WO2023155878A1

WO2023155878A1 - Methods for monitoring cell migration

Info

Publication number: WO2023155878A1
Application number: PCT/CN2023/076777
Authority: WO
Inventors: Li Yu; Yizheng Wang
Original assignee: Tsinghua University
Priority date: 2022-02-18
Filing date: 2023-02-17
Publication date: 2023-08-24

Abstract

The invention provides a method for determining the presence, amount and/or function of a retractosome generated by the cell, and the method can be used for monitoring and/or regulating cell migration and/or a cell migration related biological process. The invention also provides a method for regulating the formation and/or function of the retractosome generated by the cell, and the method can be used for regulating cell migration and/or a cell migration related biological process.

Description

METHODS FOR MONITORING CELL MIGRATION

BACKGROUND OF THE INVENTION

Cell migration is the directed movement of a single cell or a group of cells in response to chemical and/or mechanical signals. It is a fundamental cellular process that occurs throughout life, starting during embryonic development and continuing until death, and at times it can contribute to pathogenic states in disease.

In a developing embryo, cell migration is the driving factor for various morphogenetic events. For instance, during gastrulation in very early embryos, groups of cells migrate as sheets to form the three germ layers. Subsequently, cells from the germ layers migrate to various target locations, where they specialize into distinct cell populations that make up various tissues or organs in the embryo.

In adult organisms, cell migration occurs during vital cellular processes such as tissue renewal and repair, wherein old or damaged cells are replaced by the migration of newly formed cells from the underlying tissue layers. Such events are essential to maintain tissue integrity and homeostasis. Cell migration also plays a role in mediating immune responses during infections, in which phagocytic cells such as neutrophils circulating in the bloodstream migrate to the infected tissues and destroy the invading pathogens.

While on one hand, cell migration is vital for maintaining tissue health and homeostasis, on the other hand, undesirable migratory events are causative factors for a number of pathological states such as inflammatory diseases, cancers, and so on. Therefore, migration of cells has to be a tightly controlled process -both in time and space-to maintain a homeostatic state in an organism.

However, there are still many unclear areas involving the regulatory mechanisms and functions of cell migration to explore.

SUMMARY OF THE INVENTION

The present disclosure discloses a retractosome for the first time, a type of small extracellular vesicle which is generated from broken-off retraction fibers. The retractosomes are formed by the migratory cells and the formation of retractosomes is observed in various in vivo and in vitro settings. The present disclosure provides a new method for determining the presence, amount and/or function of a retractosome generated by said cell, and said method can be used for monitoring and/or regulating cell migration and/or a cell migration related biological process. The present disclosure also provides a new method for regulating the formation and/or function of the retractosome generated by said cell, and said method can be used for regulating cell migration and/or a cell migration related biological process.

In one aspect, the present disclosure provides a method for monitoring cell migration and/or a cell migration related biological process, comprising determining the presence, amount and/or function of a retractosome generated by said cell.

In some embodiments, wherein determining the presence, amount and/or function of said retractosome comprises determining the presence and/or amount of tetraspanin⁺ and/or PIGK⁺vesicles.

In some embodiments, wherein said tetraspanin comprises tetraspanin 4.

In some embodiments, wherein said vesicle is CPQ^-, NDST1^-and/or PCCA^-.

In some embodiments, wherein said vesicle is Alix^-, Tsg101^-and/or CD63^-.

In some embodiments, wherein said vesicle is an extracellular vesicle.

In some embodiments, wherein said vesicle has a diameter of about 30 nm to about 400 nm.

In some embodiments, wherein said vesicle has a diameter of about 40 nm to about 350 nm.

In some embodiments, wherein said vesicle has a diameter of about 50 nm to about 250 nm.

In some embodiments, wherein said cell comprises an epithelial cell, a cancer cell, a fibroblast, an embryonic cell, a neural cell and/or an immune cell.

In one aspect, the present disclosure provides a method for regulating cell migration and/or a cell migration related biological process, comprising:

i) monitoring said cell migration and/or said cell migration related biological process according to the present disclosure; and

ii) administering an appropriate regulator according to the result of step i) .

In one aspect, the present disclosure provides a method for characterizing a retractosome, comprising:

determining the presence and/or amount of tetraspanin and/or PIGK in a vesicle to be characterized.

In some embodiments, said method further comprising determining the presence and/or amount of CPQ, NDST1 and/or PCCA in said vesicle.

In some embodiments, said method further comprising determining the presence and/or amount of Alix, Tsg101 and/or CD63 in said vesicle.

In some embodiments, wherein said vesicle is an extracellular vesicle.

