided for screening for candidate agents that inhibit formation of integrin-mediated curved adhesions. Screening assays may further include determining the effectiveness of candidate agents in reducing cell adhesion, proliferation, cell spreading, migration, or survival of cells and screening candidate agents for efficacy in treating disorders associated with integrin-mediated formation of curved adhesions.
  • curved adhesion refers to an integrin isoform ⁇ V ⁇ 5-mediated adhesion that forms at regions of a cell membrane that are curved. Formation of curved adhesions is mediated not only by integrin ⁇ V ⁇ 5 but also curvature-sensitive proteins such as FCHo2, which localize in curved adhesions. Curved adhesions can be distinguished from other cell adhesions based on the colocalization of integrin isoform ⁇ V ⁇ 5 and FCHo2 in curved adhesions. Curved adhesions can be detected, for example, using an assay that measures the proximity of integrin isoform ⁇ V ⁇ 5 to FCHo2, as described herein.
  • the term "administering" is intended to include routes of administration which allow the agent to perform its intended function of inhibiting formation of integrin-mediated curved adhesions and/or adhesion, proliferation, cell spreading, migration, or survival of cells.
  • routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal.
  • the injection can be bolus injections or can be continuous infusion.
  • the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function.
  • the agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. Further, the agent may be coadministered with a pharmaceutically acceptable carrier.
  • the agent also may be administered as a prodrug, which is converted to its active form in vivo.
  • determining refers to both quantitative and qualitative determinations and as such, the term “determining” is used interchangeably herein with “assaying,” “measuring,” and the like.
  • substantially purified generally refers to isolation of a substance (e.g., compound, polynucleotide, protein, polypeptide, antibody, aptamer, or inhibitor) such that the substance comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample.
  • Techniques for purifying polynucleotides and polypeptides of interest are well- known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • isolated is meant, when referring to a polypeptide or peptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type.
  • isolated with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
  • a peptide is said to "interact" with a protein if it binds specifically (e.g., in a lock-and-key type mechanism), non-specifically or in some combination of specific and non-specific binding.
  • a first peptide "interacts preferentially” with a protein if it binds (non-specifically and/or specifically) to the protein with greater affinity and/or greater specificity than it binds to other proteins (e.g., binds to integrin isoform ⁇ V ⁇ 5 or FCHo2 to a greater degree than to other proteins).
  • affinity refers to the strength of binding and can be expressed quantitatively as a dissociation constant (Kd).
  • a peptide interacts preferentially with integrin isoform ⁇ V ⁇ 5 or FCHo2 but, nonetheless, may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest).
  • weak binding, or background binding is readily discernible from the preferential interaction with the compound or polypeptide of interest, e.g., by use of appropriate controls.
  • inhibitor refers to any molecule (e.g., small molecule inhibitor, protein, polypeptide, peptide, fusion protein, inhibitory nucleic acid (e.g., siRNA, miRNA, antisense nucleic acid), peptide nucleic acid, antibody, antibody mimetic, aptamer) or CRISPR system that inhibits formation of integrin-mediated curved adhesions or integrin isoform ⁇ V ⁇ 5 or FCHo2 biological activity, and/or integrin isoform «V
  • an inhibitor may reduce the activity of integrin isoform ⁇ V ⁇ 5 or FCHo2 by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels.
  • the inhibitor may reduce the number or rate of formation of integrin-mediated curved adhesions or the colocalization of integrin isoform ⁇ V ⁇ 5 and FCHo2 in integrin-mediated curved adhesions by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels.
  • an "effective amount" of an inhibitor e.g., small molecule inhibitor, protein, polypeptide, peptide, fusion protein, inhibitory nucleic acid (e.g., siRNA, miRNA, antisense nucleic acid, or peptide nucleic acid), antibody, antibody mimetic, or aptamer) or CRISPR system targeting the integrin isoform ⁇ V ⁇ 5or FCHo2 gene (e.g., Cas9, Cas12a), RNA (Cas13), or epigenome (dCas9 fusion protein) is an amount sufficient to inhibit the formation of integrin-mediated curved adhesions, for example, by inhibiting activity of integrin isoform ⁇ V ⁇ 5 or FCHo2 and/or colocalization of integrin isoform ocV ⁇ 5 and FCHo2 in integrin-mediated curved adhesions.
  • An effective amount can be administered in one or more administrations, applications, or dosages.
  • tumor refers to a cell or population of cells whose growth, proliferation or survival is greater than growth, proliferation or survival of a normal counterpart cell, e.g. a cell proliferative, hyperproliferative or differentiative disorder. Typically, the growth is uncontrolled.
  • malignancy refers to invasion of nearby tissue.
  • metastasis or a secondary, recurring or recurrent tumor, cancer or neoplasia refers to spread or dissemination of a tumor, cancer or neoplasia to other sites, locations or regions within the subject, in which the sites, locations or regions are distinct from the primary tumor or cancer.
  • Neoplasia, tumors and cancers include benign, malignant, metastatic and non- metastatic types, and include any stage (I, II, III, IV or V) or grade (G1 , G2, G3, etc.) of neoplasia, tumor, or cancer, or a neoplasia, tumor, cancer or metastasis that is progressing, worsening, stabilized or in remission.
  • carcinomas such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, and small cell carcinoma
  • cancers such as, but are not limited to, pancreatic cancer, lung cancer (non-small cell lung cancer, small cell lung cancer), gastric cancer, ovarian cancer, endometrial cancer, colorectal cancer, oral cancer, skin cancer, cholangiocarcinoma, head and neck cancer, breast cancer, ovarian cancer, melanoma, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorders, colon cancer, endometrial cancer, ependymoma, esophage
  • carcinomas such as squamous cell carcinoma, adenocarcinoma, a
  • anti-tumor activity or “anti-cancer activity” is intended a reduction in the rate of cell proliferation, and hence a decline in growth rate of an existing tumor or in a tumor that arises during therapy, and/or destruction of existing neoplastic (tumor) cells or newly formed neoplastic cells, and hence a decrease in the overall size of a tumor during therapy. Such activity can be assessed using animal models.
  • “Disorders associated with integrin-mediated formation of curved adhesions” include various types of cancer, including without limitation, glioblastoma, breast cancer, pancreatic cancer, and prostate cancer; fibrotic diseases including, without limitation, pulmonary fibrosis, cardiac fibrosis, scleroderma/systemic sclerosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, nephrogenic systemic fibrosis, retroperitoneal fibrosis, arthrofibrosis, myelofibrosis, and mediastinal fibrosis; thrombosis; autoimmune disorders, including, without limitation, multiple sclerosis, Guillain-Barre syndrome, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, autoimmune liver disease, autoimmune colitis, autoimmune thyroid disorders such as Graves’ disease and Hashimoto's thyroiditis, and autoimmune encephalomye
  • a therapeutically effective dose or amount of an inhibitor is intended an amount that, when administered as described herein, brings about a positive therapeutic response in treatment of a disorder associated with formation of curved adhesions, such as an amount that has anti- cancer, anti-inflammatory, or anti-fibrotic activity.
  • a therapeutically effective dose or amount of an inhibitor may reduce cell adhesion, proliferation, cell spreading, migration, or survival of cells. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.
  • tumor response means a reduction or elimination of all measurable lesions.
  • the criteria for tumor response are based on the WHO Reporting Criteria [WHO Offset Publication, 48-World Health Organization, Geneva, Switzerland, (1979)]. Ideally, all uni- or bidimensionally measurable lesions should be measured at each assessment. When multiple lesions are present in any organ, such measurements may not be possible and, under such circumstances, up to 6 representative lesions should be selected, if available.
  • CR complete response
  • partial response means a 50% or greater reduction from baseline in the sum of the products of the longest perpendicular diameters of all measurable disease without progression of evaluable disease and without evidence of any new lesions as determined by at least two consecutive assessments at least four weeks apart. Assessments should show a partial decrease in the size of lytic lesions, recalcifications of lytic lesions, or decreased density of blastic lesions.
  • “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • “Pharmaceutically acceptable salt” includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts.
  • salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).
  • subject any member of the subphylum Chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • antibody encompasses polyclonal antibodies, monoclonal antibodies as well as hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies.
  • the term antibody includes: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991 ) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab') 2 and F(ab) fragments; F v molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659- 2662; and Ehrlich et al.
  • the phrase "specifically (or selectively) binds" with reference to binding of an antibody to an antigen refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies.
  • the specified antibodies bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample.
  • Specific binding to an antigen under such conditions may require an antibody that is selected for its specificity for a particular antigen.
  • antibodies raised to an antigen from specific species can be selected to obtain only those antibodies that are specifically immunoreactive with the antigen and not with other proteins, except for polymorphic variants and alleles. This selection may be achieved by subtracting out antibodies that cross-react with molecules from other species.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular antigen.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane. Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • microRNA refers to endogenous or artificial non-coding RNAs that are capable of regulating gene expression. It is believed that miRNAs function via RNA interference. When used herein in the context of inactivation, the use of the term microRNAs is intended to include also long non-coding RNAs, piRNAs, siRNAs, and the like. Endogenous (e.g., naturally occurring) miRNAs are typically expressed from RNA polymerase II promoters and are generated from a larger transcript.
  • siRNA and “short interfering RNA” are interchangeable and refer to single- stranded or double-stranded RNA molecules that are capable of inducing RNA interference. SiRNA molecules typically have a duplex region that is between 18 and 30 base pairs in length.
  • PiRNA and "Piwi-interacting RNA” are interchangeable and refer to a class of small RNAs involved in gene silencing. PiRNA molecules typically are between 26 and 31 nucleotides in length.
  • snRNA and "small nuclear RNA” are interchangeable and refer to a class of small RNAs involved in a variety of processes including RNA splicing and regulation of transcription factors.
  • the subclass of small nucleolar RNAs (snoRNAs) is also included.
  • the term is also intended to include artificial snRNAs, such as antisense derivatives of snRNAs comprising antisense sequences directed against the integrin isoform ⁇ V ⁇ 5 or FCHo2 gene.
  • antisense refers to any composition containing nucleotide sequences which are complementary to a specific DNA or RNA sequence.
  • the term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the “sense” strand.
  • Antisense molecules include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation. The designation “negative” is sometimes used in reference to the antisense strand, and “positive” is sometimes used in reference to the sense strand.
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • nucleic acid molecule polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single- stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide.
  • polynucleotide examples include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • PNAs peptide nucleic acids
  • polynucleotide As well as double- and single-stranded RNA, microRNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, "caps,” substitution of one or more of the naturally occurring nucleotides with an analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl aden
  • the term also includes locked nucleic acids (e.g., comprising a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom).
  • locked nucleic acids e.g., comprising a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom.
  • homologous region refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a "homologous region” is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term “homologous, region,” as used herein, refers to the ability of nucleic acid molecules to hybridize to each other. For example, a single-stranded nucleic acid molecule can have two homologous regions which are capable of hybridizing to each other. Thus, the term “homologous region” includes nucleic acid segments with complementary sequence.
  • Homologous regions may vary in length, but will typically be between 4 and 40 nucleotides (e.g., from about 4 to about 40, from about 5 to about 40, from about 5 to about 35, from about 5 to about 30, from about 5 to about 20, from about 6 to about 30, from about 6 to about 25, from about 6 to about 15, from about 7 to about 18, from about 8 to about 20, from about 8 to about 15, etc.).
  • nucleotides e.g., from about 4 to about 40, from about 5 to about 40, from about 5 to about 35, from about 5 to about 30, from about 5 to about 20, from about 6 to about 30, from about 6 to about 25, from about 6 to about 15, from about 7 to about 18, from about 8 to about 20, from about 8 to about 15, etc.
  • Complementary and “complementarity” are interchangeable and refer to the ability of polynucleotides to form base pairs with one another.
  • Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands or regions.
  • Complementary polynucleotide strands or regions can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G).
  • 100% complementary refers to the situation in which each nucleotide unit of one polynucleotide strand or region can hydrogen bond with each nucleotide unit of a second polynucleotide strand or region.
  • Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands or two regions can hydrogen bond with each other and can be expressed as a percentage.
  • a "target site” or “target sequence” for an inhibitory nucleic acid is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by an antisense oligonucleotide or inhibitory RNA molecule.
  • transfection is used to refer to the uptake of foreign DNA or RNA by a cell.
  • a cell has been "transfected” when exogenous DNA or RNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981 ) Gene 13:197.
  • Such techniques can be used to introduce one or more exogenous DNA or RNA moieties into suitable host cells.
  • the term refers to both stable and transient uptake of the genetic material, and includes uptake, for example, of microRNA, siRNA, piRNA, IncRNA, or antisense nucleic acids.
  • a “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas") genes.
  • one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system.
  • one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
  • Cas9 encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).
  • CRISPR type II clustered regularly interspaced short palindromic repeats
  • a Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA).
  • a gRNA may comprise a sequence "complementary" to a target sequence (e.g., major or minor allele), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence).
  • the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.
  • a gRNA will bind to a substantially complementary sequence and not to unrelated sequences.
  • a gRNA that "selectively binds" to a particular allele, such as a particular mutant allele e.g., allele comprising a substitution, insertion, or deletion) denotes a gRNA that binds preferentially to the particular target allele, but to a lesser extent to a wild-type allele or other sequences.
  • a gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.
  • donor polynucleotide refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).
  • HDR homology directed repair
  • a "target site” or “target sequence” is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide.
  • gRNA guide RNA
  • the target site may be allele-specific (e.g., a major or minor allele).
  • homology arm is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell.
  • the donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA.
  • the homology arms are referred to herein as 5' and 3' (i.e. , upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide.
  • the 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence” and "3' target sequence,” respectively.
  • the nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.
  • administering a nucleic acid, such as an inhibitory or regulatory nucleic acid (e.g., microRNA, siRNA, piRNA, snRNA, antisense nucleic acid, or IncRNA), or a CRISPR system (expressing, e.g., a donor polynucleotide, guide RNA, Cas protein (e.g., Cas9, Cas12a, Cas12d, Cas13, or dCas9)) to a cell comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.
  • an inhibitory or regulatory nucleic acid e.g., microRNA, siRNA, piRNA, snRNA, antisense nucleic acid, or IncRNA
  • CRISPR system expressing, e.g., a donor polynucleotide, guide
  • Integrin-mediated curved adhesions can be distinguished from other cell adhesions based on the colocalization of integrin isoform ⁇ V ⁇ 5 and FCHo2 in curved adhesions. Accordingly, integrin-mediated curved adhesions can be detected by measuring the proximity of integrin isoform «V
  • proximity assays include, without limitation, complementation assays using a split reporter protein, fluorescence resonance energy transfer (FRET) assays, bioluminescence resonance energy transfer (BRET) assays, proximity ligation assays (PLA), and proximity-dependent initiation of hybridization chain reaction (proxHCR) assays.
  • FRET fluorescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • PHA proximity ligation assays
  • proxHCR proximity-dependent initiation of hybridization chain reaction
  • a first reporter protein fragment is linked to integrin isoform ⁇ V ⁇ 5, and a second reporter protein fragment is linked to FCHo2, wherein colocalization of the integrin isoform ⁇ V ⁇ 5 and the FCHo2 in a curved adhesion brings the two fragments of the reporter protein in close enough proximity for a reconstituted functional reporter protein to form that can be detected.
  • the split reporter protein can be any protein that can be split into two parts and reconstituted non-covalently to form a functional protein that can be detected.
  • the reconstituted reporter protein may provide, for example, a fluorescent, bioluminescent, chemiluminescent, or chromogenic signal.
  • the assay is typically performed with a split fluorescent protein, a split bioluminescent protein, or a split enzyme (e.g., a split horseradish peroxidase or a split [3-galactosidase) used with a chemiluminescent or chromogenic substrate.
  • a split fluorescent protein e.g., a split bioluminescent protein, or a split enzyme
  • a split horseradish peroxidase or a split [3-galactosidase e.g., horseradish peroxidase or a split [3-galactosidase
  • the split reporter protein is a split fluorescent protein.
  • split fluorescent proteins include, without limitation, a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, and a split sfCherry2 fluorescent protein.
  • the fluorescent signal of the reconstituted fluorescent protein can be measured, for example, using fluorescence microscopy, two-photon excitation microscopy, fluorescence confocal laser scanning microscopy, or light sheet fluorescence microscopy.
  • 35 to the FCHo2 comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split fluorescent protein connected to the FCHo2, wherein a reconstituted fluorescent protein forms when the first component and the second component of the split fluorescent protein are expressed and interact to produce a detectable fluorescent signal; and detecting the fluorescent signal to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 from the fluorescent signal, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved
  • the split reporter protein is a split bioluminescent protein.
  • split bioluminescent proteins include, without limitation, split aequorins and split luciferases, such as, but not limited to, a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, and a split nano-lantern luciferase.
  • the luminescent signal of the reconstituted bioluminescent protein can be measured, for example, using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split luminescent protein connected to the FCHo2, wherein a reconstituted luminescent protein forms when the first component and the second component of the split luminescent protein are expressed and interact to produce a detectable luminescent signal; and detecting the luminescent signal to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 from the luminescent signal, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether
  • the split luminescent protein is a split aequorin or a split luciferase such as, but not limited to, a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
  • the luminescent signal is measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
  • a FRET assay energy is transferred from a donor fluorophore to an acceptor fluorophore upon illumination at an excitation wavelength of the donor fluorophore.
  • the emission spectrum of the donor fluorophore must overlap with the excitation spectrum of the acceptor fluorophore. Fluorescence resonance energy transfer from the donor fluorophore to the acceptor fluorophore results in a fluorescent emission from the acceptor fluorophore, which emits fluorescence at a longer wavelength than the donor fluorophore.
  • FRET efficiency between a donor fluorophore and an acceptor fluorophore can be used to determine the distance between the integrin isoform «V
  • FRET efficiency can be measured, for example, using fluorescence microscopy or fluorescence confocal laser scanning microscopy.
  • the FRET donor and the FRET acceptor are fluorescent proteins or fluorescent dyes.
  • the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein
  • the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein
  • the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5)
  • the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine.
  • 35 to the FCHo2 comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a FRET donor connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the integrin isoform ⁇ V ⁇ 5 or FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a FRET acceptor connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • BRET assays are similar to FRET assays but have the advantage of not requiring external illumination for the energy transfer between a donor and an acceptor.
  • BRET efficiency between a bioluminescent donor and a bioluminescent acceptor is used to determine the distance between the integrin isoform ⁇ V ⁇ 5 and the FCHo2.
  • BRET efficiency can be measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
  • the BRET donor or the BRET acceptor is a bioluminescent protein or a luminescent quantum dot.
  • the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 with a BRET assay comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a BRET donor connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 with a BRET assay comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a BRET acceptor connected to the integrin isoform « ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • a PLA assay can also be used to determine the proximity of integrin isoform ⁇ V ⁇ 5 to FCHo2.
  • PLA uses oligonucleotides conjugated to antibodies, which are brought in proximity upon binding to colocalized antigens.
  • PLA probes comprising oligonucleotides conjugated to secondary antibodies, which specifically bind to the primary antibodies, are used for detection.
  • the oligonucleotides can be ligated and amplified.
  • a bridge oligonucleotide is used to facilitate ligation.
  • the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with a first oligonucleotide of a first PLA probe and a second portion sufficiently complementary to hybridize with a second oligonucleotide of a second PLA probe when the first oligonucleotide and the second oligonucleotide are in sufficient proximity to each other to simultaneously hybridize to the bridge oligonucleotide.
  • the ligation product can be amplified, for example, using polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification. In some embodiments quantitative PCR is used to amplify the ligation product.
  • circularizing oligonucleotides are hybridized to the oligonucleotides of the PLA probes and ligated to form a circular template for rolling circle amplification.
  • Amplicons can be detected with detectably labeled probes.
  • Detectably labeled probes that bind to the amplicons may comprise, for example, a fluorescent label, a bioluminescent label, or a chemiluminescent label.
  • PLA see, e.g., Lof et al. (2017) Curr Protoc Cytom. 81 :4.8.1-4.8.10, Gullberg et al. (2004) Proc. Natl. Acad. Sci.
  • performing the PLA comprises: contacting a cell, a population of cells, or a tissue with a first antibody that specifically binds to integrin isoform ⁇ V ⁇ 5; contacting the cell, population of cells, or tissue with a second antibody that specifically binds to FCHo2; contacting the first antibody with a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; contacting the second antibody with a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody; contacting the first probe and the second probe with a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with
  • amplifying comprises performing polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification. In some embodiments, amplifying comprises performing quantitative PCR. In some embodiments, the amplicon is detected with a detectably labeled probe that binds to the amplicon.
  • PCR polymerase chain reaction
  • isothermal amplification or rolling circle amplification.
  • amplifying comprises performing quantitative PCR.
  • the amplicon is detected with a detectably labeled probe that binds to the amplicon.
  • the first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab') 2 fragment, an F v fragment, and an scF v fragment.
  • Proximity-dependent initiation of hybridization chain reaction is similar to PLA but uses antibodies conjugated to fluorescently labeled oligonucleotides having a hairpin conformation.
  • the hairpins of the two probes are partly reversely complementary to each other and can cross-hybridize.
  • An activator oligonucleotide is used to open the secondary structure of one of the proximity probes to release it 3’-end, which then invades and unfolds the other proximity probe, thereby liberating its 3’-end. This initiates a hybridization chain reaction (HCR), which is used for signal amplification.
  • HCR hybridization chain reaction
  • the two oligonucleotide hairpin probes are conjugated to antibodies that must bind in close proximity to initiate the HCR to generate a detectable fluorescent product.
  • the proximity of integrin isoform ⁇ V ⁇ 5 to FCHo2 can be determined using this method with a pair of fluorescently labeled hairpin probes conjugated to antibodies that specifically bind to integrin isoform ⁇ V ⁇ 5 and FCHo2.
  • proxHCR see, e.g., Koos et al. (2015) Nat. Commun. 6:7294, Raykova et al. (2016) Proteomes 4(4):36; herein incorporated by reference.
  • curved adhesions are detected on a cell, a population of cells, or tissue in vitro (i.e. , grown in culture) or in vivo. Curved adhesions may be detected on any type of cell or tissue, including normal cells or tissue, diseased or damaged cells or tissue, cancerous cells or tissue, inflammatory cells or inflamed tissue, or cells or tissue at risk of future disease (e.g., cells of precancerous tissue, transplanted tissue, tissue at high risk of tumor or disease development due to an underlying genetic abnormality, mutation, or autoimmunity).
  • Cells and tissue suitable for use with the methods described herein generally include any type of cells or tissue collected from living or dead subjects, such as cells or tissue from biopsy specimens, surgical specimens, and autopsy specimens, including, but not limited to, epithelium, muscle, connective, and nervous tissue.
  • a vertical nanostructure is used to facilitate the formation of curved adhesions in assays.
  • the nanostructure is a nanobar or a nanopillar.
  • the nanostructure is coated with vitronectin or fibronectin. (See Examples.)
  • the vertical nanostructure has an end curvature diameter ranging from 100 nm to 3 pm, including any end curvature diameter in this range, such as 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 800 nm, 825 nm, 850 nm, 900 nm, 925 nm, 950 nm, 975 nm, 1 pm, 1 .5 pm, 2 pm, 2.5 pm, or 3 pm
  • fibers comprising collagen, fibronectin, or vitronectin, or a combination thereof, may be used to facilitate the formation of integrin-mediated curved adhesions, wherein the integrin-mediated curved adhesions form on the surface of the fibers.
  • a curved extracellular matrix (ECM) surface may also be used to facilitate the formation of integrin-mediated curved adhesions, wherein a cell forms the integrin-mediated curved adhesions on the curved ECM surface.
  • Integrin-mediated curved adhesions can be detected and measured based on the colocalization of integrin isoform ⁇ V ⁇ 5 and FCHo2 using a proximity assay, as described above.
  • a cell, population of cells, or a tissue is contacted with a candidate agent; and the proximity of integrin isoform ⁇ V ⁇ 5 to FCHo2 is measured in the presence and absence of the candidate agent to determine whether the inhibitor inhibits colocalization of integrin isoform «VP5 and FCHo2 in integrin-mediated curved adhesions, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • Assays can be applied to any cell or cell fragment of interest including, without limitation, cancer cells, blood cells, inflammatory cells, fibroblasts, and epithelial cells, as well as platelets.
  • proximity assays may be used for this purpose, and in many embodiments, a candidate agent will be tested in different assays to confirm inhibitory capability as well as efficacy in treating a disorder associated with integrin-mediated formation of curved adhesions.
  • proximity assays may be used to determine the ability of an agent to inhibit integrin- mediated formation of curved adhesions.
  • cell-based or tissue assays may be used for testing for the effectiveness of candidate agents in reducing cell adhesion, proliferation, cell spreading, migration, or survival of cells.
  • An inhibitor can be any molecule including, without limitation, a small molecule inhibitor, protein, polypeptide, peptide, fusion protein, nucleic acid, oligonucleotide, peptide nucleic acid, antibody or fragment thereof, antibody mimetic, aptamer, or a CRISPR system that inhibits formation of integrin-mediated curved adhesions or integrin isoform «V
  • a small molecule inhibitor e.g., protein, polypeptide, peptide, fusion protein, nucleic acid, oligonucleotide, peptide nucleic acid, antibody or fragment thereof, antibody mimetic, aptamer, or a CRISPR system that inhibits formation of integrin-mediated curved adhesions or integrin isoform «V
  • Inhibition may be complete or partial (i.e., all formation of integrin-mediated curved adhesions, some formation of integrin-mediated curved adhesions, or most formation of integrin- mediated curved adhesions; all activity, some activity, or most activity of integrin isoform «V
  • an inhibitor may reduce the activity of integrin isoform ⁇ V ⁇ 5 or FCHo2 by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels.
  • the inhibitor may reduce the number or rate of formation of integrin-mediated curved adhesions or the colocalization of integrin isoform ⁇ V ⁇ 5 and FCHo2 in integrin-mediated curved adhesions by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels.
  • any convenient format may be used for the assay, e.g., wells, plates, flasks, etc., preferably a high throughput format, such as multi-well plates.
  • Inhibitors can be identified by contacting a cell, a population of cells, or a tissue with a candidate agent; and measuring inhibition of formation of integrin-mediated curved adhesions by the candidate agent.
  • Assays may further include suitable controls (e.g., a sample comprising cells or tissue in the absence of the test agent). Generally, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
  • suitable controls e.g., a sample comprising cells or tissue in the absence of the test agent.
  • a variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc., including agents that are used to facilitate optimal binding activity and/or reduce non-specific or background activity. Reagents that improve the efficiency of the assay, such as nanostructures, fibers, or curved extracellular matrix, as described herein, protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The components of the assay mixture are added in any order that provides for the requisite activity. Incubations are performed at any suitable temperature, typically between 4°C and 40°C. Incubation periods are selected for optimum activity but may also be optimized to facilitate rapid high-throughput screening. In some embodiments, between 0.1 hour and 1 hour, between 1 hour and 2 hours, or between 2 hours and 4 hours, will be sufficient.
  • test agents encompass numerous chemical classes, e.g., small organic compounds having a molecular weight of more than 50 daltons and less than about 10,000 daltons, less than about 5,000 daltons, or less than about 2,500 daltons.
  • Test agents can comprise functional groups necessary for structural interaction with proteins, e.g., hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl or carboxyl group, or at least two of the functional chemical groups.
  • the test agents can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Test agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • screening may be directed to known pharmacologically active compounds and chemical analogs thereof, or to new agents with unknown properties such as those created through rational drug design.
  • test agents are synthetic compounds.
  • a number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. See for example WO 94/24314, hereby expressly incorporated by reference, which discusses methods for generating new compounds, including random chemistry methods as well as enzymatic methods.
  • test agents are provided as libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.
  • test agents are organic moieties.
  • test agents are synthesized from a series of substrates that can be chemically modified. “Chemically modified” herein includes traditional chemical reactions as well as enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero
  • test agents are assessed for any cytotoxic activity it may exhibit toward a living eukaryotic cell, using well-known assays, such as trypan blue dye exclusion, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide) assay, and the like. Agents that do not exhibit significant cytotoxic activity are considered candidate agents.
  • the test agent is an antibody that specifically binds to and inhibits biological activity of integrin isoform ⁇ V ⁇ 5 or FCHo2.
  • Any type of antibody may be screened for the ability to inhibit integrin isoform ⁇ V ⁇ 5 or FCHo2 by the methods described herein, including polyclonal antibodies, monoclonal antibodies, hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No.
  • F(ab') 2 and F(ab) fragments F(ab') 2 and F(ab) fragments
  • F v molecules noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096
  • single- chain Fv molecules sFv
  • sdAb single-domain antibodies
  • the test agent is an aptamer that specifically binds to and inhibits biological activity of integrin isoform ⁇ V ⁇ 5 or FCHo2.
  • Aptamers may be isolated from a combinatorial library and improved by directed mutation or repeated rounds of mutagenesis and selection.
  • Aptamers see, e.g., Aptamers: Tools for Nanotherapy and Molecular Imaging (R.N. Veedu ed., Pan Stanford, 2016), Nucleic Acid and Peptide Aptamers: Methods and Protocols (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2009), Aptamers Selected by Cell-SELEX for Theranostics (W.
  • the test agent is an antibody mimetic that specifically binds to and inhibits biological activity of integrin isoform ⁇ V ⁇ 5 or FCHo2.
  • Any type of antibody mimetic may be used as an inhibitor, including, but not limited to, affibody molecules (Nygren (2008) FEBS J. 275 (11 ):2668-2676), affilins (Ebersbach et al. (2007) J. Mol. Biol. 372 (1 ) :172-185), affimers (Johnson et al. (2012) Anal. Chem. 84 (15):6553-6560), affitins (Krehenbrink et al. (2008) J. Mol. Biol.
  • a candidate agent that inhibits formation of integrin-mediated curved adhesions is further tested for its effects on cell adhesion, proliferation, cell spreading, migration, or survival of cells in a cell-based assay.
  • a candidate agent of interest is contacted with the cells; and the effect, if any, of the candidate agent on the cells is determined.
  • a population of cells can be cultured in vitro in the presence of an effective dose of a candidate agent.
  • the effects on formation of integrin-mediated curved adhesions, cell adhesion, proliferation, cell spreading, migration, or survival, or any combination thereof, may be assayed.
  • the candidate agent is added to the culture medium, and the culture medium is maintained under conventional conditions suitable for growth of the cells.
  • Various commercially available systems have been developed for the growth of mammalian cells to provide for removal of adverse metabolic products, replenishment of nutrients, and maintenance of oxygen. By employing these systems, the medium may be maintained as a continuous medium, so that the concentrations of the various ingredients are maintained relatively constant or within a prescribed range.
  • a test compound identified as inhibiting formation of integrin- mediated curved adhesions, is further tested for its efficacy in treating a disorder associated with integrin-mediated formation of curved adhesions in vivo, e.g., in an animal such as an animal model of a disorder associated with integrin-mediated formation of curved adhesions.
  • an agent that inhibits integrin-mediated formation of curved adhesions can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified, as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • Monitoring the efficacy of agents e.g., drugs
  • monitoring the efficacy of agents e.g., drugs
  • monitoring the efficacy of agents e.g., drugs for treatment of a disorder associated with integrin-mediated formation of curved adhesions can be applied not only in basic drug screening, but also in clinical trials.
  • Test compounds can be tested for efficacy in treating various disorders associated with integrin-mediated formation of curved adhesions including, but not limited to, cancers such as glioblastoma, breast cancer, pancreatic cancer, and prostate cancer; fibrotic diseases such as pulmonary fibrosis, cardiac fibrosis, scleroderma/systemic sclerosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, nephrogenic systemic fibrosis, retroperitoneal fibrosis, arthrofibrosis, myelofibrosis, and mediastinal fibrosis; thrombosis; autoimmune disorders such as multiple sclerosis, Guillain-Barre syndrome, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, autoimmune liver disease, autoimmune colitis, autoimmune thyroid disorders such as Graves’ disease and Hashimoto's thyroiditis, and autoimmune encephalomy
  • an inhibitor may reduce integrin isoform ⁇ V ⁇ 5 or FCHo2 gene expression (e.g., transcription or translation).
  • Candidate agents are identified by contacting a cell, a population of cells, or a tissue with a candidate compound and measuring the expression of integrin isoform ⁇ V ⁇ 5 or FCHo2, as determined by, e.g., mRNA or polypeptide levels. The level of expression of integrin isoform ⁇ V ⁇ 5 or FCHo2 mRNA or protein in the presence of the candidate compound is compared to the level of expression of integrin isoform ⁇ V ⁇ 5 or FCHo2 mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified based on this comparison.
  • the candidate compound when expression of integrin isoform ⁇ V ⁇ 5 or FCHo2 mRNA or protein in cells is decreased (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an agent that inhibits integrin isoform aV ⁇ 5 or FCHo2 expression.
  • expression of integrin isoform ⁇ V ⁇ 5 or FCHo2 mRNA or protein is the same or increased (statistically significantly more) in the presence of the candidate compound than in its absence, the candidate compound is likely not an agent that inhibits integrin isoform ⁇ V ⁇ 5 or FCHo2 expression or activity.
  • an inhibitor of integrin isoform ⁇ V ⁇ 5 or FCHo2 gene expression reduces integrin isoform ⁇ V ⁇ 5 or FCHo2 mRNA or protein levels by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels.
  • Inhibitors of integrin isoform ⁇ V ⁇ 5 or FCHo2 gene expression can include, but are not limited to, antisense oligonucleotides, inhibitory RNA molecules, such as miRNAs, siRNAs, piRNAs, and snRNAs, peptide nucleic acids, small molecule inhibitors, and CRISPR systems designed for genome, RNA transcript, or epigenome editing.
  • inhibitors for inhibiting nucleic acid function are well known in the art. See e.g., International patent application WO/2012/018881 ; U.S. patent application 2011/0251261 ; U.S. patent no. 6,713,457; Kole et al. (2012) Nat. Rev. Drug Discov. 11 (2):125-40; Sanghvi (201 1 ) Curr. Protoc. Nucleic Acid Chem. Chapter 4:Unit 4.1.1-22; herein incorporated by reference in their entireties.
  • Inhibitors can be single stranded or double stranded polynucleotides and may contain one or more chemical modifications, such as, but not limited to, locked nucleic acids, peptide nucleic acids, sugar modifications, such as 2'-O-alkyl (e.g., 2'-O-methyl, 2'-0-methoxyethyl), 2'-fluoro, and 4'-thio modifications, and backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages.
  • chemical modifications such as, but not limited to, locked nucleic acids, peptide nucleic acids, sugar modifications, such as 2'-O-alkyl (e.g., 2'-O-methyl, 2'-0-methoxyethyl), 2'-fluoro, and 4'-thio modifications, and backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages.
  • inhibitory RNA molecules may have a "tail" covalently attached to their 3'- and/or 5'-end, which may be used to stabilize the RNA inhibitory molecule or enhance cellular uptake.
  • tails include, but are not limited to, intercalating groups, various kinds of reporter groups, and lipophilic groups attached to the 3' or 5' ends of the RNA molecules.
  • the RNA inhibitory molecule is conjugated to cholesterol or acridine. See, for example, the following for descriptions of syntheses of 3'- cholesterol or 3'-acridine modified oligonucleotides: Gamper, H. B., Reed, M. W., Cox, T., Virosco, J. S., Adams, A.
  • Additional lipophilic moieties that can be used, include, but are not limited to, oleyl, retinyl, and cholesteryl residues, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, 1 ,3-Bis- O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1 ,3- propanediol, heptadecyl group, palmitic acid, myristic acid, 0 3 -(oleoyl)lithocholic acid, 0 3 - (oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. Additional compounds, and methods of use, are set out in US Patent Publication Nos. 2010/0076056, 2009/0247608 and 2009/0131360; herein
  • inhibition of integrin isoform ⁇ V ⁇ 5 or FCHo2 function may be achieved by administering antisense oligonucleotides targeting the integrin isoform ⁇ V ⁇ 5 or FCHo2 gene.
