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  • 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
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  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/53—Physical structure partially self-complementary or closed
  • C12N2310/531—Stem-loop; Hairpin
• • 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
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4703—Regulators; Modulating activity
  • G01N2333/4704—Inhibitors; Supressors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

• the present invention relates to the use of caveolin-1 as a biomarker for tumor progression when expressed in tumor-associated fibroblasts.
• the present invention also relates to a method for the diagnosis and/or prognosis of tumor progression comprising the detection and/or quantification of caveolin-1 expression in the tumor-associated fibroblasts of an isolated sample.
• Microenvironment-mediated tensile forces also contribute to disease. Matrix stiffness promotes breast cancer progression via mechanoreciprocal induction of Rho-dependent cell contractility (Levental et al., 2009 Cell 139, 891-906).
• the microenvironment is also important for tumor invasion and metastasis: tumor cells (TCs) migrate along tracks made of extracellular matrix (ECM) collagen fibers (Friedl and Gilmour, 2009 Nat. Rev. Mol. Cell Biol. 10, 445-457), and whereas reticular collagen surrounding mammary glands restrains invasion, Rho mediated alignment of dense collagen fibers perpendicular to the tumor boundary promotes it (Provenzano et al., 2008 Biophys. J. 95, 5374-5384).
• ECM extracellular matrix
• Activated fibroblasts facilitate tumor cell invasion through protease- and force- dependent generation of ECM tracks (Gaggioli et al., 2007 Nat. Cell Biol. 9, 1392-1400).
• Carcinoma-associated fibroblasts also called tumor- associated fibroblasts (TAFs) and mesenchymal stem cells, via paracrine cytokine signaling, promote tumor growth, invasion, and metastasis (Karnoub et al., 2007 Nature 449, 557-563; Orimo et al., 2005 Cell 121 , 335-348).
• Caveolin-1 (Cav1 ), the major component of endocytic caveolae plasma membrane (PM) invaginations, has many functions outside caveolae (Parton and Simons, 2007 Nat. Rev. Mol. Cell Biol. 8, 185-194). Cav1 activates Rho by regulating its endogenous inhibitor p190RhoGAP (p190) and assists in focal adhesion (FA) stabilization required for directional cell migration (Goetz et al., 2008 J. Cell Biol. 180, 1261- 1275; Grande-Garcia et al., 2007 J. Cell Biol. 177, 683-694). The role of Cav1 in tumor progression remains unclear.
• the present invention provides a useful and reliable biomarker of tumor progression.
• the inventors have found that the higher expression of caveolin-1 (Cav1 ) in tumor-associated fibroblasts (TAFs) correlates with a poorer prognosis, increased tumor progression, higher invasive and metastatic tumor capacity and decreased patient survival. Also, the inventors have found that the inhibition or absence of Cav1 in the tumor microenvironment favours tumor encapsulation, and therefore prevents tumor progression and metastasis.
• Cav1 caveolin-1
• TAFs tumor-associated fibroblasts
• a first aspect of the present invention relates to the use of Cav1 in TAFs as a tumor progression biomarker.
• at least one further known biomarker for TAFs selected from smooth muscle actin (SMA), CD90 and vimentin is used.
• SMA smooth muscle actin
• CD90 CD90
• vimentin a further known biomarker for TAFs selected from smooth muscle actin (SMA), CD90 and vimentin.
• the TAFs biomarker SMA is further used in order to specifically localize Cav1 expression in TAFs.
• a second aspect of the present invention relates to a method for the diagnosis and/or the prognosis of tumor progression, comprising the following steps: (a) detection and/or quantification of an expression product of Cav1 gene in tumor- associated fibroblasts of a sample isolated from a subject; (b) comparison of the expression levels of Cav1 detected and/or quantified in step (a) with standard values; (c) finding a significant overexpression of Cav1 in the comparison of step (b); and (d) attributing the significant overexpression of Cav1 found in step (c) to a poor prognosis.
• a third aspect of the present invention relates to a kit consisting essentially of specific primers and/or probes and/or antibodies for detecting and/or quantifying caveolin-1 expression; and specific primers and/or probes and/or antibodies for detecting and/or quantifying the expression of at least one of the following genes: SMA, CD90 and vimentin, preferably of SMA.
• a fourth aspect of the present invention relates to Cav1 inhibitors for use as a medicament.
• a fifth aspect of the present invention relates to Cav1 inhibitors for use in the prevention of tumor progression.
• a sixth aspect of the present invention relates to p190RhoGAP-A (hereafter called p190) agonists for use as a medicament.
• a seventh aspect of the present invention relates to p190 agonist for use in the prevention of tumor progression.
• An eighth aspect of the present invention relates to Rho-A inhibitors for use in the prevention of tumor progression.
• FIG. 1 Cav1 -Regulated Contractility Controls Matrix-Induced Cell Morphology and Reciprocal Interaction with the 3D Microenvironment
• FDMs Fibroblast-Derived Matrices
• B CavlWT (Cav1 Wild Type) and KO (Knock Out) MEFs (Mouse Embyonic Fibroblasts) were plated (4 hr) on NIH 3T3 FDMs (3D) or FN (Fibronectin) (5 mg/ml, 2D) and labeled as indicated.
• Elliptical factors (EF) were calculated.
• C Quantification of protrusions per cell (means indicated). Representative CavlWT and CavI KO cells (asterisks mark protrusions) are shown.
• D CavlWT and KO MEFs were embedded (6 hr) in Col-I (Collagen type I) gels (1 mg/ml). EFs (Embryonic Fibroblasts) are indicated.
• E Rac1 distribution in CavlWT and KO MEFs.
• (a) Rac1 immunostaining in cells plated (4 hr) on FN. Asterisks mark Rac1 foci
• c Immunoblot showing total Rac1 expression.
• FIG. 1 Cav1 -Dependent Extracellular Environment Regulates Cell Shape, Protrusion Number, Rac1 Activity, and Maturation of Integrin-Dependent Adhesions
• A FDMs were generated from CavlWT and KO MEFs (or KO MEFs rescued with CavlWT or CavY14F) and seeded with unmodified or reconstituted MEFs as indicated.
• B EFs of CavlWT and KO MEFs seeded in the indicated FDMs.
• C EFs of CavI KO MEFs rescued by re-expression (+) of CavlWT or CavY14F and seeded in FDMs generated by similarly rescued CavI KO MEFs.
• FIG. 3 Cav1 Promotes Patterning and Stiffness of 3D Matrices and Favors Normal Tissue Architecture
• A-D FDMs were generated and after cell extraction were fixed and labeled. The orientation of all thresholded FN fibers was quantified and plotted against the modal angle (set at 0°).