In some embodiments, wherein the vesicle that is:

i) tetraspanin⁺ and/or PIGK⁺;

ii) CPQ^-, NDST1^-and/or PCCA^-; and

iii) Alix^-, Tsg101^-and/or CD63^-

is characterized as a retractosome.

In one aspect, the present disclosure provides a method for isolating and/or regulating a retractosome, comprising:

i) characterizing the retractosome according to the method of the present disclosure; and

ii) isolating the characterized retractosome, and/or administering a regulating agent to said charactrerized retractosome.

In one aspect, the present disclosure provides a method for regulating the formation and/or function of a retractosome, and/or for regulating a retractosome mediated biological process, comprising:

regulating the amount and/or function of a tetraspanin in a cell generating said retractosome;

regulating the migration of a cell generating said retractosome; and/or

regulating the formation and/or degradation of a retraction fiber (RF) by a cell generating said retractosome.

In some embodiments, said method promotes the formation of the retractosome, and comprises:

increasing the amount and/or function of the tetraspanin in the cell generating said retractosome;

reducing the amount and/or function of cholesterol in the cell generating said retractosome;

promoting the migration of the cell generating said retractosome; and/or

promoting the formation and/or inhibiting the degradation of the retraction fiber (RF) by the cell generating said retractosome.

In some embodiments, wherein increasing the amount and/or function of the tetraspanin comprises overexpressing said tetraspanin in said cell.

In some embodiments, wherein reducing the amount and/or function of the cholesterol comprises inhibiting the synthesis and/or uptake of cholesterol by said cell.

In some embodiments, wherein inhibiting the synthesis and/or uptake of cholesterol by said cell comprises administering to said cell a cholesterol lowering agent.

In some embodiments, wherein said cholesterol lowering agent comprises cholesterol absorption inhibitor and/or cholesterol synthesis inhibitor.

In some embodiments, wherein said cholesterol synthesis inhibitor comprises pravastatin.

In some embodiments, wherein inhibiting the uptake of cholesterol comprises culturing said cells in a cholesterol depletion environment.

In some embodiments, said method inhibits the formation of the retractosome, and comprises:

decreasing the amount and/or function of the tetraspanin in the cell generating said retractosome;

inhibiting the migration of the cell generating said retractosome; and/or

inhibiting the formation and/or promoting the degradation of the retraction fiber (RF) by the cell generating said retractosome.

In some embodiments, wherein decreasing the amount and/or function of the tetraspanin comprises knocking out or knocking down the expression of a gene encoding for said tetraspanin in said cell.

In some embodiments, wherein inhibiting the migration of said cell comprises administering a cell migration inhibitor to said cell.

In some embodiments, wherein said cell migration inhibitor comprises Blebbistatin.

In some embodiments, wherein said tetraspanin comprises tetraspanin 4.

In one aspect, the present disclosure provides a method for regulating a cell migration mediated biological process, comprising regulating the formation and/or function of the retractosome according to the present disclosure.

In one aspect, the present disclosure provides a method for isolating a retractosome, comprising:

providing a cell culture from which cells and cell debris have been removed;

passing said cell culture through a filter with a pore size not exceeding about 0.25 μm to obtain a filtrate containing said retractosome; and

isolating said retractosome from the filtrate.

In some embodiments, wherein said retractosome is not a migrasome.

In one aspect, the present disclosure provides an engineered cell with altered ability for generating a retractosome comparing to an unmodified corresponding cell, wherein said engineered cell has been modified to:

change the amount and/or function of a tetraspanin therein;

change the amount and/or function of cholesterol therein;

change the migration ability thereof; and/or

change its ability to form and/or degrade a retraction fiber (RF) .

In some embodiments, wherein said tetraspanin comprises tetraspanin 4.

In some embodiments, said cell comprises an epithelial cell, a cancer cell, a fibroblast, an embryonic cell, a neural cell and/or an immune cell.

In one aspect, the present disclosure provides an agent capable of determining the presence, amount and/or function of a retractosome generated by a cell, for use in monitoring the migration of said cell and/or a biological process related to the migration of said cell.

In one aspect, the present disclosure provides an agent capable of regulating the formation and/or function of a retractosome generated by a cell, for use in regulating a biological process mediated by the migration of said cell.

In one aspect, the present disclosure provides an agent capable of regulating the amount and/or function of a tetraspanin in a cell, regulating the amount and/or function of cholesterol in a cell, regulating the migration ability of a cell, and/or regulating the ability of a cell to form and/or degrade a retraction fiber (RF) , for use in regulating the formation and/or function of a retractosome.

In one aspect, the present disclosure provides an agent capable of specifically identifying tetraspanin, PIGK, CPQ, NDST1, PCCA, Alix, Tsg101 and/or CD63, for use in characterizing a retractosome.