  • the antisense oligonucleotides may be ribonucleotides or deoxyribonucleotides.
  • the antisense oligonucleotides have at least one chemical modification.
  • Antisense oligonucleotides may be comprised of one or more "locked nucleic acids".
  • LNAs Locked nucleic acids
  • the antisense oligonucleotides may comprise peptide nucleic acids (PNAs), which contain a peptide-based backbone rather than a sugar-phosphate backbone.
  • PNAs peptide nucleic acids
  • the antisense oligonucleotides may contain one or more chemical modifications, including, but are not limited to, sugar modifications, such as 2'-O-alkyl (e.g.
  • suitable antisense oligonucleotides are 2'-O- methoxyethyl "gapmers" which contain 2'-0-methoxyethyl-modified ribonucleotides on both 5' and 3' ends with at least ten deoxyribonucleotides in the center. These "gapmers” are capable of triggering RNase H-dependent degradation mechanisms of RNA targets.
  • Other modifications of antisense oligonucleotides to enhance stability and improve efficacy such as those described in U.S. Pat. No. 6,838,283, which is herein incorporated by reference in its entirety, are known in the art and are suitable for use in the methods of the invention.
  • Antisense oligonucleotides may comprise a sequence that is at least partially complementary to an integrin isoform ⁇ V ⁇ 5 or FCHo2 target sequence, e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to the integrin isoform ⁇ V ⁇ 5 or FCHo2 target sequence.
  • the antisense oligonucleotide may be substantially complementary to the integrin isoform ⁇ V ⁇ 5 or FCHo2 target sequence, that is at least about 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence.
  • the antisense oligonucleotide comprises a sequence that is 100% complementary to the integrin isoform ⁇ V ⁇ 5 or FCHo2 target sequence.
  • the inhibitor of integrin isoform ⁇ V ⁇ 5 or FCHo2 is an inhibitory RNA molecule (e.g., a miRNA, a siRNA, a piRNA, or a snRNA) having a single-stranded or double-stranded region that is at least partially complementary to the target sequence of integrin isoform ⁇ V ⁇ 5 or FCHo2, e.g., about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence of integrin isoform ⁇ V ⁇ 5 or FCHo2.
  • an inhibitory RNA molecule e.g., a miRNA, a siRNA, a piRNA, or a snRNA having a single-stranded or double-stranded region that is at least partially complementary to the target sequence of integrin isoform ⁇ V ⁇ 5 or FCHo2, e.g., about 75%, 80%,
  • the inhibitory RNA comprises a sequence that is substantially complementary to the target sequence of integrin isoform ⁇ V ⁇ 5 or FCHo2, e.g., about 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence.
  • the inhibitory RNA molecule may contain a region that has 100% complementarity to the target sequence.
  • the inhibitory molecules may target the integrin isoform ⁇ V ⁇ 5 or FCHo2 sequence of SEQ ID NO:1 .
  • the inhibitory RNA molecule may be a double-stranded, small interfering RNA or a short hairpin RNA molecule (shRNA) comprising a stem-loop structure.
  • Inhibitors can be detectably labeled by well-known techniques. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Such labeled inhibitors can be used to determine cellular uptake efficiency, quantitate binding of inhibitors at target sites, or visualize inhibitor localization.
  • a CRISPR/Cas system is used to inactivate or reduce expression of an endogenous gene expressing integrin isoform «V
  • a CRISPR/Cas system can be used to delete, inactivate, or mutate an endogenous gene expressing integrin isoform ⁇ V ⁇ 5 or FCHo2 to eliminate or reduce integrin isoform ⁇ V ⁇ 5 or FCHo2 gene expression or protein activity.
  • Genome modification can be performed, for example, using homology directed repair (HDR) with a donor polynucleotide comprising a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the target locus (e.g., integrin isoform ⁇ V ⁇ 5 or FCHo2 gene) to be edited in a cell.
  • the donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence.
  • the homology arms are referred to herein as 5' and 3' (i.e.
  • upstream and downstream homology arms which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide.
  • the 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence” and "3' target sequence,” respectively.
  • the homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus.
  • a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e. , sufficiently complementary for hybridization) by the 5' and 3' homology arms.
  • the corresponding homologous nucleotide sequences in the genomic target sequence flank a specific site for cleavage and/or a specific site for introducing the intended edit.
  • the distance between the specific cleavage site and the homologous nucleotide sequences can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides).
  • the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.
  • a homology arm can be of any length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc.
  • the 5' and 3' homology arms are substantially equal in length to one another, e.g.
  • the 5' and 3' homology arms are substantially different in length from one another, e.g. one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.
  • the donor polynucleotide is used in combination with an RNA-guided nuclease, which is targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by a guide RNA (gRNA).
  • gRNA guide RNA
  • a target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease- gRNA complex by hybridization at the target site.
  • the gRNA can be designed with a sequence complementary to a target sequence in the integrin isoform ⁇ V ⁇ 5 or FCHo2gene.
  • the gRNA is designed to with a sequence complementary to a cancer- specific integrin isoform ⁇ V ⁇ 5 or FCHo2 mutation to target the nuclease-gRNA complex to the site of a mutation in a cell.
  • the mutation may comprise an insertion, a deletion, or a substitution.
  • the mutation may include a single nucleotide variation, gene fusion, translocation, inversion, duplication, frameshift, missense, nonsense, or other mutation.
  • the targeted minor allele may be a common genetic variant or a rare genetic variant.
  • the gRNA is designed to selectively bind to a minor allele with single base-pair discrimination, for example, to allow binding of the nuclease-gRNA complex to a single nucleotide polymorphism (SNP).
  • the gRNA may be designed to target disease-relevant mutations of interest for the purpose of genome editing to delete or deactivate the integrin isoform ⁇ V ⁇ 5 or FCHo2 gene in a cell.
  • the RNA-guided nuclease used for genome modification is a CRISPR system Cas nuclease.
  • Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Cas nucleases.
  • Cas proteins include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Casi o, Cas10d, Cas12a (Cpf 1 ), Cas12b (C2c1 ), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), CasF, CasG, CasH, Csy1 , Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Cs
  • a type II CRISPR system Cas9 endonuclease is used.
  • Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks
  • the Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced.
  • Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database.
  • sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol.
  • the CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA.
  • the bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break.
  • gRNA guide RNA
  • Targeting of Cas9 typically further relies on the presence of a 5' protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.
  • PAM 5' protospacer-adjacent motif
  • the genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM).
  • the target site comprises 20-30 base pairs in addition to a 3 base pair PAM.
  • the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen.
  • Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide.
  • the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9- gRNA complex to the allele.
  • the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 nucleotides in length.
  • the guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.
  • Cpf1 the CRISPR nuclease from Prevotella and Francisella 1
  • Cas12a the CRISPR nuclease from Prevotella and Francisella 1
  • Cpf1 is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpf1 does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpf1 for targeting than Cas9.
  • Cpf 1 is capable of cleaving either DNA or RNA.
  • the PAM sites recognized by Cpf 1 have the sequences 5'-YTN-3' (where "Y” is a pyrimidine and “N” is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9.
  • Cpf1 cleavage of DNA produces double-stranded breaks with a sticky- ends having a 4 or 5 nucleotide overhang.
  • Cpf 1 see, e.g., Ledford et al. (2015) Nature. 526 (7571 ):17-17, Zetsche et al. (2015) Cell. 163 (3):759-771 , Murovec et al. (2017) Plant Biotechnol. J.
  • Cas12b (C2c1) is another class II CRISPR/Cas system RNA-guided nuclease that may be used.
  • C2c1 similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites.
  • RNA-guided Fokl nucleases comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (Fokl-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on Fokl.
  • dCas9 inactive Cas9
  • Fokl-dCas9 Fokl endonuclease
  • engineered RNA-guided Fokl nucleases see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; herein incorporated by reference.
  • RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector).
  • RNA e.g., messenger RNA
  • DNA expression vector
  • the RNA- guided nuclease and the gRNA are both provided by vectors. Both can be expressed by a single vector or separately on different vectors.
  • the vector(s) encoding the RNA-guided nuclease an gRNA may be included in a CRISPR expression system to target the integrin isoform ⁇ V ⁇ 5 or FCHo2 gene in cells.
  • Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell or organism.
  • a nucleic acid encoding an RNA-guided nuclease or reverse transcriptase can be modified to substitute codons having a higher frequency of usage in a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence.
  • the protein can be transiently, conditionally, or constitutively expressed in the cell.
  • CRISPR interference is used to repress integrin isoform ⁇ V ⁇ 5 or FCHo2 gene expression.
  • CRISPRi is performed with a complex of a catalytically inactive Cas9 (dCas9) with a guide RNA that targets the integrin isoform ⁇ V ⁇ 5 or FCHo2 gene.
  • An engineered nuclease-deactivated Cas9 (dCas9) is used to allow sequence-specific targeting without cleavage.
  • Nuclease-deactivated forms of Cas9 may be engineered by mutating catalytic residues at the active site of Cas9 to destroy nuclease activity.
  • nuclease deficient Cas9 protein from any species may be used as long as the engineered dCas9 retains gRNA-mediated sequence-specific targeting.
  • the nuclease activity of Cas9 from Streptococcus pyogenes can be deactivated by introducing two mutations (D10A and H841 A) in the RuvC1 and HNH nuclease domains.
  • Other engineered dCas9 proteins may be produced by similarly mutating the corresponding residues in other bacterial Cas9 isoforms.
  • engineered nuclease-deactivated forms of Cas9 see, e.g., Qi et al. (2013) Cell 152:1173-1183, Dominguez et al. (2016) Nat. Rev. Mol. Cell. Biol. 17(1 ):5-15; herein incorporated by reference in their entireties.
  • the dCas9 protein can be designed to target the integrin isoform ocVps or FCHo2 gene by altering its guide RNA sequence.
  • a target-specific single guide RNA comprises a nucleotide sequence that is complementary to a target site, and thereby mediates binding of the dCas9-sgRNA complex by hybridization at the target site.
  • CRISPRi can be used to sterically repress transcription by blocking either transcriptional initiation or elongation by designing a sgRNA with a sequence complementary to an integrin isoform ⁇ V ⁇ 5 or FCHo2 promoter or exonic sequence.
  • the sgRNA may be complementary to the non-template strand or the template strand, but preferably is complementary to the non-template strand to more strongly repress transcription.
  • the target site will typically comprise a nucleotide sequence that is complementary to the sgRNA, and may further comprise a protospacer adjacent motif (PAM).
  • the target site comprises 20-30 base pairs in addition to a 3 base pair PAM.
  • the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen.
  • Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide.
  • the sgRNA comprises 5-50 nucleotides, 10-30 nucleotides, 15- 25 nucleotides, 18-22 nucleotides, 19-21 nucleotides, and any length between the stated ranges, including, for example, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
  • sgRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et aL, Tetrahedron (1992) 48:2223-2311 ; and Applied Biosystems User Bulletin No. 13 (1 April 1987).
  • Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et aL, Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et aL, Meth. Enzymol. (1979) 68:109.
  • the dCas9 is fused to a transcriptional repressor domain capable of further repressing transcription of the integrin isoform ⁇ V ⁇ 5 or FCHo2 gene, e.g., by inducing heterochromatinization.
  • a Kruppel associated box KRAB
  • KRAB Kruppel associated box
  • dCas9 can be used to introduce epigenetic changes that reduce integrin isoform ⁇ V ⁇ 5 or FCHo2 gene expression by fusion of dCas9 to an epigenetic modifier such as a chromatin-modifying epigenetic enzyme.
  • the promoter for integrin isoform ⁇ V ⁇ 5 or FCHo2 can be silenced, for example, by methylation or acetylation (e.g. histone H3 lysine 9 [H3K9] methylation, histone H3 lysine 27 [H3K27] methylation, and/or DNA methylation).
  • fusion of dCas9 to a DNA methyltransferase such as DNA methyltransferase 3 alpha (DNMT3A) or a chimeric Dnmt3a/Dnmt3L methyltransferase (DNMT3A3L) allows targeted DNA methylation.
  • Fusion of dCas9 to histone demethylase LSD1 allows targeted histone demethylation (see, e.g., Liu et al. (2016) Cell 167(1 ) :233-247, Lo et al. (2017) F1000Res. 6. pii: F1000 Faculty Rev-747, and Stepper et al. (2017) Nucleic Acids Res.
  • an RNA-targeting CRISPR-Cas13 system is used to perform RNA interference to reduce integrin isoform ⁇ V ⁇ 5 or FCHo2 expression.
  • Members of the Cas13 family are RNA-guided RNases containing two HERN domains having RNase activity.
  • Cas13a (C2c2), Cas13b (C2c6), and Cas13d can be used for RNA knockdown.
  • Cas13 proteins can be made to target and cleave integrin isoform ⁇ V ⁇ 5 or FCHo2 transcribed RNA using a gRNA with complementarity to the target transcript sequence.
  • the gRNA is typically about 64 nucleotides in length with a short hairpin crRNA and a 28-30 nucleotide spacer that is complementary to the target site on the integrin isoform otV
  • Cas13 recognition and cleavage of a target transcript results in degradation of the transcript as well as nonspecific degradation of any nearby transcripts. See, e.g., Abudayyeh et al. (2017) Nature 550:280-284, Hameed et al. (2019) Microb. Pathog. 133:103551 , Wang et al. (2019) Biotechnol Adv. 37(5):708-729, Aman et al. (2016) Viruses 10(12). pii: E732, and Zhang et al. (2016) Cell 175(1 ):212-223; herein incorporated by reference. Determining Levels of Expression of Inteqrin Isoform otV
  • an "effective amount" of an integrin isoform ⁇ V ⁇ 5 or FCHo2 inhibitory nucleic acid e.g., microRNA, siRNA, piRNA, snRNA, antisense oligonucleotide
  • a CRISPR system targeting the integrin isoform ⁇ V ⁇ 5 or FCHo2 gene e.g., Cas9, Cas12a), RNA (e.g., Cas13), or epigenome (e.g., dCas9 fusion protein)
  • an amount sufficient to effect beneficial or desired results such as an amount that reduces formation of integrin-mediated formation of curved adhesions, colocalization of integrin isoform ⁇ V ⁇ 5 or FCHo2, and/or reduces integrin isoform ⁇ V ⁇ 5 or FCHo2 activity, for example, by interfering with transcription of integrin isoform ⁇ V ⁇ 5 or FCHo2 or interfering with translation of integrin isoform ⁇ V ⁇ 5 or
  • an integrin isoform ⁇ V ⁇ 5 or FCHo2 inhibitory nucleic acid or CRISPR system reduces the integrin isoform ⁇ V ⁇ 5 or FCHo2 mRNA levels or protein levels by at least about 10% to about 100%, 20% to about 100%, 30% to about 100%, 40% to about 100%, 50% to about 100%, 60% to about 100%, 70% to about 100%, 10% to about 90%, 20% to about 85%, 40% to about 84%, 60% to about 90%, including any percent within these ranges, such as but not limited to 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%.
  • Any convenient protocol may be used for evaluating integrin isoform ⁇ V ⁇ 5 or FCHo2 expression by detecting integrin isoform ⁇ V ⁇ 5 or FCHo2 protein or mRNA levels in the presence or absence of a candidate agent.
  • various antibody- based methods including without limitation immunoassays, e.g., enzyme-linked immunosorbent assays (ELISAs), immunohistochemistry, and flow cytometry (FACS) may be used.
  • ELISAs enzyme-linked immunosorbent assays
  • FACS flow cytometry
  • Any convenient antibody can be used that specifically binds to the integrin isoform ⁇ V ⁇ 5 or FCHo2 protein.
  • telomere binding refers to preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides or epitopes).
  • the affinity of one molecule for another molecule to which it specifically binds is characterized by a K d (dissociation constant) of 10 -5 M or less (e.g., 10 -6 M or less, 10 -7 M or less, 10' 8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, 10 -15 M or less, or 10 -16 M or less).
  • K d dissociation constant
  • ELISA enzyme- linked immunosorbent assay
  • one or more antibodies specific for the proteins of interest may be immobilized onto a selected solid surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate.
  • a non-specific "blocking" protein that is known to be antigenically neutral with regard to the test sample such as bovine serum albumin (BSA), casein or solutions of powdered milk.
  • BSA bovine serum albumin
  • the immobilizing surface is contacted with the sample to be tested under conditions that are conducive to immune complex (antigen/antibody) formation.
  • Such conditions include diluting the sample with diluents such as BSA or bovine gamma globulin (BGG) in phosphate buffered saline (PBS)ZTween or PBS/Triton-X 100, which also tend to assist in the reduction of nonspecific background, and allowing the sample to incubate for about 2-4 hours at temperatures on the order of about 25°-27° C.
  • an exemplary washing procedure includes washing with a solution such as PBS/Tween, PBS/Triton-X 100, or borate buffer.
  • the occurrence and amount of immunocomplex formation may then be determined by subjecting the bound immunocomplexes to a second antibody having specificity for the target that differs from the first antibody and detecting binding of the second antibody.
  • the second antibody will have an associated enzyme, e.g. urease, peroxidase, or alkaline phosphatase, which will generate a color precipitate upon incubating with an appropriate chromogenic substrate.
  • a urease or peroxidase-conjugated anti-human IgG may be employed, for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS/Tween).
  • the amount of label is quantified, for example by incubation with a chromogenic substrate such as urea and bromocresol purple in the case of a urease label or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6- sulfonic acid (ABTS) and H2O2, in the case of a peroxidase label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer. The preceding format may be altered by first binding the sample to the assay plate.
  • a chromogenic substrate such as urea and bromocresol purple in the case of a urease label or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6- sulfonic acid (ABTS) and H2O2
  • Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • the solid substrate upon which the antibody or antibodies are immobilized can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc.
  • the substrate may be chosen to maximize signal to noise ratios, to minimize background binding, as well as for ease of separation and cost.
  • Washes may be effected in a manner most appropriate for the substrate being used, for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, or rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent.
  • non-ELISA-based methods for measuring the levels of the integrin isoform ⁇ V ⁇ 5 or FCHo2 protein in a sample may be employed, and any convenient method may be used.
  • Representative examples known to one of ordinary skill in the art include but are not limited to other immunoassay techniques such as radioimmunoassays (RIA), sandwich immunoassays, fluorescent immunoassays, enzyme multiplied immunoassay technique (EMIT), capillary electrophoresis immunoassays (CEIA), and immunoprecipitation assays; mass spectrometry, or tandem mass spectrometry, proteomic arrays, xMAP microsphere technology, western blotting, immunohistochemistry, flow cytometry, cytometry by time-of-flight (CyTOF), multiplexed ion beam imaging (MIBI), and detection in body fluid by electrochemical sensor.
  • RIA radioimmunoassays
  • EMIT enzyme multiplied immunoassay technique
  • CEIA capillary
  • the quantitative level of gene products of the one or more genes of interest are detected on cells in a cell suspension by lasers.
  • antibodies e.g., monoclonal antibodies
  • antibodies that specifically bind the polypeptides encoded by the genes of interest are used in such methods.
  • electrochemical sensors may be employed.
  • a capture aptamer or an antibody that is specific for a target protein (the "analyte") is immobilized on an electrode.
  • a second aptamer or antibody, also specific for the target protein is labeled with, for example, pyrroquinoline quinone glucose dehydrogenase ((PQQ)GDH).
  • the sample of body fluid is introduced to the sensor either by submerging the electrodes in body fluid or by adding the sample fluid to a sample chamber, and the analyte allowed to interact with the labeled aptamer/antibody and the immobilized capture aptamer/antibody.
  • Glucose is then provided to the sample, and the electric current generated by (PQQ)GDH is observed, where the amount of electric current passing through the electrochemical cell is directly related to the amount of analyte captured at the electrode.
  • Flow cytometry can be used to distinguish subpopulations of cells expressing different cellular markers and to determine their frequency in a population of cells.
  • whole cells are incubated with antibodies that specifically bind to the cellular markers.
  • the antibodies can be labeled, for example, with a fluorophore, isotope, or quantum dot to facilitate detection of the cellular markers.
  • the cells are then suspended in a stream of fluid and passed through an electronic detection apparatus.
  • fluorescence-activated cell sorting FACS
  • FACS fluorescence-activated cell sorting
  • the amount or level in the sample of mRNA encoded by the integrin isoform ⁇ V ⁇ 5 or FCHo2 gene is determined.
  • Any convenient method for measuring mRNA levels in a sample may be used, e.g. hybridization-based methods, e.g. northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)), RNase protection assays (Hod, Biotechniques 13:852-854 (1992)), and PCR-based methods (e.g. reverse transcription PCR (RT-PCR) (Weis et aL, Trends in Genetics 8:263-264 (1992)).
  • RT-PCR reverse transcription PCR
  • the starting material may be total RNA, i.e. unfractionated RNA, or poly A+ RNA isolated from a suspension of cells.
  • RNA isolation can also be performed using a purification kit, buffer set and protease from commercial manufacturers, according to the manufacturer's instructions.
  • RNA from cell suspensions can be isolated using Qiagen RNeasy mini-columns, and RNA from cell suspensions or homogenized tissue samples can be isolated using the TRIzol reagent-based kits (Invitrogen), MasterPure Complete DNA and RNA Purification Kit (EPICENTRE, Madison, Wis.), Paraffin Block RNA Isolation Kit (Ambion, Inc.) or RNA Stat-60 kit (Tel-Test).
  • TRIzol reagent-based kits Invitrogen
  • MasterPure Complete DNA and RNA Purification Kit EPICENTRE, Madison, Wis.
  • Paraffin Block RNA Isolation Kit Ambion, Inc.
  • RNA Stat-60 kit Tel-Test
  • the mRNA levels may be measured by any convenient method. Examples of methods for measuring mRNA levels may be found in, e.g., the field of differential gene expression analysis.
  • One representative and convenient type of protocol for measuring mRNA levels is array-based gene expression profiling. Such protocols are hybridization assays in which a nucleic acid that displays "probe" nucleic acids for each of the genes to be assayed/profiled in the profile to be generated is employed. In these assays, a sample of target nucleic acids is first prepared from the initial nucleic acid sample being assayed, where preparation may include labeling of the target nucleic acids with a label, e.g., a member of signal producing system.
  • a label e.g., a member of signal producing system.
  • the sample is contacted with the array under hybridization conditions, whereby complexes are formed between target nucleic acids that are complementary to probe sequences attached to the array surface. The presence of hybridized complexes is then detected, either qualitatively or quantitatively.
  • an array of "probe" nucleic acids that includes a probe for each of the phenotype determinative genes whose expression is being assayed is contacted with target nucleic acids as described above. Contact is carried out under hybridization conditions, e.g., stringent hybridization conditions, and unbound nucleic acid is then removed.
  • hybridization conditions e.g., stringent hybridization conditions
  • unbound nucleic acid is then removed.
  • stringent assay conditions refers to conditions that are compatible to produce binding pairs of nucleic acids, e.g., surface bound and solution phase nucleic acids, of sufficient complementarity to provide for the desired level of specificity in the assay while being less compatible to the formation of binding pairs between binding members of insufficient complementarity to provide for the desired specificity. Stringent assay conditions are the summation or combination (totality) of both hybridization and wash conditions.
  • the resultant pattern of hybridized nucleic acid provides information regarding expression for each of the genes that have been probed, where the expression information is in terms of whether or not the gene is expressed and, typically, at what level, where the expression data, i.e., expression profile (e.g., in the form of a transcriptosome), may be both qualitative and quantitative.
  • non-array based methods for quantitating the level of one or more nucleic acids in a sample may be employed. These include those based on amplification protocols, e.g., Polymerase Chain Reaction (PCR)-based assays, including quantitative PCR, reverse-transcription PCR (RT-PCR), real-time PCR, and the like, e.g. TaqMan, RT-PCR, MassARRAY System, BeadArray technology, and Luminex technology; and those that rely upon hybridization of probes to filters, e.g. Northern blotting and in situ hybridization.
  • PCR Polymerase Chain Reaction
  • Inhibitors that inhibit formation of integrin-mediated curved adhesions can be formulated into pharmaceutical compositions optionally comprising one or more pharmaceutically acceptable excipients.
  • exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.
  • Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants.
  • a carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient.
  • carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.
  • the excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphat
  • a composition of the invention can also include an antimicrobial agent for preventing or deterring microbial growth.
  • antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.
  • An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the inhibitor, or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof. [00207] A surfactant can be present as an excipient.
  • Exemplary surfactants include: polysorbates, such as “Tween 20” and “Tween 80,” and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.
  • Acids or bases can be present as an excipient in the composition.
  • acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof.
  • Suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.
  • the amount of the inhibitor (e.g., when contained in a drug delivery system) in the composition will vary depending on a number of factors but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial).
  • a therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.
  • the amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred.
  • compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • solutions and suspensions are envisioned.
  • Additional preferred compositions include those for oral, ocular, or localized delivery.
  • compositions herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use.
  • the compositions comprising one or more inhibitors that inhibit formation of integrin-mediated curved adhesions, or nucleic acids encoding them are in unit dosage form, meaning an amount of a conjugate or composition of the invention appropriate for a single dose, in a premeasured or pre- packaged form.
  • compositions herein may optionally include one or more additional agents, such as one or more other drugs for treating a disorder associated with integrin-mediated formation of curved adhesions, or other medications.
  • additional agents such as one or more other drugs for treating a disorder associated with integrin-mediated formation of curved adhesions, or other medications.
  • compounded preparations may include at least one inhibitor that inhibits formation of integrin-mediated curved adhesions and one or more other drugs for treating a disorder associated with integrin-mediated formation of curved adhesions such as a cancer, a fibrotic disease, thrombosis, an autoimmune disease, or a microbial infection; or other medications used to treat a subject for a condition or disease.
  • such agents can be contained in a separate composition from the composition comprising the inhibitor that inhibits formation of integrin-mediated curved adhesions and co- administered concurrently, before, or after the composition comprising the inhibitor that inhibits formation of integrin-mediated curved adhesions. Kits
  • kits for use in the methods described herein include agents for detecting integrin-mediated formation of curved adhesions.
  • kits may include reagents for performing a proximity assay to determine the proximity of integrin isoform ⁇ V ⁇ 5 to FCHo2, such as reagents for performing a complementation assay using a split reporter protein (e.g., split fluorescent protein or split bioluminescent protein), a fluorescence resonance energy transfer (FRET) assay, a bioluminescence resonance energy transfer (BRET) assay, an antibody-based proximity ligation assay (PLA), or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay).
  • a split reporter protein e.g., split fluorescent protein or split bioluminescent protein
  • FRET fluorescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • PDA antibody-based proximity ligation assay
  • proxHCR proximity-dependent initiation of hybridization chain reaction
  • Such assay reagents may include but are not limited to, split fluorescent proteins or split bioluminescent proteins conjugated to integrin isoform ⁇ V ⁇ 5 and FCHo2, FRET/BRET donors and acceptors conjugated to integrin isoform aV ⁇ 5 and FCHo2, or antibodies that specifically bind to integrin isoform ⁇ V ⁇ 5 and the FCHo2, which may be conjugated to oligonucleotides, PLA probes, bridge oligonucleotides, circularization oligonucleotides, amplification reagents for PGR, isothermal amplification, or rolling circle amplification, detection probes, primers, and the like.
  • the kit may further include buffers, cells, microtiter plates, culturing plates, tubes, flasks, culture media, and the like.
  • Kits may further include control reagents and samples including but not limited to, e.g., control samples (e.g., positive control samples, negative control samples, etc.) calibration reagents (e.g., fluorescent calibration reagents, etc.).
  • the kit further comprises a vertical nanostructure (e.g., nanobar or a nanopillar) having an end curvature diameter ranging from 100 nm to 3 pm.
  • the nanostructure is coated with vitronectin or fibronectin.
  • the kit further comprises fibers comprising collagen, fibronectin, or vitronectin, or a combination thereof.
  • the kit further comprises a curved ECM surface.
  • the kit comprises a reporter system for measuring curved adhesions, the reporter system comprising a cell expressing a split fluorescent or luminescent protein comprising a first component of a fluorescent or luminescent protein and a second component of the fluorescent or luminescent protein, wherein the cell comprises: a first nucleic acid comprising a coding sequence encoding a first fusion protein comprising the first component of the fluorescent or luminescent protein connected to integrin isoform «Vp5; and a second nucleic acid comprising a coding sequence encoding a second fusion protein comprising the second component of the fluorescent or luminescent protein connected to FCHo2; wherein a reconstituted fluorescent or luminescent protein forms when the first component and the second component of the split fluorescent or luminescent protein are expressed and interact to produce a detectable fluorescent or luminescent signal indicative of the proximity of the integrin isoform ⁇ V ⁇ 5 and the FCHo2 in the cell.
  • the reporter system comprising a cell expressing a split fluorescent or lumin
  • the above components may be present in separate containers or one or more components may be combined into a single container, e.g., a glass or plastic vial or tube.
  • the kit further comprises a fluorescence microscope, a two-photon excitation microscope, a fluorescence confocal laser scanning microscope, or a light sheet fluorescence microscope.
  • the kit further comprises a luminescence microscope, luminescence digital imaging microscope, time-gated luminescence microscope, or a luminometer.
  • the subject kits may further include (in certain embodiments) instructions for practicing the subject methods.
  • These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like.
  • Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, flash drive, and the like, on which the information has been recorded.
  • Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.
  • a method of screening for an inhibitor that inhibits formation of integrin-mediated curved adhesions comprising: contacting a cell, a population of cells, or a tissue with a candidate agent; and measuring proximity of integrin isoform «vp5 to FCHo2 in presence of the candidate agent to determine whether the inhibitor inhibits colocalization of integrin isoform aV 5 and FCHo2 in integrin-mediated curved adhesions, wherein the proximity of the ⁇ xVp5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • the method of aspect 1 further comprising providing fibers comprising collagen, fibronectin, or vitronectin, or a combination thereof, wherein the cell, population of cells, or tissue forms the integrin-mediated curved adhesions on the surface of the fibers.
  • measuring the proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 comprises performing a complementation assay using a split reporter protein, a fluorescence resonance energy transfer (FRET) assay, a bioluminescence resonance energy transfer (BRET) assay, an antibody-based proximity ligation assay (PLA), or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay.
  • FRET fluorescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • PDA antibody-based proximity ligation assay
  • proxHCR proximity-dependent initiation of hybridization chain reaction
  • said measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 with a complementation assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split fluorescent protein connected to the FCHo2, wherein a reconstituted fluorescent protein forms when the first component and the second component of the split fluorescent protein are expressed and interact to produce a detectable fluorescent signal; and detecting the fluorescent signal to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 from the fluorescent signal, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin-
  • the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 with a complementation assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split luminescent protein connected to the FCHo2, wherein a reconstituted luminescent protein forms when the first component and the second component of the split luminescent protein are expressed and interact to produce a detectable luminescent signal; and detecting the luminescent signal to determine proximity of the integrin isoform «V
  • split luminescent protein is a split luciferase or a split aequorin.
  • the split luciferase is a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 with a FRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a FRET donor connected to the integrin isoform «vp5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 with a FRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a FRET acceptor connected to the integrin isoform «VP5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the «VP5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein
  • the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein
  • the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5)
  • the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 with a BRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising BRET donor connected to the integrin isoform ⁇ V ⁇ 5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the ⁇ V ⁇ 5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • measuring proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2 with a BRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a BRET acceptor connected to the integrin isoform «VP5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform ⁇ V ⁇ 5 to the FCHo2, wherein the proximity of the «VP5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
  • the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein.
  • said performing the PLA comprises: contacting the cell, population of cells, or tissue with a first antibody that specifically binds to ⁇ V ⁇ 5; contacting the cell, population of cells, or tissue with a second antibody that specifically binds to FCHo2; contacting the first antibody with a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; contacting the second antibody with a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody; contacting the first probe and the second probe with a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with the first oligonucleotide and a second portion sufficiently complementary to hybridize with the second oligonucleotide when the first oligonucleotide and the second oligonucleotide when the
  • first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab') 2 fragment, an F v fragment, and an scF v fragment.
  • any one of aspects 1 -32 further comprising: contacting a population of cancer cells with the candidate agent if the candidate agent inhibits the formation of the integrin- mediated curved adhesions; and measuring adhesion, proliferation, cell spreading, migration, or survival of the population of cancer cells, wherein reduced adhesion, proliferation, cell spreading, migration, or survival in presence of the candidate agent compared to that in a negative control population of cancer cells that are not treated with the candidate agent indicates that the candidate agent has anti-cancer activity.
  • the candidate agent is a small molecule, a peptide, a protein, an aptamer, an antibody, an antibody mimetic, an inhibitory nucleic acid, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a F v fragment, and a nanobody.
  • inhibitory nucleic acid is selected from the group consisting of a small interfering RNA (siRNA), a microRNA (miRNA), a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), an antisense oligonucleotide, and a peptide nucleic acid.
  • siRNA small interfering RNA
  • miRNA microRNA
  • piRNA Piwi-interacting RNA
  • snRNA small nuclear RNA
  • antisense oligonucleotide an antisense oligonucleotide
  • the CRISPR system targets a gene or an RNA transcript encoding the integrin isoform ⁇ V ⁇ 5 or the FCHo2, or makes epigenetic changes that reduce expression of the integrin isoform ⁇ V ⁇ 5 or the FCHo2.
  • the CRISPR system comprises Cas9, Cas12a, Cas12d, Cas13a, Cas13b, Cas13d, or a dead Cas9 (dCas9).
  • composition comprising the inhibitor of aspect 41 and a pharmaceutically acceptable excipient.
  • composition of aspect 42 further comprising a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment.
  • a reporter system for measuring curved adhesion comprising a cell expressing a split fluorescent or luminescent protein comprising a first component of a fluorescent or luminescent protein and a second component of the fluorescent or luminescent protein, wherein the cell comprises: a first nucleic acid comprising a coding sequence encoding a first fusion protein comprising the first component of the fluorescent or luminescent protein connected to integrin isoform ⁇ V ⁇ 5; and a second nucleic acid comprising a coding sequence encoding a second fusion protein comprising the second component of the fluorescent or luminescent protein connected to FCHo2; wherein a reconstituted fluorescent or luminescent protein forms when the first component and the second component of the split fluorescent or luminescent protein are expressed and interact to produce a detectable fluorescent or luminescent signal indicative of the proximity of the integrin isoform ⁇ V ⁇ 5 and the FCHo2 in the cell.
  • the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
  • the reporter system of aspect 45 wherein the split luminescent protein is a split luciferase or a split aequorin.
  • split luciferase is a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
  • kits comprising the reporter system of any one of aspects 44-47 and instructions for detecting integrin-mediated curved adhesions.
  • a system for screening a candidate agent to determine its effects on formation of integrin-mediated curved adhesions by a cell comprising a vertical nanostructure having an end curvature diameter ranging from 100 nm to 3 pm, wherein the cell forms the integrin-mediated curved adhesions on the surface of the nanostructure.
  • nanostructure is a nanobar or a nanopillar.
  • the cell or cell fragment of interest is a cancer cell, a blood cell, a platelet, an inflammatory cell, a fibroblast, or an epithelial cell.
  • any one of aspects 49-53 further comprising: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to integrin isoform «V
  • the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
  • the system of aspect 54 or 55 further comprising a fluorescence microscope, a two-photon excitation microscope, a fluorescence confocal laser scanning microscope, or a light sheet fluorescence microscope.
  • any one of aspects 49-53 further comprising: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to integrin isoform «V
  • the split luminescent protein is a split luciferase or a split aequorin.