• E Atomic force microscopy was used to plot point-by-point force versus distance along fibers. The chart shows quantification of Young modulus.
• F Skin sections of WT and CavI KO mice stained with Masson's trichrome and picrosirius red (PR). Polarized light highlights fibrillar collagen.
• FIG. 4 Cav1 Promotes Force-Dependent Microenvironment Remodeling via Rho GTPase Activation
• A Indicated MEFs expressing GFP-tagged WT Cav1 , RhoV14, or empty vector (see immunoblot) were plated on FN (24 hr). FN remodeling was quantified by thresholding for bright FN fibrils.
• B CavI KO MEFs stably expressing scrambled or p190 shRNA were plated as in (A) and analyzed for (C) Rac1 -GTPase activity and (D) Col-I gel contraction.
• E FN staining of FDMs generated from the indicated MEFs.
• FIG. 1 Stroma of Human Breast, Kidney, and Colon Carcinomas and Melanoma Metastases Are Enriched in Cav1 -Expressing Fibroblasts
• A Representative images of normal and breast cancer tissue stained for Cav1 and SMA.
• C Kaplan-meier curve of progression-free survival for patients sorted by stromal Cav1 expression.
• A EFs of ATCC-231 , LM-4175, and BM-1833 metastatic cells seeded in CavlWT or CavI KO FDMs.
• B ATCC-231 cells grown in the indicated FDMs (6 hr) were monitored for 12 hr. Samples were labeled for Rac and FN to reveal cell morphology and FDM structure. Example migration tracks are depicted. Charts show quantification of cell velocity and directionality.
• C Col-I gels containing prelabeled TCs (tumor cells) and MEFs were placed under cell-free gel (scheme) and 3D TC invasion was quantified.
• FIG. 7 Cav1 -Dependent 3D Microenvironment Stimulates In Vivo Tumor Cell Invasion and Increases Metastatic Potency.
• A Orthotopic mammary gland allografts, (a) Experimental scheme, (b) Bioluminescence detection of TCs (representative images). Scale depicts the photon flux (photons per second), (c) MPE-SHG of primary tumor explants. Arrows mark invading and encapsulated TCs. (d) Quantification of the angle of collagen fibers to the tumor boundary by SHG.
• B Orthotopic mammary gland xenografts and tumor transplantation in nude mice, (a) Experimental scheme.
• C Identification and quantification of ITRAQ (Isobaric tag for relative and absolute quantitation)-labeled ECM proteins by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS),
• LC liquid chromatography
• MS/MS tandem mass spectrometry
• D Skin sections of 3-week-old WT and CavI KO mice stained with Masson's trichrome and picrosirius red (PR). Polarized (orthogonal) light highlights fibrillar collagen.
• E Multiphoton excitation microscopy of intact fixed mammary glands from CavlWT and KO mice. Images show second harmonic generation (SHG) and autofluorescence signals of surrounding stroma. Arrows mark curly and straight collagen fibers devoid of SHG signal. Data are represented as mean ⁇ standard error of the mean (SEM).
• E Immunoblot showing the effectiveness of three independent p190RhoGAP shRNA sequences on p190RhoGAP expression in CavI KO MEFs. Cav1 and tubulin blots are shown as internal controls.
• FIG. 1 (A) Paired paraffin-embedded sections of normal kidney and renal tumor tissue from a representative patient. Sections are stained for Cav1 , a- SMA, and Collagen deposition (Masson's trichrome) as indicated.
• B Fibroblasts from renal tumor tissue (CAFs) were isolated and infected with lentivirus encoding scrambled or Cav1 -siRNA. Immunoblot shows Cav1 silencing efficiency. Tubulin shows equal loading. Collagen gel contraction assay confirms impaired contraction by Cav1 -silenced CAFs.
• C Hmb45 (white) and Cav1 (red) staining in distant tumor-associated stroma of metastases from melanoma samples.
• the present invention identifies Cav1 as a regulator of ECM remodeling and desmoplastic processes.
• CAFs the main cell component of the desmoplastic stroma of solid cancers and their metastases, influence TC growth through paracrine secretion of growth factors and recruit endothelial progenitors to promote angiogenesis.
• CAFs deposit ECM and remodel the tumor stroma.
• ECM fiber tracks align at the tumor margin, and force- and protease-mediated ECM remodeling by fibroblasts favors TC invasion (Gaggioli et al., 2007 Nat. Cell Biol. 9, 1392-1400; Provenzano et al., 2008 Biophys. J. 95, 5374-5384).
• the inventors show that the stroma of breast, renal, and colon carcinoma and melanoma metastases is enriched in Cav1 -expressing CAFs, and contractility of renal CAFs is reduced by lowering Cav1 levels.
• Cell-free 3D matrices generated by CavlWT fibroblasts are stiffer and more aligned than matrices derived from CavI KO fibroblast, thus better stimulating TC elongation, velocity, and directional migration.
• Orthotopic implantation in CavI KO mice impairs tumor invasiveness and metastatic potency. In cocultures fibroblast Cav1 favors TC invasion, and coinjection of these cells into nude mice increases metastasis. Both effects depend on Cav1 -regulated p190 activity. Results from the experimental metastasis assay show the important role of fibroblast Cav1 in events before intravasation.
• CAF expression of Cav1 favors tumor progression via biomechanical remodeling of the primary tumor-associated stroma.
• Cav1 has a complex role in tumor stroma, and the present invention a last provides a way to use of Cav1 for prognosis.
• the experimental results with orthotopic grafts and the stroma-dependent tumorigenicity assay show that increased fibroblast Cav1 promotes local invasiveness and metastasis via remodeling of the stromal ECM. Cav1 also regulates other (nonfibroblast) tumor-associated stromal components.
• the stroma provides environmental cues essential for tissue architecture.
• Cav1 loss in stromal cells produces benign stromal lesions causing abnormal epithelium growth and differentiation.
• the inventors have found that CavlWT mammary glands show markedly higher SHG signal intensity and numbers of straight collagen fibers which favour TC migration, invasion and metastasis.
• the correlation between disorganized mammary stroma and impaired growth, invasion, and metastatic potency of orthotopic grafts shows that Cav1 - positive stroma is permissive for tumor progression. Together, this shows that Cav1 -dependent impaired architecture of native stroma affects tumor progression.
• the FDMs generated in the present invention model the physiological microenvironment of cell interactions and reveal that the matrix generated by Cav1 fibroblasts increases cell elongation. Morphology of all cells tested was more strongly influenced by the surrounding 3D architectural organization than by its "intrinsic" shape in 2D. Cav1 phosphorylation at Tyr14 coordinates Rho GTPase activity (Grande-Garcia et al., 2007 J. Cell Biol. 177, 683-694).