In one aspect, the present disclosure provides an isolated retractosome, which is a tetraspanin⁺and/or PIGK⁺ vesicle.

In some embodiments, said retractosome is not a migrasome.

In some embodiments, said retractosome is CPQ^-, NDST1^-and/or PCCA^-.

In some embodiments, said retractosome is Alix^-, Tsg101^-and/or CD63^-.

In some embodiments, said retractosome is an extracellular vesicle.

In some embodiments, said retractosome has a diameter of about 30 nm to about 400 nm.

In some embodiments, wherein said tetraspanin comprises tetraspanin 4.

In one aspect, the present disclosure provides a composition, comprising the engineered cell according to the present disclosure, the agent according to the present disclosure, and/or the isolated retractosome according to the present disclosure.

In some embodiments, wherein said composition is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable excipient.

In one aspect, the present disclosure provides a use of the agent according to the present disclosure, in the preparation of a reagent for characterizing the retractosome.

In one aspect, the present disclosure provides a use of the agent according to the present disclosure, in the preparation of a reagent for regulating the formation and/or function of a retractosome.

In one aspect, the present disclosure provides a use of the agent according to the present disclosure and/or the engineered cell according to the present disclosure, in the preparation of a reagent for regulating a biological process mediated by the migration of said cell.

In one aspect, the present disclosure provides a use of an agent according to the present disclosure, in the preparation of a reagent for monitoring the migration of said cell and/or a biological process related to the migration of said cell.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “FIG. ” , “Fig. ” and “FIG. ” herein) , of which:

FIG. 1A illustrates breakage of retraction fibers. Tspan4-mCherry-expressing L929 cells were cultured for 6 h, and time-lapse images were acquired at 7.5 min per frame with an Olympus FV3000 confocal microscope. Scale bar, 10 μm for main images (top) and 2 μm for enlarged images (bottom) .

FIG. 1B illustrates SEM analysis of L929 cells expressing Tspan4-GFP. Tspan4-GFP-expressing L929 cells were grown on a 35 mm confocal chamber for 12 hr and observed by field emission scanning electron microscopy. The boxed area in the main image is enlarged at top right. The boxed area in the top right panel is enlarged at bottom right. Scale bar, 10 μm.

FIG. 1C illustrates TEM analysis of L929 cells expressing Tspan4-GFP. TEM image (left) and enlarged image (right) of an ultra-thin section of a Tspan4-GFP-expressing L929 cell cultured on a 35 mm confocal chamber for 12 hr. Scale bar, 500 nm.

FIG. 1D illustrates cryo-TEM analysis of L929 cells expressing Tspan4-GFP. Cells were cultured on a 3 mm sapphire disc, then fixed by high pressure freezing. Freeze substitution was started at -90℃and the samples were slowly warmed to 0℃ with 2%osmium tetroxide in acetone. The samples were washed with acetone, then infiltrated and embedded in Pon812.70-nm sections were checked under TEM. Scale bar, 1 μm.

FIG. 1E illustrates retractosomes from FIG. 1C were quantified for the diameter. Data shown represent the mean ± s.e.m. n = 150 retractosomes from 3 independent experiments.

FIG. 1F illustrates cryo-TEM analysis of the “beads-on-a string” structure. Wild-type L929 cells were cultured on a 3 mm sapphire disc, then fixed by high pressure freezing. Freeze substitution was started at -90℃ and the samples were slowly warmed to 0℃ with 2%osmium tetroxide in acetone. The samples were washed with acetone, then infiltrated and embedded in Pon812.70-nm sections were checked under TEM. Scale bar, 1 μm.

FIG. 1G illustrates time-lapse images of RFs of a Tspan4-GFP-expressing cell obtained in resonant scanning mode. The arrowhead indicates a retraction fiber breaking to form a retractosome. Scale bar, 1 μm.

FIG. 1H illustrates representative image of wild-type and Tspan4-mCherry-expressing L929 cells dyed with WGA488. Cells were cultured for 18 hr, dyed with WGA488 and imaged with an Olympus FV3000 confocal microscope. Scale bar, 10 μm.

FIG. 1I illustrates cells from FIG. 1H were quantified for the number of migrasomes per cell. Data shown represent the mean ± s.e.m. n =66 cells from 3 independent experiments.

FIG. 1J illustrates cells from FIG. 1H were quantified for the number of retractosomes per cell. Data shown represent the mean ± s.e.m. n =66 cells from 3 independent experiments.

FIG. 1K illustrates TEM image of an ultra-thin section of a Tspan4-GFP-expressing L929 cell cultured on a 35 mm confocal chamber in cholesterol depletion medium (DFBS) without or with pravastatin for 12 hr. Scale bar, 1 μm.