  • the split luciferase is a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
  • 61 The system of any one of aspects 49-53, wherein the system comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) donor connected to integrin isoform ⁇ V ⁇ 5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to FCHo2.
  • FRET fluorescence resonance energy transfer
  • any one of aspects 49-53 wherein the system comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) acceptor connected to the integrin isoform otV(35; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2.
  • FRET fluorescence resonance energy transfer
  • the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein
  • the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein
  • the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5)
  • the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine.
  • system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) donor connected to the integrin isoform ⁇ V ⁇ 5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2.
  • BRET bioluminescence resonance energy transfer
  • system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) acceptor connected to the integrin isoform «vp5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2.
  • BRET bioluminescence resonance energy transfer
  • the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein.
  • reagents for performing an antibody-based proximity ligation assay PLA
  • proxHCR proximity-dependent initiation of hybridization chain reaction
  • the reagents for performing the PLA comprise: a first antibody that specifically binds to ⁇ V ⁇ 5; a second antibody that specifically binds to FCHo2; a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; and a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody.
  • PLA antibody-based proximity ligation assay
  • proxHCR proximity-dependent initiation of hybridization chain reaction
  • the system of aspect 72 further comprising a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with the first oligonucleotide and a second portion sufficiently complementary to hybridize with the second oligonucleotide when the first oligonucleotide and the second oligonucleotide are in sufficient proximity to each other to simultaneously hybridize to the bridge oligonucleotide.
  • first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab') 2 fragment, an F v fragment, and an scF v fragment.
  • 76 The system of any one of aspects 72-75, further comprising reagents for performing polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification.
  • PCR polymerase chain reaction
  • 79 The system of aspect 78, wherein the probe comprises a fluorescent label, a bioluminescent label, or a chemiluminescent label.
  • 80. A kit comprising the system of any one of aspects 49-80 and instructions for detecting integrin-mediated curved adhesions.
  • T o control membrane curvature we engineered nanobars on quartz substrates by electron beam lithography patterning and anisotropic etching (FIG. 1 A and FIG. 7A). These nanobars are vertically aligned with 200-nm width, 2-pm length, 1.4-pm height, and 5-pm spacing.
  • the half cylinders at the nanobar ends mimic the curvatures of ECM fibres, and the flat side walls of nanobars serve as an internal control to differentiate curvature effects from other effects that might be introduced by the vertical structures (FIG. 1 B).
  • the plasma membrane is locally deformed to accommodate the geometry of nanobars and thus generates well-defined local curvatures (FIG. 1C) 19 .
  • Each quartz substrate has multiple well-separated nanopattern areas (1x1 mm 2 each) with the rest being flat areas, which allows us to compare cells on nanostructures with cells on flat areas in the same culture.
  • integrins In mammals, integrins have 18 a and 8 p subunits forming 24 distinct op heterodimers that bind various ECM proteins 20 .
  • fibronectin and vitronectin to probe [33, ⁇ 5, [36 and [38- containing integrins, and laminin to probe the [34-containing integrin.
  • ⁇ 5-GFP showed strong accumulation at the end of fibronectin- or vitronectin-coated nanobars, while co-expressed FusionRed-CaaX was relatively evenly distributed on the same nanobars (FIG. 1 F).
  • FIG. 1 F We quantified the nanobar end/side ratio of [3 subunits which were normalized against the FusionRed-CaaX.
  • FIG. 2E centrifugation- based force assay 25.
  • cells were cultured on vitronectin-coated nanopillar substrates. After the cells fully spread, the substrates were placed face down in a 3D-printed support (FIG. 10D) and spun for 15 min in a centrifuge tube filled with buffer solution. If the cell detachment force (see Methods for calculation) was greater than the maximum force that cell adhesions can withstand, the cells would be removed from the substrate.
  • nanopillar arrays were designed with fiducial markers, which allowed us to image the same fields covering both nanopillar (left half) and flat (right half) areas before and after centrifugation for a direct comparison (FIG. 2F).
  • centrifugation speed increased from 200 to 1500 relative centrifugal field (RCF)
  • the percentage of remaining cells (green arrows) on both flat areas and nanopillar areas decreased dramatically.
  • at speeds of 500, 1000 and 1500 RCF there were significantly more cells remaining on nanopillar areas than on flat areas of the same substrates (FIG. 2G). This indicated that there was a greater adhesion force to nanopillar areas than to flat areas.
  • Curved adhesions involve a subset of focal adhesion proteins to anchor actin filaments.
  • FCHol/2 early-stage endocytic proteins that can sense membrane curvature through an N-terminal FBAR domain 738 .
  • FCHo1/2 have not been reported to be involved in cell adhesion before.
  • ⁇ 5 and FCHo2 showed distinct intracellular distributions on flat surfaces, they showed strong colocalization at nanopillars (FIG. 4E), unlike 35 and AP2 (FIG. 4C).
  • the FCHo2 intensity positively correlated with 35 accumulation at nanopillars with a correlation coefficient of 0.62 (FIG. 4F).
  • shRNA KD of FCHol/2 significantly reduced the 35 accumulation at nanobar ends (FIG. 4D).
  • FCHo2 is crucial for the formation of curved adhesions and is an integral component of stable curved adhesions.
  • the natural ECM is enriched with fibrous structures, which may locally deform the cell membrane.
  • ECM fibres with collagen, vitronectin and fibronectin on coverslips. Colocalizations of Alexa Fluor 647- labeled collagen, Cy3-labeled vitronectin, and immunolabeled fibronectin confirmed the presence of aV35 ligands vitronectin and fibronectin in the fibre structures (FIG. 17A).
  • Natural ECMs are often soft 3D networks of fibres. Focal adhesions, the most well-known adhesions, are rare in 3D cultures, due to their reliance on force-dependent talin activation 15 . The curvature preference of aV ⁇ 5 is independent of talin. We thus hypothesized that curved adhesions may be prevalent in 3D cultures. To test it, we cultured cells on a thick layer of 3D ECM fibres. Cells in 3D cultures showed extensive ⁇ 5 accumulations on ECM fibres, which colocalized with FCHo2 (FIG. 5D), while the ⁇ 5 and FCHo2 colocalization was extremely rare on 2D surfaces coated with the same composition of ECM proteins (FIG. 18B).
  • Natural ECM fibres present a wide range of thicknesses from tens of nanometres to tens of micrometres in diameter 5 .
  • we engineered gradient bars whose end curvature diameters vary from 100 nm to 5 pm (FIG. 5F and FIG. 7C).
  • GFP-CaaX showed that the cell membrane wrapped around all nanobars, ⁇ 5 exhibited a clear preference to the ends of thin nanobars.
  • Quantification of the ⁇ 5 end/side ratios showed that curved adhesions preferred sharp curvatures with curvature diameters no more than 3 pm (FIG. 5G). Discussions
  • curved adhesion a new type of cell-matrix adhesion that selectively forms on curved ECM surfaces and is exclusively mediated by the integrin isoform ⁇ V ⁇ 5.
  • Curved adhesions engage a unique subset of focal adhesion adaptor proteins, including talin, paxillin, and zyxin, but not vinculin or pFAK.
  • focal adhesions whose assemblies are stabilized by the mechanical force through talin 2 14
  • curvature-sensing proteins FCHo1/2 (FIG. 6).
  • Integrin ⁇ V ⁇ 5 and many of its ligands are widely expressed 40 .
  • the upregulation of integrin aV ⁇ 5 is associated with poor prognosis of glioblastoma, gastric, pancreas, and breast cancers 9-11 41 .
  • Our finding uncovers a mechanism for cells to grab onto delicate ECM fibres that can be easily deformed by mechanical forces required for focal adhesion development. The discovery of curved adhesions will hopefully lead to new perspectives regarding the cell and ECM coupling in a broad spectrum of biological and pathological conditions.
  • the patterns of nanobar, nanopillar, and gradient bar arrays were designed using an open- source python package.
  • the fabrication method was adapted from a previous study 42 . Briefly, a 4-inch quartz wafer (Silicon Materials, 04Q 525-25-1 F-SO) was diced and cleaned with acetone and isopropanol under sonication. Their surfaces were then coated with 275-nm CSAR 6200 (AllResist) and 100-nm Electra 92 (AllResist). The desired patterns were exposed to e-beam using the JEOL JBX-6300FS system.
  • a 120 nm-thick layer of chromium mask was deposited on the post-exposure chips using the AJA e-beam evaporator and then immediately lifted off with acetone and isopropanol.
  • the chips were then etched anisotropically by reactive ion etching (Plasma Therm Versaline LL ICP Dielectric Etcher, PT-Ox) with a mixture of C4F8, H 2 and Ar for 3 min. Finally, the chips were immersed in chromium etchant 1020 (Transene) for 30 min to remove the Cr mask.
  • chromium etchant 1020 Transene
  • U2OS cells ATCC® HTB-96TM
  • HeLa cells ATCC® CCL-2TM
  • Mouse Embryonic Fibroblasts (ATCC® CRL-2991 TM) were cultured in complete cell-culture media, Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplied with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich), and 1 % (v/v) penicillin and streptomycin (Gibco).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin and streptomycin Gibco
  • HEK293T cells (ATCC® CRL-3216TM) were cultured in complete cell-culture media supplemented with 1 10 pg/mL sodium pyruvate (Gibco) for lentivirus generation. All cell lines were maintained at 37°C in a 5% CO 2 atmosphere.
  • Gelatin (Sigma-Aldrich, G9391 ) was added into water 283 and autoclaved to make a 1 mg/mL gelatin solution. The gelatin solution was then mixed with an equal volume of 0.2 M sodium bicarbonate buffer (Sigma-Aldrich). For vitronectin labelling, the buffer of human multimeric vitronectin solution (Molecular Innovations Inc, HVN-U) was changed to 0.1 M sodium bicarbonate using a Zeba spin desalting column (Thermo Scientific, 89882).
  • Rat- tail collagen type I (Corning, 354236) was labelled with Alexa Fluor 647 NHS ester (Thermo Scientific, A37573) and CF568 NHS ester (Sigma-Aldrich, SCJ4600027) to generate AF647- collagen and CF568-collagen, respectively.
  • the protocol was adapted from previous studies 1643 .
  • collagen type I was first diluted to a final concentration of 4 mg/ml in ice-cold 20 mM acetic acid.
  • the collagen solution (1 mL) was then neutralized with a pre-chilled mixture of 20 pL 1 M NaOH, 200 pL 10x DMEM, 200 pL 10 mM HEPES, and 580 pL water.
  • the neutralized collagen was dropped in a pre-chilled 10-cm petri dish and incubated stilly for 1 h at RT for gelation.
  • the collagen gel was washed with 10 mL of PBS 3 times for 10 min each and then washed with 0.1 M sodium bicarbonate 3 times for 10 min each.
  • the dye was diluted in 0.1 M sodium bicarbonate to a final concentration of 2 pg/mL and added into the collagen gel. After 30- min incubation at RT, the free dye was washed away with PBS 5 times for 15 min each.
  • the collagen gel was dissolved in 500 pL of 500 mM acetic acid by overnight incubation at 4°C. The collagen solution was then dialyzed against 4 L of 20 mM acetic acid overnight at 4°C. If possible, samples were handled in dark to avoid photobleaching from the ambient light.
  • the nanochips were first treated with air plasma (Harrick Plasma) for 15 min and then incubated with 0.1 mg/mL PLL (Sigma-Aldrich, P5899) in PBS at 37°C for 1 h. The chips were then washed with PBS 3 times. For PLL surface coating, we stopped here. To coated with other ECM proteins, the nanochips were then incubated with 0.5% (v/v) glutaraldehyde (Sigma-Aldrich, G6257) in PBS at RT for 15 min.
  • the substrates were immediately incubated in PBS with 1 mg/mL gelatin, 1 mg/mL Atto 647-gelatin (this study), 0.25 mg/ml human plasma fibronectin (Sigma-Aldrich, 341635), 0.25 mg/ml vitronectin (PeproTech, 140-09), Cy3- vitronectin (this study), 0.25 mg/ml laminin 511 (Sigma-Aldrich, CC160), or BSA (Sigma-Aldrich, A9418), at 37°C for 1 h. The chips were then washed with PBS 3 times. Substrates with fluorescent labelling were immediately imaged. Before cell plating, the substrates were treated with 1 mg/ml sodium borohydride (Sigma-Aldrich, 452882) in PBS for 5 min to eliminate autofluorescence and then washed with PBS 5 times.
  • 1 mg/ml sodium borohydride Sigma-Aldrich, 452882
  • the nanostructures were imaged by SEM (FEI Nova NanoSEM 328450 and FEI Magellan 400 XHR).
  • SEM FEI Nova NanoSEM 328450 and FEI Magellan 400 XHR.
  • the cells on nanostructures were washed with PBS and fixed in 0.1 M sodium cacodylate buffer (pH 7.2) supplemented with 2% glutaraldehyde (Electron Microscopy Sciences) and 4% paraformaldehyde (PFA) for 20 min at RT and then overnight at 4°C.
  • the cells were then incubated in 0.1 M sodium cacodylate buffer supplied with 1% OsO 4 and 0.8% potassium ferricyanide (Electron Microscopy Sciences), for 2 h at 4°C.
  • the samples were dehydrated sequentially with 50%, 70%, 95%, 100%, and 100% (v/v) ethanol in water for 10 min each, and then dried by critical point drying.
  • the samples were sputter-coated with 3 nm-thick Cr and imaged by SEM.
  • the DNA fragment encoding human integrin ⁇ 5 was amplified from the pCX-EGFP beta5 integrin receptor (a gift from Raymond Birge, Addgene #14996), and then inserted into the pEGFP-N1 vector (Clontech) for the expression of ⁇ 5-GFP.
  • the DNA fragments encoding human integrin p3 and [38 were amplified from the complementary DNA (cDNA) of LI2OS cells and were cloned into the pEGFP-N1 vector for the expression of [33-GFP and [38-GFP, respectively.
  • the DNA fragment encoding integrin [34 was amplified from pcDNA3.1/Myc-His beta4 (a gift from Filippo Giancotti, Addgene #16039), and then inserted into the pEGFP-N1 vector for the expression of [34-GFP.
  • the expression vector of integrin [36-GFP was a gift from Dean Sheppard (Addgene #13593).
  • the expression vector of mCherry-FCHo2 and Eps15-mCherry were gifts from Christien Merrifield (Addgene #27686 and #27696).
  • the expression vector of talin-mCherry was a gift from Michael Davidson (Addgene #55139).
  • the DNA fragments encoding human zyxin, a-actinin 1 , and a-actinin 4 were amplified from the cDNA of U2OS cells and then inserted into the pmCherry-N1 vector (Clontech) for the expression of zyxin-mCherry, a-actinin 1 -mCherry, and a-actinin 4- mCherry, respectively.
  • the DNA fragments encoding GFP-351 CaaX and FusionRed- CaaX were generated by fusing the DNA fragment encoding the CaaX motif of K-Ras protein (GKKKKKKSKTKCVIM, SEQ ID NO:21 ) to the 3'-end of the DNA fragments encoding EGFP and FusionRed.
  • the DNA fragment of FusionRed-CaaX was inserted into the pEGFP-C1 vector (Clontech) to replace the EGFP for the mammal expression.
  • the DNA fragment encoding GFP- CaaX was inserted into pLenti pRRL-SV40(puro)_CMV for lentivirus packaging.
  • the expression vectors of LifeAct-RFP were gifts from Michael Davidson (Addgene #54491 ).
  • the DNA fragment encoding SNAP-tag was amplified from the pSNAP-tag (m) vector (a gift from New England Biolabs & Ana Egana, Addgene #101 135), and then inserted into the pDisplay vector (Invitrogen, V66020) for the expression of cell-surface SNAP-tag.
  • the vector for scramble shRNA transcription was a gift from David Sabatini (Addgene #1864).
  • the shRNA vectors for the KDs of human integrin ⁇ 5, AP2-p, clathrin heavy chain, FCHol , FCHo2, Eps15, Eps15R, and intersectin 1 and 2 were from Sigma-Aldrich.
  • the clone IDs of shRNA vectors used in this study are listed in Table 1.
  • the shRNAs are cloned in the pLKO.1 vector for their transcription in mammal cells.
  • the pLKO.1 vector is designed for lentivirus transfection and subsequent puromycin selection. For transient transfection, the puromycin resistant sequence in pLKO.1 vector was replaced with a sequence encoding EBFP2 to fluorescently label shRNA transcription cells.
  • U2OS cells and MEFs were transfected with plasmids encoding proteins or shRNAs by electroporation.
  • Cells were grown in 6-well plates at -80% confluency before transfection.
  • One well of cells were detached, collected, and then resuspended in an electroporation buffer containing 100 pL Electroporation buffer II (88 mM KH 2 PO 4 and 14 mM NaHCO 3 , pH 7.4), 2 pL Electroporation buffer I (360 mM ATP + 600 mM MgCI 2 ), 374 and ⁇ 2 j g plasmids.
  • the cells were electroporated in 0.2-cm gap electroporation cuvettes by Amaxa Nucleofector II (Lonza) using protocols pre-installed by the manufacturer.
  • Amaxa Nucleofector II Loxza
  • the transfected cells were maintained in complete cell-culture media for ⁇ 72 h before being plated on desired substrates.
  • HEK293T cells at ⁇ 80% confluency in 35-mm dishes were transfected with 1.5 pg 3 rd generation lentivirus package vector encoding GFP- CaaX or shRNAs, 0.8 pg psPAX2 (a gift from D. Trono, Addgene #12260), and 0.7 pg pMD2.G (a gift from D. Trono, Addgene #12259), mixed with 9 pL lipofectamine 2000 transfection reagent in 2 mL Opti-MEM-I medium. The transfection media were replaced with 2 mL of HEK293T culture media 6 h after transfection.
  • the transfected cells were maintained for 24 h. Then, the virus containing supernatants were collected, and the cell debris was removed by spin at 400 RCF for 5 min followed by filtration through 0.45-pm PVDF syringe filter units (Millipore).
  • the GFP-CaaX and integrin ⁇ 5-knockdown stable cell lines were generated by lentivirus transduction of wild-type U2OS cells followed by antibiotic selection with 2.5 pg/mL puromycin in complete cell-culture media. The antibiotic pressure was released 3 days before experiments. Immunofluorescence labelling
  • integrin [31 and av ⁇ 5 were first snap-chilled in ice- cold HEPES-buffered hanks balanced salt solution (HBSS), which is 1X HBSS (Gibco, 24020117) buffered with 10 mM HEPES (Gibco, 15630106). The samples were then incubated with either mouse anti-activated integrin [31 antibody 397 (Sigma-Aldrich, MAB2079Z) or mouse anti-integrin av ⁇ 5 antibody (R&D Systems, MAB2528), which were 1 :100 diluted in HHBSS, for 30 min at 4°C.
  • HBSS HEPES-buffered hanks balanced salt solution
  • the samples were then washed with ice-cold HHBSS 3 times for 5 min each and immersed with 4% ice-cold PFA in PBS to fix for 15 min at RT.
  • the fixed samples were washed and permeabilized with 0.1 % Triton-X (Sigma-Aldrich, T8787) in PBS for 15 min at RT.
  • the permeabilized samples were washed and incubated in a blocking buffer (5% BSA in PBS) for 1 h at RT.
  • the primary antibody-labelled samples were then washed with the blocking buffer 3 times for 15 min each and incubated in fluorescently labelled secondary antibodies 1 :500 diluted in blocking buffer for 1 h at RT.
  • the samples were washed with the blocking buffer 3 times for 15 min each and then with PBS 3 times before imaging.
  • the secondary antibodies used are listed in Table 2.
  • Cell-surface SNAP-tags were fluorescently labelled before fixation.
  • Cells were incubated in CO 2 -balanced pre-warmed media supplied with 1 pM O6-benzylguanine (BG)-coupled Alexa Fluor 647 (NEB, S9136S) for 15 min at 37°C in a 5% CO 2 atmosphere. The cells were then washed 5 times with cell culture media before fixation.
  • BG O6-benzylguanine
  • the centrifugation supports were printed using a 3D printer (Original Prusa i3).
  • Cells stably expressing GFP-CaaX were grown on substrates (1 cm 2 square quartz chips). The cells were incubated in complete cell-culture media supplied with 1 pg/mL Hoechst 33342 (Thermo Scientific, 62249) for 15 min at 37°C in a 5% C0 2 atmosphere. The samples were washed with PBS 5 times and imaged before spin. The samples were then flipped upside down and put in centrifugation support. The support was gently inserted into a 50-mL centrifuge tube (Corning, 352070) filled with 20 mL of HHBSS. The samples were spun at 200, 500, 1000, or 1500 RCF for 15 min. The samples were then gently removed from the support and fixed for imaging after spin.
  • Unlabeled and fluorescently labelled collagen was mixed in a 4:1 (w/w) ratio at a final concentration of 2 mg/ml in 20 mM acetic acid.
  • 50 pL collagen mixture was neutralized with a mixture of 1 pL 1 M NaOH, 10 pL 10x DMEM, 10 pL 10 mM HEPES, and 29 pL water on ice.
  • the neutralized collagen was dropped in Lab-Tek II chambered coverglass (Sigma-Aldrich, Z734853) for 20 pL per well, and incubated at RT for 5 min.
  • the non- polymerized collagen was then washed away with 0.5 mL 20 mM phosphate buffer (at pH 7.4) 5 times.
  • the collagen fibres were incubated in 100 pL of 20 mM phosphate buffer supplied with 50 pg/mL human plasma fibronectin (Sigma-Aldrich, 341635), and 50 pg/ml multimeric vitronectin (Molecular Innovations Inc, HVN-U) or Cy3-vitronectin (this study), for 1 h at RT.
  • the collagen fibres were washed with 20 mM phosphate buffer 3 times before immunofluorescence labelling or cell plating.
  • cells were detached using the enzyme-free cell dissociation buffer and collected by spin. The cells were then resuspended in CO 2 -balanced pre-warmed media and immediately seeded on collagen fibres. The cells were grown on fibres for 45 min before the next treatment.
  • Fluorescence images were acquired in an epi-fluorescence microscope (Leica DMI 6000B) using a 100x or 10x objective.
  • a stage top incubator Tokai Hit, INUBSF-ZILCS.
  • Time-lapse images were taken at 15 s/frame using 500 ms exposure times.
  • Confocal images were acquired in a Nikon A1 R confocal microscope using a 60X objective.
  • Standard localization-based approaches fitted the shape of the PSF of emitters to a 2D Gaussian to yield highly precise XY positions.
  • a double helix phase mask was inserted in the Fourier plane of the microscope to modify the shape of the standard PSF to now have two lobes that rotate as a function of Z.
  • the localization precision was calculated from the detected photons using a formula calibrated specifically for our microscope using previously described approaches 45 .
  • the bright-field images were used to generate arrays of square masks with bars at the centre, using MATLAB code written for this study. All mask regions of the plasma membrane channel within each cell were averaged. Based on the average, the bar-end and bar-side region- of-interests (ROIs) were identified manually using Fiji. The ROIs were then applied to each bar in integrin and membrane channels and the mean intensities in each ROI were measured. For each bar, the integrin intensity was normalized by the membrane intensity and the end/side ratio was then calculated. The normalized end/side ratios of individual nanobars in each cell were averaged and reported as mean ⁇ SD. For cells on gradient bars, the normalized end/side ratios in each imaging field were averaged and reported, because very few b 486 ars with the same diameter were covered by one single cell.
  • the square masks with nanopillars at the centre were generated by using the same MATLAB above. Based on the average mask region of the plasma membrane channel within each cell, the ROIs for “at nanopillar” and “flat regions surrounding nanopillars” were defined as 0-9 and 10-20 pixel from the centre of a nanopillar, respectively. The ROIs were then applied to each mask region in both integrin and membrane channels. Mean intensities in each ROI were measured. The p5/membrane ratio of each ROI was calculated and normalized by the mean ⁇ 5/membrane ratio in each cell. The normalized ⁇ 5/membrane ratios of individual ROIs were reported as mean ⁇ SD.
  • the cell nuclei were counted to determine cell numbers before and after spin.
  • the images in the Hoechst channel were auto-thresholded using the ‘Moments’ method in Fiji to identify cell nuclei.
  • the nuclei clusters were segmented using the watershed binary tool in Fiji.
  • the nuclei were then counted using the analyse particles tool in Fiji. Based on images in the GFP-CaaX channel, the cells that cover both nanopillars and the flat area were excluded.
  • F (pcell - pmedium) ' Vcell ‘ RCF in which F is the average detachment force for each cell; pcell is the density of the cell (1.07 g/cm 3 ); pmedium is the density of HHBSS (1 .00 g/cm 3 ); and V ce n is the average cell volume of U2OS ( ⁇ 1700 pm 3 ) 25 .
  • the F at 200, 500, 1000, and 1500 g (RFC) were calculated to be 233, 583, 1 166, and 1749 pN, respectively.
  • the square masks for nanopillars were first generated as described above.
  • the mean intensities of LifeAct at nanopillars (0-9 pixel to the nanopillar centres) in a time- lapse image stack were measured using MATLAB code written for this study.
  • the mean intensities of ⁇ 5 at nanopillars were first measured and ranked.
  • F-actin dynamics the nanopillars with top 25% and bottom 25% in ⁇ 5 were separated into two groups: high-35 and low-35 nanopillars, respectively.
  • FCHo dynamics the nanopillars with top 25% and bottom 75% in 35 were separated into two groups: high-35 and low-35 nanopillars, respectively.
  • the mean intensities at each group of nanopillars in a time-lapse image stack were measured. The temporal standard deviations of either LifeAct or FCHo at individual nanopillars were reported as mean ⁇ SD.
  • the fibre, FCHo, and ⁇ 5/membrane ratiometric images were auto-thresholded using the ‘Moments’ method in Fiji.
  • the resulting binary images were multiplied to generate ROIs for curved adhesions, whose number and size per cell were reported as mean ⁇ SD.
  • FCHo2 F-BAR domain a dimerizing and membrane recruitment module that effects membrane curvature. Structure 15, 839-852 (2007).
  • FCHo proteins are nucleators of clathrin-mediated endocytosis.

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Abstract

Compositions, methods, and kits are provided for screening for candidate agents that inhibit formation of integrin-mediated curved adhesions. Screening assays may further include determining the effectiveness of candidate agents in reducing cell adhesion, proliferation, cell spreading, migration, or survival of cells and screening candidate agents for efficacy in treating disorders associated with integrin-mediated formation of curved adhesions.

Description

SCREENING FOR INHIBITORS OF INTEGRIN-MEDIATED CURVED ADHESION
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[001] This invention was made with Government support under contract GM141598 awarded by the National Institutes of Health. The Government has certain rights in the invention.
INCORPORATION BY REFERENCE OF A SEQUENCE LISTING
[002] A Sequence Listing is provided herewith as a Sequence Listing XML file, “STAN-1951 WO” created on May 10, 2023 and having a size of 35,554 bytes. The contents of the Sequence Listing XML file are incorporated by reference herein in their entirety.
BACKGROUND OF THE INVENTION
[003] Dysregulation of integrin signaling contributes to the development of various diseases, including thrombosis, autoimmune disorders, fibrotic diseases, microbial infections, and many types of cancer. Integrins have long been identified as therapeutic targets. Integrins are heterodimers composed of alpha and beta subunits forming 24 distinct integrins, which recognize a broad spectrum of ligands in the extracellular matrix (ECM). In the last two decades, six integrin inhibitors targeting four integrins (αIIbβa,α4β,α4β7, and α Lβ2) have been approved by The United States Food and Drug Administration (FDA) for the treatment of thrombosis, inflammatory bowel disease, multiple sclerosis, psoriasis, and dry eye disease. However, drug developments targeting integrins that are prominently implicated in fibrosis and cancer, such as the αv subfamily including integrins αvβ1, αvβ3, αvβ5, and αvβ6, have faced significant challenges. These molecules often exhibited encouraging in vitro results, but failed to show significant therapeutic benefits in clinical trials. One famous example is the failure of Cilengitide (a potent inhibitor of αvβ3 and αvβ5 at subnanomolar regime in binding assays) in a Phase III clinical trial for glioblastoma treatment. The discordance between preclinical and clinical studies strongly indicates that our understanding of integrin adhesions and their signaling in the human body remains limited. It is possible that these drugs are inhibiting only some of the integrin functions under physiological conditions.
[004] Thus, there remains a need to improve the understanding of the different integrins and to provide better methods of screening for inhibitors to treat diseases associated with dysregulated integrin function. SUMMARY OF THE INVENTION
[005] Compositions, methods, and kits are provided for screening for candidate agents that inhibit formation of integrin-mediated curved adhesions. Screening assays may further include determining the effectiveness of candidate agents in reducing cell adhesion, proliferation, cell spreading, migration, or survival of cells and screening candidate agents for efficacy in treating disorders associated with integrin-mediated formation of curved adhesions.
[006] In one aspect, a method of screening for an inhibitor that inhibits formation of integrin- mediated curved adhesions is provided, the method comprising: contacting a cell, a population of cells, or a tissue with a candidate agent; and measuring proximity of integrin isoform αVβ5 to FCHo2 in the presence of the candidate agent to determine whether the inhibitor inhibits colocalization of integrin isoform αVβ5 and FCHo2 in integrin-mediated curved adhesions, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
[007] In certain embodiments, the method further comprises providing a vertical nanostructure having an end curvature diameter ranging from 100 nm to 3 pm, wherein the cell forms the integrin-mediated curved adhesions on the surface of the nanostructure.
[008] In certain embodiments, the nanostructure is a nanobar or a nanopillar. In some embodiments, the nanostructure is coated with vitronectin or fibronectin.
[009] In certain embodiments, the method further comprises providing fibers comprising collagen, fibronectin, or vitronectin, or a combination thereof, wherein the cell forms the integrin- mediated curved adhesions on the surface of the fibers.
[0010] In certain embodiments, the method further comprises providing a curved extracellular matrix (ECM) surface, wherein the cell forms the integrin-mediated curved adhesions on the curved ECM surface.
[0011] In certain embodiments, the method further comprises detecting talin or paxillin in the integrin-mediated curved adhesions.
[0012] In certain embodiments, measuring the proximity of the integrin isoform αVβ5 to the FCHo2 comprises performing a complementation assay using a split reporter protein, a fluorescence resonance energy transfer (FRET) assay, a bioluminescence resonance energy transfer (BRET) assay, an antibody-based proximity ligation assay (PLA), or a proximity- dependent initiation of hybridization chain reaction (proxHCR) assay. [0013] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split fluorescent protein connected to the FCHo2, wherein a reconstituted fluorescent protein forms when the first component and the second component of the split fluorescent protein are expressed and interact to produce a detectable fluorescent signal; and detecting the fluorescent signal to determine proximity of the integrin isoform αVβ5 to the FCHo2 from the fluorescent signal, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion. In some embodiments, the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein. In some embodiments, the fluorescent signal is measured using fluorescence microscopy two-photon excitation microscopy, fluorescence confocal laser scanning microscopy, or light sheet fluorescence microscopy.
[0014] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split luminescent protein connected to the FCHo2, wherein a reconstituted luminescent protein forms when the first component and the second component of the split luminescent protein are expressed and interact to produce a detectable luminescent signal; and detecting the luminescent signal to determine proximity of the integrin isoform αVβ5 to the FCHo2 from the luminescent signal, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion. In some embodiments, the split luminescent protein is a split aequorin or a split luciferase such as, but not limited to, a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase. In some embodiments, the luminescent signal is measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
[0015] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) donor connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the integrin isoform αVβ5 or FCHo2 indicates whether a cellular adhesion is an integrin- mediated curved adhesion.
[0016] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) acceptor connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
[0017] In certain embodiments, FRET efficiency is measured using fluorescence microscopy or fluorescence confocal laser scanning microscopy.
[0018] In certain embodiments, the FRET donor and the FRET acceptor are fluorescent proteins or fluorescent dyes. In some embodiments, the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein, the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein, the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5), or the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine. [0019] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) donor connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin- mediated curved adhesion.
[0020] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) acceptor connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin- mediated curved adhesion.
[0021] In certain embodiments, the BRET efficiency is measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
[0022] In certain embodiments, the BRET donor or the BRET acceptor is a bioluminescent protein or a luminescent quantum dot. In some embodiments, the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein.
[0023] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 comprises performing an antibody-based proximity ligation assay (PLA) or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay. In some embodiments, performing the PLA comprises: contacting the cell, population of cells, or tissue with a first antibody that specifically binds to αVβ5; contacting the cell, population of cells, or tissue with a second antibody that specifically binds to FCHo2; contacting the first antibody with a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; contacting the second antibody with a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody; contacting the first probe and the second probe with a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with the first oligonucleotide and a second portion sufficiently complementary to hybridize with the second oligonucleotide when the first oligonucleotide and the second oligonucleotide are in sufficient proximity to each other to simultaneously hybridize to the bridge oligonucleotide; and ligating the first oligonucleotide to the second oligonucleotide to produce a ligation product; amplifying the ligation product to produce an amplicon; and detecting the amplicon. In some embodiments, amplifying comprises performing polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification. In some embodiments, amplifying comprises performing quantitative PCR. In some embodiments, the amplicon is detected with a detectably labeled probe that binds to the amplicon. In some embodiments, the probe comprises a fluorescent label, a bioluminescent label, or a chemiluminescent label.
[0024] In certain embodiments, the first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an Fv fragment, and an scFv fragment.
[0025] In certain embodiments, the method further comprises quantifying the number and area of the integrin-mediated curved adhesions.
[0026] In certain embodiments, the method further comprises contacting a population of cancer cells with the candidate agent if the candidate agent inhibits the formation of the integrin-mediated curved adhesions; and measuring cell adhesion, proliferation, cell spreading, migration, or survival of the population of cancer cells, wherein reduced proliferation, cell spreading, migration, or survival in presence of the candidate agent compared to that in a negative control population of cancer cells that are not treated with the candidate agent indicates that the candidate agent has anti-cancer activity. [0027] In certain embodiments, the candidate agent is a small molecule, a peptide, a protein, an aptamer, an antibody, an antibody mimetic, an inhibitory nucleic acid, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.
[0028] In certain embodiments, the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fv fragment, and a nanobody, wherein the antibody specifically binds to the integrin isoform αVβ5 or the FCHo2.
[0029] In certain embodiments, the inhibitory nucleic acid is selected from the group consisting of a small interfering RNA (siRNA), a microRNA (miRNA), a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), an antisense oligonucleotide, and a peptide nucleic acid, wherein the inhibitory nucleic acid inhibits transcription or protein translation of the integrin isoform αVβ5 or the FCHo2.
[0030] In certain embodiments, the CRISPR system targets a gene or an RNA transcript encoding the integrin isoform αVβ5 or the FCHo2, or makes epigenetic changes that reduce expression of the integrin isoform αVβ5 or the FCHo2. In some embodiments, the CRISPR system comprises Cas9, Cas12a, Cas12d, Cas13a, Cas13b, Cas13d, or a dead Cas9 (dCas9).
[0031] In another aspect, an inhibitor of integrin-mediated curved adhesion, identified by a method described herein, is provided.
[0032] In another aspect, a composition comprising an inhibitor of integrin-mediated curved adhesion, identified by a method described herein, is provided. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment.
[0033] In another aspect, a reporter system for measuring curved adhesion is provided, the reporter system comprising a cell expressing a split fluorescent or luminescent protein comprising a first component of a fluorescent or luminescent protein and a second component of the fluorescent or luminescent protein, wherein the cell comprises: a first nucleic acid comprising a coding sequence encoding a first fusion protein comprising the first component of the fluorescent or luminescent protein connected to integrin isoform ocVJ35; and a second nucleic acid comprising a coding sequence encoding a second fusion protein comprising the second component of the fluorescent or luminescent protein connected to FCHo2; wherein a reconstituted fluorescent or luminescent protein forms when the first component and the second component of the split fluorescent or luminescent protein are expressed and interact to produce a detectable fluorescent or luminescent signal indicative of the proximity of the integrin isoform αVβ5 and the FCHo2 in the cell.