• the inventors show that Cav1 regulates p190 phosphorylation and partitioning into ordered domains, and that lack of Cav1 generates Rac accumulation in these sites.
• the inventors show that Cav1 -expressing fibroblasts increase alignment of ECM fibers through Rho- and force-dependent matrix reorganization. In turn these matrices enhance cell elongation, mature integrin adhesions, and reduce cell protrusions. They also show that tumor cell elongation, velocity, and migration directionality are enhanced by Cav1 FDMs, as. coculture with Cav1 fibroblasts favors TC elongation and invasiveness in vivo and in vitro.
• the inventors have found that the tumor-stroma interface of mammary tumors in Cav1 -expressing mice is more proinvasive than in their CavI KO counterparts, with increased collagen fiber alignment.
• the inventors have found a strong correlation between metastatic potency and intratumoral matrix alignment.
• the results provided by the inventors identify a critical role for Cav1 in growth and invasion, highlighting a function of Cav1 in the regulation of matrix-dependent cell behavior.
• Cav1 regulates Rho GTPase activity by modulating membrane partitioning of p190 and thereby its phosphorylation.
• Fibroblast expression of Cav1 in vitro and in vivo favors an organized 3D stromal architecture that promotes spindle morphology, facilitates TC invasion, and increases p190-dependent metastatic potency.
• a first aspect of the present invention relates to the use of caveolin-1 in tumor-associated fibroblasts as biomarker of tumor progression.
• Caveolin-1 is a protein that in humans is encoded by the Cav1 gene (caveolin- 1). It is a scaffolding protein and it is the main component of the caveolae plasma membranes found in most cell types. Cav1 has been considered a tumor-suppressor gene, although its role is controversial.
• TAFs tumor-associated fibroblasts
• CAFs carcinoma-associated fibroblasts
• TAFs can be identified de visu by specialized histologists when employing common histology stainings or can be stained with specific markers known in the art.
• the preferred TAF markers are smooth muscle actin (SMA), CD90 and vimentin, and SMA is the most preferred one.
• tumor progression refers to tumor growth as well as to cell migration to produce both local invasion and distant metastasis.
• tumor refers to a benign, pre-malignant or malignant neoplasm, including cancer.
• At least one further biomarker selected from: smooth muscle actin, CD90 and vimentin is used.
• SMA is further used.
• a preferred embodiment of the first aspect of the present invention is therefore the use of Cav1 and SMA as biomarker of tumor progression.
• the results of the present invention show that intrinsic fibroblast Cav1 and the Cav1 -regulated microenvironment cooperate to enhance cell elongation, migration, and invasion through force-dependent organization of the surrounding 3D environment. Cav1 mediates these effects by regulating Rho activity via changes in p190 localization and phosphorylation. These data show that this Cav1 -dependent bidirectional cell/matrix mechanical crosstalk is critical for normal tissue homeostasis and architecture, and for tumor invasion and metastasis.
• a second aspect of the present invention relates to a method for the diagnosis and/or prognosis of tumor progression comprising the following steps:
• step (a) comparison of the expression levels of caveolin-1 detected and/or quantified in step (a) with standard values
• step (c) attributing the significant overexpression of caveolin-1 found in step (c) to a poor prognosis.
• the expression "detection and/or quantification" of an expression product of Cav1 gene in TAFs of a sample isolated from a subject refers to the detection of the presence and/or the measure of the quantity or the concentration of said expression product, preferably in a semiquantitative or quantitative manner.
• the measure can be direct or indirect.
• a direct measure refers to the measure of the quantity or the concentration of the Cav1 gene expression products in TAFs based on a signal that is directly related to the number of molecules of said expression products present in the TAFs of the sample. This signal can be measured by measuring the intensity of a physical or chemical property of the expression product.
• An indirect measure includes a measure obtained by using other components or a biological measuring system, such as measuring a cell response, a ligand or a tag or enzymatic reaction products.
• the detection is not intended to be correct in a 100% of the analyzed samples. Nevertheless, it requires a statistically significant amount of the analyzed samples to be classified correctly.
• the amount that is statistically significant can be established by a person skilled in the art by means of different statistical tools, as for example, but not limited to, determining the confidence intervals, the p value, a Student's t test or a Mann- Whitney test or Fisher functions.
• the confidence intervals are at least of 90% or at least of 95%, at least of 97%, at least of 98%, or at least of 99%.
• the p value is smaller than 0.1 , than 0.05, than 0.01 , than 0.005, or than 0.001 .
• the present invention allows the correct determination of the prognosis of at least 60%, at least 70%, at least 80% or in at least 90% of the subjects of a certain group or analyzed population.
• expression product of Cav1 gene refers to either the messenger RNA or the protein generated when said gene is expressed.
• a person skilled in the art is aware of many methods that can be employed to detect and/or quantify such expression products, as for example, but not limited to, in situ hybridization, PCR, qPCR, northern blot, immunological methods such as immunohistochemistry, immunocytochemistry or western blot.
• a person skilled in the art knows how to design specific primers and or probes and/or antibodies against Cav1 gene expression products for their detection and/or quantification.
• sample refers to an isolated biological sample that has been isolated from an organism as the human or animal body and comprises cells and physiological fluids.
• the biological sample can be a tissue, for example, but not limited to, a biopsy or a fine needle aspiration biopsy.
• the isolated sample of step (a) is a solid tissue.
• the biological sample can be, for example, but not limited to, fresh, frozen, fixed or paraffin- embedded.
• the term “comparison” refers to the action of comparing Cav1 expression levels in the TAFs of the sample isolated from a subject that is supposed to have a tumor with Cav1 expression levels in the stroma of an isolated sample of a healthy or normal tissue, preferably with Cav1 expression levels of the fibroblasts of said healthy or normal tissue.
• the "standard values” are therefore the amount of Cav1 expression products in the stroma of a healthy or normal tissue, preferably in the stromal fibroblasts.
• a reference sample may be used. This reference sample may be analyzed simultaneously or consecutively with the sample isolated in step (a).
• the comparison of step (b) may be carried out manually or by computerized means.
• amount refers to the absolute or relative quantity of Cav1 gene expression products.
• subject refers to a vertebrate animal, preferably to a mammal, more preferably to a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, and rodents.
• step (c) refers to the statistically significant difference found in step (c) when comparing the levels of Cav1 expression products in the TAFs of the isolated sample with the levels of Cav1 expression products in the reference sample.
• a statistically significant different means that the p value is at least smaller than 0.5, smaller than 0.1 , than 0.05, than 0.01 , than 0.005, or than 0.001.
• overexpression refers to the levels of Cav1 expression in the TAFs of the isolated sample of step (a) being higher than the levels of Cav1 expression in the reference sample.