FIG. 1L illustrates Tspan4-GFP-expressing L929 cells were cultured in control medium or cholesterol depletion medium without or with pravastatin for 12 h, and images were acquired with an Olympus FV3000 confocal microscope. The number of retractosomes per cell was quantified. Data shown represent the mean ± s.e.m. n =70 cells from 3 independent experiments.

FIG. 1M illustrates cells from FIG. 1L were quantified for the number of retractosomes per μm retraction fiber. Data shown represent the mean ± s.e.m. n =70 cells from 3 independent experiments.

FIG. 1N illustrates typical negative staining EM image of purified small EVs, migrasomes and retractosomes. Scale bar, 0.5 μm.

FIG. 1O illustrates purified small EVs and retractosomes from FIG. 1N were quantified for diameter. Data shown represent the mean ± s.e.m. n =150 small EVs or retractosomes from 3 independent experiments.

FIG. 1P illustrates samples from cell bodies and purified migrasomes, small EVs and retractosomes were analyzed by western blotting using antibodies against the identified migrasome-specific markers CPQ, PIGK, NDST1 and PCCA; the plasma membrane marker ITGAV; the exosomal markers TSG101 and CD63; the ER marker calnexin; the mitochondrion marker TIM23; and the nuclear marker H3.

FIG. 1Q illustrates principal components analysis (PCA) of the protein composition of cell bodies, migrasomes, small EVs and retractosomes from D2SC cells.

FIG. 1R illustrates immunostaining of endogenous Ccl2 in D2SC cells. The enlarged images on the right show migrasomes (top) and retractosomes (bottom) , Scale bar, 10 μm.

FIG. 1S illustrates representative image of an L929 cell stained with WGA488 and SYTO14. The enlarged images on the right show migrasomes (top) and retractosomes (bottom) . Scale bar, 10 μm.

FIG. 1T illustrates quality control western blot of purified migrasomes, retractosomes and small EVs in FIG. 1U.

FIG. 1U illustrates ratio of small EVs to retractosomes based on total protein amount. Data shown represent the mean ± s.e.m. from 5 independent experiments.

FIG. 1V illustrates extracellular vesicles purified form culture medium do not contain a significant amount of retractosomes. “Retractosome” indicates retractosomes purified from cultured cells, and “Crude EV” indicates the crude preparation of extracellular vesicles, collected by pelleting the culture medium at 120,000 g. Small EVs were further isolated from this crude EV fraction by treating with or without filtration, 10,000 g centrifugation or 18,000 g centrifugation.

FIG. 2A illustrates retractosome formation is impaired by blebbistatin. WT L929 cells were cultured for 12 h with DMSO or 10 μM blebbistatin, and images were acquired with an Olympus FV3000 confocal microscope.

FIG. 2B illustrates cells from FIG. 2A were quantified for the number of migrasomes per cell. Data shown represent the mean ± s.e.m. ; n =60 cells for each genotype, each pooled from 3 independent experiments

FIG. 2C illustrates cells from FIG. 2A were quantified for the number of retractosomes per cell. Data shown represent the mean ± s.e.m. ; n = 60 cells for each genotype, each pooled from 3 independent experiments.

FIG. 2D illustrates retractosomes can be detected in different cell lines in the absence of Tspan4 overexpression. U2OS (human bone osteosarcoma epithelial cells) , NRK (normal rat kidney epithelial cells) , MGC803 (human gastric carcinoma cells) , MEF (mouse embryonic fibroblasts) , HT1080 (human fibrosarcoma cells) , D2SC (mouse dendritic cells) , NMuMG (mouse normal mammary gland cells) and BV2 (mouse microglial cells) were cultured for 12 h, dyed by WGA488 and observed by confocal microscopy. Insets show enlarged regions of interest. Scale bar, 10 μm.

FIG. 2E illustrates cells from FIG. 1H were quantified for the length of retraction fibers per cell. Data shown represent the mean ± s.e.m. ; n = 60 cells for each genotype, each pooled from 3 independent experiments.

FIG. 3A illustrates retractosome generation in zebrafish embryos. A single blastomere of an embryo at 8 h post-fertilization was injected with 100 pg PLMT–GFP mRNA to label plasma membranes. Time-lapse images were acquired at 27.88 s per frame with a Nikon A1 confocal microscope. 4.78 μm Z-stack images are shown here. Arrowhead indicates retractosomes. Scale bar, 10 μm.

FIG. 3B illustrates retractosome generation by mouse neutrophils. Neutrophils were labeled with anti-mouse Ly-6G (Gr-1) PE, and blood vessels were labeled by AF647-WGA. Arrow indicates migrasomes and arrowhead indicates retractosomes. Scale bar, 10 μm.