[0034] In certain embodiments, the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split- mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
[0035] In certain embodiments, the split luminescent protein is a split aequorin or a split luciferase such as, but not limited to, a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
[0036] In another aspect, a kit comprising a reporter system described herein and instructions for detecting integrin-mediated curved adhesions is provided.
[0037] In another aspect, a system for screening a candidate agent to determine its effects on formation of integrin-mediated curved adhesions by a cell is provided, the system comprising a vertical nanostructure having an end curvature diameter ranging from 100 nm to 3 pm, wherein the cell forms the integrin-mediated curved adhesions on the surface of the nanostructure.
[0038] In certain embodiments, the nanostructure is a nanobar or a nanopillar.
[0039] In certain embodiments, the nanostructure is coated with vitronectin or fibronectin.
[0040] In certain embodiments, the system further comprises a cell or cell fragment of interest such as, but not limited to, a cancer cell, a blood cell, a platelet, an inflammatory cell, a fibroblast, or an epithelial cell.
[0041] In certain embodiments, the system further comprises reagents for measuring the proximity of the integrin isoform αVβ5 to the FCHo2 with a complementation assay using a split reporter protein, a fluorescence resonance energy transfer (FRET) assay, a bioluminescence resonance energy transfer (BRET) assay, an antibody-based proximity ligation assay (PLA), or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay.
[0042] In certain embodiments, the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to integrin isoform αVβ5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split fluorescent protein connected to FCHo2, wherein a reconstituted fluorescent protein forms when the first component and the second component of the split fluorescent protein are expressed and interact to produce a detectable fluorescent signal. In some embodiments, the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, or a split yellow-green fluorescent protein. In some embodiments, the system further comprises a fluorescence microscope, a two-photon excitation microscope, a fluorescence confocal laser scanning microscope, or a light sheet fluorescence microscope.
[0043] In certain embodiments, the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to integrin isoform αVβ5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split luminescent protein connected to FCHo2, wherein a reconstituted luminescent protein forms when the first component and the second component of the split luminescent protein are expressed and interact to produce a detectable luminescent signal. In some embodiments, the split luminescent protein is a split aequorin or a split luciferase such as, but not limited to, a split firefly luciferase, a Renilla luciferase, an Elateroidea luciferase, a Metridia luciferase, a Vibrio luciferase, a dinoflagellate luciferase, or a nano-lantern luciferase. In some embodiments, the system further comprises a luminescence microscope, luminescence digital imaging microscope, time-gated luminescence microscope, or a luminometer.
[0044] In certain embodiments, the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) donor connected to integrin isoform αVβ5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to FCHo2.
[0045] In certain embodiments, the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) acceptor connected to the integrin isoform αVβ5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2.
[0046] In certain embodiments, the system further comprises a fluorescence microscope or a fluorescence confocal laser scanning microscope.
[0047] In certain embodiments, the FRET donor and the FRET acceptor are fluorescent proteins or fluorescent dyes. In some embodiments, the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein, the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein, the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5), or the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine.
[0048] In certain embodiments, the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) donor connected to the integrin isoform αVβ5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2.
[0049] In certain embodiments, the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) acceptor connected to the integrin isoform αVβ5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2.
[0050] In certain embodiments, the system further comprises a luminescence microscope, a luminescence digital imaging microscope, a time-gated luminescence microscope, or a luminometer.
[0051 ] In certain embodiments, the BRET donor or the BRET acceptor is a bioluminescent protein or a luminescent quantum dot. In some embodiments, the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein.
[0052] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 comprises performing an antibody-based proximity ligation assay (PLA) or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay.
[0053] In certain embodiments, the system further comprises reagents for performing the PLA. In some embodiments, the system comprises: a first antibody that specifically binds to αVβ5; a second antibody that specifically binds to FCHo2; a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; and a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody. In some embodiments, the system further comprises a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with the first oligonucleotide and a second portion sufficiently complementary to hybridize with the second oligonucleotide when the first oligonucleotide and the second oligonucleotide are in sufficient proximity to each other to simultaneously hybridize to the bridge oligonucleotide. In some embodiments, the system further comprises a ligase. In some embodiments, the system further comprises reagents for performing polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification.
[0054] In certain embodiments, the first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an Fv fragment, and an scFv fragment.
[0055] In certain embodiments, the system further comprises a detectably labeled probe that binds to the amplicon. In some embodiments, the detectably labeled probe comprises a fluorescent label, a bioluminescent label, or a chemiluminescent label
[0056] In another aspect, a kit comprising a system described herein and instructions for detecting integrin-mediated curved adhesions is provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIGS. 1A-1 G. Positive membrane curvature induces selective accumulation of integrin αVβ5. FIG. 1A, Scanning electron microscopy (SEM) image of nanobars viewed at 45o angle. FIG. 1 B, SEM images of a nanobar viewed at 45o angle (top) or from the top (bottom). FIG. 1C, SEM image viewed at 45° angle showing nanobars deforming the cell membrane. FIG. 1 D, A chart of integrin p subunits probed in this study, which form heterodimers with different a 703 subunits to bind specific ECM proteins. FIG. 1 E, Top: schematic illustration of integrins binding to the ECM protein-coated surface. Bottom: bright-field (left) and fluorescence (right) images of nanobars coated with Atto647-gelatin. FIG. 1F, Zoom-in fluorescence images of isoforms and a membrane marker FusionRed-CaaX on nanobar substrates coated with different ECM proteins. The 5 shows preferential accumulation toward the nanobar ends, on both vitronectin and fibronectin coatings. Additional images of [31 , |33, |36, [38 isoforms on different ECMs are included in FIG. 9A. FIG. 1G, Quantification of the curvature preferences of |3 isoforms by measuring their nanobar end/side ratio normalized by the membrane marker. The end/side ratios of -100 nanobars per cell were individually calculated and then averaged. N = 12 cells from two independent experiments were quantified for each condition. Data are mean ± standard deviation (SD). Scale bars: 5 μm (FIGS. 1A, 1 C, 1 E, 1 F); 1 pm (1 B).
[0058] FIGS. 2A-2I: αVβ5 forms curved adhesions to extracellular ligands and bear forces. FIG. 2A, SEM images of nanopillars. FIG. 2B, Left: top-view SEM image of a cell on nanopillars. Right: schematic drawing of the interface between the cell membrane and a nanopillar. FIG. 2C, EDTA treatment induces a dramatic reduction of [35-GFP accumulation at nanopillars in a live cell labelled with CellMask membrane dye. Bottom: the normalized [35/membrane ratio is shown in the Parula colour scale. FIG. 2D, Quantification of the normalized β5/membrane ratio at nanopillars and their surrounding flat regions. N = 968/691 nanopillars before/after EDTA, from three independent cells. FIG. 2E, Schematic drawing of the centrifugation adhesion assay. FIG. 2F, Nucleus staining shows that more cells remain on nanopillar areas than on flat areas after spin. FIG. 2G, Quantification of the percentage of remaining cells after centrifugation. N = 6 imaging fields, from two independent experiments. FIG. 2H, Nanopillars induce β5 accumulation and promote early-stage (30 min after seeding) cell spreading. β5 KD largely abolished the nanopillar-induced early cell spreading. FIG. 2I, Quantification of the cell area on nanopillar/flat areas from N = 25/22 wildtype (WT), 22/21 β5-KD cells (vitronectin coating); N = 25/19 WT, 21/20 β5-KD cells (fibronectin coating); N = 21/22, 20/17 and 18/20 WT cells (respective gelatin, PLL and BSA coatings). Data are mean ± SD. P values calculated using Kruskal-Wallis test with Dunn’s multiple-comparison (FIG. 2D), unpaired multiple t-test with Holm-Sidak multiple- comparison (FIG. 2G), one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (I vitronectin, fibronectin) or unpaired two-tailed t-test (I gelatin, PLL, BSA). Scale bars: 1 pm (FIG. 2A); 5 pm (FIGS. 2B, 2C); 10pm (FIG. 2H); 200 pm (FIG. 2F).
[0059] FIGS. 3A-3F: Curved adhesions involve a subset of focal adhesion proteins to anchor F- actin. FIG. 3A, At vitronectin-coated nanopillars, β5-GFP accumulation (β5/membrane, green) spatially correlates with talin and paxillin, but not with vinculin or pFAK (magenta). Talin was tagged with mCherry. Endogenous paxillin, vinculin and FAK were immunolabeled. FIG. 3B, Both 35-GFP and LifeAct-mCherry, a marker for F-actin, strongly accumulate at nanopillars, but their intensities are not spatially correlated. At nanopillars, β5-GFP accumulation correlates with zyxin, but not with a-actinin 1 or a-actinin 4 (mCherry-tagged, magenta). The larger images of FIGS. 3A, 3B are shown in FIG. 13C, Pearson correlation coefficients between β5/membrane and focal adhesion proteins at nanopillars. N = 12 cells with each cell interfacing with -300 nanopillars were quantified for each condition, from two independent experiments. FIGS. 3D, 3E, Dynamics of F- actin at nanopillars coated with vitronectin (FIG. 3D) or gelatin (FIG. 3E) imaged at 15 s/frame for 15 min. Top: representative images of β5-GFP and LifeAct-RFP. Blue and red boxes indicate representative nanopillars with respective high and low/no β5 accumulations. Bottom: trajectories of LifeAct intensity at nanopillars indicated by the boxes in top pictures. FIG. 3F, The temporal standard deviations of LifeAct at N = 103 high-35, N=103 low-|35 nanopillars (vitronectin coating), and N = 153 nanopillars (gelatin coating). Data are mean ± SD. Rvalues calculated using Kruskal- Wallis test with Dunn’s multiple-comparison (FIG. 3F). Scale bars, 10 pm.
[0060] FIGS. 4A-4H: The formation of curved adhesion requires curvature-sensing proteins FCHol /2. FIG.4A, Mutant β5 (Y752A Y770A) that does not bind talin still accumulates at the ends of vitronectin coated nanobars in cells co-expressing Fusion Red-CaaX. FIG. 4B, Quantifications of the end/side ratios show that β5 (Y752A Y770A) mutant and WT β5 are not statistically different in sensing membrane curvatures. FIG. 4C, Top: β5 (green) colocalizes with clathrin plaques, marked by AP2 (magenta), on vitronectin- but not on fibronectin-coated flat surfaces. Bottom: at vitronectin coated nanopillars, 35 accumulation (35/membrane) does not spatially correlate with AP2. FIG. 4D, The shRNA KD of FCHo1/2 but not CHC, AP2-p, Eps15/R or intersectin 1/2 significantly reduces the nanobar end/side ratio of 35, compared with treatment with negative control shRNA. FIG. 4E, Top: 5 (green) does not colocalize with FCHo2 (magenta) on vitronectin-coated flat surfaces. Bottom: 35 accumulation (35/membrane) strongly correlates with FCHo2 at vitronectin-coated nanopillars. FIG. 4F, Pearson correlation coefficients of AP2 and FCHo2 against 35/membrane at vitronectin-coated nanopillars. FIG. 4G, Dynamics of 35 and FCHo2 at vitronectin-coated nanopillars imaged at 15 s/frame for 20 min. Left: representative fluorescence images. Right: kymographs generated with nanopillars indicated by the boxes in left images. FIG. 4H, The temporal standard deviations of FCHo2 at high-35/low-35 nanopillars (N = 104/313). Data are mean ± SD. For FIGS. 4B, 4D, 4F, N = 12 cells were quantified for each condition from two independent experiments, -100 nanobars per cell. P values calculated using unpaired two-tailed t-test (FIG. 4B), one-way ANOVA with Bonferroni’s multiple-comparison (FIG. 4D) or Mann-Whitney test (FIG. 4H). Scale bars, 10 pm.
[0061] FIGS. 5A-5G: Cells form curved adhesions on ECM fibres. FIG. 5A, β5-GFP accumulates at places where the plasma membrane is deformed along sparse ECM fibres. FIG. 5B, Intensity profiles of fibre (blue), (35 (green) and membrane (magenta) vertically crossing the membrane- wrapped fibre segment indicated in FIG. 5A. Lines 776 and shaded areas indicate means and SD, respectively, along the fibre segment. FIG. 5C, Quantification of β5 accumulation (β5/membrane) on sparse fibres made of pure collagen or ECM fibres with aVβ5 ligands fibronectin and vitronectin. N = 8/1 1/9 fibre fragments. FIG. 5D, On dense 3D ECM fibres, curved adhesions marked by the colocalizations of β5 and FCHo2 are abundant, while focal adhesions marked by the colocalization of β5 and vinculin are almost absent. FIG. 5E, Quantifications of the number and area of focal adhesions and curved adhesions on 2D flat surfaces and 3D ECM fibres. N = 12 cells for each condition. FIG. 5F, Probing the range of curvatures that facilitate the formation of curved adhesions using the gradient nanobar array. First row: bright-field (BF) image of the gradient nanobars. Second row: fluorescent images of the gradient bars coated with Cy3- vitronectin (Vn). Third and fourth rows: immunofluorescence of β5 (third row) in cells stably expressing membrane marker GFP-CaaX (fourth row) on vitronectin-coated gradient bars. FIG. 5G, Quantification of the end/side ratio of β5/membrane on gradient bars. N = 8 imaging fields. Data are mean ± SD, from two independent experiments. P values calculated using one-way ANOVA with Tukey's (FIGS. 5C, 5E) or Bonferroni’s (FIG. 5G) multiple comparison test. Scale bars, 10 pm.
[0062] FIG. 6: Schematic illustrations of focal adhesion and curved adhesion structures.
[0063] FIGS. 7A-7C: Characterizations of nanobars, nanopillars and gradient bars. FIG. 7A, Representative SEM images of a 200 nm-wide vertical nanobar array. The same sample was either viewed from the top (left) or tilted 45° (right). Scale bar, 5 pm. Statistical analysis of the geometrical dimensions for nanobars is shown in the table. Data are mean ± SD. FIG. 7B, Representative SEM images of a vertical nanopillar array. The sample was tilted 45°. Zoom-in image shows one single nanopillar. The top, middle, and bottom diameters of each nanopillar were measured. Scale bars, 1 pm. Statistical analysis of the geometrical dimensions for nanopillars is shown in the table. Data are mean ± SD. FIG. 7C, Representative SEM images of gradient bar array with designed end curvature diameters that range from 100 to 5000 nm. The sample was tilted 45°. Scale bars, 10 pm. Inset: the top view of a 100 nm-wide nanobar. Scale bar, 1 pm. Statistical analysis of the geometrical dimensions for gradient bars is shown in the table. Data are mean ± SD.
[0064] FIGS. 8A-8B: The ECM protein-coated quartz surfaces can support integrins to form adhesion structures. FIG. 8A, The mRNA levels of the integrin a and β subunits (presented in FIG. 1 D) in U2OS cells are listed. The mRNA levels are shown in the unit of transcripts per million (TPM)21. FIG. 8B, Fluorescence images of p subunits on flat surfaces that were coated with different ECM proteins (either gelatin, laminin, fibronectin or vitronectin). Endogenous pi (in the activated state) was immunolabelled; [33, [34, β5, [36, and P8 were tagged with GFP and transiently expressed. Scale bar, 10 pm.
[0065] FIGS. 9A-9G: Curvature induces the preferential accumulation of αVβ5 exclusively, related to FIG. 1 . FIG. 9A, Fluorescence images of integrin isoforms in U2OS cells 816 expressing a plasma membrane marker Fusion Red-CaaX on 200-nm nanobar arrays that were coated with different ECM proteins. Endogenous pi (in the activated state) was immunolabelled; [33, [36, and P8 were tagged with GFP and transiently expressed. The p subunits above show no preference for the ends of nanobars where the membranes are positively curved. Quantifications of their curvature preferences are presented in FIG. 1G. Scale bar, 5 pm. FIG. 9B, Fluorescence images of immunolabelled P5 (top) and αVβ5 (bottom) in U2OS cells on vitronectin-coated flat surfaces. Scale bar, 10 pm. FIG. 9C, Fluorescence images showing that immunolabelled P5 (top) and αVβ5 (bottom) preferentially accumulate at the ends of vitronectin-coated nanobars in U2OS cells expressing a plasma membrane marker FusionRed-CaaX. Scale bar, 5 pm. FIG. 9D, The curvature preferences of p5 and αVβ5 quantified, as in FIG. 1 G, by the nanobar end/side ratio of integrin/membrane. The ratios for individual nanobars were averaged to calculate the ratio for each cell. N = 12 cells for each condition, from two independent experiments. FIG. 9E, Fluorescence images of P5-GFP in MEFs (top) and Hela (bottom) cells on vitronectin-coated flat surfaces. Scale bar, 10 pm. FIG. 9F, Fluorescence images shows that P5-GFP preferentially accumulates at the ends of vitronectin-coated nanobars in MEFs (top) and Hela (bottom) cells transiently expressing a plasma membrane marker FusionRed-CaaX. Scale bar, 5 pm. FIG. 9G, The curvature preferences of P5 in MEFs and Hela cells quantified by the nanobar end/side ratio of integrin/membrane, as in FIG. 9D and FIG. 1 G. N = 12 cells for each condition, from two independent experiments. Data are mean ± SD.
[0066] FIGS. 10A-10G: αVβ5 accumulates at vitronectin-coated nanopillars to form curve adhesions, related to FIG. 2. FIGS. 10A-10B, Fluorescence images of β5-GFP and plasma membrane marker Fusion Red-CaaX on flat surfaces (top) and nanopillars (bottom) coated with vitronectin (FIG. 10A) or gelatin (FIG. 10B). Ratiometric images of β5/membrane (normalized by its mean per cell) are shown in the Parula colour scale. Scale bars, 10 pm. FIG. 10C, Quantification of the normalized 841 β5/membrane ratio at individual nanopillars and flat surrounding regions coated with vitronectin or gelatin. N = 926 and 863 nanopillars coated with vitronectin and gelatin, respectively, from three independent cells. FIG. 10D, Photo of a 3D-printed support for cell centrifugation assay. FIG. 10E, Quantification of the cell areas. Cells were cultured for 12 h on vitronectin-coated nanopillars and flat surfaces. N = 24 cells for each condition, from two independent experiments. FIG. 10F, Immunofluorescence of pFAK in cells on vitronectin- coated nanopillars (left) and flat surfaces (right). Scale bar, 10 pm. FIG. 10G, The percentage of focal adhesion area in total cell area, for cells treated as in FIG. 10F. The focal adhesions are defined as regions with pFAK accumulation. N = 21 and 17 cells on nanopillars and flat surfaces, respectively, from two independent experiments. Data are mean ± SD. Rvalues calculated using Kruskal-Wallis test with Dunn’s multiple-comparison (FIG. 10C) and unpaired two-tailed t test (FIGS. 10E, 10G).
[0067] FIGS. 11A-11 C: Curved adhesions promote early-stage cell spreading. FIGS. 11A-11 B, Overlay of the bright-field and fluorescence 854 images showing wild type (left) and β5-KD (right) cells stably expressing a plasma membrane marker GFP-CaaX on both nanopillars (left half) and flat surfaces (right half) in the same imaging fields. The cells were seeded on substrates coated with vitronectin (FIG. 11 A) or fibronectin (FIG. 11 B) 30 min before fixation. Scale bars, 50 pm. FIG. 11C, Overlay of bright-field and fluorescence images showing wild type cells stably expressing GFP-CaaX on both nanopillar arrays (left half) and flat surfaces (right half) in the same imaging fields. The cells were seeded on substrates coated with gelatin (left), poly-L-lysine (middle), or BSA (right) 30 min before fixation. Scale bar, 50 pm. Quantifications of the areas of cells described in this figure are presented in FIG. 2I.
[0068] FIGS. 12A-12H: β5 colocalizes with focal adhesion proteins on flat surfaces. FIGS. 12A- 12H, Fluorescence images show that β5-GFP colocalizes with focal adhesion proteins talin (FIG. 12A), paxillin (FIG. 12B), vinculin (FIG. 12C), pFAK (FIG. 12D), LifeAct (FIG. 12E), zyxin (FIG. 12F), a-Actinin 1 (FIG. 12G), and a-Actinin 4 (FIG. 12H) at focal adhesion-like patches on vitronectin-coated flat surfaces. Endogenous paxillin, vinculin, and pFAK were immunolabelled. Talin, LifeAct, zyxin, a-Actinin 1 , and a-Actinin 4 were tagged with mCherry and transiently expressed. Scale bars, 10 pm. The zoom-in images of β5 (green) and focal adhesion proteins (magenta) are shown. Scale bars, 5 pm.
[0069] FIGS. 13A-13H: Nanopillar-induced β5 accumulation correlate with a subset of focal adhesion proteins. FIGS. 13A-13H, Fluorescence images showing that at vitronectin-coated nanopillars, |35-GFP accumulation spatially correlates with talin (FIG. 13A), paxillin (FIG. 13B) and zyxin (FIG. 13F), but not with vinculin (FIG. 13C), pFAK (FIG. 13D), LifeAct (FIG. 13E), a- Actinin 1 (FIG. 13G), and a-Actinin 4 (FIG. 13H). Endogenous paxillin, vinculin, and pFAK were immunolabelled. Talin, LifeAct, zyxin, a-Actinin 1 , and a-Actinin 4 were tagged with mCherry and transiently expressed. The cell membrane was 878 marked with transiently expressed surface SNAP-tag conjugated with Alexa Fluor 647. Scale bars, 10 pm. The zoom-in images of |35/membrane ratio and focal adhesion proteins in the white box-indicated areas are shown in FIGS. 3A-3B.
[0070] FIGS. 14A-14E: Curved adhesions are static. FIG. 14A, Fluorescence images of a live cell expressing β5-GFP and a plasma membrane marker Fusion Red-CaaX on vitronectin-coated nanopillars. The ratiometric image of β5/membrane (normalized by its mean per cell) is shown in the Parula colour scale. Scale bar, 10 pm. FIG. 14B, Kymograph of the normalized β5/membrane ratio at nanopillars indicated by the white box in FIG. 14A. Images were acquired at 15 s/frame for 80 min. Scale bar, 1 pm. c, Representative trajectories of the normalized β5/membrane ratio dynamics at nanopillars where curved adhesions assemble (nanopillar 1 in FIG. 14A) or disassemble (nanopillar 2 in FIG. 14A). FIGS. 14D-14E, Time-lapse images of β5-GFP and FusionRed-CaaX at nanopillar 1 (FIG. 14D) and 2 (FIG. 14E). At nanopillar 1 , the curved adhesion gradually assembled. At nanopillar 2, the curved adhesion gradually disassembled. Scale bars, 1 pm.
[0071] FIGS. 15A-15E: The curvature preference of aVβ5 is determined by FCHo proteins, but not by talin or clathrin lattices, related to FIG. 4. FIG. 15A, Fluorescence images showing that mutant β5 (Y752A Y770A) does not colocalize with talin897 mCherry at focal adhesions. Scale bar, 10 pm. FIG. 15B, Fluorescence images showing that both β5-GFP and AP2-a, a marker for clathrin lattices, strongly accumulate at vitronectin-coated nanopillars, but their intensities are not spatially correlated. The cell membrane was marked with transiently expressed surface SNAP- tag conjugated with Alexa Fluor 647. The zoom-in images of β5/membrane ratio and AP2-a in the white box-indicated area are shown in FIG. 4C. FIG. 15C, Scatter plot of the |35/membrane ratio (normalized by its mean within 902 the cell) against AP2-a intensity at nanopillars. N = 299 pillars covered by the cell in FIG. 15B. 15D, Fluorescence images showing that 35-GFP accumulation spatially correlates with FCHo2-mCherry at vitronectin-coated nanopillars. The cell membrane was marked with transiently expressed surface SNAP-tag conjugated with Alexa Fluor 647. The zoom-in images of β5/membrane ratio and FCHo2 in the white box-indicated area are shown in FIGS. 4E. FIG. 15E, Scatter plot of the 35/membrane ratio (normalized by its mean within the cell) against FCHo2 intensity at nanopillars. N = 305 pillars covered by the cell in FIG. 15D.
[0072] FIG. 16: FCHo1/2 KD reduces the curvature preference of β5. Representative fluorescence images showing that KD of FCHol/2 but not AP2-p, clathrin heavy chain (CHC), Eps15/R, intersectin 1/2, or negative control (scramble shRNA treatment) reduces the β5 accumulation at the two ends of vitronectin-coated nanobars. The transcription of shRNA in cells was indicated by the expression of blue fluorescent protein. Cells transiently expressed FusionRed-CaaX as a plasma membrane marker. Scale bar, 10 nm. Quantifications of the 35 curvature preference under conditions described in this figure are presented in FIG. 4D.
[0073] FIGS. 17A-17C: aV918 35 ligands incorporation and super-resolution analysis of ECM fibres. FIG. 17A, Fluorescence images showing the colocalization of Alexa Fluor 647-collagen with fibronectin (immunolabelled with Alexa Fluor 488) and Cy3-vitronectin in sparse (left) and dense (right) ECM fibres. Scale bars, 10 pm. FIG. 17B, 3D super-resolution reconstruction of Alexa Fluor 647-collagen fibres. The widefield diffraction-limited image (top left) depicts collagen fibres. The XY projection of the 3D super-resolution reconstruction (right) reveals 924 higher resolution structures of the collagen fibres in the diffraction-limited image. Yellow arrows point to examples of individual collagen fibres. Colour encodes the z position. Scale bars, 5 pm. FIG. 17C, Extracting the diameter of individual fibres, (i) The image shows an example of a resolved individual fibre. Transverse line profiles (representative example shown at top) are taken across the fibre to estimate its diameter. Scale bar, 1 pm. (ii) Localizations from the line profile are binned into a histogram which is then fit to a Gaussian function (orange line). The full width at half maximum is extracted to estimate the diameter. The line profile in (i) yields the histogram and fit depicted in (ii).
[0074] FIGS. 18A-18C: The formation of curved adhesions on ECM fibres requires ligand, curvature, and FCHol/2, related to FIG. 5. FIG. 18A, Top: fluorescence images showing that 35- GFP does not accumulate at places where the plasma membrane is deformed along sparse pure collagen fibres in WT cells. Bottom: fluorescence images showing that FCHol/2 KD reduces the 35-GFP accumulation at places where the plasma membrane is deformed along sparse ECM collagen fibres with aVβ5 ligands. Scale bar, 10 pm. FIG. 18B, Intensity profiles of fibre (blue), β5 (green) and membrane (magenta) vertically crossing the membrane-wrapped fibre segments indicated in FIG. 18A. Lines and shaded areas indicate means and SD, respectively, along the fibre segments. FIG. 18C, Top: fluorescence images showing that β5-GFP does not colocalize with FCHo2-mCherry on 2D surfaces coated with the same components of ECM fibres (i.e., collagen, fibronectin and vitronectin). Bottom: fluorescence images showing that β5-GFP colocalize with vinculin at focal adhesion-like patches on 2D surfaces coated with the same components of ECM fibres. Scale bar, 10 pm.
DETAILED DESCRIPTION
[0075] Compositions, methods, and kits are provided for screening for candidate agents that inhibit formation of integrin-mediated curved adhesions. Screening assays may further include determining the effectiveness of candidate agents in reducing cell adhesion, proliferation, cell spreading, migration, or survival of cells and screening candidate agents for efficacy in treating disorders associated with integrin-mediated formation of curved adhesions.
[0076] Before the methods and compositions are provided for screening candidate agents for inhibition of formation of integrin-mediated curved adhesions are further described, it is to be understood that this invention is not limited to a particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0077] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0078] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0079] It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.
[0080] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
[0081] As used herein the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the inhibitor" includes reference to one or more inhibitors and equivalents thereof, e.g., antagonists, known to those skilled in the art, and so forth.
[0082] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
[0083] The term "about," particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.
[0084] As used herein, the term “curved adhesion” refers to an integrin isoform αVβ5-mediated adhesion that forms at regions of a cell membrane that are curved. Formation of curved adhesions is mediated not only by integrin αVβ5 but also curvature-sensitive proteins such as FCHo2, which localize in curved adhesions. Curved adhesions can be distinguished from other cell adhesions based on the colocalization of integrin isoform αVβ5 and FCHo2 in curved adhesions. Curved adhesions can be detected, for example, using an assay that measures the proximity of integrin isoform αVβ5 to FCHo2, as described herein.
[0085] The term "administering" is intended to include routes of administration which allow the agent to perform its intended function of inhibiting formation of integrin-mediated curved adhesions and/or adhesion, proliferation, cell spreading, migration, or survival of cells. Examples of routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. Further, the agent may be coadministered with a pharmaceutically acceptable carrier. The agent also may be administered as a prodrug, which is converted to its active form in vivo.
[0086] As used herein, the term “determining” refers to both quantitative and qualitative determinations and as such, the term “determining” is used interchangeably herein with “assaying,” “measuring,” and the like.
[0087] "Substantially purified" generally refers to isolation of a substance (e.g., compound, polynucleotide, protein, polypeptide, antibody, aptamer, or inhibitor) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well- known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
[0088] By "isolated" is meant, when referring to a polypeptide or peptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type. The term "isolated" with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
[0089] A peptide is said to "interact" with a protein if it binds specifically (e.g., in a lock-and-key type mechanism), non-specifically or in some combination of specific and non-specific binding. A first peptide "interacts preferentially" with a protein if it binds (non-specifically and/or specifically) to the protein with greater affinity and/or greater specificity than it binds to other proteins (e.g., binds to integrin isoform αVβ5 or FCHo2 to a greater degree than to other proteins). The term "affinity" refers to the strength of binding and can be expressed quantitatively as a dissociation constant (Kd). It is to be understood that specific binding does not necessarily require interaction between specific amino acid residues and/or motifs of each peptide. For example, in certain embodiments, a peptide interacts preferentially with integrin isoform αVβ5 or FCHo2 but, nonetheless, may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest). Typically, weak binding, or background binding, is readily discernible from the preferential interaction with the compound or polypeptide of interest, e.g., by use of appropriate controls.
[0090] The term “inhibitor" as used herein refers to any molecule (e.g., small molecule inhibitor, protein, polypeptide, peptide, fusion protein, inhibitory nucleic acid (e.g., siRNA, miRNA, antisense nucleic acid), peptide nucleic acid, antibody, antibody mimetic, aptamer) or CRISPR system that inhibits formation of integrin-mediated curved adhesions or integrin isoform αVβ5 or FCHo2 biological activity, and/or integrin isoform «V|35 or FCHo2 expression (e.g., transcription or translation). Inhibition may be complete or partial (i.e. , all formation of integrin-mediated curved adhesions, some formation of integrin-mediated curved adhesions, or most formation of integrin- mediated curved adhesions; all activity, some activity, or most activity of integrin isoform αVβ5 or FCHo2 is blocked by an inhibitor). For example, an inhibitor may reduce the activity of integrin isoform αVβ5 or FCHo2 by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels. The inhibitor may reduce the number or rate of formation of integrin-mediated curved adhesions or the colocalization of integrin isoform αVβ5 and FCHo2 in integrin-mediated curved adhesions by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels.
[0091] An "effective amount" of an inhibitor (e.g., small molecule inhibitor, protein, polypeptide, peptide, fusion protein, inhibitory nucleic acid (e.g., siRNA, miRNA, antisense nucleic acid, or peptide nucleic acid), antibody, antibody mimetic, or aptamer) or CRISPR system targeting the integrin isoform αVβ5or FCHo2 gene (e.g., Cas9, Cas12a), RNA (Cas13), or epigenome (dCas9 fusion protein) is an amount sufficient to inhibit the formation of integrin-mediated curved adhesions, for example, by inhibiting activity of integrin isoform αVβ5 or FCHo2 and/or colocalization of integrin isoform ocVβ5 and FCHo2 in integrin-mediated curved adhesions. An effective amount can be administered in one or more administrations, applications, or dosages.
[0092] The terms "tumor," "cancer" and "neoplasia" are used interchangeably and refer to a cell or population of cells whose growth, proliferation or survival is greater than growth, proliferation or survival of a normal counterpart cell, e.g. a cell proliferative, hyperproliferative or differentiative disorder. Typically, the growth is uncontrolled. The term "malignancy" refers to invasion of nearby tissue. The term "metastasis" or a secondary, recurring or recurrent tumor, cancer or neoplasia refers to spread or dissemination of a tumor, cancer or neoplasia to other sites, locations or regions within the subject, in which the sites, locations or regions are distinct from the primary tumor or cancer. Neoplasia, tumors and cancers include benign, malignant, metastatic and non- metastatic types, and include any stage (I, II, III, IV or V) or grade (G1 , G2, G3, etc.) of neoplasia, tumor, or cancer, or a neoplasia, tumor, cancer or metastasis that is progressing, worsening, stabilized or in remission. In particular, the terms "tumor," "cancer" and "neoplasia" include carcinomas, such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, and small cell carcinoma, and include cancers such as, but are not limited to, pancreatic cancer, lung cancer (non-small cell lung cancer, small cell lung cancer), gastric cancer, ovarian cancer, endometrial cancer, colorectal cancer, oral cancer, skin cancer, cholangiocarcinoma, head and neck cancer, breast cancer, ovarian cancer, melanoma, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorders, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumors, extrahepatic bile duct cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gestational trophoblastic tumors, hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, leukemia, lip cancer, oral cavity cancer, liver cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic squamous neck cell carcinoma, multiple myeloma and other plasma cell neoplasms, mycosis fungoides and the Sezary syndrome, myelodysplastic syndromes, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, bone cancers, including osteosarcoma and malignant fibrous histiocytoma of bone, paranasal sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumors, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilm's tumor and other childhood kidney tumors.
[0093] By "anti-tumor activity" or “anti-cancer activity” is intended a reduction in the rate of cell proliferation, and hence a decline in growth rate of an existing tumor or in a tumor that arises during therapy, and/or destruction of existing neoplastic (tumor) cells or newly formed neoplastic cells, and hence a decrease in the overall size of a tumor during therapy. Such activity can be assessed using animal models.
[0094] “Disorders associated with integrin-mediated formation of curved adhesions” include various types of cancer, including without limitation, glioblastoma, breast cancer, pancreatic cancer, and prostate cancer; fibrotic diseases including, without limitation, pulmonary fibrosis, cardiac fibrosis, scleroderma/systemic sclerosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, nephrogenic systemic fibrosis, retroperitoneal fibrosis, arthrofibrosis, myelofibrosis, and mediastinal fibrosis; thrombosis; autoimmune disorders, including, without limitation, multiple sclerosis, Guillain-Barre syndrome, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, autoimmune liver disease, autoimmune colitis, autoimmune thyroid disorders such as Graves’ disease and Hashimoto's thyroiditis, and autoimmune encephalomyelitis; and microbial infections.
[0095] By "therapeutically effective dose or amount" of an inhibitor is intended an amount that, when administered as described herein, brings about a positive therapeutic response in treatment of a disorder associated with formation of curved adhesions, such as an amount that has anti- cancer, anti-inflammatory, or anti-fibrotic activity. In addition, a therapeutically effective dose or amount of an inhibitor may reduce cell adhesion, proliferation, cell spreading, migration, or survival of cells. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.
[0096] The term "tumor response" as used herein means a reduction or elimination of all measurable lesions. The criteria for tumor response are based on the WHO Reporting Criteria [WHO Offset Publication, 48-World Health Organization, Geneva, Switzerland, (1979)]. Ideally, all uni- or bidimensionally measurable lesions should be measured at each assessment. When multiple lesions are present in any organ, such measurements may not be possible and, under such circumstances, up to 6 representative lesions should be selected, if available.