• poor prognosis refers to an increased progression of the tumor (including local invasion and distant metastasis), and to decreased patient survival.
• step (a) further comprises the detection and/or quantification of the expression product of at least one of the following biomarkers: SMA, CD90 and vimentin.
• step (a) comprises the detection and/or quantification of the expression product of SMA.
• the expression product is an mRNA or a protein.
• the reference of the sequence of human Cav1 mRNA is GenBank accession number NM_001753, and the reference of the sequence of human Cav1 protein is GenBank accession number NP_001744.2.
• the tumor is an epithelial tumor.
• the tumor is of breast, kidney, colon or melanoma.
• a third aspect of the present invention relates to a kit consisting essentially in specific primers and/or probes and/or antibodies for detecting and/or quantifying caveolin-1 expression; and specific primers and/or probes and/or antibodies for detecting and/or quantifying the expression of at least one of the following genes: SMA, CD90 and vimentin, preferably of SMA.
• the kit further comprises other reagents or materials as for example, but not limited to, buffers, complex forming proteins such as avidin, streptavidin and biotin, enzymes, labeled or unlabeled secondary antibodies, enzyme substrates, tubes, plates or any other additional materials needed to carry on any of the already described protocols for analyzing gene expression.
• buffers complex forming proteins such as avidin, streptavidin and biotin
• enzymes labeled or unlabeled secondary antibodies
• enzyme substrates enzyme substrates, tubes, plates or any other additional materials needed to carry on any of the already described protocols for analyzing gene expression.
• a fourth aspect of the present invention relates to a Caveolin-1 inhibitor for use as a medicament.
• the term "inhibitor” refers to a substance or molecule capable of reducing or eliminating Cav1 protein activity, either by blocking its function or by reducing or eliminating its presence.
• the activity of Cav1 can indirectly be determined by a person skilled in the art using different methods already described in the art, as for example, but not limited to, measuring Rho GTPase activity by a Rho Pull down assay as described (Grande-Garcia et al., 2007 J. Cell Biol. 177, 683- 694) and/or measuring cell-contractile ability of collagen gels as described in the present document.
• Direct determination of Cav1 can be determined by the quantification of Cav1 mRNA or protein.
• the Caveolin-1 inhibitor further comprises an excipient.
• excipient refers to a component of a pharmaceutical composition or a medicament that is not an active compound but a diluent, a vehicle or a carrier, that is considered “pharmaceutically acceptable” when it is safe, non toxic and does not present adverse effects.
• excipient refers to a substance that helps the absorption of the compound, stabilizes it or helps in the preparation of the medicament to give it consistency or provide flavors that make it nicer.
• excipients may serve to maintain the ingredients together, such as starch, sugars o cellulose, to sweeten, to give color, to protect the medicament as for example to isolate it from air and/or humidity, to work as a filler in a pill, capsule or any other form of presentation as for example, dicalcium phosphate, to favor disintegration of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.
• the medicament comprises a therapeutically effective amount of the Caveolin-1 inhibitor.
• therapeutically effective amount refers to an amount that, administered in doses and during the necessary period of time, is effective in achieving the desired prophylactic or therapeutic effect.
• a “therapeutically effective amount” of the Caveolin-1 inhibitor of the invention may vary with the stage of the disease, the age, sex or weight of the subject, and refers to an amount that does not present adverse effects nor toxicity, and that us capable of achieving the desired prophylactic or therapeutic effect.
• the Caveolin-1 inhibitors are preferably administered by parental administration, in the form of a sterile injectable aqueous or oleaginous suspension.
• parenteral refers to introduction into the body by way of an injection (i.e., administration by injection), including, for example, subcutaneously (i.e., an injection beneath the skin), intramuscularly (i.e., an injection into a muscle); intravenously (i.e., an injection into a vein), intrathecally (i.e., an injection into the space around the spinal cord), intrasternal injection, or infusion techniques.
• a parenterally administered composition of the described invention is delivered using a needle, e.g., a surgical needle.
• Injectable preparations such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the administration of the medicament of the present invention is preferably administered locally, preferably by guided therapy directed to the tumor stroma.
• the medicament further comprises another active ingredient.
• the Caveolin-1 inhibitors may be combined with another active ingredient.
• An "active ingredient” is all matter of human, animal, vegetable, chemical o other origin whose activity is appropriate to constitute a medicament.
• a fifth aspect of the present invention relates to a Caveolin-1 inhibitor for use in the prevention of tumor progression.
• the tumor is epithelial.
• the tumor is of breast, kidney, colon or melanoma.
• the Caveolin-1 inhibitor is an interfering RNA or a blocking antibody.
• the Caveolin-1 inhibitor is a small interfering RNA. More preferably, it is SEQ ID NO: 1 or SEQ ID NO: 2.
• SEQ ID NO: 1 is the DNA sequence corresponding to a siRNA duplex corresponding to bases 301-319 from the open reading frame of the murine caveolin-1 mRNA.
• SEQ ID NO: 2 is the DNA sequence corresponding to a small interfering RNA (siRNA) corresponding to bases 254-277 of the human Cav1 .
• siRNA small interfering RNA
• a small interfering RNA or short interfering RNA is a small double strained RNA molecule of 20-25 nucleotides that interferes with the translation of an mRNA, therefore inhibiting the production of the peptide or polypeptide codified by said mRNA, causing its deficiency.
• RNA interference can also be achieved by short or small hairpin RNAs that are delivered into cells by a vector and produce siRNAs inside the cell.
• antibody refers to immunoglobulin molecules and immunologically active portions thereof, that is molecules that comprise a specific binding site in the antigen. There are five isotypes or main classes of immunoglobulins: IgM, IgD, IgG, IgA and IgE.
• a Caveolin-1 blocking antibody binds specifically to Caveolin-1 and inhibits its function and activity.
• a sixth aspect of the present invention relates to p190RhoGAP (hereafter called p190) agonists for use as a medicament.
• the p190 agonists are p190 mRNA or p190 protein.
• Other p190 agonists can be determined by a skilled in the art by performing the following assay: a Rho Pull down assay to measure Rho Activity as described (Grande-Garcia et al., 2007 J. Cell Biol. 177, 683-694). The higher the activity of p190, the lower the activity or Rho.
• An alternative assay is to measure the cell-contractile ability of collagen gels as described in the present document. The higher activity of p190, the lower the ability of cells embedded in collagen gels to contract them.
• a seventh aspect of the present invention relates to p190 agonist for use in the prevention of tumor progression.
• the inventors have shown that p190 promotes tumor encapsulation by controlling the reorganization of at least FN and collagen in the tumor stroma. Also, the inventors have shown a correlation between the alignment of this ECM components and the number of metastasis.