FIG. 4A illustrates Tspan4-GFP-expressing L929 cells were cultured for 12 h, fixed with PFA, dyed with Filipin III to detect cholesterol, and imaged with a Nikon A1 confocal microscope. The panels underneath show migrasomes (left) and retractosomes (right) . Scale bar, 10 μm.

FIG. 4B illustrates retractosomes and migrasomes from FIG. 4A were quantified for the ratio of Filipin III signal to Tspan4 signal. Data shown represent the mean ± s.e.m. n = 60 retractosomes or migrasomes from 3 independent experiments.

FIG. 4C illustrates Tspan4-GFP-expressing L929 cells were cultured in normal medium or cholesterol depletion medium without or with pravastatin for 12 h, and images were acquired with an Olympus FV3000 confocal microscope. Scale bar, 10 μm.

FIG. 4D illustrates cells from FIG. 4C were quantified for the number of migrasomes per cell. Data shown represent the mean ± s.e.m. n = 60 cells from 3 independent experiments.

FIG. 5A illustrates scheme for purification of retractosomes.

FIG. 5B illustrates volcano blot showing the label-free mass spectrometry-based protein quantification of retractosomes vs cell bodies. The red dots represent retractosome: cell abundance ≥2, P<0.05; the blue dots represent retractosome: cell abundance ≤ 0.5, P<0.05. n=3 biologically independent experiments. P values were calculated using a two-tailed, two-sample unequal variance t-test using Excel.

FIG. 5C illustrates immunostaining of endogenous Ccl9 in D2SC cells. The bottom panels show migrasomes (left) and retractosomes (right) . Scale bar, 10 μm.

FIG. 6 illustrates samples from cell bodies and purified migrasomes, retractosomes and small EVs were analyzed by western blotting using antibodies against the migrasome-specific markers PIGK and PCCA; the small EV markers Flotillin-1, Alix, Syntenin-1 and TSG101; the plasma membrane marker ITGAV; the ER markers Calnexin and Serca2; the mitochondrion marker TIM23; and the nuclear marker H3.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

As used herein, the term “CCR2” generally refers to C-C Motif Chemokine Receptor 2, which is a seven-transmembrane domain G-protein coupled chemotactic receptor. CCR2 is capable of binding to MCP-1, CCL8 (MCP-2) , CCL7 (MCP-3) and/or CCL13 (MCP-4) . CCR2 is also known as CMKBR2 and CKR2. Two alternatively-spliced forms of the CCR2, CCR2A and CCR2B, have been cloned which differ in their C-termini. The protein encoded by human CCR2 has the accession number of P41597 in UniProtKB/Swiss-Prot. The term also encompasses CCR2 or a fragment thereof coupled to, for example, a tag (e.g., a histidine tag) , mouse or human Fc, or a signal sequence. The term CCR2 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof. The term CCR2 encompasses the CCR2 gene or protein from any species, such as human, or a non-human animal, e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.

As used herein, the terms “cell migration mediated biological process” and “cell migration related biological process” may be used interchangeably and generally refer to a biological process which is associated with cell migration. For example, the process that are vital for an organism to live during cell migration. The term “cell migration” generally refers to a directed movement of a cell. The term includes an active cell migration and/or passive cell migration. Such cell migration mediated biological process or cell migration related biological process can include, for example, embryogenesis, damage repair, cancer metastasis.

As used herein, the term “cytokine” generally refers to the general class of biological molecules which effect/affect cells of the immune system. The definition is meant to include, but is not limited to, those biological molecules that act locally or may circulate in the blood, and which may serve to regulate or modulate an individual's immune response. Exemplary cytokines for use in practicing the inventions of the present disclosure include but are not limited to interferons, interleukins, tumor necrosis factors, erythropoietin (EPO) , MIP3a, monocyte chemotactic protein (MCP) -1, intracellular adhesion molecule (ICAM) , macrophage colony stimulating factor (M-CSF) , granulocyte colony stimulating factor (G-CSF) , granulocyte-macrophage colony stimulating factor (GM-CSF) and chemokines.

As used herein, the term "engineered" generally refers to one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome, of a polypeptide, or of other components. The term "engineered" can refer to alterations, additions, and/or deletions of the genes, polypeptides or other components. The term "engineered cell" generally refers to a modified cell of human or non-human origin. For example, an engineered cell can refer to a cell with an added, deleted and/or altered gene, polypeptide or other components.