[0097] The term "complete response" (CR) as used herein means a complete disappearance of all clinically detectable malignant disease, determined by 2 assessments at least 4 weeks apart.
[0098] The term "partial response" (PR) as used herein means a 50% or greater reduction from baseline in the sum of the products of the longest perpendicular diameters of all measurable disease without progression of evaluable disease and without evidence of any new lesions as determined by at least two consecutive assessments at least four weeks apart. Assessments should show a partial decrease in the size of lytic lesions, recalcifications of lytic lesions, or decreased density of blastic lesions.
[0099] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
[00100] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).
[00101] By "subject" is meant any member of the subphylum Chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
[00102] The term "antibody" encompasses polyclonal antibodies, monoclonal antibodies as well as hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies. The term antibody includes: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991 ) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab')2 and F(ab) fragments; Fv molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659- 2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (scFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11 :3287-3303, Vincke et al. (2012) Methods Mol Biol 911 :15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31 :1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule.
[00103] The phrase "specifically (or selectively) binds" with reference to binding of an antibody to an antigen refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample. Specific binding to an antigen under such conditions may require an antibody that is selected for its specificity for a particular antigen. For example, antibodies raised to an antigen from specific species such as rat, mouse, or human can be selected to obtain only those antibodies that are specifically immunoreactive with the antigen and not with other proteins, except for polymorphic variants and alleles. This selection may be achieved by subtracting out antibodies that cross-react with molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane. Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
[00104] The terms "microRNA," "miRNA," and MiR" are interchangeable and refer to endogenous or artificial non-coding RNAs that are capable of regulating gene expression. It is believed that miRNAs function via RNA interference. When used herein in the context of inactivation, the use of the term microRNAs is intended to include also long non-coding RNAs, piRNAs, siRNAs, and the like. Endogenous (e.g., naturally occurring) miRNAs are typically expressed from RNA polymerase II promoters and are generated from a larger transcript.
[00105] The terms "siRNA" and "short interfering RNA" are interchangeable and refer to single- stranded or double-stranded RNA molecules that are capable of inducing RNA interference. SiRNA molecules typically have a duplex region that is between 18 and 30 base pairs in length.
[00106] The terms "piRNA" and "Piwi-interacting RNA" are interchangeable and refer to a class of small RNAs involved in gene silencing. PiRNA molecules typically are between 26 and 31 nucleotides in length.
[00107] The terms "snRNA" and "small nuclear RNA" are interchangeable and refer to a class of small RNAs involved in a variety of processes including RNA splicing and regulation of transcription factors. The subclass of small nucleolar RNAs (snoRNAs) is also included. The term is also intended to include artificial snRNAs, such as antisense derivatives of snRNAs comprising antisense sequences directed against the integrin isoform αVβ5 or FCHo2 gene.
[00108] The term "antisense", as used herein, refers to any composition containing nucleotide sequences which are complementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. Antisense molecules include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation. The designation "negative" is sometimes used in reference to the antisense strand, and "positive" is sometimes used in reference to the sense strand.
[00109] The terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" are used herein to include a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single- stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. There is no intended distinction in length between the terms "polynucleotide,'1 "oligonucleotide," "nucleic acid" and "nucleic acid molecule," and these terms will be used interchangeably. Thus, these terms include, for example, 3'-deoxy-2',5'- DNA, oligodeoxyribonucleotide N3' P5' phosphoramidates, 2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, microRNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5- propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide. The term also includes locked nucleic acids (e.g., comprising a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom). See, for example, Kurreck et al. (2002) Nucleic Acids Res. 30: 1911 -1918; Elayadi et al. (2001 ) Curr. Opinion Invest. Drugs 2: 558-561 ; Orum et al. (2001 ) Curr. Opinion Mol. Ther. 3: 239-243; Koshkin et al. (1998) Tetrahedron 54: 3607-3630; Obika et al. (1998) Tetrahedron Lett. 39: 5401 -5404.
[00110] The term "homologous region" refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a "homologous region" is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term "homologous, region," as used herein, refers to the ability of nucleic acid molecules to hybridize to each other. For example, a single-stranded nucleic acid molecule can have two homologous regions which are capable of hybridizing to each other. Thus, the term "homologous region" includes nucleic acid segments with complementary sequence. Homologous regions may vary in length, but will typically be between 4 and 40 nucleotides (e.g., from about 4 to about 40, from about 5 to about 40, from about 5 to about 35, from about 5 to about 30, from about 5 to about 20, from about 6 to about 30, from about 6 to about 25, from about 6 to about 15, from about 7 to about 18, from about 8 to about 20, from about 8 to about 15, etc.).
[00111] The term "complementary" and "complementarity" are interchangeable and refer to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands or regions. Complementary polynucleotide strands or regions can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G). 100% complementary refers to the situation in which each nucleotide unit of one polynucleotide strand or region can hydrogen bond with each nucleotide unit of a second polynucleotide strand or region. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands or two regions can hydrogen bond with each other and can be expressed as a percentage.
[00112] A "target site" or "target sequence" for an inhibitory nucleic acid is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by an antisense oligonucleotide or inhibitory RNA molecule.
[00113] The term "transfection" is used to refer to the uptake of foreign DNA or RNA by a cell. A cell has been "transfected" when exogenous DNA or RNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981 ) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA or RNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake, for example, of microRNA, siRNA, piRNA, IncRNA, or antisense nucleic acids.
[00114] A “CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
[00115] The term "Cas9" as used herein encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).
[00116] A Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA). For purposes of Cas9 targeting, a gRNA may comprise a sequence "complementary" to a target sequence (e.g., major or minor allele), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.
[00117] By "selectively binds" with reference to a guide RNA is meant that the guide RNA binds preferentially to a target sequence of interest or binds with greater affinity to the target sequence than to other genomic sequences. For example, a gRNA will bind to a substantially complementary sequence and not to unrelated sequences. A gRNA that "selectively binds" to a particular allele, such as a particular mutant allele e.g., allele comprising a substitution, insertion, or deletion), denotes a gRNA that binds preferentially to the particular target allele, but to a lesser extent to a wild-type allele or other sequences. A gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.
[00118] The term "donor polynucleotide" refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).
[00119] A "target site" or "target sequence" is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide. The target site may be allele-specific (e.g., a major or minor allele).
[00120] By "homology arm" is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA. The homology arms are referred to herein as 5' and 3' (i.e. , upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively. The nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.
[00121] "Administering" a nucleic acid, such as an inhibitory or regulatory nucleic acid (e.g., microRNA, siRNA, piRNA, snRNA, antisense nucleic acid, or IncRNA), or a CRISPR system (expressing, e.g., a donor polynucleotide, guide RNA, Cas protein (e.g., Cas9, Cas12a, Cas12d, Cas13, or dCas9)) to a cell comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.
Detecting Integrin-Mediated Curved Adhesions
[00122] Integrin-mediated curved adhesions can be distinguished from other cell adhesions based on the colocalization of integrin isoform αVβ5 and FCHo2 in curved adhesions. Accordingly, integrin-mediated curved adhesions can be detected by measuring the proximity of integrin isoform «V|35 to FCHo2 in a cell adhesion, wherein the proximity of integrin isoform αVβ5 to FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion. Any suitable proximity assay known in the art may be used for detecting the proximity of integrin isoform αVβ5 to FCHo2 in cell adhesions. Exemplary proximity assays include, without limitation, complementation assays using a split reporter protein, fluorescence resonance energy transfer (FRET) assays, bioluminescence resonance energy transfer (BRET) assays, proximity ligation assays (PLA), and proximity-dependent initiation of hybridization chain reaction (proxHCR) assays.
[00123] In a complementation assay using a split reporter protein, a first reporter protein fragment is linked to integrin isoform αVβ5, and a second reporter protein fragment is linked to FCHo2, wherein colocalization of the integrin isoform αVβ5 and the FCHo2 in a curved adhesion brings the two fragments of the reporter protein in close enough proximity for a reconstituted functional reporter protein to form that can be detected. The split reporter protein can be any protein that can be split into two parts and reconstituted non-covalently to form a functional protein that can be detected. The reconstituted reporter protein may provide, for example, a fluorescent, bioluminescent, chemiluminescent, or chromogenic signal. The assay is typically performed with a split fluorescent protein, a split bioluminescent protein, or a split enzyme (e.g., a split horseradish peroxidase or a split [3-galactosidase) used with a chemiluminescent or chromogenic substrate. For a description of split reporter protein assays, see, e.g., Shekhawat et al. (2011) Curr. Opin. Chem. Biol. 15(6):789-797, Kerppola et al. (2006) Nat. Protoc. 1 :1278-1286, Biewenga et al. (2020) Biochem. Soc. Trans. 48(6):2643-2655, Azad et al. (2014) Anal Bioanal Chem. 406(23):5541 -60, Feng et al. (2017) Nat. Common. 8(1 ):370, Rowe et al. (2009) Anal. Chem. 81 :8662-8668, and Lang et al. (2019) Curr. Protoc. Toxicol. 82(1):e90); herein incorporated by reference.
[00124] In some embodiments, the split reporter protein is a split fluorescent protein. Exemplary split fluorescent proteins include, without limitation, a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, and a split sfCherry2 fluorescent protein. The fluorescent signal of the reconstituted fluorescent protein can be measured, for example, using fluorescence microscopy, two-photon excitation microscopy, fluorescence confocal laser scanning microscopy, or light sheet fluorescence microscopy.
[00125] In certain embodiments, measuring proximity of the integrin isoform «V|35 to the FCHo2 comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split fluorescent protein connected to the FCHo2, wherein a reconstituted fluorescent protein forms when the first component and the second component of the split fluorescent protein are expressed and interact to produce a detectable fluorescent signal; and detecting the fluorescent signal to determine proximity of the integrin isoform αVβ5 to the FCHo2 from the fluorescent signal, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
[00126] In some embodiments, the split reporter protein is a split bioluminescent protein. Exemplary split bioluminescent proteins include, without limitation, split aequorins and split luciferases, such as, but not limited to, a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, and a split nano-lantern luciferase. The luminescent signal of the reconstituted bioluminescent protein can be measured, for example, using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
[00127] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split luminescent protein connected to the FCHo2, wherein a reconstituted luminescent protein forms when the first component and the second component of the split luminescent protein are expressed and interact to produce a detectable luminescent signal; and detecting the luminescent signal to determine proximity of the integrin isoform αVβ5 to the FCHo2 from the luminescent signal, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion. In some embodiments, the split luminescent protein is a split aequorin or a split luciferase such as, but not limited to, a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase. In some embodiments, the luminescent signal is measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
[00128] In a FRET assay, energy is transferred from a donor fluorophore to an acceptor fluorophore upon illumination at an excitation wavelength of the donor fluorophore. For fluorescence resonance energy transfer to occur, the emission spectrum of the donor fluorophore must overlap with the excitation spectrum of the acceptor fluorophore. Fluorescence resonance energy transfer from the donor fluorophore to the acceptor fluorophore results in a fluorescent emission from the acceptor fluorophore, which emits fluorescence at a longer wavelength than the donor fluorophore. FRET efficiency between a donor fluorophore and an acceptor fluorophore can be used to determine the distance between the integrin isoform «V|35 and the FCHo2. Illumination with light at an excitation wavelength of the donor fluorophore results in radiative energy transfer from the donor fluorophore to the acceptor fluorophore when the donor fluorophore and the acceptor fluorophore are in proximity (i.e., less than 10 nm distance apart), FRET efficiency can be measured, for example, using fluorescence microscopy or fluorescence confocal laser scanning microscopy. For a description of methods of using FRET, see, e.g., Helms (2008) "Fluorescence Resonance Energy Transfer". Principles of Computational Cell Biology. Weinheim: Wiley-VCH. p. 202; Schneckenburger, Herbert (2019-11 -27). "Forster resonance energy transfer-what can we learn and how can we use it?". Methods and Applications in Fluorescence. 8 (1): 013001 ; Hochreiter et al. (2015) Sensors (Basel). 2015 Oct 16;15(10):26281 - 314; Ergin et al. (2016) Curr. Pharm. Biotechnol. 17(14):1222-1230; Hirata et al. (2016) Biophys J. 111 (6):1103-1111 ; Sun et al. (2013) Cytometry A 83(9):780-93; herein incorporated by reference.
[00129] In certain embodiments, the FRET donor and the FRET acceptor are fluorescent proteins or fluorescent dyes. In some embodiments, the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein, the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein, the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5), or the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine.
[00130] In certain embodiments, measuring proximity of the integrin isoform «V|35 to the FCHo2 comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a FRET donor connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the integrin isoform αVβ5 or FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
[00131] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a FRET acceptor connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
[00132] BRET assays are similar to FRET assays but have the advantage of not requiring external illumination for the energy transfer between a donor and an acceptor. In a BRET assay, BRET efficiency between a bioluminescent donor and a bioluminescent acceptor is used to determine the distance between the integrin isoform αVβ5 and the FCHo2. BRET efficiency can be measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer. In certain embodiments, the BRET donor or the BRET acceptor is a bioluminescent protein or a luminescent quantum dot. In some embodiments, the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein. For a description of methods of using BRET, see, e.g., Bevan et al. (2006) "Pharmaceutical Applications of GFP and RCFP". In Chalfie M, Kain SR (eds.). Green Fluorescent Protein: Properties, Applications and Protocols. Methods of Biochemical Analysis. Vol. 47 (2nd ed.). Hoboken, NJ: John Wiley & Sons. pp. 361 -90; Pfleger et al. (2006). "Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET)". Nature Methods. 3 (3): 165-74; and Dale et al. (2019) Front. Bioeng. Biotechnol. 7:56; herein incorporated by reference.
[00133] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a BRET assay comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a BRET donor connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion. [00134] In certain embodiments, measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a BRET assay comprises: transfecting a cell, a population of cells, or a tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a BRET acceptor connected to the integrin isoform «αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
[00135] A PLA assay can also be used to determine the proximity of integrin isoform αVβ5 to FCHo2. PLA uses oligonucleotides conjugated to antibodies, which are brought in proximity upon binding to colocalized antigens. To detect proximity of integrin isoform αVβ5 to FCHo2, two primary antibodies are used: one that specifically binds to integrin isoform αVβ5 and a second that specifically binds to FCHo2. PLA probes comprising oligonucleotides conjugated to secondary antibodies, which specifically bind to the primary antibodies, are used for detection. If the PLA probes are in proximity because of the primary antibodies binding to integrin isoform αVβ5 and FCHo2 (which are in proximity because of colocalization at a curved adhesion), the oligonucleotides can be ligated and amplified. In some embodiments, a bridge oligonucleotide is used to facilitate ligation. The bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with a first oligonucleotide of a first PLA probe and a second portion sufficiently complementary to hybridize with a second oligonucleotide of a second PLA probe when the first oligonucleotide and the second oligonucleotide are in sufficient proximity to each other to simultaneously hybridize to the bridge oligonucleotide. The ligation product can be amplified, for example, using polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification. In some embodiments quantitative PCR is used to amplify the ligation product. In some embodiments, circularizing oligonucleotides are hybridized to the oligonucleotides of the PLA probes and ligated to form a circular template for rolling circle amplification. Amplicons can be detected with detectably labeled probes. Detectably labeled probes that bind to the amplicons may comprise, for example, a fluorescent label, a bioluminescent label, or a chemiluminescent label. For a description of PLA, see, e.g., Lof et al. (2017) Curr Protoc Cytom. 81 :4.8.1-4.8.10, Gullberg et al. (2004) Proc. Natl. Acad. Sci. USA 101 (22):8420-4, Soderberg et al. (2006) Nature Methods 3 (12): 995-1000; herein incorporated by reference. [00136] In some embodiments, performing the PLA comprises: contacting a cell, a population of cells, or a tissue with a first antibody that specifically binds to integrin isoform αVβ5; contacting the cell, population of cells, or tissue with a second antibody that specifically binds to FCHo2; contacting the first antibody with a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; contacting the second antibody with a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody; contacting the first probe and the second probe with a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with the first oligonucleotide and a second portion sufficiently complementary to hybridize with the second oligonucleotide when the first oligonucleotide and the second oligonucleotide are in sufficient proximity to each other to simultaneously hybridize to the bridge oligonucleotide; and ligating the first oligonucleotide to the second oligonucleotide to produce a ligation product; amplifying the ligation product to produce an amplicon; and detecting the amplicon. In some embodiments, amplifying comprises performing polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification. In some embodiments, amplifying comprises performing quantitative PCR. In some embodiments, the amplicon is detected with a detectably labeled probe that binds to the amplicon.
[00137] In certain embodiments, the first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an Fv fragment, and an scFv fragment.
[00138] Proximity-dependent initiation of hybridization chain reaction (proxHCR) is similar to PLA but uses antibodies conjugated to fluorescently labeled oligonucleotides having a hairpin conformation. The hairpins of the two probes are partly reversely complementary to each other and can cross-hybridize. An activator oligonucleotide is used to open the secondary structure of one of the proximity probes to release it 3’-end, which then invades and unfolds the other proximity probe, thereby liberating its 3’-end. This initiates a hybridization chain reaction (HCR), which is used for signal amplification. The two oligonucleotide hairpin probes are conjugated to antibodies that must bind in close proximity to initiate the HCR to generate a detectable fluorescent product. The proximity of integrin isoform αVβ5 to FCHo2 can be determined using this method with a pair of fluorescently labeled hairpin probes conjugated to antibodies that specifically bind to integrin isoform αVβ5 and FCHo2. For a description of proxHCR, see, e.g., Koos et al. (2015) Nat. Commun. 6:7294, Raykova et al. (2016) Proteomes 4(4):36; herein incorporated by reference.
[00139] In some embodiments, curved adhesions are detected on a cell, a population of cells, or tissue in vitro (i.e. , grown in culture) or in vivo. Curved adhesions may be detected on any type of cell or tissue, including normal cells or tissue, diseased or damaged cells or tissue, cancerous cells or tissue, inflammatory cells or inflamed tissue, or cells or tissue at risk of future disease (e.g., cells of precancerous tissue, transplanted tissue, tissue at high risk of tumor or disease development due to an underlying genetic abnormality, mutation, or autoimmunity). Cells and tissue suitable for use with the methods described herein generally include any type of cells or tissue collected from living or dead subjects, such as cells or tissue from biopsy specimens, surgical specimens, and autopsy specimens, including, but not limited to, epithelium, muscle, connective, and nervous tissue.
[00140] Cells may fail to form curved adhesions on flat surfaces in the absence of an extracellular matrix. Therefore, in some embodiments, a vertical nanostructure is used to facilitate the formation of curved adhesions in assays. In certain embodiments, the nanostructure is a nanobar or a nanopillar. In some embodiments, the nanostructure is coated with vitronectin or fibronectin. (See Examples.)
[00141] In certain embodiments, the vertical nanostructure has an end curvature diameter ranging from 100 nm to 3 pm, including any end curvature diameter in this range, such as 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 800 nm, 825 nm, 850 nm, 900 nm, 925 nm, 950 nm, 975 nm, 1 pm, 1 .5 pm, 2 pm, 2.5 pm, or 3 pm, wherein the cell forms the integrin-mediated curved adhesions on the surface of the nanostructure.
[00142] Alternatively, fibers comprising collagen, fibronectin, or vitronectin, or a combination thereof, may be used to facilitate the formation of integrin-mediated curved adhesions, wherein the integrin-mediated curved adhesions form on the surface of the fibers. A curved extracellular matrix (ECM) surface may also be used to facilitate the formation of integrin-mediated curved adhesions, wherein a cell forms the integrin-mediated curved adhesions on the curved ECM surface. Screening for Inhibitors that Inhibit Formation of Inteqrin-Mediated Curved Adhesions
[00143] Methods of screening for inhibitors that inhibit formation of integrin-mediated curved adhesions are also provided, Integrin-mediated curved adhesions can be detected and measured based on the colocalization of integrin isoform αVβ5 and FCHo2 using a proximity assay, as described above. Accordingly, a cell, population of cells, or a tissue is contacted with a candidate agent; and the proximity of integrin isoform αVβ5 to FCHo2 is measured in the presence and absence of the candidate agent to determine whether the inhibitor inhibits colocalization of integrin isoform «VP5 and FCHo2 in integrin-mediated curved adhesions, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion. Assays can be applied to any cell or cell fragment of interest including, without limitation, cancer cells, blood cells, inflammatory cells, fibroblasts, and epithelial cells, as well as platelets.
[00144] A variety of proximity assays may be used for this purpose, and in many embodiments, a candidate agent will be tested in different assays to confirm inhibitory capability as well as efficacy in treating a disorder associated with integrin-mediated formation of curved adhesions. For example, proximity assays may be used to determine the ability of an agent to inhibit integrin- mediated formation of curved adhesions. In addition, cell-based or tissue assays may be used for testing for the effectiveness of candidate agents in reducing cell adhesion, proliferation, cell spreading, migration, or survival of cells.
[00145] An inhibitor can be any molecule including, without limitation, a small molecule inhibitor, protein, polypeptide, peptide, fusion protein, nucleic acid, oligonucleotide, peptide nucleic acid, antibody or fragment thereof, antibody mimetic, aptamer, or a CRISPR system that inhibits formation of integrin-mediated curved adhesions or integrin isoform «V|35 or FCHo2 biological activity or colocalization, and/or integrin isoform αVβ5 or FCHo2 expression (e.g., transcription or translation). Inhibition may be complete or partial (i.e., all formation of integrin-mediated curved adhesions, some formation of integrin-mediated curved adhesions, or most formation of integrin- mediated curved adhesions; all activity, some activity, or most activity of integrin isoform «V|35 and/or FCHo2 is blocked by an inhibitor). For example, an inhibitor may reduce the activity of integrin isoform αVβ5 or FCHo2 by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels. The inhibitor may reduce the number or rate of formation of integrin-mediated curved adhesions or the colocalization of integrin isoform αVβ5 and FCHo2 in integrin-mediated curved adhesions by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels.
[00146] Any convenient format may be used for the assay, e.g., wells, plates, flasks, etc., preferably a high throughput format, such as multi-well plates. Inhibitors can be identified by contacting a cell, a population of cells, or a tissue with a candidate agent; and measuring inhibition of formation of integrin-mediated curved adhesions by the candidate agent.
[00147] Assays may further include suitable controls (e.g., a sample comprising cells or tissue in the absence of the test agent). Generally, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
[00148] A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc., including agents that are used to facilitate optimal binding activity and/or reduce non-specific or background activity. Reagents that improve the efficiency of the assay, such as nanostructures, fibers, or curved extracellular matrix, as described herein, protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The components of the assay mixture are added in any order that provides for the requisite activity. Incubations are performed at any suitable temperature, typically between 4°C and 40°C. Incubation periods are selected for optimum activity but may also be optimized to facilitate rapid high-throughput screening. In some embodiments, between 0.1 hour and 1 hour, between 1 hour and 2 hours, or between 2 hours and 4 hours, will be sufficient.
[00149] A variety of different test agents may be screened. Candidate agents encompass numerous chemical classes, e.g., small organic compounds having a molecular weight of more than 50 daltons and less than about 10,000 daltons, less than about 5,000 daltons, or less than about 2,500 daltons. Test agents can comprise functional groups necessary for structural interaction with proteins, e.g., hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl or carboxyl group, or at least two of the functional chemical groups. The test agents can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Test agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. [00150] Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Moreover, screening may be directed to known pharmacologically active compounds and chemical analogs thereof, or to new agents with unknown properties such as those created through rational drug design.
[00151] In some embodiments, test agents are synthetic compounds. A number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. See for example WO 94/24314, hereby expressly incorporated by reference, which discusses methods for generating new compounds, including random chemistry methods as well as enzymatic methods.
[00152] In another embodiment, the test agents are provided as libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.
[00153] In some embodiments, the test agents are organic moieties. In this embodiment, test agents are synthesized from a series of substrates that can be chemically modified. “Chemically modified” herein includes traditional chemical reactions as well as enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleosides. Chemical (including enzymatic) reactions may be done on the moieties to form new substrates or candidate agents which can then be tested using the present invention.
[00154] In some embodiments test agents are assessed for any cytotoxic activity it may exhibit toward a living eukaryotic cell, using well-known assays, such as trypan blue dye exclusion, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide) assay, and the like. Agents that do not exhibit significant cytotoxic activity are considered candidate agents.
[00155] In some embodiments, the test agent is an antibody that specifically binds to and inhibits biological activity of integrin isoform αVβ5 or FCHo2. Any type of antibody may be screened for the ability to inhibit integrin isoform αVβ5 or FCHo2 by the methods described herein, including polyclonal antibodies, monoclonal antibodies, hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab')2 and F(ab) fragments; Fv molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single- chain Fv molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11 :3287-3303, Vincke et al. (2012) Methods Mol Biol 911 :15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31 :1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule.
[00156] In other embodiments, the test agent is an aptamer that specifically binds to and inhibits biological activity of integrin isoform αVβ5 or FCHo2. Aptamers may be isolated from a combinatorial library and improved by directed mutation or repeated rounds of mutagenesis and selection. For a description of methods of producing aptamers, see, e.g., Aptamers: Tools for Nanotherapy and Molecular Imaging (R.N. Veedu ed., Pan Stanford, 2016), Nucleic Acid and Peptide Aptamers: Methods and Protocols (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2009), Aptamers Selected by Cell-SELEX for Theranostics (W. Tan, X. Fang eds., Springer, 2015), Cox et al. (2001 ) Bioorg. Med. Chem. 9(10):2525-2531 ; Cox et al. (2002) Nucleic Acids Res. 30(20): e108, Kenan et al. (1999) Methods Mol. Biol. 1 18:217-231 ; Platella et al. (2016) Biochim. Biophys. Acta Nov 16 pii: S0304-4165(16)30447-0, and Lyu et al. (2016) Theranostics 6(9) :1440-1452; herein incorporated by reference in their entireties.
[00157] In yet other embodiments, the test agent is an antibody mimetic that specifically binds to and inhibits biological activity of integrin isoform αVβ5 or FCHo2. Any type of antibody mimetic may be used as an inhibitor, including, but not limited to, affibody molecules (Nygren (2008) FEBS J. 275 (11 ):2668-2676), affilins (Ebersbach et al. (2007) J. Mol. Biol. 372 (1 ) :172-185), affimers (Johnson et al. (2012) Anal. Chem. 84 (15):6553-6560), affitins (Krehenbrink et al. (2008) J. Mol. Biol. 383 (5):1058-1068), alphabodies (Desmet et al. (2014) Nature Communications 5:5237), anticalins (Skerra (2008) FEBS J. 275 (11 ):2677-2683), avimers (Silverman et al. (2005) Nat. BiotechnoL 23 (12):1556-1561 ), darpins (Stumpp et al. (2008) Drug Discov. Today 13 (15- 16):695-701 ), fynomers (Grabulovski et al. (2007) J. Biol. Chem. 282 (5) :3196-3204), and monobodies (Koide et al. (2007) Methods Mol. Biol. 352:95-109).
[00158] In some embodiments, a candidate agent that inhibits formation of integrin-mediated curved adhesions is further tested for its effects on cell adhesion, proliferation, cell spreading, migration, or survival of cells in a cell-based assay. In these embodiments, a candidate agent of interest is contacted with the cells; and the effect, if any, of the candidate agent on the cells is determined.
[00159] For example, a population of cells can be cultured in vitro in the presence of an effective dose of a candidate agent. The effects on formation of integrin-mediated curved adhesions, cell adhesion, proliferation, cell spreading, migration, or survival, or any combination thereof, may be assayed. The candidate agent is added to the culture medium, and the culture medium is maintained under conventional conditions suitable for growth of the cells. Various commercially available systems have been developed for the growth of mammalian cells to provide for removal of adverse metabolic products, replenishment of nutrients, and maintenance of oxygen. By employing these systems, the medium may be maintained as a continuous medium, so that the concentrations of the various ingredients are maintained relatively constant or within a prescribed range.
[00160] In some embodiments, a test compound, identified as inhibiting formation of integrin- mediated curved adhesions, is further tested for its efficacy in treating a disorder associated with integrin-mediated formation of curved adhesions in vivo, e.g., in an animal such as an animal model of a disorder associated with integrin-mediated formation of curved adhesions. For example, an agent that inhibits integrin-mediated formation of curved adhesions, identified as described herein, can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified, as described herein, can be used in an animal model to determine the mechanism of action of such an agent. Monitoring the efficacy of agents (e.g., drugs) for treatment of a disorder associated with integrin-mediated formation of curved adhesions can be applied not only in basic drug screening, but also in clinical trials.
[00161] Test compounds can be tested for efficacy in treating various disorders associated with integrin-mediated formation of curved adhesions including, but not limited to, cancers such as glioblastoma, breast cancer, pancreatic cancer, and prostate cancer; fibrotic diseases such as pulmonary fibrosis, cardiac fibrosis, scleroderma/systemic sclerosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, nephrogenic systemic fibrosis, retroperitoneal fibrosis, arthrofibrosis, myelofibrosis, and mediastinal fibrosis; thrombosis; autoimmune disorders such as multiple sclerosis, Guillain-Barre syndrome, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, autoimmune liver disease, autoimmune colitis, autoimmune thyroid disorders such as Graves’ disease and Hashimoto's thyroiditis, and autoimmune encephalomyelitis; and microbial infections.
Screening for Inhibitors of Expression of Inteqrin Isoform αVβ5 or FCHo2
[00162] Alternatively, an inhibitor may reduce integrin isoform αVβ5 or FCHo2 gene expression (e.g., transcription or translation). Candidate agents are identified by contacting a cell, a population of cells, or a tissue with a candidate compound and measuring the expression of integrin isoform αVβ5 or FCHo2, as determined by, e.g., mRNA or polypeptide levels. The level of expression of integrin isoform αVβ5 or FCHo2 mRNA or protein in the presence of the candidate compound is compared to the level of expression of integrin isoform αVβ5 or FCHo2 mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified based on this comparison. For example, when expression of integrin isoform αVβ5 or FCHo2 mRNA or protein in cells is decreased (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an agent that inhibits integrin isoform aVβ5 or FCHo2 expression. Alternatively, when expression of integrin isoform αVβ5 or FCHo2 mRNA or protein is the same or increased (statistically significantly more) in the presence of the candidate compound than in its absence, the candidate compound is likely not an agent that inhibits integrin isoform αVβ5 or FCHo2 expression or activity.
[00163] In some embodiments, an inhibitor of integrin isoform αVβ5 or FCHo2 gene expression reduces integrin isoform αVβ5 or FCHo2 mRNA or protein levels by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels. Inhibitors of integrin isoform αVβ5 or FCHo2 gene expression can include, but are not limited to, antisense oligonucleotides, inhibitory RNA molecules, such as miRNAs, siRNAs, piRNAs, and snRNAs, peptide nucleic acids, small molecule inhibitors, and CRISPR systems designed for genome, RNA transcript, or epigenome editing. Various types of inhibitors for inhibiting nucleic acid function are well known in the art. See e.g., International patent application WO/2012/018881 ; U.S. patent application 2011/0251261 ; U.S. patent no. 6,713,457; Kole et al. (2012) Nat. Rev. Drug Discov. 11 (2):125-40; Sanghvi (201 1 ) Curr. Protoc. Nucleic Acid Chem. Chapter 4:Unit 4.1.1-22; herein incorporated by reference in their entireties.
[00164] Inhibitors can be single stranded or double stranded polynucleotides and may contain one or more chemical modifications, such as, but not limited to, locked nucleic acids, peptide nucleic acids, sugar modifications, such as 2'-O-alkyl (e.g., 2'-O-methyl, 2'-0-methoxyethyl), 2'-fluoro, and 4'-thio modifications, and backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages. In addition, inhibitory RNA molecules may have a "tail" covalently attached to their 3'- and/or 5'-end, which may be used to stabilize the RNA inhibitory molecule or enhance cellular uptake. Such tails include, but are not limited to, intercalating groups, various kinds of reporter groups, and lipophilic groups attached to the 3' or 5' ends of the RNA molecules. In certain embodiments, the RNA inhibitory molecule is conjugated to cholesterol or acridine. See, for example, the following for descriptions of syntheses of 3'- cholesterol or 3'-acridine modified oligonucleotides: Gamper, H. B., Reed, M. W., Cox, T., Virosco, J. S., Adams, A. D., Gall, A., Scholler, J. K., and Meyer, R. B. (1993) Facile Preparation and Exonuclease Stability of 3'-Modified Oligodeoxynucleotides. Nucleic Acids Res. 21 145-150; and Reed, M. W., Adams, A. D., Nelson, J. S., and Meyer, R. B., Jr. (1991 ) Acridine and Cholesterol- Derivatized Solid Supports for Improved Synthesis of 3'-Modified Oligonucleotides. Bioconjugate Chem. 2 217-225 (1993); herein incorporated by reference in their entireties. Additional lipophilic moieties that can be used, include, but are not limited to, oleyl, retinyl, and cholesteryl residues, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, 1 ,3-Bis- O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1 ,3- propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03- (oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. Additional compounds, and methods of use, are set out in US Patent Publication Nos. 2010/0076056, 2009/0247608 and 2009/0131360; herein incorporated by reference in their entireties.
[00165] In one embodiment, inhibition of integrin isoform αVβ5 or FCHo2 function may be achieved by administering antisense oligonucleotides targeting the integrin isoform αVβ5 or FCHo2 gene. The antisense oligonucleotides may be ribonucleotides or deoxyribonucleotides. Preferably, the antisense oligonucleotides have at least one chemical modification. Antisense oligonucleotides may be comprised of one or more "locked nucleic acids". "Locked nucleic acids" (LNAs) are modified ribonucleotides that contain an extra bridge between the 2' and 4' carbons of the ribose sugar moiety resulting in a "locked" conformation that confers enhanced thermal stability to oligonucleotides containing the LNAs. Alternatively, the antisense oligonucleotides may comprise peptide nucleic acids (PNAs), which contain a peptide-based backbone rather than a sugar-phosphate backbone. The antisense oligonucleotides may contain one or more chemical modifications, including, but are not limited to, sugar modifications, such as 2'-O-alkyl (e.g. 2'-O- methyl, 2'-0-methoxyethyl), 2'-fluoro, and 4' thio modifications, and backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages (see, for example, U.S. Pat. Nos. 6,693,187 and 7,067,641 , which are herein incorporated by reference in their entireties). In some embodiments, suitable antisense oligonucleotides are 2'-O- methoxyethyl "gapmers" which contain 2'-0-methoxyethyl-modified ribonucleotides on both 5' and 3' ends with at least ten deoxyribonucleotides in the center. These "gapmers" are capable of triggering RNase H-dependent degradation mechanisms of RNA targets. Other modifications of antisense oligonucleotides to enhance stability and improve efficacy, such as those described in U.S. Pat. No. 6,838,283, which is herein incorporated by reference in its entirety, are known in the art and are suitable for use in the methods of the invention. Antisense oligonucleotides may comprise a sequence that is at least partially complementary to an integrin isoform αVβ5 or FCHo2 target sequence, e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to the integrin isoform αVβ5 or FCHo2 target sequence. In some embodiments, the antisense oligonucleotide may be substantially complementary to the integrin isoform αVβ5 or FCHo2 target sequence, that is at least about 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence. In one embodiment, the antisense oligonucleotide comprises a sequence that is 100% complementary to the integrin isoform αVβ5 or FCHo2 target sequence.