• An eighth aspect of the present invention relates to Rho-A inhibitors for use in the prevention of tumor progression.
• the Rho-A inhibitor is selected from the dominant negative form RhoN19, C3 toxin, an interfering RNA and a blocking antibody.
• Rho-A inhibitors that can be used in the tumor stroma, preferably in the fibroblasts of the tumor stroma, to prevent tumor progression.
• Rho Kinase inhibitor Y-27632 may be used as Rho-A pathway inhibitor for use as a medicament and in the prevention of tumor progression.
• Rho Kinase is a direct Rho effector regulating this pathway downstream of Rho.
• the administration of the medicaments of the present invention is preferably administered locally, preferably by guided therapy directed to the tumor stroma, preferably directed to the fibroblast of the tumor stroma, preferably to SMA expressing fibroblasts in the tumor stroma.
• Example 1 Cav1 Regulates Matrix- Induced Cell Morphology and Reciprocal Interaction with the 3D Microenvironment through Contraction
• FDMs fibroblast-derived 3D matrices
• FN fibronectin
• Figure 1A, Figure 8A we seeded FDMs with Cav1 wild-type (CavlWT) and Cav1 knockout (CavI KO) mouse embryonic fibroblasts (MEFs) and analyzed cell morphology.
• Cav1 deficiency increased PM targeting of Rac1 and its downstream effector phospho-S141-Pak1 ( Figure 1 E and Figure 8D).
• phosphorylation of myosin light chain-2 (pMLC) was decreased, suggesting that Cav1 influences cell-induced matrix contractility.
• CavlWT MEFs contracted Col-I gels more effectively than CavI KO MEFS at all cell concentrations tested ( Figure 1 F and Figure 8E).
• Re-expression of unmodified Cav1 , but not its nonphosphorylatable mutant Cav1Y14F rescued Rac1 localization, cell elongation, gel contraction, and pMLC levels in CavI KO MEFs ( Figure 1 G).
• Cav1 thus regulates features of fibroblasts that influence mechanical 3D microenvironment remodeling.
• Example 2 Cav1 -Dependent Microenvironment Regulates Cell Shape, Protrusion Number. Rac1 Activity, and Morphology of Inteqrin-Dependent Adhesion Structures
• Cav1 through residue Tyr14, favors fibroblast elongation directly through endogenous expression and indirectly through cell dependent 3D microenvironment remodeling, indicating that endogenous Cav1 and the Cav1 -dependent microenvironment cooperate to enhance cell polarity.
• CavlWT FDMs also reduced the number of cell protrusions (Figure 2E and Figure 9C) and Rac-GTPase activity (Figure 2F) independently of Cav1 expression by seeded cells, confirming the ability of Cav1 -dependent ECM to favor in wVo-like spindle morphology.
• CavlWT and CavI KO FDMs both decreased adhesion-targeted pY397FAK levels compared to a 2D FN substrate (Figure 9D).
• the longest 3D-matrix adhesions were obtained when CavlWT MEFs were plated in CavlWT FDMs (1 1 .14 ⁇ 0.48 mm) ( Figure 2G and Figure 9E), indicating that both matrix and cells are important for determining adhesion length.
• the matrix adhesion marker vinculin localizes to 3D-matrix adhesions and its recruitment is force dependent, and the mobile vinculin fraction, determined by fluorescence recovery after photobleaching (FRAP), is an index of adhesion-dependent force increase.
• FRAP fluorescence recovery after photobleaching
• Example 3 Cav1 Promotes Patterning and Stiffness of 3D Matrices thus Regulating Normal Tissue Architecture
• Tissues also contained collagen fibers devoid of SHG signal, which were often kinked in CavI KO mammary glands but almost all straight in CavlWT ( Figure 3Ga and Figure 10E). PR staining confirmed disturbed collagen fiber organization in CavI KO mammary glands, highlighting deficiency in fibrillar collagen around the acinus ( Figure 3Gb).
• Example 4 Cav1 Promotes Force-Dependent Remodeling of the Surrounding Environment via Rho GTPase Activation
• FN fibrillogenesis relies on Rho GTPase-dependent cell contractility, which generates tensile forces that expose cryptic selfassembly sites in stretched FN molecules. Although their fibrillogenesis was delayed, CavI KO MEFs were able to remodel the FN coating at later time points ( Figure 1 1A) with similar FN expression levels as CavlWT MEFs ( Figure 11 B). Absence of Cav1 thus delays but does not negate FN remodeling. Re-expression of Cav1 in CavI KO MEFs restored cell-mediated FN fibrillogenesis (Figure 4A); re-expression of Cav1Y14F had no effect (Figure 1 1 C).
• Cav1 deficiency decreases Rho activity via the endogenous inhibitor p190 (Grande-Garcia et al., 2007 J. Cell Biol. 177, 683-694).
• Constitutive activation of Rho GTPase in CavI KO MEFs rescued the FN remodeling phenotype but had no effect on CavlWT MEFs ( Figure 4A).
• Defective FN remodeling in CavI KO MEFs was also reversed by stable silencing of p190 (Figure 4B and Figure 1 1 Da), which decreased Rac activity and PM localization (Figure 4C and Figure 11 Db).
• p190 knockdown (KD) also increased pMLC levels (not shown) and rescued cell contractility (Figure 4D).
• p190 activation is associated with its phosphorylation and partitioning into PM- ordered domains, where it inhibits Rho activity.
• p190 PM targeting was independent of Cav1 expression ( Figure 4G).
• p190 phosphorylation at the PM was higher in the absence of Cav1 ( Figure 4H). Cav1 absence also promoted p190 partitioning into PM-ordered domains, an effect reversed by Cav1 re-expression (Figure 4I).
• Cav1 thus appears to remodel the 3D microenvironment by stimulating Rho-dependent cell contractility and to regulate Rho activity by controlling the phosphorylation and partitioning of p190 into membrane-ordered domains.
• Example 5 Stroma of Human Breast, Kidney, and Colon Carcinoma and Melanoma Metastases Are Enriched in Cav1 -Expressing Fibroblasts
• tubules (epithelium) Clear cell
• fibroblast Cav1 expression in colorectal cancer stroma is one of the most common causes of cancer death in Western countries and its outcome is determined by the occurrence of metastatic dissemination.
• Cav1 is expressed in smooth muscle cells of the muscularis mucosae, in blood vessels, and to a lower extent in the stroma underlying the epithelium (Figure 5J).
• Analysis of a tissue microarray of 84 colorectal carcinomas detected Cav1 expression in tumor-associated stroma but not in the epithelial TCs (approx. 98% of cases).
• Cav1 staining intensity scores revealed strong expression in 75% of tumor-associated stroma ( Figure 5J).