As used herein, the term “extracellular vesicle” or “EV” generally refers to a membrane-delimited (such as lipid-bilayer delimited) particle that is released from a cell or artificially generated. Unlike a cell, an extracellular vesicle generally cannot replicate. EVs range in diameter from near the size of the smallest physically possible unilamellar liposome (around 20-30 nanometers) to as large as 4000nm or more. EVs can be divided according to size and synthesis route into exosomes, microvesicles, apoptotic bodies, migrasomes and retractosomes. They may carry a cargo of proteins, nucleic acids, lipids, metabolites, and even organelles from the parent cell. A wide variety of EV subtypes have been proposed, defined variously by size, biogenesis pathway, cargo, cellular source, and function. The term “small extracellular vesicle” or “small EV” generally refers to the EV recovered by differential ultracentrifugation and density gradient from the conditioned medium. As a non-limiting example, the small EV may comprise exosome ectosomes and/or microvesicle.

As used herein, the term “knock down” generally refers to a measurable reduction in the expression of a target mRNA or the corresponding protein in a genetically modified cell or organism as compared to the expression of the target mRNA or the corresponding protein in a counterpart control cell or organism that does not contain the genetic modification to reduce expression. Those skilled in the art will readily appreciate how to use various genetic approaches, e.g., siRNA, shRNA, microRNA, antisense RNA, or other RNA-mediated inhibition techniques, to knock down a target polynucleotide sequence.

As used herein, the term “knock out” generally includes deleting all or a portion of the target polynucleotide sequence in a way that interferes with the function of the target polynucleotide sequence. For example, a knock-out can be achieved by altering a target polynucleotide sequence by inducing a deletion in the target polynucleotide sequence in a functional domain of the target polynucleotide sequence. Those skilled in the art will readily appreciate how to use various genetic approaches, e.g., CRISPR/Cas systems, ZFN, TALEN, TgAgo, to knock out a target polynucleotide sequence or a portion thereof based upon the details described herein.

As used herein, the term “migrasome” generally refers to a membrane-bound cellular structure derived from or generated by a migrating cell. The term “migrasome” encompasses an organelle (also known as “pomegranate-like structure” or PLS) attached to a retraction fiber generated by a migrating cell. In some cases, the term “migrasome” also refers to a vesicle (e.g., an extracellular vesicle) already detached from the cell generating it. In the present disclosure, the term “migrasome” also refers to a vesicle (e.g., an artificial vesicle) with similar functions and/or compositions as such a vesicle or organelle derived from, and/or generated by migrating cells.

As used herein, the term “migrasome mediated biological process” generally refers to a biological process mediated by the formation, movement, function, degradation, and/or disintegration of a migrasome.

As used herein, the terms “migrating cell” and “migratory cell” are used interchangeably, and generally refer to a cell moving from one location to another location. In some cases, a migrating cell is a cell whose relative position, space, and/or contour has changed or is changing with time. A circulating cell comprises a cell circulating in the body fluid (e.g., blood or lymph) of an organism.

As used herein, the term “pharmaceutically acceptable excipient” generally refers to any material, which is inert in the sense that it substantially does not have a therapeutic and/or prophylactic effect per se. Such an excipient is added with the purpose of making it possible to obtain a pharmaceutical composition having acceptable technical properties.

As used herein, the term “retraction fiber” or “RF” generally refers to actin-rich fibers exposed as the cell margin retracts. For example, the retraction fiber may include tubular strands left behind a cell during cell migration. During migration, RF may be pulled out at the trailing edge of cells, and migrasomes may form on the tips or branch points of the RF.

As used herein, the term “retractosome” generally refers to a migration-dependent small extracellular vesicle. A retractosome may have sizes ranging from about 50 nm to about 250 nm. During cell migration, the RF may go through a stage of morphological transformation, with sections of the RF bulging and broken off into small vesicles, which may be referred to as retractosomes. For example, tetraspanins may be enriched and raft lipids such as cholesterol are deficient in retractosomes.

As used herein, the term “tetraspanin” generally refers to a membrane protein, which is also known as the transmembrane 4 superfamily (TM4SF) protein, and may have four transmembrane alpha-helices and two extracellular domains. For example, the term “tetraspanin” may encompass various isoforms of the tetraspanin, as well as the naturally-occurring allelic and processed forms thereof.

As used herein, the term “Tetraspanin 4 (TSPAN4) ” generally refers to a TSPAN4 gene and/or a protein that is encoded by the TSPAN4 gene. For example, the NCBI Entrez Gene for TSPAN4 may be 7106. For example, the UniProtKB/Swiss-Prot number for Tetraspanin 4 may be O14817. For example, the term “Tetraspanin 4” may encompass various isoforms of the Tetraspanin 4, the naturally-occurring allelic and processed forms thereof. The term also encompasses TSPAN4 or a fragment thereof coupled to, for example, a tag (e.g., a histidine tag) , mouse or human Fc, or a signal sequence. The term TSPAN4 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof. The term TSPAN4 encompasses the TSPAN4 gene or protein from any species, such as human, or a non-human animal, e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.