[00166] In another embodiment, the inhibitor of integrin isoform αVβ5 or FCHo2 is an inhibitory RNA molecule (e.g., a miRNA, a siRNA, a piRNA, or a snRNA) having a single-stranded or double-stranded region that is at least partially complementary to the target sequence of integrin isoform αVβ5 or FCHo2, e.g., about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence of integrin isoform αVβ5 or FCHo2. In some embodiments, the inhibitory RNA comprises a sequence that is substantially complementary to the target sequence of integrin isoform αVβ5 or FCHo2, e.g., about 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence. In other embodiments, the inhibitory RNA molecule may contain a region that has 100% complementarity to the target sequence. The inhibitory molecules may target the integrin isoform αVβ5 or FCHo2 sequence of SEQ ID NO:1 . In certain embodiments, the inhibitory RNA molecule may be a double-stranded, small interfering RNA or a short hairpin RNA molecule (shRNA) comprising a stem-loop structure.
[00167] Inhibitors can be detectably labeled by well-known techniques. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Such labeled inhibitors can be used to determine cellular uptake efficiency, quantitate binding of inhibitors at target sites, or visualize inhibitor localization.
CRISPR System for Targeting Integrin Isoform ocV|35 or FCHo2 in Cells
[00168] In some embodiments, a CRISPR/Cas system is used to inactivate or reduce expression of an endogenous gene expressing integrin isoform «V|35 or FCHo2 in a cell, a population of cells, or a tissue of interest. For example, a CRISPR/Cas system can be used to delete, inactivate, or mutate an endogenous gene expressing integrin isoform αVβ5 or FCHo2 to eliminate or reduce integrin isoform αVβ5 or FCHo2 gene expression or protein activity. Genome modification can be performed, for example, using homology directed repair (HDR) with a donor polynucleotide comprising a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the target locus (e.g., integrin isoform αVβ5 or FCHo2 gene) to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence. The homology arms are referred to herein as 5' and 3' (i.e. , upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively.
[00169] The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e. , sufficiently complementary for hybridization) by the 5' and 3' homology arms.
[00170] In certain embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the "5' target sequence" and "3' target sequence") flank a specific site for cleavage and/or a specific site for introducing the intended edit. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. In a preferred embodiment, the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.
[00171] A homology arm can be of any length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some instances, the 5' and 3' homology arms are substantially equal in length to one another, e.g. one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5' and 3' homology arms are substantially different in length from one another, e.g. one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.
[00172] The donor polynucleotide is used in combination with an RNA-guided nuclease, which is targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by a guide RNA (gRNA). A target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease- gRNA complex by hybridization at the target site. For example, the gRNA can be designed with a sequence complementary to a target sequence in the integrin isoform αVβ5 or FCHo2gene. In some embodiments, the gRNA is designed to with a sequence complementary to a cancer- specific integrin isoform αVβ5 or FCHo2 mutation to target the nuclease-gRNA complex to the site of a mutation in a cell. The mutation may comprise an insertion, a deletion, or a substitution. For example, the mutation may include a single nucleotide variation, gene fusion, translocation, inversion, duplication, frameshift, missense, nonsense, or other mutation. The targeted minor allele may be a common genetic variant or a rare genetic variant. In certain embodiments, the gRNA is designed to selectively bind to a minor allele with single base-pair discrimination, for example, to allow binding of the nuclease-gRNA complex to a single nucleotide polymorphism (SNP). In particular, the gRNA may be designed to target disease-relevant mutations of interest for the purpose of genome editing to delete or deactivate the integrin isoform αVβ5 or FCHo2 gene in a cell.
[001 3] In certain embodiments, the RNA-guided nuclease used for genome modification is a CRISPR system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Cas nucleases. Examples of Cas proteins include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Casi o, Cas10d, Cas12a (Cpf 1 ), Cas12b (C2c1 ), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), CasF, CasG, CasH, Csy1 , Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx15, Csf1 , Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof. [00174] In certain embodiments, a type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP 038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP_002344900), Campylobacter coli (WP 060786116); Campylobacter fetus (WP 059434633); Corynebacterium ulcerans (NC 015683, NC 017317); Corynebacterium diphtheria (NC 016782, NC 016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721 ); Streptococcus thermophilus (YP 820832), Streptococcus mutans (WP 061046374, WP 024786433); Listeria innocua (NP 472073); Listeria monocytogenes (WP 061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP 001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP 048482595, WP 032965177); and Neisseria meningitidis (WP 061704949, YP 002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385- 397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091 -6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.
[00175] The CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA. The bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break. Targeting of Cas9 typically further relies on the presence of a 5' protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.
[00176] The genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In certain embodiments, the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9- gRNA complex to the allele.
[00177] In certain embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.
[00178] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpf1 , also known as Cas12a) is used. Cpf1 is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpf1 does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpf1 for targeting than Cas9. Cpf 1 is capable of cleaving either DNA or RNA. The PAM sites recognized by Cpf 1 have the sequences 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9. Cpf1 cleavage of DNA produces double-stranded breaks with a sticky- ends having a 4 or 5 nucleotide overhang. For a discussion of Cpf 1 , see, e.g., Ledford et al. (2015) Nature. 526 (7571 ):17-17, Zetsche et al. (2015) Cell. 163 (3):759-771 , Murovec et al. (2017) Plant Biotechnol. J. 15(8) :917-926, Zhang et al. (2017) Front. Plant Sci. 8:177, Fernandes et al. (2016) Postepy Biochem. 62(3) :315-326; herein incorporated by reference. [00179] Cas12b (C2c1) is another class II CRISPR/Cas system RNA-guided nuclease that may be used. C2c1 , similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. For a description of Cas12b, see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.
[00180] In yet another embodiment, an engineered RNA-guided Fokl nuclease may be used. RNA-guided Fokl nucleases comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (Fokl-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on Fokl. For a description of engineered RNA-guided Fokl nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; herein incorporated by reference.
[00181] An RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector). In some embodiments, the RNA- guided nuclease and the gRNA are both provided by vectors. Both can be expressed by a single vector or separately on different vectors. The vector(s) encoding the RNA-guided nuclease an gRNA may be included in a CRISPR expression system to target the integrin isoform αVβ5 or FCHo2 gene in cells.
[00182] Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell or organism. For example, a nucleic acid encoding an RNA-guided nuclease or reverse transcriptase can be modified to substitute codons having a higher frequency of usage in a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the RNA-guided nuclease is introduced into cells, the protein can be transiently, conditionally, or constitutively expressed in the cell.
[00183] In another embodiment, CRISPR interference (CRISPRi) is used to repress integrin isoform αVβ5 or FCHo2 gene expression. CRISPRi is performed with a complex of a catalytically inactive Cas9 (dCas9) with a guide RNA that targets the integrin isoform αVβ5 or FCHo2 gene. An engineered nuclease-deactivated Cas9 (dCas9) is used to allow sequence-specific targeting without cleavage. Nuclease-deactivated forms of Cas9 may be engineered by mutating catalytic residues at the active site of Cas9 to destroy nuclease activity. Any such nuclease deficient Cas9 protein from any species may be used as long as the engineered dCas9 retains gRNA-mediated sequence-specific targeting. In particular, the nuclease activity of Cas9 from Streptococcus pyogenes can be deactivated by introducing two mutations (D10A and H841 A) in the RuvC1 and HNH nuclease domains. Other engineered dCas9 proteins may be produced by similarly mutating the corresponding residues in other bacterial Cas9 isoforms. For a description of engineered nuclease-deactivated forms of Cas9, see, e.g., Qi et al. (2013) Cell 152:1173-1183, Dominguez et al. (2016) Nat. Rev. Mol. Cell. Biol. 17(1 ):5-15; herein incorporated by reference in their entireties.
[00184] The dCas9 protein can be designed to target the integrin isoform ocVps or FCHo2 gene by altering its guide RNA sequence. A target-specific single guide RNA (sgRNA) comprises a nucleotide sequence that is complementary to a target site, and thereby mediates binding of the dCas9-sgRNA complex by hybridization at the target site. CRISPRi can be used to sterically repress transcription by blocking either transcriptional initiation or elongation by designing a sgRNA with a sequence complementary to an integrin isoform αVβ5 or FCHo2 promoter or exonic sequence. The sgRNA may be complementary to the non-template strand or the template strand, but preferably is complementary to the non-template strand to more strongly repress transcription.
[00185] The target site will typically comprise a nucleotide sequence that is complementary to the sgRNA, and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide.
[00186] In certain embodiments, the sgRNA comprises 5-50 nucleotides, 10-30 nucleotides, 15- 25 nucleotides, 18-22 nucleotides, 19-21 nucleotides, and any length between the stated ranges, including, for example, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
[00187] The sgRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et aL, Tetrahedron (1992) 48:2223-2311 ; and Applied Biosystems User Bulletin No. 13 (1 April 1987). Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et aL, Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et aL, Meth. Enzymol. (1979) 68:109. [00188] In some embodiments, the dCas9 is fused to a transcriptional repressor domain capable of further repressing transcription of the integrin isoform αVβ5 or FCHo2 gene, e.g., by inducing heterochromatinization. For example, a Kruppel associated box (KRAB) can be fused to dCas9 to repress transcription of a target gene in human cells (see, e.g., Gilbert et al. (2013) Cell. 154 (2): 442-45, O'Geen et al. (2017) Nucleic Acids Res. 45(17):9901 -9916; herein incorporated by reference).
[00189] Alternatively, dCas9 can be used to introduce epigenetic changes that reduce integrin isoform αVβ5 or FCHo2 gene expression by fusion of dCas9 to an epigenetic modifier such as a chromatin-modifying epigenetic enzyme. The promoter for integrin isoform αVβ5 or FCHo2 can be silenced, for example, by methylation or acetylation (e.g. histone H3 lysine 9 [H3K9] methylation, histone H3 lysine 27 [H3K27] methylation, and/or DNA methylation). For example, fusion of dCas9 to a DNA methyltransferase such as DNA methyltransferase 3 alpha (DNMT3A) or a chimeric Dnmt3a/Dnmt3L methyltransferase (DNMT3A3L) allows targeted DNA methylation. Fusion of dCas9 to histone demethylase LSD1 allows targeted histone demethylation (see, e.g., Liu et al. (2016) Cell 167(1 ) :233-247, Lo et al. (2017) F1000Res. 6. pii: F1000 Faculty Rev-747, and Stepper et al. (2017) Nucleic Acids Res. 45(4):1703-1713; herein incorporated by reference). [00190] In yet other embodiments, an RNA-targeting CRISPR-Cas13 system is used to perform RNA interference to reduce integrin isoform αVβ5 or FCHo2 expression. Members of the Cas13 family are RNA-guided RNases containing two HERN domains having RNase activity. In particular, Cas13a (C2c2), Cas13b (C2c6), and Cas13d can be used for RNA knockdown. Cas13 proteins can be made to target and cleave integrin isoform αVβ5 or FCHo2 transcribed RNA using a gRNA with complementarity to the target transcript sequence. The gRNA is typically about 64 nucleotides in length with a short hairpin crRNA and a 28-30 nucleotide spacer that is complementary to the target site on the integrin isoform otV|35 or FCHo2 RNA transcript. Cas13 recognition and cleavage of a target transcript results in degradation of the transcript as well as nonspecific degradation of any nearby transcripts. See, e.g., Abudayyeh et al. (2017) Nature 550:280-284, Hameed et al. (2019) Microb. Pathog. 133:103551 , Wang et al. (2019) Biotechnol Adv. 37(5):708-729, Aman et al. (2018) Viruses 10(12). pii: E732, and Zhang et al. (2018) Cell 175(1 ):212-223; herein incorporated by reference. Determining Levels of Expression of Inteqrin Isoform otV|35 or FCHo2
[00191] An "effective amount" of an integrin isoform αVβ5 or FCHo2 inhibitory nucleic acid (e.g., microRNA, siRNA, piRNA, snRNA, antisense oligonucleotide) or a CRISPR system targeting the integrin isoform αVβ5 or FCHo2 gene (e.g., Cas9, Cas12a), RNA (e.g., Cas13), or epigenome (e.g., dCas9 fusion protein) is an amount sufficient to effect beneficial or desired results, such as an amount that reduces formation of integrin-mediated formation of curved adhesions, colocalization of integrin isoform αVβ5 or FCHo2, and/or reduces integrin isoform αVβ5 or FCHo2 activity, for example, by interfering with transcription of integrin isoform αVβ5 or FCHo2 or interfering with translation of integrin isoform αVβ5 or FCHo2. In some embodiments, an integrin isoform αVβ5 or FCHo2 inhibitory nucleic acid or CRISPR system reduces the integrin isoform αVβ5 or FCHo2 mRNA levels or protein levels by at least about 10% to about 100%, 20% to about 100%, 30% to about 100%, 40% to about 100%, 50% to about 100%, 60% to about 100%, 70% to about 100%, 10% to about 90%, 20% to about 85%, 40% to about 84%, 60% to about 90%, including any percent within these ranges, such as but not limited to 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%.
[00192] Any convenient protocol may be used for evaluating integrin isoform αVβ5 or FCHo2 expression by detecting integrin isoform αVβ5 or FCHo2 protein or mRNA levels in the presence or absence of a candidate agent. For measuring protein levels in a sample, various antibody- based methods, including without limitation immunoassays, e.g., enzyme-linked immunosorbent assays (ELISAs), immunohistochemistry, and flow cytometry (FACS) may be used. Any convenient antibody can be used that specifically binds to the integrin isoform αVβ5 or FCHo2 protein. The terms "specifically binds" or "specific binding" as used herein refer to preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides or epitopes). In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a Kd (dissociation constant) of 10-5 M or less (e.g., 10-6 M or less, 10-7 M or less, 10'8 M or less, 10-9 M or less, 10-10 M or less, 10-11 M or less, 10-12 M or less, 10-13 M or less, 10-14 M or less, 10-15 M or less, or 10-16 M or less). By "Affinity" it is meant the strength of binding, increased binding affinity being correlated with a lower Kd.
[00193] While a variety of different manners of assaying for protein levels are known in the art, one representative and convenient type of protocol for assaying protein levels is the enzyme- linked immunosorbent assay (ELISA). In ELISA and ELISA-based assays, one or more antibodies specific for the proteins of interest may be immobilized onto a selected solid surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, the assay plate wells are coated with a non- specific "blocking" protein that is known to be antigenically neutral with regard to the test sample such as bovine serum albumin (BSA), casein or solutions of powdered milk. This allows for blocking of non-specific adsorption sites on the immobilizing surface, thereby reducing the background caused by non-specific binding of antigen onto the surface. After washing to remove unbound blocking protein, the immobilizing surface is contacted with the sample to be tested under conditions that are conducive to immune complex (antigen/antibody) formation. Such conditions include diluting the sample with diluents such as BSA or bovine gamma globulin (BGG) in phosphate buffered saline (PBS)ZTween or PBS/Triton-X 100, which also tend to assist in the reduction of nonspecific background, and allowing the sample to incubate for about 2-4 hours at temperatures on the order of about 25°-27° C. (although other temperatures may be used). Following incubation, the antisera-contacted surface is washed so as to remove non- immunocomplexed material. An exemplary washing procedure includes washing with a solution such as PBS/Tween, PBS/Triton-X 100, or borate buffer. The occurrence and amount of immunocomplex formation may then be determined by subjecting the bound immunocomplexes to a second antibody having specificity for the target that differs from the first antibody and detecting binding of the second antibody. In certain embodiments, the second antibody will have an associated enzyme, e.g. urease, peroxidase, or alkaline phosphatase, which will generate a color precipitate upon incubating with an appropriate chromogenic substrate. For example, a urease or peroxidase-conjugated anti-human IgG may be employed, for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS/Tween). After such incubation with the second antibody and washing to remove unbound material, the amount of label is quantified, for example by incubation with a chromogenic substrate such as urea and bromocresol purple in the case of a urease label or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6- sulfonic acid (ABTS) and H2O2, in the case of a peroxidase label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer. The preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with specificity for the primary antibody. [00194] The solid substrate upon which the antibody or antibodies are immobilized can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate may be chosen to maximize signal to noise ratios, to minimize background binding, as well as for ease of separation and cost. Washes may be effected in a manner most appropriate for the substrate being used, for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, or rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent.
[00195] Alternatively, non-ELISA-based methods for measuring the levels of the integrin isoform αVβ5 or FCHo2 protein in a sample may be employed, and any convenient method may be used. Representative examples known to one of ordinary skill in the art include but are not limited to other immunoassay techniques such as radioimmunoassays (RIA), sandwich immunoassays, fluorescent immunoassays, enzyme multiplied immunoassay technique (EMIT), capillary electrophoresis immunoassays (CEIA), and immunoprecipitation assays; mass spectrometry, or tandem mass spectrometry, proteomic arrays, xMAP microsphere technology, western blotting, immunohistochemistry, flow cytometry, cytometry by time-of-flight (CyTOF), multiplexed ion beam imaging (MIBI), and detection in body fluid by electrochemical sensor. In, for example, flow cytometry methods, the quantitative level of gene products of the one or more genes of interest are detected on cells in a cell suspension by lasers. As with ELISAs and immunohistochemistry, antibodies (e.g., monoclonal antibodies) that specifically bind the polypeptides encoded by the genes of interest are used in such methods.
[00196] As another example, electrochemical sensors may be employed. In such methods, a capture aptamer or an antibody that is specific for a target protein (the "analyte") is immobilized on an electrode. A second aptamer or antibody, also specific for the target protein, is labeled with, for example, pyrroquinoline quinone glucose dehydrogenase ((PQQ)GDH). The sample of body fluid is introduced to the sensor either by submerging the electrodes in body fluid or by adding the sample fluid to a sample chamber, and the analyte allowed to interact with the labeled aptamer/antibody and the immobilized capture aptamer/antibody. Glucose is then provided to the sample, and the electric current generated by (PQQ)GDH is observed, where the amount of electric current passing through the electrochemical cell is directly related to the amount of analyte captured at the electrode.
[00197] Flow cytometry can be used to distinguish subpopulations of cells expressing different cellular markers and to determine their frequency in a population of cells. Typically, whole cells are incubated with antibodies that specifically bind to the cellular markers. The antibodies can be labeled, for example, with a fluorophore, isotope, or quantum dot to facilitate detection of the cellular markers. The cells are then suspended in a stream of fluid and passed through an electronic detection apparatus. In addition, fluorescence-activated cell sorting (FACS) can be used to sort a heterogeneous mixture of cells into separate containers. (See, e.g., Shapiro Practical Flow Cytometry, Wiley-Liss, 4th edition, 2003; Loken Immunofluorescence Techniques in Flow Cytometry and Sorting, Wiley, 2nd edition, 1990; Flow Cytometry: Principles and Applications, (ed. Macey), Humana Press 1 st edition, 2007; herein incorporated by reference in their entireties.)
[00198] In other embodiments, the amount or level in the sample of mRNA encoded by the integrin isoform αVβ5 or FCHo2 gene is determined. Any convenient method for measuring mRNA levels in a sample may be used, e.g. hybridization-based methods, e.g. northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)), RNase protection assays (Hod, Biotechniques 13:852-854 (1992)), and PCR-based methods (e.g. reverse transcription PCR (RT-PCR) (Weis et aL, Trends in Genetics 8:263-264 (1992)).
[00199] For measuring mRNA levels, the starting material may be total RNA, i.e. unfractionated RNA, or poly A+ RNA isolated from a suspension of cells. General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et aL, Current Protocols of Molecular Biology, John Wiley and Sons (1997). RNA isolation can also be performed using a purification kit, buffer set and protease from commercial manufacturers, according to the manufacturer's instructions. For example, RNA from cell suspensions can be isolated using Qiagen RNeasy mini-columns, and RNA from cell suspensions or homogenized tissue samples can be isolated using the TRIzol reagent-based kits (Invitrogen), MasterPure Complete DNA and RNA Purification Kit (EPICENTRE, Madison, Wis.), Paraffin Block RNA Isolation Kit (Ambion, Inc.) or RNA Stat-60 kit (Tel-Test).
[00200] The mRNA levels may be measured by any convenient method. Examples of methods for measuring mRNA levels may be found in, e.g., the field of differential gene expression analysis. One representative and convenient type of protocol for measuring mRNA levels is array-based gene expression profiling. Such protocols are hybridization assays in which a nucleic acid that displays "probe" nucleic acids for each of the genes to be assayed/profiled in the profile to be generated is employed. In these assays, a sample of target nucleic acids is first prepared from the initial nucleic acid sample being assayed, where preparation may include labeling of the target nucleic acids with a label, e.g., a member of signal producing system. Following target nucleic acid sample preparation, the sample is contacted with the array under hybridization conditions, whereby complexes are formed between target nucleic acids that are complementary to probe sequences attached to the array surface. The presence of hybridized complexes is then detected, either qualitatively or quantitatively.
[00201] Specific hybridization technology which may be employed in the subject methods includes that described in U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661 ,028; 5,800,992; the disclosures of which are herein incorporated by reference; as well as WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373 203; and EP 785 280. In these methods, an array of "probe" nucleic acids that includes a probe for each of the phenotype determinative genes whose expression is being assayed is contacted with target nucleic acids as described above. Contact is carried out under hybridization conditions, e.g., stringent hybridization conditions, and unbound nucleic acid is then removed. The term "stringent assay conditions" as used herein refers to conditions that are compatible to produce binding pairs of nucleic acids, e.g., surface bound and solution phase nucleic acids, of sufficient complementarity to provide for the desired level of specificity in the assay while being less compatible to the formation of binding pairs between binding members of insufficient complementarity to provide for the desired specificity. Stringent assay conditions are the summation or combination (totality) of both hybridization and wash conditions.
[00202] The resultant pattern of hybridized nucleic acid provides information regarding expression for each of the genes that have been probed, where the expression information is in terms of whether or not the gene is expressed and, typically, at what level, where the expression data, i.e., expression profile (e.g., in the form of a transcriptosome), may be both qualitative and quantitative.
[00203] Additionally or alternatively, non-array based methods for quantitating the level of one or more nucleic acids in a sample may be employed. These include those based on amplification protocols, e.g., Polymerase Chain Reaction (PCR)-based assays, including quantitative PCR, reverse-transcription PCR (RT-PCR), real-time PCR, and the like, e.g. TaqMan, RT-PCR, MassARRAY System, BeadArray technology, and Luminex technology; and those that rely upon hybridization of probes to filters, e.g. Northern blotting and in situ hybridization. Serial Analysis Gene Expression (SAGE) can also be used to determine RNA abundances in a cell sample. See, e.g., Velculescu et aL, 1995, Science 270:484-7; Carulli, et aL, 1998, Journal of Cellular Biochemistry Supplements 30/31 :286-96; herein incorporated by reference in their entireties. SAGE analysis does not require a special device for detection, can be used for simultaneously detecting the expression of large numbers of transcription products.
Pharmaceutical Compositions
[00204] Inhibitors that inhibit formation of integrin-mediated curved adhesions, identified by the screening methods described herein, can be formulated into pharmaceutical compositions optionally comprising one or more pharmaceutically acceptable excipients. Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.
[00205] A composition of the invention can also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.
[00206] An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the inhibitor, or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof. [00207] A surfactant can be present as an excipient. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.
[00208] Acids or bases can be present as an excipient in the composition. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.
[00209] The amount of the inhibitor (e.g., when contained in a drug delivery system) in the composition will vary depending on a number of factors but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial). A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.
[00210] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred. These foregoing pharmaceutical excipients along with other excipients are described in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), the "Physician’s Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.
[00211] The compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are envisioned. Additional preferred compositions include those for oral, ocular, or localized delivery.
[00212] The pharmaceutical preparations herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use. Preferably, the compositions comprising one or more inhibitors that inhibit formation of integrin-mediated curved adhesions, or nucleic acids encoding them, are in unit dosage form, meaning an amount of a conjugate or composition of the invention appropriate for a single dose, in a premeasured or pre- packaged form.
[00213] The compositions herein may optionally include one or more additional agents, such as one or more other drugs for treating a disorder associated with integrin-mediated formation of curved adhesions, or other medications. For example, compounded preparations may include at least one inhibitor that inhibits formation of integrin-mediated curved adhesions and one or more other drugs for treating a disorder associated with integrin-mediated formation of curved adhesions such as a cancer, a fibrotic disease, thrombosis, an autoimmune disease, or a microbial infection; or other medications used to treat a subject for a condition or disease. Alternatively, such agents can be contained in a separate composition from the composition comprising the inhibitor that inhibits formation of integrin-mediated curved adhesions and co- administered concurrently, before, or after the composition comprising the inhibitor that inhibits formation of integrin-mediated curved adhesions. Kits
[00214] Also provided are kits for use in the methods described herein. The subject kits include agents for detecting integrin-mediated formation of curved adhesions. For example, kits may include reagents for performing a proximity assay to determine the proximity of integrin isoform αVβ5 to FCHo2, such as reagents for performing a complementation assay using a split reporter protein (e.g., split fluorescent protein or split bioluminescent protein), a fluorescence resonance energy transfer (FRET) assay, a bioluminescence resonance energy transfer (BRET) assay, an antibody-based proximity ligation assay (PLA), or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay). Such assay reagents may include but are not limited to, split fluorescent proteins or split bioluminescent proteins conjugated to integrin isoform αVβ5 and FCHo2, FRET/BRET donors and acceptors conjugated to integrin isoform aVβ5 and FCHo2, or antibodies that specifically bind to integrin isoform αVβ5 and the FCHo2, which may be conjugated to oligonucleotides, PLA probes, bridge oligonucleotides, circularization oligonucleotides, amplification reagents for PGR, isothermal amplification, or rolling circle amplification, detection probes, primers, and the like. The kit may further include buffers, cells, microtiter plates, culturing plates, tubes, flasks, culture media, and the like. Kits may further include control reagents and samples including but not limited to, e.g., control samples (e.g., positive control samples, negative control samples, etc.) calibration reagents (e.g., fluorescent calibration reagents, etc.).
[00215] In some embodiments, the kit further comprises a vertical nanostructure (e.g., nanobar or a nanopillar) having an end curvature diameter ranging from 100 nm to 3 pm. In some embodiments, the nanostructure is coated with vitronectin or fibronectin. In some embodiments, the kit further comprises fibers comprising collagen, fibronectin, or vitronectin, or a combination thereof.
[00216] In some embodiments, the kit further comprises a curved ECM surface.
[00217] In some embodiments, the kit comprises a reporter system for measuring curved adhesions, the reporter system comprising a cell expressing a split fluorescent or luminescent protein comprising a first component of a fluorescent or luminescent protein and a second component of the fluorescent or luminescent protein, wherein the cell comprises: a first nucleic acid comprising a coding sequence encoding a first fusion protein comprising the first component of the fluorescent or luminescent protein connected to integrin isoform «Vp5; and a second nucleic acid comprising a coding sequence encoding a second fusion protein comprising the second component of the fluorescent or luminescent protein connected to FCHo2; wherein a reconstituted fluorescent or luminescent protein forms when the first component and the second component of the split fluorescent or luminescent protein are expressed and interact to produce a detectable fluorescent or luminescent signal indicative of the proximity of the integrin isoform αVβ5 and the FCHo2 in the cell.
[00218] The above components may be present in separate containers or one or more components may be combined into a single container, e.g., a glass or plastic vial or tube.
[00219] In some embodiments, the kit further comprises a fluorescence microscope, a two-photon excitation microscope, a fluorescence confocal laser scanning microscope, or a light sheet fluorescence microscope.
[00220] In some embodiments, the kit further comprises a luminescence microscope, luminescence digital imaging microscope, time-gated luminescence microscope, or a luminometer.
[00221] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.
Examples of Non-Limiting Aspects of the Disclosure
[00222] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1 -80 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below: 1 . A method of screening for an inhibitor that inhibits formation of integrin-mediated curved adhesions, the method comprising: contacting a cell, a population of cells, or a tissue with a candidate agent; and measuring proximity of integrin isoform «vp5 to FCHo2 in presence of the candidate agent to determine whether the inhibitor inhibits colocalization of integrin isoform aV 5 and FCHo2 in integrin-mediated curved adhesions, wherein the proximity of the <xVp5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
2. The method of aspect 1 , further comprising providing a vertical nanostructure having an end curvature diameter ranging from 100 nm to 3 pm, wherein the cell, population of cells, or tissue forms the integrin-mediated curved adhesions on the surface of the nanostructure.
3. The method of aspect 2, wherein the nanostructure is a nanobar or a nanopillar.
4. The method of aspect 2 or 3, wherein the nanostructure is coated with vitronectin or fibronectin.
5. The method of aspect 1 , further comprising providing fibers comprising collagen, fibronectin, or vitronectin, or a combination thereof, wherein the cell, population of cells, or tissue forms the integrin-mediated curved adhesions on the surface of the fibers.
6. The method of aspect 1 , further comprising providing a curved extracellular matrix (ECM) surface, wherein the cell, population of cells, or tissue forms the integrin-mediated curved adhesions on the curved ECM surface.
7. The method of any one of aspects 1 -6, further comprising detecting talin or paxillin in the integrin-mediated curved adhesions.
8. The method of any one of aspects 1 -7, wherein said measuring the proximity of the integrin isoform αVβ5 to the FCHo2 comprises performing a complementation assay using a split reporter protein, a fluorescence resonance energy transfer (FRET) assay, a bioluminescence resonance energy transfer (BRET) assay, an antibody-based proximity ligation assay (PLA), or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay.
9. The method of aspect 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a complementation assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split fluorescent protein connected to the FCHo2, wherein a reconstituted fluorescent protein forms when the first component and the second component of the split fluorescent protein are expressed and interact to produce a detectable fluorescent signal; and detecting the fluorescent signal to determine proximity of the integrin isoform αVβ5 to the FCHo2 from the fluorescent signal, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
10. The method of aspect 8, wherein the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
11 . The method of aspect 9 or 10, wherein the fluorescent signal is measured using fluorescence microscopy two-photon excitation microscopy, fluorescence confocal laser scanning microscopy, or light sheet fluorescence microscopy.
12. The method of aspect 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a complementation assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split luminescent protein connected to the FCHo2, wherein a reconstituted luminescent protein forms when the first component and the second component of the split luminescent protein are expressed and interact to produce a detectable luminescent signal; and detecting the luminescent signal to determine proximity of the integrin isoform «V|35 to the FCHo2 from the luminescent signal, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
13. The method of aspect 12, wherein the split luminescent protein is a split luciferase or a split aequorin.
14. The method of aspect 13, wherein the split luciferase is a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
15. The method of any one of aspects 12-14, wherein the luminescent signal is measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
16. The method of aspect 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a FRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a FRET donor connected to the integrin isoform «vp5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion. 17. The method of aspect 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a FRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a FRET acceptor connected to the integrin isoform «VP5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the «VP5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
18. The method of aspect 16 or 17, wherein the FRET efficiency is measured using fluorescence microscopy or fluorescence confocal laser scanning microscopy.
19. The method of any one of aspects 16-18, wherein the FRET donor and the FRET acceptor are fluorescent proteins or fluorescent dyes.
20. The method of aspect 19, wherein the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein, the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein, the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5), or the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine.
21 . The method of aspect 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a BRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising BRET donor connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
22. The method of aspect 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a BRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a BRET acceptor connected to the integrin isoform «VP5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the «VP5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
23. The method of aspect 21 or 22, wherein the BRET efficiency is measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
24. The method of any one of aspects 21-23, wherein the BRET donor or the BRET acceptor is a bioluminescent protein or a luminescent quantum dot.
25. The method of aspect 24, wherein the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein.
26. The method of aspect 8, wherein said performing the PLA comprises: contacting the cell, population of cells, or tissue with a first antibody that specifically binds to αVβ5; contacting the cell, population of cells, or tissue with a second antibody that specifically binds to FCHo2; contacting the first antibody with a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; contacting the second antibody with a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody; contacting the first probe and the second probe with a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with the first oligonucleotide and a second portion sufficiently complementary to hybridize with the second oligonucleotide when the first oligonucleotide and the second oligonucleotide are in sufficient proximity to each other to simultaneously hybridize to the bridge oligonucleotide; and ligating the first oligonucleotide to the second oligonucleotide to produce a ligation product; amplifying the ligation product to produce an amplicon; and detecting the amplicon.
27. The method of aspect 26, wherein said amplifying comprises performing polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification.
28. The method of aspect 27, wherein the PCR is quantitative PCR.
29. The method of any one of aspects 26-28, wherein the first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an Fv fragment, and an scFv fragment.
30. The method of any one of aspects 26-29, wherein the amplicon is detected with a detectably labeled probe that binds to the amplicon.
31. The method of aspect 30, wherein the probe comprises a fluorescent label, a bioluminescent label, or a chemiluminescent label. 32. The method of any one of aspects 1-31 , further comprising quantifying number and area of the integrin-mediated curved adhesions.
33. The method of any one of aspects 1 -32, further comprising: contacting a population of cancer cells with the candidate agent if the candidate agent inhibits the formation of the integrin- mediated curved adhesions; and measuring adhesion, proliferation, cell spreading, migration, or survival of the population of cancer cells, wherein reduced adhesion, proliferation, cell spreading, migration, or survival in presence of the candidate agent compared to that in a negative control population of cancer cells that are not treated with the candidate agent indicates that the candidate agent has anti-cancer activity.
34. The method of any one of aspects 1 -33, wherein the candidate agent is a small molecule, a peptide, a protein, an aptamer, an antibody, an antibody mimetic, an inhibitory nucleic acid, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.
35. The method of aspect 34, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fv fragment, and a nanobody.
36. The method of aspect 34 or 35, wherein the antibody specifically binds to the integrin isoform αVβ5 or the FCHo2.
37. The method of aspect 34, wherein the inhibitory nucleic acid is selected from the group consisting of a small interfering RNA (siRNA), a microRNA (miRNA), a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), an antisense oligonucleotide, and a peptide nucleic acid.
38. The method of aspect 37, wherein the inhibitory nucleic acid inhibits transcription or protein translation of the integrin isoform αVβ5 or the FCHo2.
39. The method of aspect 34, wherein the CRISPR system targets a gene or an RNA transcript encoding the integrin isoform αVβ5 or the FCHo2, or makes epigenetic changes that reduce expression of the integrin isoform αVβ5 or the FCHo2. 40. The method of aspect 34 or 39, wherein the CRISPR system comprises Cas9, Cas12a, Cas12d, Cas13a, Cas13b, Cas13d, or a dead Cas9 (dCas9).
41 . An inhibitor of integrin-mediated curved adhesion identified by the method of any one of aspects 1-40.
42. A composition comprising the inhibitor of aspect 41 and a pharmaceutically acceptable excipient.
43. The composition of aspect 42, further comprising a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment.
44. A reporter system for measuring curved adhesion, the reporter system comprising a cell expressing a split fluorescent or luminescent protein comprising a first component of a fluorescent or luminescent protein and a second component of the fluorescent or luminescent protein, wherein the cell comprises: a first nucleic acid comprising a coding sequence encoding a first fusion protein comprising the first component of the fluorescent or luminescent protein connected to integrin isoform αVβ5; and a second nucleic acid comprising a coding sequence encoding a second fusion protein comprising the second component of the fluorescent or luminescent protein connected to FCHo2; wherein a reconstituted fluorescent or luminescent protein forms when the first component and the second component of the split fluorescent or luminescent protein are expressed and interact to produce a detectable fluorescent or luminescent signal indicative of the proximity of the integrin isoform αVβ5 and the FCHo2 in the cell.
45. The reporter system of aspect 44, wherein the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
46. The reporter system of aspect 45, wherein the split luminescent protein is a split luciferase or a split aequorin.
47. The reporter system of aspect 46, wherein the split luciferase is a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
48. A kit comprising the reporter system of any one of aspects 44-47 and instructions for detecting integrin-mediated curved adhesions.
49. A system for screening a candidate agent to determine its effects on formation of integrin-mediated curved adhesions by a cell, the system comprising a vertical nanostructure having an end curvature diameter ranging from 100 nm to 3 pm, wherein the cell forms the integrin-mediated curved adhesions on the surface of the nanostructure.