• Cav1 was also expressed in the peritumoral stroma of all metastatic foci examined in a set of human melanoma metastatic explants, with >80% of fibroblasts Cav1 positive (Figure 5K). Cav1 was found in 79% of fibroblasts expressing SMA and 82% expressing CD90, a marker of human CAFs (Figure 5K). Cav1 expression was detected in very few metastatic (Hmb- 45+) TCs and in only 36% of infiltrated (CD45+) leukocytes ( Figure 5K and Figure 12C). Cav1 -expressing CAFs were excluded from tumor nests but were accompanied by heavy collagen and light FN deposits in the immediate peritumoral stroma ( Figure 12D).
• Fibroblasts positive for CD90 and Cav1 were detected in distant tumor-associated stroma but were absent from normal connective or muscle tissue. Cav1 in nontumor tissue was localized exclusively in blood vessels. Stroma of metastatic foci is thus enriched in Cav1 -positive CAFs, showing that stromal Cav1 is instrumental in both primary and secondary tumor niches.
• Example 6 Cav1 -Dependent 3D Microenvironment Stimulates In Vitro Tumor Cell Migration and Invasion
• MDA-MB-231 parental breast cancer cells (here called ATCC-231 ) and derived lines selected for in vivo tropism to lung (LM-4175) and bone (BM-1833). All three lines express high levels of Cav1 .
• Metastatic cells seeded in FDMs generated by CavlWT MEFs had a higher EF than when seeded in CavI KO FDMs ( Figure 6A).
• CavlWT matrices also supported higher directionality and velocity of migration by TCs ( Figure 6B).
• Example 7 Cav1 -Dependent 3D Microenvironment Stimulates In Vivo Tumor Cell Invasion and Increases Metastatic Potency
• TCs were localized in regions surrounded by few kinked and disorganized collagen fibers ( Figures 7Ac and 7Ad).
• tumors grown in CavlWT mice were collagen-rich, with invading TCs in intimate contact with aligned straight and curly collagen fibers ( Figures 7Ab and 7Ac).
• the angle of collagen fibers was significantly more perpendicular in WT than in CavI KO hosts ( Figure 7Ad), showing higher invasivity in WT environment.
• Table 5 Quantification of primary tumors stained for FN, SMA, and nuclei. The angles of single thresholded FN-labeled fibers were calculated and the percent of fibers within ⁇ 20° compared. EF and angle distribution of SMA positive cells is shown.
• Orthotopic mammary gland tumors were obtained by injecting LM475 human TCs into lethally irradiated CavlWT or CavI KO mice ( Figure 7B). After 9 days, equal-size tumors were either processed or transplanted together with their surrounding stroma into nude mice ( Figure 7Ba). In this system, Cav1 -dependent stroma is the only variable likely to affect TC invasiveness and metastatic potency. Using MPE-SHG imaging and histology, we evaluated Cav1 -dependent matrix remodeling at the tumor-stroma interface of early tumors (8 days). Tumors grown in CavI KO mice were minimally invasive.
• Collagen fibers were stretched and distributed tangentially along the tumor boundary, constraining tumor growth and consistent with previously described TACS-2 (Tumor-associated collagen signature-2) ( Figures 14Ba and 14Bb). Indeed, tumors generated in CavI KO mammary glands grew slower before and after transplantation into nude mice ( Figure 14Bc). In contrast, WT tumors had an invasive morphology, with both TCs and collagen fibers aligned in the direction of invasion (approx. 90° to the tumor boundary), typical of TACS-3. After transplant, such tumors generated significantly more metastases in most organs analyzed (Figure 7Bb).
• Ascorbic acid, ammonium hydroxide, and Triton X-100 were from Sigma.
• mice were cultured as described (Cerezo et al., 2009 Mol. Cell. Biol. 29, 5046-5059).
• ATCC-231 , LM-4175, and BM-1833 were cultured as described (Minn et al., 2005 Nature 436, 518-524).
• PC3 and E0771 TCs were cultured as recommended.
• Cav1 -deficient mice STOCK Cav1tm1 Mls/J
• WT B6129SF2/J controls were from The Jackson Laboratory (USA).
• Cav1 -deficient mice and WT littermates B6.Cg-CAV1tm1 mls/J were used for in vivo experiments.
• Female athymic nude mice were obtained from Harlan.
• Three-dimensional matrices reminiscent of in vivo ECM were prepared. Briefly, 2.5 x 10 5 cells/ml were plated on chemically crosslinked gelatin on tissue culture dishes or coverslips and maintained in confluence for 6-8 days. Cells were supplemented every 48 hr with 50 mg/ml fresh L-ascorbic acid (AA) to stabilize ECM components thus facilitate collagen production and polymerization. The resulting 3D cultures were checked for quality by indirect immunofluorescence or cells were removed from the matrix by alkaline detergent extraction, yielding cell-free 3D matrices for further analysis.
• AA L-ascorbic acid
• 1 .2 x 10 5 fibroblasts (alternatively, 6 x 10 4 CAFs/NFs) were mixed with NaOH- titrated collagen I (PureCol, INAMED) to a final collagen I concentration of 1 mg/ml.
• 4 x 10 5 , 1 x 10 6 , or 1 .5 x 10 6 fibroblasts were used ( Figure 8).
• the mixture was immediately transferred to a 24-well plate and lattices were allowed to solidify for 20 min at room temperature. Serum- containing medium was added to each well and gels were manually detached by circular movements using a sterile pipette tip. Gels were placed at 37°C and contraction was documented. Four or five fibroblast-containing gels were assayed for each condition. Gel contraction index was calculated with Metamorph software from the gel surface area measured on acquired images, and reported as the percentage of contraction of the initial surface area.
• the protocol is based on (Gaggioli et al., 2007 Nat. Cell Biol. 9, 1392-1400).
• ATCC-231 tumor cells were labeled (30 min) with calcein and mixed 1 :1 with MEFs.
• the mixes were embedded in serum-free collagen I gels (15 ml, 1 mg/ml) in the well centers of an 8-well IBIDI chamber.
• the gel plug containing the cells was embedded in a second serum-containing collagen gel (150 ml, 1 mg/ml) and covered with serum-containing medium supplemented with SDF-1 a (50 ng/ml).
• gels were fixed, labeled with fluorescently conjugated phalloidin, imaged, and analyzed. Invasiveness was quantified as the area invaded by tumor cells.