Unless otherwise specified, “a” , “an” , “the” and “at least one” are used interchangeably and refer to one or more than one.

Unless otherwise specified, the term "about" or “approximately" as used herein is understood to be within normal tolerances in the art, e.g., within 2 standard deviations of the mean. About may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01%of the stated value. Unless obvious from the context, all values provided herein are approximately modified by the term.

In the present disclosure, the term “comprise” also encompasses “is” , “has” and “consist of” . For example, “a composition comprising X and Y” may be understood to encompass a composition that comprises at least X and Y. It shall also be understood to disclose a composition that only comprises X and Y (i.e., a composition consisting of X and Y) .

Method

In some embodiments, wherein said tetraspanin comprises tetraspanin 4.

For example, the method for monitoring cell migration and/or a cell migration related biological process, comprising determining the presence and/or amount of tetraspanin 4⁺ and/or PIGK⁺ vesicles generated by said cell.

In some embodiments, wherein said vesicle is CPQ^-, NDST1^-and/or PCCA^-.

For example, wherein said vesicle is tetraspanin⁺, PIGK⁺ and CPQ^-.

For example, wherein said vesicle is tetraspanin⁺, PIGK⁺, CPQ^-and NDST1^-.

For example, wherein said vesicle is tetraspanin⁺, PIGK⁺, CPQ^-, NDST1^-and/or PCCA^-.

In some embodiments, wherein said vesicle is Alix^-, Tsg101^-and/or CD63^-.

For example, wherein said vesicle is tetraspanin⁺, PIGK⁺ and Alix^-.

For example, wherein said vesicle is tetraspanin⁺, PIGK⁺, Alix^-and Tsg101^-.

For example, wherein said vesicle is tetraspanin⁺, PIGK⁺, CPQ^-, Alix^-, Tsg101^-and CD63^-.

For example, wherein said vesicle is tetraspanin⁺, PIGK⁺, CPQ^-and Alix^-.

For example, wherein said vesicle is tetraspanin⁺, PIGK⁺, CPQ^-, NDST1^-, Alix^-and Tsg101^-.

For example, wherein said vesicle is tetraspanin⁺, PIGK⁺, CPQ^-, NDST1^-, PCCA^-, Alix^-, Tsg101^-and CD63^-.

In some embodiments, wherein said vesicle is an extracellular vesicle.

For example, the method for monitoring cell migration and/or a cell migration related biological process, comprising determining the presence and/or amount of extracellular vesicles generated by said cell; wherein said vesicle may be:

i) tetraspanin 4⁺ and/or PIGK⁺;

ii) CPQ^-, NDST1^-and/or PCCA^-; and

iii) Alix^-, Tsg101^-and/or CD63^-.

For example, said vesicle may have a diameter of about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 110nm, about 120nm, about 130nm, about 140nm, about 150nm, about 160nm, about 170nm, about 180nm, about 190nm, about 200nm, about 210nm, about 220nm, about 230nm, about 240nm, about 250nm, about 260nm, about 270nm, about 280nm, about 290nm, about 300nm, about 310nm, about 320nm, about 330nm, about 340nm, about 350nm, about 360nm, about 370nm, about 380nm, about 390nm, or about 400nm.

i) tetraspanin 4⁺ and/or PIGK⁺;

ii) CPQ^-, NDST1^-and/or PCCA^-;

iii) Alix^-, Tsg101^-and/or CD63^-; and

said vesicle may have a diameter of about 50 nm to about 250 nm.

For example, said cell may be any cell capable of migrating and/or producing retractosomes during and/or after the migration. For example, said cell may comprise cells of human or animal origin, such as rat, mouse, chicken, zebrafish. For example, said cell may comprise but not limited to L929 cells, D2SC cells, mouse neutrophils, zebrafish embryonic cells.

ii) administering an appropriate regulator according to the result of step i) .

For example, the method may comprise: i) determining the presence, amount and/or function of a retractosome generated by said cell; and ii) administering an appropriate regulator according to the result of step i) .

For example, when a higher amount and/or function of a retractosome generated by said cell than normal is detected, an inhibitor may be applied to said cell. For example, when not detected or the amount and/or function of a retractosome generated by said cell is lower than normal, a promoter can be applied to said cells.

For example, the method for characterizing a retractosome, comprising determining the presence and/or amount of tetraspanin and/or PIGK in a vesicle to be characterized.