50. The system of aspect 49, wherein the nanostructure is a nanobar or a nanopillar.
51 . The system of aspect 49 or 50, wherein the nanostructure is coated with vitronectin or fibronectin.
52. The system of any one of aspects 49-51 , further comprising a cell or cell fragment of interest.
53. The system of aspect 52, wherein the cell or cell fragment of interest is a cancer cell, a blood cell, a platelet, an inflammatory cell, a fibroblast, or an epithelial cell.
54. The system of any one of aspects 49-53, further comprising: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to integrin isoform «V|35; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split fluorescent protein connected to FCHo2, wherein a reconstituted fluorescent protein forms when the first component and the second component of the split fluorescent protein are expressed and interact to produce a detectable fluorescent signal.
55. The system of aspect 54, wherein the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
56. The system of aspect 54 or 55, further comprising a fluorescence microscope, a two-photon excitation microscope, a fluorescence confocal laser scanning microscope, or a light sheet fluorescence microscope.
57. The system of any one of aspects 49-53, further comprising: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to integrin isoform «V|35; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split luminescent protein connected to FCHo2, wherein a reconstituted luminescent protein forms when the first component and the second component of the split luminescent protein are expressed and interact to produce a detectable luminescent signal.
58. The system of aspect 44, wherein the split luminescent protein is a split luciferase or a split aequorin. 59. The system of aspect 45, wherein the split luciferase is a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
60. The system of any one of aspects 44-46, further comprising a luminescence microscope, luminescence digital imaging microscope, time-gated luminescence microscope, or a luminometer.
61 . The system of any one of aspects 49-53, wherein the system comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) donor connected to integrin isoform αVβ5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to FCHo2.
62. The system of any one of aspects 49-53, wherein the system comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) acceptor connected to the integrin isoform otV(35; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2.
63. The system of aspect 61 or 62, further comprising a fluorescence microscope or a fluorescence confocal laser scanning microscope.
64. The system of any one of aspects 61 -63, wherein the FRET donor and the FRET acceptor are fluorescent proteins or fluorescent dyes.
65. The system of aspect 64, wherein the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein, the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein, the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5), or the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine.
66. The system of any one of aspects 49-53, wherein the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) donor connected to the integrin isoform αVβ5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2.
67. The system of any one of aspects 49-53, wherein the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) acceptor connected to the integrin isoform «vp5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2.
68. The system of aspect 66 or 67, wherein the system further comprises a luminescence microscope, a luminescence digital imaging microscope, a time-gated luminescence microscope, or a luminometer.
69. The system of any one of aspects 66-68, wherein the BRET donor or the BRET acceptor is a bioluminescent protein or a luminescent quantum dot.
70. The system of aspect 69, wherein the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein.
71. The system of any one of aspects 49-53, further comprising reagents for performing an antibody-based proximity ligation assay (PLA) or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay. 72. The system of aspect 71 , wherein the reagents for performing the PLA comprise: a first antibody that specifically binds to αVβ5; a second antibody that specifically binds to FCHo2; a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; and a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody.
73. The system of aspect 72, further comprising a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with the first oligonucleotide and a second portion sufficiently complementary to hybridize with the second oligonucleotide when the first oligonucleotide and the second oligonucleotide are in sufficient proximity to each other to simultaneously hybridize to the bridge oligonucleotide.
74. The system of aspect 73, further comprising a ligase.
75. The system of any one of aspects 72-74, wherein the first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an Fv fragment, and an scFv fragment.
76. The system of any one of aspects 72-75, further comprising reagents for performing polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification.
77. The system of aspect 76, wherein the PCR is quantitative PCR.
78. The system of any one of aspects 72-77, further comprising a detectably labeled probe that binds to the amplicon.
79. The system of aspect 78, wherein the probe comprises a fluorescent label, a bioluminescent label, or a chemiluminescent label. 80. A kit comprising the system of any one of aspects 49-80 and instructions for detecting integrin-mediated curved adhesions.
EXPERIMENTAL
[00223] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
[00224] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
[00225] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.
Example 1
A Curvature-Dependent Integrin Adhesion Mediates Cell Attachment to Matrix Fibres
[00226] Previous studies using two-dimensional (2D) substrates have shown that the assembly of integrin adhesions, such as focal adhesions, depends on mechanical forces (Kechagia et al. Nat. Rev. Mol. Cell Biol. 20, 457-473 (2019)) and the spatial distribution of ligands (Cavalcanti-Adam et al. Eur. J. Cell Biol. 85, 219-224 (2006); Oria, et al. Nature 552, 219-224 (2017)). In this process, integrins that transiently bind to their extracellular ligands are activated and stabilized by force- sensitive regulatory proteins (Kechagia et al., supra), especially talin (del Rio, et al. Science 323, 638-641 (2009)). Talin activation at adhesion sites requires a minimum substrate rigidity of 5 kPa (Elosegui-Artola et al. Nat. Cell Biol. 18, 540-548 (2016)), which is higher than the rigidity of most natural extracellular matrix (ECM) fibres (Doyle et al. Nat. Commun. 6, 8720 (2015)). Therefore, focal adhesions are rare in 3D cultures (Harunaga et al. Matrix Biol. 30, 363-368 (2011 )). A fundamental question in mechanobiology is how cells adhere to soft ECM fibres in physiologically relevant environments. The key geometrical feature of ECM fibres is their cylindrical and curved surface, which has been suggested to be important for cell migration in 3D environments (Harunaga et aL, supra; Vogel et al. Nat. Rev. Mol. Cell Biol. 7, 265-275 (2006); Elkhatib et al. Science 356, (2017); Reversal et al. Nature 582, 582-585 (2020)). However, it is technically challenging to observe, let alone to manipulate, membrane curvatures at the cell-ECM interface. Thus, a mechanism that links membrane curvature to integrin adhesion is yet to be identified.
[00227] By using nanoengineering, we systematically examined the curvature dependence of different integrin isoforms and identified a new adhesion structure that we term curved adhesion.
Positive membrane curvature induces selective accumulation of integrin αVβ5.
[00228] T o control membrane curvature, we engineered nanobars on quartz substrates by electron beam lithography patterning and anisotropic etching (FIG. 1 A and FIG. 7A). These nanobars are vertically aligned with 200-nm width, 2-pm length, 1.4-pm height, and 5-pm spacing. The half cylinders at the nanobar ends mimic the curvatures of ECM fibres, and the flat side walls of nanobars serve as an internal control to differentiate curvature effects from other effects that might be introduced by the vertical structures (FIG. 1 B). When adherent cells are cultured on the nanobars, the plasma membrane is locally deformed to accommodate the geometry of nanobars and thus generates well-defined local curvatures (FIG. 1C) 19. Each quartz substrate has multiple well-separated nanopattern areas (1x1 mm2 each) with the rest being flat areas, which allows us to compare cells on nanostructures with cells on flat areas in the same culture.
[00229] In mammals, integrins have 18 a and 8 p subunits forming 24 distinct op heterodimers that bind various ECM proteins 20. We investigated the effect of curvature on integrin isoforms by using nanobar substrates that were coated with defined ECM proteins (FIG. 1 D). Specifically, we used hydrolysed collagen (gelatin), laminin, fibronectin, and vitronectin to probe the large [31 - containing integrin family. We used fibronectin and vitronectin to probe [33, β5, [36 and [38- containing integrins, and laminin to probe the [34-containing integrin. In this study, we did not investigate [32 and [37, the leukocyte-specific receptors that mainly mediate cell-cell adhesion. To coat the surface of quartz substrates, we first applied poly-L-lysine (PLL) and then crosslinked it to specific ECM proteins using glutaraldehyde. This protocol generated uniform ECM coatings as demonstrated by fluorescent gelatin (FIG. 1 E). We used immunofluorescence to probe endogenous [31 and β5 that are highly expressed in LI2OS cells, and green fluorescent protein (GFP) tagging to probe [33, [34, β5, [36, and [38 (FIG. 8A) 21.
[00230] On flat areas, [31 , [33, β5, and [36 formed focal adhesion-like structures on their respective ECM coatings, [34 formed hemidesmosomes on laminin 22, and [38 appeared diffusive due to the lack of talin binding domains 23 (FIG. 8B); these observations agree with previous studies. On nanobar areas (FIG. 1 F and FIG. 9A), [31 appeared in focal adhesions adjacent to nanobars but showed little signal at nanobars. Even when [31 showed signals at nanobars, it exhibited no preference for the nanobar ends. The GFP-tagged [33, [34, [36, and [38 exhibited enhanced signals at nanobars with a relatively even distribution along the nanobar length, in a way like the membrane marker FusionRed-CaaX that was co-expressed to indicate membrane wrapping. Strikingly, β5-GFP showed strong accumulation at the end of fibronectin- or vitronectin-coated nanobars, while co-expressed FusionRed-CaaX was relatively evenly distributed on the same nanobars (FIG. 1 F). We quantified the nanobar end/side ratio of [3 subunits which were normalized against the FusionRed-CaaX. The quantifications confirmed that β5 was the only isoform that showed a strong preference for high curvature, with an average end/side ratio of ~1.9 on both vitronectin and fibronectin-coated nanobars (FIG. 1 G). For subsequent studies, we primarily used vitronectin instead of fibronectin because fibronectin is also a potent ligand for a5[31 , which is highly expressed in U2OS cells 21. The end/side ratios of other |3 subunits were -1.0 on their respective ligands, indicating no curvature preference. Integrin β5 interacts with only the aV subunit to form heterodimer adhesion receptor aVβ5. When immunostaining endogenous integrins with either an antibody that recognizes the β5 subunit or an aVβ5 antibody that recognizes the heterodimeric complex, we observed that both anti-β5 and anti-aVβ5 strongly accumulated at the nanobar ends, suggesting that curvature-recruited β5 was in the complex with the aV subunit (FIGS. 9B-9D). In addition to U2OS cells, |35-GFP accumulated at the nanobar ends in both MEF and HeLa cells, indicating that the curvature preference of αVβ5 was not limited to one specific cell type (FIGS. 9E-9G). αVβ5 forms curved adhesions to extracellular ligands and can bear strong forces.
[00231] To determine whether the aV|35 that accumulates on curved membranes participates in cell-matrix adhesions, we engineered quartz nanopillar arrays that induce more curvatures per cell than nanobars (FIGS. 2A, 2B and FIG. 7B). We found that P5-GFP accumulated strongly on vitronectin-coated nanopillars, but not on gelatin-coated nanopillars, since gelatin is not a ligand for αVβ5 (FIGS. 10A-10C). When the extracellular Ca2+ necessary for the ligand binding of integrins 24 was sequestered with ethylenediaminetetraacetic acid (EDTA), we observed a dramatic decrease of P5-GFP accumulation at vitronectin-coated nanopillars (FIG. 2C). The ratio of 5/membrane was ~1.6 at nanopillars before Ca2+ sequestration and decreased to -1 after (FIG. 2d). These results indicate that the accumulation of αVβ5 on curved membranes requires extracellular binding to its ECM ligands, suggesting the formation of cell adhesions. We name this curvature-dependent and αVβ5-mediated cell adhesion curved adhesion.
[00232] To determine whether curved adhesions can bear forces, we designed a centrifugation- based force assay (FIG. 2E) 25. First, cells were cultured on vitronectin-coated nanopillar substrates. After the cells fully spread, the substrates were placed face down in a 3D-printed support (FIG. 10D) and spun for 15 min in a centrifuge tube filled with buffer solution. If the cell detachment force (see Methods for calculation) was greater than the maximum force that cell adhesions can withstand, the cells would be removed from the substrate. For this study, nanopillar arrays were designed with fiducial markers, which allowed us to image the same fields covering both nanopillar (left half) and flat (right half) areas before and after centrifugation for a direct comparison (FIG. 2F). As the centrifugation speed increased from 200 to 1500 relative centrifugal field (RCF), the percentage of remaining cells (green arrows) on both flat areas and nanopillar areas decreased dramatically. Interestingly, at speeds of 500, 1000 and 1500 RCF, there were significantly more cells remaining on nanopillar areas than on flat areas of the same substrates (FIG. 2G). This indicated that there was a greater adhesion force to nanopillar areas than to flat areas. Using phosphorylated-focal adhesion kinase (pFAK) to mark focal adhesions, we observed that even though cells on nanopillars and on flat surfaces exhibited similar cell areas, cells on nanopillars had smaller focal adhesion areas (FIGS. 10E-10G). In combination, these results suggest that curved adhesions are responsible for the strong adhesion forces on nanopillars. Besides bearing forces, we found that curved adhesions promote early-stage cell spreading, a characteristic of functional cell adhesions26. Immunofluorescence showed strong β5 accumulation at vitronectin-coated nanopillars 30 minutes after cell plating, before the formation of focal adhesions (FIG. 2H). Additionally, cell areas on nanopillars were visibly larger than those on flat surfaces (FIGS. 2H, 21, see FIG. 11 A for large images covering both nanopillar and flat areas). Similar results were also observed for cells cultured on fibronectin-coated substrates, but not on gelatin, pLL, or BSA-coated substrates (FIGS. 11 B, 11 C and FIG. 2I). The knockdown (KD) of β5 by shRNAs largely eliminated nanopillar-induced early cell spreading on both vitronectin and fibronectin-coated substrates (FIGS. 2H, 2I and FIGS. 11 A, 11 B). Therefore, nanopillar-assisted early-stage cell spreading is specific to aVβ5 and its ECM ligands. The results in this section collectively demonstrate that curved adhesions are functional cell-matrix adhesions that can bear strong adhesion forces and promote cell spreading.
Curved adhesions involve a subset of focal adhesion proteins to anchor actin filaments.
[00233] To explore whether curved adhesions contain protein complexes that are involved in focal adhesions, we examined the spatial and temporal correlations between β5 and key focal adhesion proteins including talin, paxillin, vinculin, and pFAK. On flat areas of vitronectin-coated substrates, talin, paxillin, vinculin, and pFAK, all strongly colocalized with β5-marked focal adhesion patches (FIGS. 12A-12D), agreeing with previous reports. At nanopillars, however, only talin and paxillin showed strong correlation with curved adhesions marked with high |35/membrane ratio, while vinculin and pFAK did not (FIG. 3A and FIGS. 13A-13D). The Pearson correlation coefficients between β5/membrane ratio and different adhesion components at nanopillars confirmed that curved adhesions contain talin and paxillin, but not vinculin or pFAK (FIG. 3C). In U2OS cells co- expressing β5-GFP and Lifeact-143 RFP, we observed that actin filaments (F-actin) were anchored to |35-marked focal adhesion patches on flat areas (FIG. 12E). Both β5 and F-actin preferentially accumulated at nanopillars (FIG. 3B and FIG. 13E). However, to our great surprise, nanopillars with β5 accumulations were not the same ones with high intensities of F-actin. The Pearson correlation coefficient between β5 accumulation and F148 actin at nanopillars was calculated to be -0.18, indicating very weak correlation (FIG. 3C). F-actin regulators such as zyxin, o-Actinin 1 , and a-Actinin 4 are associated with different F-actin populations2728. Though all three proteins colocalized with β5 at focal adhesions on flat areas (FIGS. 12F-12H), only zyxin showed a strong correlation with curved adhesions with a Pearson coefficient of -0.65, while a- actinin 1 and a-actinin 4 showed weak correlations with curved adhesions with Pearson coefficients of ~0.2 (FIGS. 3B, 3C). From the studies of actin and actin regulators, we hypothesized that curved adhesions involved a subpopulation of F-actin that accumulate at nanopillars. We measured the dynamic correlation between 35-GFP and LifeAct-RFP in live cells. The curved adhesions at vitronectin-coated nanopillars were highly stable with few assembly and disassembly events over 80 minutes (FIG. 14). Interestingly, we observed two distinct populations of the F-actin that accumulated at nanopillars: a stable population at high-β5 nanopillars that did not show significant fluctuations, and a dynamic population of F-actin at low-35 nanopillars that rapidly assembled and disassembled in 1 -2 minutes (FIG. 3D). This dynamic population of F-actin was often of higher intensity, which resembles the previously reported F-actin on gelatin-coated nanopillars29. Indeed, when we used gelatin-coated nanopillars that could not induce β5 accumulation, we only observed this highly dynamic population of F-actin at nanopillars (FIG. 3E). Quantification of the dynamic fluctuation confirmed the two F-actin populations (FIG. 3F). In combination, the results indicate that curved adhesions involve a subset of regulatory adhesion proteins and a stable population of F-actin at nanopillars.
The formation of curved adhesion requires curvature-sensing proteins FCHo1/2.
[00234] We then hypothesized that an “inside-out” signaling mechanism may underlie why avβ5 accumulates on curved membranes when its ligands are uniformly distributed. To explore the mechanism, we first examined whether the formation of curved adhesion required talin, a key protein that regulates the formation of focal adhesions in a force-dependent manner2 14. We constructed a β5 (Y752A Y770A) double mutant that does not bind talin and observed that the mutant still accumulated strongly at the nanobar ends (FIGS. 4A-4B), even though it was largely excluded from talin patches on flat areas (FIG. 15A). These results indicate that unlike focal adhesions, 35 stabilization in curved adhesions is not dependent on talin-mediated mechanotransduction2.
[00235] We then examined the relationship between curved adhesions and clathrin lattices. The latter has been reported to recruit 31 , P3, and 35-containing integrins on flat surfaces1730 36 and has been shown to accumulate at nanostructure-induced membrane curvatures37. On flat areas, we found that 35 accumulated in clathrin lattices, marked by clathrin-adaptor protein AP2 a subunit (AP2-a), only on vitronectin- but not on fibronectin-coated surfaces (FIG. 4C), agreeing with a recent study35. This is different from curved adhesions since curved adhesions can form on both vitronectin and fibronectin coatings (FIGS. 1 F-1 G). In addition, we separately performed knocked down key proteins involved in clathrin-lattices using validated shRNAs. We found that KD of either clathrin heavy chain (CHC), AP2 p subunit (AP2-p), adaptor proteins Eps15/R, or intersectin 1/2 did not affect the curvature preference of β5 (FIG. 4D and FIG. 16). Furthermore, although both β5 and AP2 preferentially accumulated at nanopillars, intensities of β5 and AP2 showed little correlation (FIG. 4C) with a Pearson correlation coefficient of ~0.2 (FIG. 4F). In combination, these results demonstrate that curved adhesions are not related to clathrin lattices. [00236] In search of the key protein(s) that is responsible for recruiting 35 to curved membranes, we came upon FCHol/2, early-stage endocytic proteins that can sense membrane curvature through an N-terminal FBAR domain738. FCHo1/2 have not been reported to be involved in cell adhesion before. We observed that though β5 and FCHo2 showed distinct intracellular distributions on flat surfaces, they showed strong colocalization at nanopillars (FIG. 4E), unlike 35 and AP2 (FIG. 4C). The FCHo2 intensity positively correlated with 35 accumulation at nanopillars with a correlation coefficient of 0.62 (FIG. 4F). Furthermore, shRNA KD of FCHol/2 significantly reduced the 35 accumulation at nanobar ends (FIG. 4D). Interestingly, some nanopillars without 35 also showed FCHo2 accumulations. Dynamic measurements further revealed that the curvature-induced FCH02 accumulations were strong and static with 35, but weak and dynamic without 35 (FIGS. 4G, 4H). The latter FCHo2 population exhibited frequent assembly and disassembly, which were likely involved in endocytosis or other dynamic processes. These results suggest that FCHo2 is crucial for the formation of curved adhesions and is an integral component of stable curved adhesions.
Cells form curved adhesions on ECM fibres.
[00237] The natural ECM is enriched with fibrous structures, which may locally deform the cell membrane. To determine whether curved adhesions form on ECM fibres, we assembled ECM fibres with collagen, vitronectin and fibronectin on coverslips. Colocalizations of Alexa Fluor 647- labeled collagen, Cy3-labeled vitronectin, and immunolabeled fibronectin confirmed the presence of aV35 ligands vitronectin and fibronectin in the fibre structures (FIG. 17A). As the fibre diameter is often below the diffraction limit, we characterized the ECM fibres using 3D super resolution microscopy39, which showed the most single-fibre diameter to be -1 15 nm (FIG. 17B).
[00238] We then examined cells that spread on sparsely distributed ECM fibres laid on coverslips. We found that 35-GFP strongly accumulated on ECM fibres only at places where the fibre bended the plasma membrane (FIGS. 5A-5B), but not on pure collagen fibres even though they sometimes also bended the plasma membrane (FIG. 18A). Furthermore, KD of FCHo2 significantly reduced β5 accumulation on ECM fibres (FIG. 18A). Quantifications of β5/membrane confirmed that enhanced accumulation of β5 on fibre-induced curved membranes required both ligands and FCHo1/2 (FIG. 5C). These results indicate that ECM fibres can indeed induce curved adhesions.
[00239] Natural ECMs are often soft 3D networks of fibres. Focal adhesions, the most well-known adhesions, are rare in 3D cultures, due to their reliance on force-dependent talin activation15. The curvature preference of aVβ5 is independent of talin. We thus hypothesized that curved adhesions may be prevalent in 3D cultures. To test it, we cultured cells on a thick layer of 3D ECM fibres. Cells in 3D cultures showed extensive β5 accumulations on ECM fibres, which colocalized with FCHo2 (FIG. 5D), while the β5 and FCHo2 colocalization was extremely rare on 2D surfaces coated with the same composition of ECM proteins (FIG. 18B). These results indicated the prevalence of curved adhesions in 3D cultures. Concurrently, we observed that β5 accumulations on 3D ECM fibres were largely absent of vinculin a marker of focal adhesion, whereas vinculin strongly colocalized with β5 at focal adhesions on 2D surfaces. Our observation that focal adhesions were largely missing in 3D cultures is consistent with previous reports8. We quantified curved adhesions by measuring the colocalization of β5 and FCHo2, and focal adhesions by measuring the colocalization of β5 and vinculin. The quantifications further demonstrated that curved adhesions, not focal adhesions, dominated cell-matrix interactions in soft 3D physiological environments (FIG. 5E).
[00240] Natural ECM fibres present a wide range of thicknesses from tens of nanometres to tens of micrometres in diameter5. To determine the range of curvatures that facilitate the formation of curved adhesions, we engineered gradient bars whose end curvature diameters vary from 100 nm to 5 pm (FIG. 5F and FIG. 7C). While GFP-CaaX showed that the cell membrane wrapped around all nanobars, β5 exhibited a clear preference to the ends of thin nanobars. We also observed that the preference gradually diminished as the nanobar width became thicker (FIG. 5F). Quantification of the β5 end/side ratios showed that curved adhesions preferred sharp curvatures with curvature diameters no more than 3 pm (FIG. 5G). Discussions
[00241] Here, we report the identification of curved adhesion, a new type of cell-matrix adhesion that selectively forms on curved ECM surfaces and is exclusively mediated by the integrin isoform αVβ5. We engineered well-defined nanostructures to uncouple the curvature effect from other physical and biochemical varieties of substrates. Curved adhesions engage a unique subset of focal adhesion adaptor proteins, including talin, paxillin, and zyxin, but not vinculin or pFAK. Unlike focal adhesions whose assemblies are stabilized by the mechanical force through talin2 14, curved adhesions are stabilized by curvature-sensing proteins FCHo1/2 (FIG. 6). This study reveals a novel function of FCHo1/2 in cell adhesions besides its well-known role in initiating endocytosis38. Since talin participates in curved adhesions, it is possible that curvature and force act in synergy in the formation of curved adhesions, but further investigation into this is needed.
[00242] Integrin αVβ5 and many of its ligands, e.g., vitronectin and fibronectin, are widely expressed40. The upregulation of integrin aVβ5 is associated with poor prognosis of glioblastoma, gastric, pancreas, and breast cancers9-11 41. Our finding uncovers a mechanism for cells to grab onto delicate ECM fibres that can be easily deformed by mechanical forces required for focal adhesion development. The discovery of curved adhesions will hopefully lead to new perspectives regarding the cell and ECM coupling in a broad spectrum of biological and pathological conditions.
Methods
Nanofabrication
[00243] The patterns of nanobar, nanopillar, and gradient bar arrays were designed using an open- source python package. The fabrication method was adapted from a previous study42. Briefly, a 4-inch quartz wafer (Silicon Materials, 04Q 525-25-1 F-SO) was diced and cleaned with acetone and isopropanol under sonication. Their surfaces were then coated with 275-nm CSAR 6200 (AllResist) and 100-nm Electra 92 (AllResist). The desired patterns were exposed to e-beam using the JEOL JBX-6300FS system. A 120 nm-thick layer of chromium mask was deposited on the post-exposure chips using the AJA e-beam evaporator and then immediately lifted off with acetone and isopropanol. The chips were then etched anisotropically by reactive ion etching (Plasma Therm Versaline LL ICP Dielectric Etcher, PT-Ox) with a mixture of C4F8, H2 and Ar for 3 min. Finally, the chips were immersed in chromium etchant 1020 (Transene) for 30 min to remove the Cr mask. Cell culture
[00244] U2OS cells (ATCC® HTB-96™), HeLa cells (ATCC® CCL-2™), and Mouse Embryonic Fibroblasts (MEFs) (ATCC® CRL-2991 ™) were cultured in complete cell-culture media, Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplied with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich), and 1 % (v/v) penicillin and streptomycin (Gibco). HEK293T cells (ATCC® CRL-3216™) were cultured in complete cell-culture media supplemented with 1 10 pg/mL sodium pyruvate (Gibco) for lentivirus generation. All cell lines were maintained at 37°C in a 5% CO2 atmosphere.
ECM protein labelling
[00245] Gelatin (Sigma-Aldrich, G9391 ) was added into water 283 and autoclaved to make a 1 mg/mL gelatin solution. The gelatin solution was then mixed with an equal volume of 0.2 M sodium bicarbonate buffer (Sigma-Aldrich). For vitronectin labelling, the buffer of human multimeric vitronectin solution (Molecular Innovations Inc, HVN-U) was changed to 0.1 M sodium bicarbonate using a Zeba spin desalting column (Thermo Scientific, 89882). Atto 647 NHS Ester (Sigma- Aldrich, 07376) and Cy3 Mono NHS Ester (Sigma-Aldrich, GEPA13101 ) were separately dissolved in DMSO to make 1 mg/ml stock solutions and then diluted with 0.1 M sodium bicarbonate solutions to a final concentration of 1 ug/mL The dyes were then mixed with proteins with a 1 :1 molecular ratio to make Atto 647-gelatin and Cy3-vitronectin. The mixtures were incubated for 1 h at room temperature (RT). Then, free dyes were removed, and the buffer was changed to 1x phosphate buffered saline (PBS, Gibco), using Zeba spin desalting columns. Rat- tail collagen type I (Corning, 354236) was labelled with Alexa Fluor 647 NHS ester (Thermo Scientific, A37573) and CF568 NHS ester (Sigma-Aldrich, SCJ4600027) to generate AF647- collagen and CF568-collagen, respectively. The protocol was adapted from previous studies1643.
[00246] Briefly, collagen type I was first diluted to a final concentration of 4 mg/ml in ice-cold 20 mM acetic acid. The collagen solution (1 mL) was then neutralized with a pre-chilled mixture of 20 pL 1 M NaOH, 200 pL 10x DMEM, 200 pL 10 mM HEPES, and 580 pL water. The neutralized collagen was dropped in a pre-chilled 10-cm petri dish and incubated stilly for 1 h at RT for gelation. The collagen gel was washed with 10 mL of PBS 3 times for 10 min each and then washed with 0.1 M sodium bicarbonate 3 times for 10 min each. The dye was diluted in 0.1 M sodium bicarbonate to a final concentration of 2 pg/mL and added into the collagen gel. After 30- min incubation at RT, the free dye was washed away with PBS 5 times for 15 min each. The collagen gel was dissolved in 500 pL of 500 mM acetic acid by overnight incubation at 4°C. The collagen solution was then dialyzed against 4 L of 20 mM acetic acid overnight at 4°C. If possible, samples were handled in dark to avoid photobleaching from the ambient light.
Surface coating and cell plating
[00247] The nanochips were first treated with air plasma (Harrick Plasma) for 15 min and then incubated with 0.1 mg/mL PLL (Sigma-Aldrich, P5899) in PBS at 37°C for 1 h. The chips were then washed with PBS 3 times. For PLL surface coating, we stopped here. To coated with other ECM proteins, the nanochips were then incubated with 0.5% (v/v) glutaraldehyde (Sigma-Aldrich, G6257) in PBS at RT for 15 min. After washed with PBS 3 times, the substrates were immediately incubated in PBS with 1 mg/mL gelatin, 1 mg/mL Atto 647-gelatin (this study), 0.25 mg/ml human plasma fibronectin (Sigma-Aldrich, 341635), 0.25 mg/ml vitronectin (PeproTech, 140-09), Cy3- vitronectin (this study), 0.25 mg/ml laminin 511 (Sigma-Aldrich, CC160), or BSA (Sigma-Aldrich, A9418), at 37°C for 1 h. The chips were then washed with PBS 3 times. Substrates with fluorescent labelling were immediately imaged. Before cell plating, the substrates were treated with 1 mg/ml sodium borohydride (Sigma-Aldrich, 452882) in PBS for 5 min to eliminate autofluorescence and then washed with PBS 5 times.
[00248] Before being seeded on substrates, cells were maintained in a 6-well plate reaching -50% confluency. Cells were detached with 10-min incubation in an enzyme-free cell dissociation buffer (Gibco, 13151014). The detached cells were collected by spin at 300 RCF for 3 min and then resuspended in CO2-balanced pre-warmed cell-culture media. The cells were then plated on substrates. For cell spreading assays, cells were maintained for 30 min before the next treatment. For other experiments, cells were maintained for 4-6 hours before the next treatment.
Electron microscopy
[00249] The nanostructures were imaged by SEM (FEI Nova NanoSEM 328450 and FEI Magellan 400 XHR). For cell imaging, the cells on nanostructures were washed with PBS and fixed in 0.1 M sodium cacodylate buffer (pH 7.2) supplemented with 2% glutaraldehyde (Electron Microscopy Sciences) and 4% paraformaldehyde (PFA) for 20 min at RT and then overnight at 4°C. The cells were then incubated in 0.1 M sodium cacodylate buffer supplied with 1% OsO4 and 0.8% potassium ferricyanide (Electron Microscopy Sciences), for 2 h at 4°C. The samples were dehydrated sequentially with 50%, 70%, 95%, 100%, and 100% (v/v) ethanol in water for 10 min each, and then dried by critical point drying. The samples were sputter-coated with 3 nm-thick Cr and imaged by SEM.
Plasmid construction
[00250] The DNA fragment encoding human integrin β5 was amplified from the pCX-EGFP beta5 integrin receptor (a gift from Raymond Birge, Addgene #14996), and then inserted into the pEGFP-N1 vector (Clontech) for the expression of β5-GFP. The DNA fragments encoding human integrin p3 and [38 were amplified from the complementary DNA (cDNA) of LI2OS cells and were cloned into the pEGFP-N1 vector for the expression of [33-GFP and [38-GFP, respectively. The DNA fragment encoding integrin [34 was amplified from pcDNA3.1/Myc-His beta4 (a gift from Filippo Giancotti, Addgene #16039), and then inserted into the pEGFP-N1 vector for the expression of [34-GFP. The expression vector of integrin [36-GFP was a gift from Dean Sheppard (Addgene #13593). The expression vector of mCherry-FCHo2 and Eps15-mCherry were gifts from Christien Merrifield (Addgene #27686 and #27696). The expression vector of talin-mCherry was a gift from Michael Davidson (Addgene #55139). The DNA fragments encoding human zyxin, a-actinin 1 , and a-actinin 4 were amplified from the cDNA of U2OS cells and then inserted into the pmCherry-N1 vector (Clontech) for the expression of zyxin-mCherry, a-actinin 1 -mCherry, and a-actinin 4- mCherry, respectively. The DNA fragments encoding GFP-351 CaaX and FusionRed- CaaX were generated by fusing the DNA fragment encoding the CaaX motif of K-Ras protein (GKKKKKKSKTKCVIM, SEQ ID NO:21 ) to the 3'-end of the DNA fragments encoding EGFP and FusionRed. The DNA fragment of FusionRed-CaaX was inserted into the pEGFP-C1 vector (Clontech) to replace the EGFP for the mammal expression. The DNA fragment encoding GFP- CaaX was inserted into pLenti pRRL-SV40(puro)_CMV for lentivirus packaging. The expression vectors of LifeAct-RFP were gifts from Michael Davidson (Addgene #54491 ). The DNA fragment encoding SNAP-tag was amplified from the pSNAP-tag (m) vector (a gift from New England Biolabs & Ana Egana, Addgene #101 135), and then inserted into the pDisplay vector (Invitrogen, V66020) for the expression of cell-surface SNAP-tag.
[00251] The vector for scramble shRNA transcription was a gift from David Sabatini (Addgene #1864). The shRNA vectors for the KDs of human integrin β5, AP2-p, clathrin heavy chain, FCHol , FCHo2, Eps15, Eps15R, and intersectin 1 and 2 were from Sigma-Aldrich. The clone IDs of shRNA vectors used in this study are listed in Table 1. The shRNAs are cloned in the pLKO.1 vector for their transcription in mammal cells. The pLKO.1 vector is designed for lentivirus transfection and subsequent puromycin selection. For transient transfection, the puromycin resistant sequence in pLKO.1 vector was replaced with a sequence encoding EBFP2 to fluorescently label shRNA transcription cells.
Cell transfection
[00252] U2OS cells and MEFs were transfected with plasmids encoding proteins or shRNAs by electroporation. Cells were grown in 6-well plates at -80% confluency before transfection. One well of cells were detached, collected, and then resuspended in an electroporation buffer containing 100 pL Electroporation buffer II (88 mM KH2PO4 and 14 mM NaHCO3, pH 7.4), 2 pL Electroporation buffer I (360 mM ATP + 600 mM MgCI2), 374 and <2 j g plasmids. The cells were electroporated in 0.2-cm gap electroporation cuvettes by Amaxa Nucleofector II (Lonza) using protocols pre-installed by the manufacturer. For HeLa cells transfection, cells at -50% confluency in 6-well plates were incubated with 2 mL Opti-MEM-I medium (Gibco, 31985062) supplied with 6 pL Lipofectamine 2000 transfection reagent (Invitrogen) and <2 pg plasmids, for 2 h. The transfected cells were maintained in complete cell-culture media for ~72 h before being plated on desired substrates.
Lentiviral transduction and stable cell line generation
[00253] For lentivirus packaging, HEK293T cells at ~80% confluency in 35-mm dishes (Corning, 353001 ) were transfected with 1.5 pg 3rd generation lentivirus package vector encoding GFP- CaaX or shRNAs, 0.8 pg psPAX2 (a gift from D. Trono, Addgene #12260), and 0.7 pg pMD2.G (a gift from D. Trono, Addgene #12259), mixed with 9 pL lipofectamine 2000 transfection reagent in 2 mL Opti-MEM-I medium. The transfection media were replaced with 2 mL of HEK293T culture media 6 h after transfection. The transfected cells were maintained for 24 h. Then, the virus containing supernatants were collected, and the cell debris was removed by spin at 400 RCF for 5 min followed by filtration through 0.45-pm PVDF syringe filter units (Millipore). The GFP-CaaX and integrin β5-knockdown stable cell lines were generated by lentivirus transduction of wild-type U2OS cells followed by antibiotic selection with 2.5 pg/mL puromycin in complete cell-culture media. The antibiotic pressure was released 3 days before experiments. Immunofluorescence labelling
[00254] To label integrin [31 and avβ5 with primary antibodies, cells were first snap-chilled in ice- cold HEPES-buffered hanks balanced salt solution (HBSS), which is 1X HBSS (Gibco, 24020117) buffered with 10 mM HEPES (Gibco, 15630106). The samples were then incubated with either mouse anti-activated integrin [31 antibody 397 (Sigma-Aldrich, MAB2079Z) or mouse anti-integrin avβ5 antibody (R&D Systems, MAB2528), which were 1 :100 diluted in HHBSS, for 30 min at 4°C. The samples were then washed with ice-cold HHBSS 3 times for 5 min each and immersed with 4% ice-cold PFA in PBS to fix for 15 min at RT. The fixed samples were washed and permeabilized with 0.1 % Triton-X (Sigma-Aldrich, T8787) in PBS for 15 min at RT. Then, the permeabilized samples were washed and incubated in a blocking buffer (5% BSA in PBS) for 1 h at RT.