• Equal numbers of GFP-expressing PC-3 cells and fibroblasts were mixed, loaded into the well centers of Matrigel-coated IBIDI angiogenesis wells, and covered with a 1 :1 mix of Matrigel and medium (1 1 ml). After polymerization, chambers were filled with medium containing 2% FBS. Alternatively, PC-3 cells and fibroblasts were separated by a thin Matrigel layer. For this, PC-3 cells were first seeded into Matrigel and a thin Matrigel layer was overlaid three hours later and left to polymerize before seeding MEFs. During the 6 day invasion period, 5 ml medium containing 10% serum was added to the chambers every second day.
• the cells were fixed and labeled with phalloidin and Hoechst, and imaged by confocal microscopy. Invasiveness of each cell type was quantified as the area invaded. Total numbers of MEFs at the end point were quantified by automated counting of nuclei in all the acquired planes (ImageJ) and subtraction of GFP-positive (PC3) cells.
• the spheroid invasion assay was adapted from Gaggioli et al. (2007) (Nat. Cell Biol. 9, 1392-1400) After 6 days, invaded gels were fixed, labeled with fluorescently conjugated phalloidin, imaged, and analyzed. Invasiveness was quantified as the area invaded by tumor cells. Col-I gel contraction assays were performed as published. Four or five fibroblast-containing gels were assayed for each condition. Gel contraction index was calculated with Metamorph software from the gel surface area measured on acquired images and reported as the percentage of contraction of the initial surface area.
• E0771 C57BL/6 mammary adenocarcinoma cells were lentivirally infected a vector encoding GFP and luciferase.
• GFP-positive cells (4 3 106 in 200 ml PBS:Matrigel 1 :1 ) were injected into mammary glands of WT and CavI KO mice (strain B6.Cg- CAV1tm1 mls/J).
• the same cells (1 3 106 in 100 ml PBS) were injected into tail veins of WT and CavI KO mice (strain B6.Cg-CAV1tm1 mls/J) in an experimental metastasis assay.
• LM-4175 cells prepared as before were injected into mammary glands of lethally irradiated WT and CavI KO mice (strain STOCK Cav1tm1 Mls/J). After 7 days, tumors and surrounding stroma were extracted and subcutaneously transplanted to nude mice. Alternatively, tumor architecture was analyzed using SHG imaging.
• 106 LM-4175 tumor cells were unmixed or mixed 1 :1 with CavlWT or CavI KO pMEFs, or with CavI KO pMEFs infected with p190RhoGAPshRNA lentiviral vector (+p190shKO MEFs).
• Cells were resuspended in a 1 :1 mix of PBS and Matrigel (200 ml), and injected subcutaneously into anaesthetized nude mice using a 25- gauge needle.
• mice were injected with luciferin (17.5 mg/ml), and after 20 min were placed in the IVIS Imaging System and ventral views captured.
• Tumor growth and metastasis formation were monitored at regular intervals.
• front limbs were secured with tape and the lower portion of the animal was shielded to block bioluminescence from the primary tumors.
• Exposure time for photon flux quantification was 0.2 s, but ranged from 0.2 s to 2min for metastasis detection.
• luciferin-injected mice were killed and the organs extracted and reimaged ex vivo to detect metastatic foci. Images acquired from multiple exposure times were used to manually quantify every visible metastatic focus. Small metastatic foci could be detected by adjusting the scale of photon flux in Living Image 3.2 software.
• Extracted primary tumors were frozen in tissue-Tec and prepared for histology.
• LM-4175 alone or mixed with pMEFs, was subcutaneously injected.
• Tumor growth and metastasis were assessed by bioluminescence with the IVIS Imaging System. Briefly, mice were injected with luciferin (17.5 mg/ml) and after 20 min were placed in the IVIS Imaging System and ventral views captured. Tumor growth and metastasis formation were monitored at regular intervals. Exposure time for photon flux quantification was 0.2 s but ranged from 0.2 s to 2 min for metastasis detection. At the end of the in vivo analysis, luciferin-injected mice were killed and the organs extracted and reimaged ex vivo to detect metastatic foci. Images acquired from multiple exposure times were used to manually quantify every visible metastatic focus. Small metastatic foci could be detected by adjusting the scale of photon flux in Living Image 3.2. Extracted primary tumors were frozen in tissue-Tec and prepared for histology. Alternatively, tumor architecture was analyzed by SHG imaging.
• RNA Interference-Mediated Knockdown of p190RhoGAP and Cav1 Sequences targeting mouse p190RhoGAP were cloned into short hairpin RNA (shRNA) vector pSuper.Retro.Neo+GFP (Oligoengine).
• shRNA short hairpin RNA
• sequences obtained from GenBank/EMBL/DDBJ under accession number NM_172739, were as follows: sequenced (SEQ ID NO: 3), nucleotides 2935-2953, 5'- GTTATGGACGCAACATTAA-3'; sequence#2 (SEQ ID NO: 4), nucleotides 1225-1243, 5'-ACAGGAACTTCGATGATCA-3'; and sequence#3 (SEQ ID NO: 5), nucleotides 1955-1963, 5'-GACACCAACCTTCCAACCC-3'.
• the nontargeting control (scrambled) sequence was SEQ ID NO: 6: 5'- GCGCGCTTTGTAGGATTCG-3'.
• Retroviral supernatants were generated by transfecting 293T/17 cells with each shRNA and pSVc2 vector (White et al. J Virology, 1999, 73 (4) 2832-2840) using the Fugene 6 transfection reagent (Roche). CavI KO MEFs were infected with retroviral supernatants and high GFP-expressing cells were sorted (approx. 15% of the cell population).
• sequence#1 (SEQ ID NO: 3) was cloned into pLVX- shRNA2, which contains a ZsGreenl reporter (Clontech), and primary KO MEFs were infected with supernatants derived from HEK293T cells. Infection efficiency was monitored by ZsGreenl expression. A representative immunoblot confirming silencing efficiency is presented in Figure 7 Ca.
• An alternative Cav1 siRNA was designed (sequence #2 (SEQ ID NO: 2), Fig.12B: 5'- GACGTGGTCAAGATTGACTTT-3') corresponding to bases 254-277 of the human Cav1 .
• ITRAQ Isobaric tag for relative and absolute quantitation
• FDMs from WT and KO MEFS were extracted as described above (Method #A).
• FDMs were extracted with three successive treatments with hypotonic PBS (diluted 1 :5) containing 0.5% w/v sodium deoxycholate (DOC) (Method #B).
• Extracted FDMs were extensively washed with PBS, scraped off the plate, and proteolyzed with 4 mg trypsin (30 mM Tris, pH8) overnight at 37°C. The resulting soluble protein extracts were quantified by Bradford assay. Each sample was then denatured and digested as described in the iTRAQ reagents protocol.
• modified porcine trypsin Promega was added at a final trypsin: protein ratio of 1 :50.
• samples were labeled with iTRAQ tags as follows: proteins obtained by "Method A” were labeled with WT-iTRAQ 1 15 and KO-iTRAQ 1 14; proteins prepared by "Method B” were labeled with WT-iTRAQ 1 16 and KO-iTRAQ 1 17.
• the samples were vacuum dried and dissolved in buffer A (0.5% acetic acid) for desalting and removal of excess iTRAQ reagent in reverse- phase (RP) C-18 cartridges.
• the resulting tryptic peptide mixtures were separated by nano-liquid chromatography coupled to mass spectrometry for protein identification.
• Peptides were eluted from the RP nano-column at a flow rate of 300 nl/min into an emitter nanospray needle for real-time ionization and peptide fragmentation on an LTQ-Orbitrap mass spectrometer (Thermo Fisher, San Jose, CA, USA).
• An enhanced FT- resolution spectrum (resolution 60000) followed by the MS/MS spectra of the three most intense parent ions were analyzed during the chromatographic run (180 min).
• Each parent ion was fragmented by two dissociation methods: CID for peptide sequence analysis and HCD for quantification of the iTRAQ reporter low mass signals. Dynamic exclusion was set at 0.5 min.
• ECM stiffness is several orders of magnitude smaller than that of the AFM probe, so that E «EECM- Equation 1 requires transformation of the cantilever deflection dependence on sample displacement into force versus indentation curves. This is accomplished by determining the indentation from
• MEFs transfected with vinculin-GFP were plated overnight on the assorted FDMs.
• Two prebleach events were acquired before bleaching by stimulation with the SP5 scanner at 488 nm. Fluorescence recovery was monitored at 4 s intervals until the intensity reached a plateau. Fluorescence during recovery was normalized to the prebleach intensity. Relative recovery of vinculin-GFP at 3D-matrix adhesions and FAs was evaluated by comparing the half-times of fluorescence recovery toward the asymptote. Mobile and immobile fractions were calculated from comparison of intensity ratios in the bleached area before bleaching and after recovery. Graphs are representative of a minimum of three independent experiments in which between 6 and 15 adhesion structures were bleached.
• Alignment was analyzed using Curvelet-Based Alignment Analysis software (www.loci.wisc.edu/software/curvelet-based-alignment-analysis), in which a line was drawn at the tumor/stroma boundary and collagen angles determined relative to that line. Quantitative data were graphed and statistically analyzed with Graph Pad software.
• Paraffin-embedded sections were deparaffinized in xylene, rehydrated through a graded series of ethanol and water and boiled in 10 mM sodium citrate-buffer for antigen retrieval. Sections were incubated with assorted antibodies overnight at 4°C. HRP secondary antibodies were detected with the Liquid DAB Substrate Pack (Biogenex, San Ramon, CA, USA) according to the manufacturer's instructions. Sections were counterstained with Harris modified hematoxylin (Thermo Fisher Scientific). Negative controls included omission of primary antibody and its substitution with normal rabbit or mouse IgG (Sigma-Aldrich).
• Thin sections (4 mm) of cryopreserved tissue were first blocked for 10 min with 1 % human immunoglobulins and then incubated for 1 hr with a mixture of primary antibodies from different species (for example, rabbit polyclonal antiserum against Cav-1 , and HMB-45 monoclonal antibodies, or isotype- matched control antibodies).
• Primary antibodies were used at 1-5 mg/ml, followed by incubation with Cy5-labeled antimouse and Cy3-labeled antirabbit secondary antibodies. After blocking with 10% mouse immunoglobulins, samples were incubated with FITC-labeled anti-CD90 or anti-CD45, and with DAPI to visualize nuclei.
• TMAs breast tumor microarrays
• Fresh surgical specimens (paired normal kidney and renal tumor tissue or nonpatient matched normal and tumor breast tissues) were minced and dissociated by incubation overnight at 37°C in 0.2% collagenase with agitation at 200 rpm. Fibroblasts were isolated by 10 min centrifugation at 200 g. Pellets were resuspended and serially filtered through 500 mm nylon mesh and 100 mm and 40 mm cell strainers.
• Isolated fibroblasts were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM; Mediatech, Inc.) supplemented with 15% fetal bovine serum (FBS; Hyclone), 100 units/ml penicillin, and 100 mg/ml streptomycin (Mediatech, Inc.). Nonadherent material was removed after 8 hr. After characterization, kidney fibroblasts were immortalized using a combination of hTERT and bmi-1 , while breast fibroblasts were used as primary fibroblasts. Both immortalized (at early passages) and primary cells were forced to produce their own matrix and subsequently grown in this 3D matrix (as opposed to a 2D plastic substrate) to allow them to reproduce their in vivo features.
• DMEM Dulbecco's modified Eagle medium
• FBS fetal bovine serum
• Mediatech, Inc. Mediatech, Inc.
• Nonadherent material was removed after 8 hr.
• kidney fibroblasts were immortal
• MEFs were scraped off the plate and resuspended at 4°C in 2 ml Mes buffered saline (MBS) (25 mM MES, pH 6.5, 0.15 M NaCI, 1 mM PMSF plus 1 % Triton X-100).
• MBS Mes buffered saline
• Cells were homogenized on ice by a minimum of 10 strokes through a syringe (0.5 3 16 mm). The homogenate was adjusted to 40% sucrose by the addition of 2ml 80% sucrose in MBS (4 ml in total), and was transferred to a Beckman SW40 13 ml Ultraclear tube.
• sucrose in MBS 4 ml each were successively overlaid to form a 40-30%-5% discontinuous sucrose gradient.
• Homogenates were separated by centrifugation at 200,000 g for 18 hr a SW40 rotor (Beckman) at 4°C.
• Most Cav1 was contained in a light, scattered band confined to the 30%-5% sucrose interface, which excluded most cell proteins.
• Twelve 1 ml fractions were collected from the bottom of the tube. Proteins in each fraction were precipitated by addition of 1 ml of cold acetone and incubation overnight at 4°C. Samples were centrifuged at 16,000 g in a microcentrifuge, and the protein pellets were dried for 2 hr to eliminate all traces of acetone. Precipitated proteins were analyzed by SDS- PAGE and immunoblot.
• MEF plasma membranes were resuspended in lysis buffer (5mM Tris-HCI pH 7.5, 100 mM NaCI, 0.5% sodium deoxycholate, 0.1 % SDS, 1 % NP-40 containing 0.5mMPMSF, 1 mMNa3V04, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and l OmMNaF) and incubated overnight with 4 mg anti-p190RhoGAP antibody. Fifty microliter protein G-agarose beads were added and samples incubated for 3 hr at 4°C. After washing and boiling in sample buffer, eluted proteins were analyzed by 7.5% SDS-PAGE and immunoblot. Statistics

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