[00255] To label other proteins with primary antibodies, cells or collagen fibres were fixed with 4% PFA in PBS for 15 min at RT. The samples were then washed and permeabilized with 0.1% Triton- X in PBS for 15 min at RT. The permeabilized samples were washed and incubated in blocking buffer for 1 h at RT and incubated in primary antibodies 1 :250 diluted in blocking buffer for 1 h at 37°C and then 1 h at RT. The primary antibodies used are listed in Table 2.
[00256] The primary antibody-labelled samples were then washed with the blocking buffer 3 times for 15 min each and incubated in fluorescently labelled secondary antibodies 1 :500 diluted in blocking buffer for 1 h at RT. The samples were washed with the blocking buffer 3 times for 15 min each and then with PBS 3 times before imaging. The secondary antibodies used are listed in Table 2.
SNAP-tag labelling
[00257] Cell-surface SNAP-tags were fluorescently labelled before fixation. Cells were incubated in CO2-balanced pre-warmed media supplied with 1 pM O6-benzylguanine (BG)-coupled Alexa Fluor 647 (NEB, S9136S) for 15 min at 37°C in a 5% CO2 atmosphere. The cells were then washed 5 times with cell culture media before fixation.
Centrifugation assay
[00258] The centrifugation supports were printed using a 3D printer (Original Prusa i3). Cells stably expressing GFP-CaaX were grown on substrates (1 cm2 square quartz chips). The cells were incubated in complete cell-culture media supplied with 1 pg/mL Hoechst 33342 (Thermo Scientific, 62249) for 15 min at 37°C in a 5% C02 atmosphere. The samples were washed with PBS 5 times and imaged before spin. The samples were then flipped upside down and put in centrifugation support. The support was gently inserted into a 50-mL centrifuge tube (Corning, 352070) filled with 20 mL of HHBSS. The samples were spun at 200, 500, 1000, or 1500 RCF for 15 min. The samples were then gently removed from the support and fixed for imaging after spin.
ECM fibre generation
[00259] Unlabeled and fluorescently labelled collagen was mixed in a 4:1 (w/w) ratio at a final concentration of 2 mg/ml in 20 mM acetic acid. For collagen polymerization, 50 pL collagen mixture was neutralized with a mixture of 1 pL 1 M NaOH, 10 pL 10x DMEM, 10 pL 10 mM HEPES, and 29 pL water on ice. The neutralized collagen was dropped in Lab-Tek II chambered coverglass (Sigma-Aldrich, Z734853) for 20 pL per well, and incubated at RT for 5 min. The non- polymerized collagen was then washed away with 0.5 mL 20 mM phosphate buffer (at pH 7.4) 5 times. For aVβ5 ligands incorporation, the collagen fibres were incubated in 100 pL of 20 mM phosphate buffer supplied with 50 pg/mL human plasma fibronectin (Sigma-Aldrich, 341635), and 50 pg/ml multimeric vitronectin (Molecular Innovations Inc, HVN-U) or Cy3-vitronectin (this study), for 1 h at RT. The collagen fibres were washed with 20 mM phosphate buffer 3 times before immunofluorescence labelling or cell plating. To be plated on fibres, cells were detached using the enzyme-free cell dissociation buffer and collected by spin. The cells were then resuspended in CO2-balanced pre-warmed media and immediately seeded on collagen fibres. The cells were grown on fibres for 45 min before the next treatment.
Fluorescence microscopy
[00260] Fluorescence images were acquired in an epi-fluorescence microscope (Leica DMI 6000B) using a 100x or 10x objective. During live-cell imaging, cells were maintained in phenol red-free DMEM (Gibco) supplied with 10% FBS at 37°C with 5% of CO2 in a stage top incubator (Tokai Hit, INUBSF-ZILCS). Time-lapse images were taken at 15 s/frame using 500 ms exposure times. Confocal images were acquired in a Nikon A1 R confocal microscope using a 60X objective.
Super-resolution microscopy
[00261] Our method is adapted from protocols described previously3944. The 3D single-molecule data was acquired using a custom-built wide-field double-helix point-spread function (PSF) inverted microscope. The samples were incubated in a blinking buffer solution containing 10% (w/v) glucose (BD Difco), 100 mM tri(hydroxymethyl)aminomethane-HCI (Thermo Fisher), 2 pL/mL catalase, 560 pg/mL glucose oxidase, and 10 mM of cysteamine (all Sigma Aldrich), allowing for a low emitter concentration during data acquisition. The focus was first set to the coverslip using a bead as a reference. Standard localization-based approaches fitted the shape of the PSF of emitters to a 2D Gaussian to yield highly precise XY positions. To extract the Z position, a double helix phase mask was inserted in the Fourier plane of the microscope to modify the shape of the standard PSF to now have two lobes that rotate as a function of Z. We fitted the double-helix PSF to two Gaussian functions and extracted the midpoint to estimate the XY position. From the fit, we determined the lobe angle of the double-helix PSF. Then, using a carefully calibrated curve that relates lobe angle to Z position, we determined the Z position of the emitter. The localization precision was calculated from the detected photons using a formula calibrated specifically for our microscope using previously described approaches45. After processing, poorly localized emitters (XY precision > 20 nm or Z precision > 40 nm or lobe distance > 8 pixels) were removed from the reconstruction. Localizations were merged to correct for over counting as previously reported46 before quantification of any individual fibre diameters. The localized single-molecule positions were rendered with the Vutara SRX program (Bruker).
Image analysis
[00262] All images were prepared and processed using Imaged (Fiji). Quantifications were performed using both Imaged and MATLAB 2017a (MathWorks).
Nanostructure measurements
[00263] The following parameters were measured in Fiji by counting pixels: the height, width, and length of bars; the height, diameters (top, middle, and bottom) of nanopillars. If the samples were tilted 45°, a correction factor was applied to the Z dimension. The diameters of curvatures at bar ends were measured using the Kappa curvature analysis plugin (open source) in Fiji.
Quantification of nanobar end/side ratio
[00264] The bright-field images were used to generate arrays of square masks with bars at the centre, using MATLAB code written for this study. All mask regions of the plasma membrane channel within each cell were averaged. Based on the average, the bar-end and bar-side region- of-interests (ROIs) were identified manually using Fiji. The ROIs were then applied to each bar in integrin and membrane channels and the mean intensities in each ROI were measured. For each bar, the integrin intensity was normalized by the membrane intensity and the end/side ratio was then calculated. The normalized end/side ratios of individual nanobars in each cell were averaged and reported as mean ± SD. For cells on gradient bars, the normalized end/side ratios in each imaging field were averaged and reported, because very few b 486 ars with the same diameter were covered by one single cell.
Quantification of p5/membrane ratio
[00265] The square masks with nanopillars at the centre were generated by using the same MATLAB above. Based on the average mask region of the plasma membrane channel within each cell, the ROIs for “at nanopillar” and “flat regions surrounding nanopillars” were defined as 0-9 and 10-20 pixel from the centre of a nanopillar, respectively. The ROIs were then applied to each mask region in both integrin and membrane channels. Mean intensities in each ROI were measured. The p5/membrane ratio of each ROI was calculated and normalized by the mean β5/membrane ratio in each cell. The normalized β5/membrane ratios of individual ROIs were reported as mean ± SD.
Calculation of cell and focal adhesion areas
[00266] The background of all images was subtracted using a rolling ball (radius, 1000 pixel) tool in Fiji. For cell area measurements, the membrane signal was auto-thresholded using the ‘Li’ method in Fiji to identify whole cell bodies which were then selected as ROIs by the ‘wand’ tool. The area of these ROIs was measured and reported as cell area. If the focal adhesion areas were measured later, the ROIs in the pFAK or vinculin channel were separately saved and auto- thresholded using the ‘Moments’ method in Fiji to identify focal adhesion areas. The number, size or fraction of focal adhesion area per cell was calculated and reported as mean ± SD.
Cell counting for centrifugation assay
[00267] The cell nuclei were counted to determine cell numbers before and after spin. The images in the Hoechst channel were auto-thresholded using the ‘Moments’ method in Fiji to identify cell nuclei. The nuclei clusters were segmented using the watershed binary tool in Fiji. The nuclei were then counted using the analyse particles tool in Fiji. Based on images in the GFP-CaaX channel, the cells that cover both nanopillars and the flat area were excluded.
Detachment force calculation
[00268] The average detachment force on each cell was estimated by the following equation:
F = (pcell - pmedium) ' Vcell ‘ RCF in which F is the average detachment force for each cell; pcell is the density of the cell (1.07 g/cm3); pmedium is the density of HHBSS (1 .00 g/cm3); and Vcen is the average cell volume of U2OS (~1700 pm3)25. The F at 200, 500, 1000, and 1500 g (RFC) were calculated to be 233, 583, 1 166, and 1749 pN, respectively.
Pearson correlation
[00269] To analyze the correlation between a protein of interest (POI) and the 35/membrane ratio at nanopillars, the square masks for nanopillars were first generated as described above. The means of POI, 5 and membrane at each nanopillar (0-9 pixel to the nanopillar centre) were measured. The Pearson correlation coefficients at nanopillars in each cell were calculated using Prism 6. To analyze the correlation between the β5/membrane ratio and FCHo2 on flat surfaces, the β5/membrane ratiometric images were auto-thresholded using the ‘Otsu’ method in Fiji to identify adhesion regions, which were then saved as ROIs. The β5/membrane ratio and intensity of FCHo2 were then measured to get their mean values in the ROIs. The Pearson correlation coefficients per cell were then calculated.
Dynamic measurements of F-actin and FCHo
[00270] The square masks for nanopillars were first generated as described above. For cells on gelatin, the mean intensities of LifeAct at nanopillars (0-9 pixel to the nanopillar centres) in a time- lapse image stack were measured using MATLAB code written for this study. For F-actin and FCHo dynamics in cells on vitronectin, the mean intensities of β5 at nanopillars were first measured and ranked. For F-actin dynamics, the nanopillars with top 25% and bottom 25% in β5 were separated into two groups: high-35 and low-35 nanopillars, respectively. For FCHo dynamics, the nanopillars with top 25% and bottom 75% in 35 were separated into two groups: high-35 and low-35 nanopillars, respectively. The mean intensities at each group of nanopillars in a time-lapse image stack were measured. The temporal standard deviations of either LifeAct or FCHo at individual nanopillars were reported as mean ± SD.
Quantification of curved adhesion on fibres
[00271] Line selections along membrane-wrapped segments of sparse fibres were created by using a MATLAB code written for this study. The intensities of β5, membrane and fibre along 2500 nm long transverse lines crossing a line selection were measured. They were separately averaged along the line selection and then normalized by the minimum of their own average values. The results were plotted as means ± SD against distance (2500 nm). The plot of fibre intensities along each transverse line has one major peak in the middle. The baseline width of the peak was used to define the fibre section of each transverse line. The linear fibre sections were summed up along the line selections to get fibre area. The normalized β5/membrane in the fibre areas were reported as mean ± SD. To quantify curved adhesions on dense fibres, the fibre, FCHo, and β5/membrane ratiometric images were auto-thresholded using the ‘Moments’ method in Fiji. The resulting binary images were multiplied to generate ROIs for curved adhesions, whose number and size per cell were reported as mean ± SD.
Quantification of fibre diameters
[00272] Individual collagen fibres were isolated from the 3D super-resolution reconstruction. Multiple transverse line profiles that are 100-nm thick were drawn along each of the isolated fibres. The 3D localizations were projected along these lines into 30-nm bin widths. The histogram was fitted to a Gaussian, and the full width half maximum was extracted to estimate the fibre diameter.
Statistics and reproducibility
[00273] All data were displayed and statistically analysed by using Prism 6 (GraphPad software). All experiments were repeated independently at least two times to ensure reproducibility. The normal distribution of each sample group was verified using the D'Agostino-Pearson normality test (a = 0.05). For parametric datasets, we used unpaired two-tailed Student’s t-test to compare two groups, and one-way ANOVA with Tukey’s or Bonferroni’s multiple-comparison to compare more than two groups. For nonparametric datasets, we used Mann-Whitney test to compare two groups, and Kruskal-Wallis test with Dunn’s multiple-comparison to compare more than two groups. Unpaired multiple t-test with Holm-Sidak multiple-comparison was used to compare cells remaining after spin at different speeds. The exact P values and sample sizes (N) are denoted in the figures and figure legends. Significance was considered at P< 0.05. The test statistics for null hypothesis testing including confidence levels and degrees of freedom are listed in Table 3.
References:
[00274] 1. Geiger, B., Spatz, J. P. & Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21 -33 (2009).
[00275] 2. Kechagia, J. Z., Ivaska, J. & Roca-Cusachs, P. Integrins as biomechanical sensors of the microenvironment. Nat. Rev. Mol. Cell Biol. 20, 457-473 (2019).
[00276] 3. Walma, D. A. C. & Yamada, K. M. The extracellular matrix in development.
Development 147, (2020).
[00277] 4. Kai, F., Drain, A. P. & Weaver, V. M. The Extracellular Matrix Modulates the Metastatic
Journey. Dev. Cell 49, 332-346 (2019).
[00278] 5. Young, J. L., Hoile, A. W. & Spatz, J. P. Nanoscale and mechanical properties of the physiological cell-ECM microenvironment. Exp. Cell Res. 343, 3-6 (2016).
[00279] 6. Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat.
Rev. Mol. Cell Biol. 7, 265-275 (2006).
[00280] 7. Henne, W. M. et al. Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effects membrane curvature. Structure 15, 839-852 (2007).
[00281] 8. Harunaga, J. S. & Yamada, K. M. Cell-matrix adhesions in 3D. Matrix Biol. 30, 363-368
(2011 ).
[00282] 9. Bianchi-Smiraglia, A., Paesante, S. & Bakin, A. V. Integrin β5 contributes to the tumorigenic potential of breast cancer cells through the Src-FAK and MEK-ERK signaling pathways. Oncogene 32, 3049-3058 (2013).
[00283] 10. Boger, C. et al. Integrins av|33 and ov|35 as prognostic, diagnostic, and therapeutic targets in gastric cancer. Gastric Cancer 18, 784-795 (2015).
[00284] 11. Zhang, L.-Y. et al. Integrin Beta 5 Is 622 a Prognostic Biomarker and Potential
Therapeutic Target in Glioblastoma. Front. Oncol. 9, 904 (2019).
[00285] 12. Cavalcanti- Adam, E. A. et al. Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly. Eur. J. Cell Biol. 85, 219-224 (2006).
[00286] 13. Oria, R. et al. Force loading explains spatial sensing of ligands by cells. Nature 552,
219-224 (2017). [00287] 14. del Rio, A. et al. Stretching single talin rod molecules activates vinculin binding.
Science 323, 638-641 (2009).
[00288] 15. Elosegui-Artola, A. et al. Mechanical regulation of a molecular clutch defines force transmission and transduction in response to matrix rigidity. Nat. Cell Biol. 18, 540-548 (2016).
[00289] 16. Doyle, A. D., Carvajal, N., Jin, A., Matsumoto, K. & Yamada, K. M. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility- dependent adhesions. Nat. Commun. 6, 8720 (2015).
[00290] 17. Elkhatib, N. et al. T ubular clathrin/AP-2 lattices pinch collagen fibers to support 3D cell migration. Science 356, (2017).
[00291] 18. Reversal, A. et al. Cellular locomotion using environmental topography. Nature 582,
582-585 (2020).
[00292] 19. Santoro, F. et al. Revealing the Cell-Material Interface with Nanometer Resolution by
Focused Ion Beam/Scanning Electron Microscopy. ACS Nano 11 , 8320-8328 (2017).
[00293] 20. Humphries, J. D., Byron, A. & Humphries, M. J. Integrin ligands at a glance. J. Cell
Sci. 119, 3901 -3903 (2006).
[00294] 21 . Thul, P. J. et al. A subcellular map of the human proteome. Science 356, (2017).
[00295] 22. 645 Dowling, J., Yu, Q. C. & Fuchs, E. Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival. J. Cell Biol. 134, 559-572 (1996).
[00296] 23. Legate, K. R. & Fassler, R. Mechanisms that regulate adaptor binding to beta-integrin cytoplasmic tails. J. Cell Sci. 122, 187-198 (2009).
[00297] 24. Kirchhofer, D., Grzesiak, J. & Pierschbacher, M. D. Calcium as a potential physiological regulator of integrin-mediated cell adhesion. J. Biol. Chem. 266, 4471 -4477 (1991 ).
[00298] 25. Reyes, C. D. & Garcia, A. J. A centrifugation cell adhesion assay for high-throughput screening of biomaterial surfaces. Journal of Biomedical Materials Research PartA vo\. 67A 328- 333 (2003).
[00299] 26. Changede, R., Cai, H., Wind, S. J. & Sheetz, M. P. Integrin nanoclusters can bridge thin matrix fibres to form cell-matrix adhesions. Nat. Mater. 18, 1366-1375 (2019).
[00300] 27. Smith, M. A. etal. A zyxin-mediated mechanism for actin stress fiber maintenance and repair. Dev. Cell 19, 365-376 (2010).
[00301] 28. Shiu, J.-Y., Aires, L., Lin, Z. & Vogel, V. Nanopillar force measurements reveal actin- cap mediated YAP mechanotransduction. Nat. Cell Biol. 20, 262-271 (2018). [00302] 29. Lou, H.-Y. et al. Membrane curvature underlies actin reorganization in response to nanoscale surface topography. Proc. Natl. Acad. Sci. U. S. A. 116, 23143-23151 (2019).
[00303] 30. Tawil, N., Wilson, P. & Carbonetto, S. Integrins in point contacts mediate cell spreading: factors that regulate integrin accumulation in point contacts vs. focal contacts. J. Cell Biol. 120, 261 -271 (1993).
[00304] 31. Yu, C.-H. et al. Integrin-beta3 clusters recruit clathrin-mediated endocytic machinery in the absence of traction force. Nat. Commun. 6, 8672 (2015).
[00305] 32. Leyton-Puig, D. etal. Flat clathrin lattices are dynamic actin-controlled hubs for clathrin mediated endocytosis and signalling of 668 specific receptors. Nat. Commun. 8, 16068 (2017).
[00306] 33. Baschieri, F. et al. Frustrated endocytosis controls contractility-independent mechanotransduction at clathrin-coated structures. Nat. Commun. 9, 3825 (2018).
[00307] 34. Lock, J. G. et al. Reticular adhesions are a distinct class of cell-matrix adhesions that mediate attachment during mitosis. Nat. Cell Biol. 20, 1290-1302 (2018).
[00308] 35. Zuidema, A. et al. Mechanisms of integrin aVβ5 clustering in flat clathrin lattices. J.
Cell Sci. 131 , (2018).
[00309] 36. Lock, J. G. et al. Clathrin-containing adhesion complexes. J. Cell Biol. 218, 2086-2095
(2019).
[00310] 37. Zhao, W. et al. Nanoscale manipulation of membrane curvature for probing endocytosis in live cells. Nat. Nanotechnol. 12, 750-756 (2017).
[00311] 38. Henne, W. M. et al. FCHo proteins are nucleators of clathrin-mediated endocytosis.
Science 328, 1281 -1284 (2010).
[00312] 39. Pavani, S. R. P. et al. Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc. Natl. Acad. Sci. U. S. A. 106, 2995-2999 (2009).
[00313] 40. Uhlen, M. et al. Proteomics. Tissue-based map of the human proteome. Science 347,
1260419 (2015).
[00314] 41. de Mendoza, T. H. et al. Tumor-penetrating therapy for 5 integrin-rich pancreas cancer. Nat. Commun. 12, 1 -13 (2021 ).
[00315] 42. Li, X. et al. A nanostructure platform for live-cell manipulation of membrane curvature.
Nat. Protoc. 14, 1772-1802 (2019).
[00316] 43. Doyle, A. D. Generation of 3D collagen gels with controlled diverse architectures. Curr.
Protoc. Cell Biol. 72, 10.20.1 -10.20.16 (2016). [00317] 44. Mockl, L. et al. Quantitative Super-Resolution Microscopy of the Mammalian
Glycocalyx. Dev. Cell 50, 57-72. e6 (2019).
[00318] 45. Gahlmann, A. et al. Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions. Nano Lett. 13, 987-993 (2013).
[00319] 46. Roy, A. R. etal. Exploring cell surface-nanopillar interactions with 3D super-resolution microscopy. doi:10.1 101/2021.06.21.449280.
Table 1 : Information of shRNAs
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001

Claims

WHAT IS CLAIMED IS:
1 . A method of screening for an inhibitor that inhibits formation of integrin-mediated curved adhesions, the method comprising: contacting a cell, a population of cells, or a tissue with a candidate agent; and measuring proximity of integrin isoform αVβ5 to FCHo2 in presence of the candidate agent to determine whether the inhibitor inhibits colocalization of integrin isoform αVβ5 and FCHo2 in integrin-mediated curved adhesions, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
2. The method of claim 1 , further comprising providing a vertical nanostructure having an end curvature diameter ranging from 100 nm to 3 pm, wherein the cell, population of cells, or tissue forms the integrin-mediated curved adhesions on the surface of the nanostructure.
3. The method of claim 2, wherein the nanostructure is a nanobar or a nanopillar.
4. The method of claim 2 or 3, wherein the nanostructure is coated with vitronectin or fibronectin.
5. The method of claim 1 , further comprising providing fibers comprising collagen, fibronectin, or vitronectin, or a combination thereof, wherein the cell, population of cells, or tissue forms the integrin-mediated curved adhesions on the surface of the fibers.
6. The method of claim 1 , further comprising providing a curved extracellular matrix (ECM) surface, wherein the cell, population of cells, or tissue forms the integrin-mediated curved adhesions on the curved ECM surface.
7. The method of any one of claims 1 -6, further comprising detecting talin or paxillin in the integrin-mediated curved adhesions.
8. The method of any one of claims 1 -7, wherein said measuring the proximity of the integrin isoform αVβ5 to the FCHo2 comprises performing a complementation assay using a split reporter protein, a fluorescence resonance energy transfer (FRET) assay, a bioluminescence resonance energy transfer (BRET) assay, an antibody-based proximity ligation assay (PLA), or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay.
9. The method of claim 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a complementation assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split fluorescent protein connected to the FCHo2, wherein a reconstituted fluorescent protein forms when the first component and the second component of the split fluorescent protein are expressed and interact to produce a detectable fluorescent signal; and detecting the fluorescent signal to determine proximity of the integrin isoform αVβ5 to the FCHo2 from the fluorescent signal, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
10. The method of claim 8, wherein the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
11. The method of claim 9 or 10, wherein the fluorescent signal is measured using fluorescence microscopy two-photon excitation microscopy, fluorescence confocal laser scanning microscopy, or light sheet fluorescence microscopy.
12. The method of claim 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a complementation assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split luminescent protein connected to the FCHo2, wherein a reconstituted luminescent protein forms when the first component and the second component of the split luminescent protein are expressed and interact to produce a detectable luminescent signal; and detecting the luminescent signal to determine proximity of the integrin isoform αVβ5 to the FCHo2 from the luminescent signal, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
13. The method of claim 12, wherein the split luminescent protein is a split luciferase or a split aequorin.
14. The method of claim 13, wherein the split luciferase is a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
15. The method of any one of claims 12-14, wherein the luminescent signal is measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
16. The method of claim 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a FRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a FRET donor connected to the integrin isoform otV(35; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
17. The method of claim 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a FRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a FRET acceptor connected to the integrin isoform «VP5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2; and measuring FRET efficiency between the FRET donor and the FRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the «VP5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
18. The method of claim 16 or 17, wherein the FRET efficiency is measured using fluorescence microscopy or fluorescence confocal laser scanning microscopy.
19. The method of any one of claims 16-18, wherein the FRET donor and the FRET acceptor are fluorescent proteins or fluorescent dyes.
20. The method of claim 19, wherein the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein, the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein, the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5), or the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine.
21. The method of claim 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a BRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising BRET donor connected to the integrin isoform «VP5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the «VP5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
22. The method of claim 8, wherein said measuring proximity of the integrin isoform αVβ5 to the FCHo2 with a BRET assay comprises: transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a BRET acceptor connected to the integrin isoform αVβ5; transfecting the cell, population of cells, or tissue with a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2; and measuring BRET efficiency between the BRET donor and the BRET acceptor to determine proximity of the integrin isoform αVβ5 to the FCHo2, wherein the proximity of the αVβ5 to the FCHo2 indicates whether a cellular adhesion is an integrin-mediated curved adhesion.
23. The method of claim 21 or 22, wherein the BRET efficiency is measured using luminescence microscopy, luminescence digital imaging microscopy, time-gated luminescence microscopy, or a luminometer.
24. The method of any one of claims 21 -23, wherein the BRET donor or the BRET acceptor is a bioluminescent protein or a luminescent quantum dot.
25. The method of claim 24, wherein the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein.
26. The method of claim 8, wherein said performing the PLA comprises: contacting the cell, population of cells, or tissue with a first antibody that specifically binds to αVβ5; contacting the cell, population of cells, or tissue with a second antibody that specifically binds to FCHo2; contacting the first antibody with a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; contacting the second antibody with a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody; contacting the first probe and the second probe with a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with the first oligonucleotide and a second portion sufficiently complementary to hybridize with the second oligonucleotide when the first oligonucleotide and the second oligonucleotide are in sufficient proximity to each other to simultaneously hybridize to the bridge oligonucleotide; and ligating the first oligonucleotide to the second oligonucleotide to produce a ligation product; amplifying the ligation product to produce an amplicon; and detecting the amplicon.
27. The method of claim 26, wherein said amplifying comprises performing polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification.
28. The method of claim 27, wherein the PCR is quantitative PCR.
29. The method of any one of claims 26-28, wherein the first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an Fv fragment, and an scFv fragment.
30. The method of any one of claims 26-29, wherein the amplicon is detected with a detectably labeled probe that binds to the amplicon.
31. The method of claim 30, wherein the probe comprises a fluorescent label, a bioluminescent label, or a chemiluminescent label.
32. The method of any one of claims 1 -31 , further comprising quantifying number and area of the integrin-mediated curved adhesions.
33. The method of any one of claims 1-32, further comprising: contacting a population of cancer cells with the candidate agent if the candidate agent inhibits the formation of the integrin- mediated curved adhesions; and measuring adhesion, proliferation, cell spreading, migration, or survival of the population of cancer cells, wherein reduced adhesion, proliferation, cell spreading, migration, or survival in presence of the candidate agent compared to that in a negative control population of cancer cells that are not treated with the candidate agent indicates that the candidate agent has anti-cancer activity.
34. The method of any one of claims 1 -33, wherein the candidate agent is a small molecule, a peptide, a protein, an aptamer, an antibody, an antibody mimetic, an inhibitory nucleic acid, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.
35. The method of claim 34, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fv fragment, and a nanobody.
36. The method of claim 34 or 35, wherein the antibody specifically binds to the integrin isoform αVβ5 or the FCHo2.
37. The method of claim 34, wherein the inhibitory nucleic acid is selected from the group consisting of a small interfering RNA (siRNA), a microRNA (miRNA), a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), an antisense oligonucleotide, and a peptide nucleic acid.
38. The method of claim 37, wherein the inhibitory nucleic acid inhibits transcription or protein translation of the integrin isoform α p5 or the FCHo2.
39. The method of claim 34, wherein the CRISPR system targets a gene or an RNA transcript encoding the integrin isoform αVβ 5 or the FCHo2, or makes epigenetic changes that reduce expression of the integrin isoform ocV(35 or the FCHo2.
40. The method of claim 34 or 39, wherein the CRISPR system comprises Cas9, Cas12a, Cas12d, Cas13a, Cas13b, Cas13d, or a dead Cas9 (dCas9).
41 . An inhibitor of integrin-mediated curved adhesion identified by the method of any one of claims 1-40.
42. A composition comprising the inhibitor of claim 41 and a pharmaceutically acceptable excipient.
43. The composition of claim 42, further comprising a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment.
44. A reporter system for measuring curved adhesion, the reporter system comprising a cell expressing a split fluorescent or luminescent protein comprising a first component of a fluorescent or luminescent protein and a second component of the fluorescent or luminescent protein, wherein the cell comprises: a first nucleic acid comprising a coding sequence encoding a first fusion protein comprising the first component of the fluorescent or luminescent protein connected to integrin isoform αVβ5; and a second nucleic acid comprising a coding sequence encoding a second fusion protein comprising the second component of the fluorescent or luminescent protein connected to FCHo2; wherein a reconstituted fluorescent or luminescent protein forms when the first component and the second component of the split fluorescent or luminescent protein are expressed and interact to produce a detectable fluorescent or luminescent signal indicative of the proximity of the integrin isoform «Vp5 and the FCHo2 in the cell.
45. The reporter system of claim 44, wherein the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
46. The reporter system of claim 45, wherein the split luminescent protein is a split luciferase or a split aequorin.
47. The reporter system of claim 46, wherein the split luciferase is a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
48. A kit comprising the reporter system of any one of claims 44-47 and instructions for detecting integrin-mediated curved adhesions.
49. A system for screening a candidate agent to determine its effects on formation of integrin-mediated curved adhesions by a cell, the system comprising a vertical nanostructure having an end curvature diameter ranging from 100 nm to 3 pm, wherein the cell forms the integrin-mediated curved adhesions on the surface of the nanostructure.
50. The system of claim 49, wherein the nanostructure is a nanobar or a nanopillar.
51 . The system of claim 49 or 50, wherein the nanostructure is coated with vitronectin or fibronectin.
52. The system of any one of claims 49-51 , further comprising a cell or cell fragment of interest.
53. The system of claim 52, wherein the cell or cell fragment of interest is a cancer cell, a blood cell, a platelet, an inflammatory cell, a fibroblast, or an epithelial cell.
54. The system of any one of claims 49-53, further comprising: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split fluorescent protein connected to integrin isoform αVβ5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split fluorescent protein connected to FCHo2, wherein a reconstituted fluorescent protein forms when the first component and the second component of the split fluorescent protein are expressed and interact to produce a detectable fluorescent signal.
55. The system of claim 54, wherein the split fluorescent protein is a split green fluorescent protein, a split enhanced green fluorescent protein, a split red fluorescent protein, a split blue fluorescent protein, a split cyan fluorescent protein, a split violet fluorescent protein, a split orange fluorescent protein, a split yellow fluorescent protein, a split yellow-green fluorescent protein, a split-mNeonGreen2 fluorescent protein, or a split sfCherry2 fluorescent protein.
56. The system of claim 54 or 55, further comprising a fluorescence microscope, a two-photon excitation microscope, a fluorescence confocal laser scanning microscope, or a light sheet fluorescence microscope.
57. The system of any one of claims 49-53, further comprising: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a first component of a split luminescent protein connected to integrin isoform «V|35; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a second component of the split luminescent protein connected to FCHo2, wherein a reconstituted luminescent protein forms when the first component and the second component of the split luminescent protein are expressed and interact to produce a detectable luminescent signal.
58. The system of claim 44, wherein the split luminescent protein is a split luciferase or a split aequorin.
59. The system of claim 45, wherein the split luciferase is a split firefly luciferase, a split Renilla luciferase, a split Elateroidea luciferase, a split Metridia luciferase, a split Vibrio luciferase, a split dinoflagellate luciferase, or a split nano-lantern luciferase.
60. The system of any one of claims 44-46, further comprising a luminescence microscope, luminescence digital imaging microscope, time-gated luminescence microscope, or a luminometer.
61 . The system of any one of claims 49-53, wherein the system comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) donor connected to integrin isoform αVβ5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET acceptor connected to FCHo2.
62. The system of any one of claims 49-53, wherein the system comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a fluorescence resonance energy transfer (FRET) acceptor connected to the integrin isoform «vp5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a FRET donor connected to the FCHo2.
63. The system of claim 61 or 62, further comprising a fluorescence microscope or a fluorescence confocal laser scanning microscope.
64. The system of any one of claims 61 -63, wherein the FRET donor and the FRET acceptor are fluorescent proteins or fluorescent dyes.
65. The system of claim 64, wherein the FRET donor is cyan fluorescent protein and the FRET acceptor is yellow fluorescent protein, the FRET donor is blue fluorescent protein and the FRET acceptor is green fluorescent protein, the FRET donor is cyanine dye 3 (Cy3) and the FRET acceptor is cyanine dye 5 (Cy5), or the FRET donor is fluorescein and the FRET acceptor is tetramethylrhodamine.
66. The system of any one of claims 49-53, wherein the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) donor connected to the integrin isoform «V|35; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET acceptor connected to the FCHo2.
67. The system of any one of claims 49-53, wherein the system further comprises: a vector comprising an expression cassette comprising a coding sequence encoding a first fusion protein comprising a bioluminescence resonance energy transfer (BRET) acceptor connected to the integrin isoform «vp5; and a vector comprising an expression cassette comprising a coding sequence encoding a second fusion protein comprising a BRET donor connected to the FCHo2.
68. The system of claim 66 or 67, wherein the system further comprises a luminescence microscope, a luminescence digital imaging microscope, a time-gated luminescence microscope, or a luminometer.
69. The system of any one of claims 66-68, wherein the BRET donor or the BRET acceptor is a bioluminescent protein or a luminescent quantum dot.
70. The system of claim 69, wherein the BRET donor is a Renilla luciferase or an Oplophorus luciferase and the BRET acceptor is a green fluorescent protein, a yellow fluorescent protein, or an orange fluorescent protein.
71 . The system of any one of claims 49-53, further comprising reagents for performing an antibody-based proximity ligation assay (PLA) or a proximity-dependent initiation of hybridization chain reaction (proxHCR) assay.
72. The system of claim 71 , wherein the reagents for performing the PLA comprise: a first antibody that specifically binds to αVβ5; a second antibody that specifically binds to FCHo2; a first probe comprising a first oligonucleotide conjugated to a first secondary antibody, wherein the first secondary antibody specifically binds to the first antibody; and a second probe comprising a second oligonucleotide conjugated to a second secondary antibody, wherein the second secondary antibody specifically binds to the second antibody.
73. The system of claim 72, further comprising a bridge oligonucleotide, wherein the bridge oligonucleotide comprises a first portion sufficiently complementary to hybridize with the first oligonucleotide and a second portion sufficiently complementary to hybridize with the second oligonucleotide when the first oligonucleotide and the second oligonucleotide are in sufficient proximity to each other to simultaneously hybridize to the bridge oligonucleotide.
74. The system of claim 73, further comprising a ligase.
75. The system of any one of claims 72-74, wherein the first antibody, the second antibody, the first secondary antibody, or the second secondary antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an Fv fragment, and an scFv fragment.
76. The system of any one of claims 72-75, further comprising reagents for performing polymerase chain reaction (PCR), isothermal amplification, or rolling circle amplification.
77. The system of claim 76, wherein the PCR is quantitative PCR.
78. The system of any one of claims 72-77, further comprising a detectably labeled probe that binds to the amplicon.
79. The system of claim 78, wherein the probe comprises a fluorescent label, a bioluminescent label, or a chemiluminescent label.
80. A kit comprising the system of any one of claims 49-80 and instructions for detecting integrin-mediated curved adhesions.
PCT/US2023/067339 2022-05-25 2023-05-23 Screening for inhibitors of integrin-mediated curved adhesion Ceased WO2023230461A2 (en)

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