For example, the method for characterizing a retractosome, comprising determining the presence and/or amount of tetraspanin, PIGK, CPQ, NDST1, PCCA, Alix, Tsg101 and/or CD63 in a vesicle to be characterized.

For example, the method for characterizing a retractosome, comprising determining the presence and/or amount of tetraspanin and PIGK in a vesicle to be characterized.

For example, the method for characterizing a retractosome, comprising determining the presence and/or amount of tetraspanin, PIGK and CPQ in a vesicle to be characterized.

For example, the method for characterizing a retractosome, comprising determining the presence and/or amount of tetraspanin, PIGK, CPQ and NDST1 in a vesicle to be characterized.

For example, the method for characterizing a retractosome, comprising determining the presence and/or amount of tetraspanin, PIGK, CPQ, NDST1 and PCCA in a vesicle to be characterized.

For example, the method for characterizing a retractosome, comprising determining the presence and/or amount of tetraspanin, PIGK, CPQ, NDST1, PCCA and Alix in a vesicle to be characterized.

For example, the method for characterizing a retractosome, comprising determining the presence and/or amount of tetraspanin, PIGK, CPQ, NDST1, PCCA, Alix and Tsg101 in a vesicle to be characterized.

For example, the method for characterizing a retractosome, comprising determining the presence and/or amount of tetraspanin, PIGK, CPQ, NDST1, PCCA, Alix, Tsg101 and CD63 in a vesicle to be characterized.

In some embodiments, wherein said tetraspanin comprises tetraspanin 4.

In some embodiments, wherein said vesicle is an extracellular vesicle.

In some embodiments, wherein the vesicle that is:

i) tetraspanin⁺ and/or PIGK⁺;

ii) CPQ^-, NDST1^-and/or PCCA^-; and

iii) Alix^-, Tsg101^-and/or CD63^-

is characterized as a retractosome.

For example, said vesicle characterized as a retractosome may have a diameter of about 50 nm to about 250 nm, and may be:

i) tetraspanin⁺ and/or PIGK⁺;

ii) CPQ^-, NDST1^-and/or PCCA^-; and

iii) Alix^-, Tsg101^-and/or CD63^-.

In some embodiments, wherein said isolating the characterized retractosome comprises isolating of retractosomes from small extracellular vesicles (small EVs) , cell bodies, large debris and/or migrasomes.

In some embodiments, said method comprises isolating the retractosomes from the small EVs by washing said cells.

In some embodiments, said method comprises isolating the retractosomes from cell bodies and/or large debris by differential ultracentrifugation.

In some embodiments, wherein said migrasomes were removed by passing the sample through a filter. In some embodiments, said filter is a 0.22 μm filter.

For example, said isolating the characterized retractosome may comprises:

i) removing the culture medium and washing cells with PBS to remove small EVs;

ii) collecting the cells and performing two steps of low-speed centrifugation to remove cell bodies and large debris;

iii) passing the sample through a 0.22 μm filter to remove migrasomes; and

iv) subjecting the flow-through to high-speed centrifugation to collect retractosomes.

In some embodiments, wherein in step ii) : said cells and said large debris are removed by centrifugation at 1000 g for 10 min followed by twice at 4000g for 10 min.

In some embodiments, wherein in step iv) comprises collecting retractosomes by centrifugation at 18,000 g for 60 min at 4℃.

regulating the migration of a cell generating said retractosome; and/or

promoting the migration of the cell generating said retractosome; and/or

In some embodiments, wherein said tetraspanin comprises tetraspanin 4.

For example, the method for promoting the formation and/or function of a retractosome, and/or for regulating a retractosome mediated biological process, comprising overexpressing said tetraspanin such TSPAN4 in said cell.

For example, the method for promoting the formation and/or function of a retractosome, and/or for regulating a retractosome mediated biological process, comprising inhibiting the synthesis and/or uptake of cholesterol by said cell.

In some embodiments, wherein said cholesterol lowering agent comprises cholesterol absorption inhibitor (e.g., ezetimibe) and/or cholesterol synthesis inhibitor.

For example, the method for promoting the formation and/or function of a retractosome, and/or for regulating a retractosome mediated biological process, comprising administering to said cell a cholesterol synthesis inhibitor such as pravastatin.

For example, the method for promoting the formation and/or function of a retractosome, and/or for regulating a retractosome mediated biological process, comprising culturing said cells in a cholesterol depletion environment.

For example, the method for promoting the formation and/or function of a retractosome, and/or for regulating a retractosome mediated biological process, comprising overexpressing said tetraspanin such TSPAN4 in said cell and culturing said cells in a cholesterol depletion environment.

inhibiting the migration of the cell generating said retractosome; and/or

In some embodiments, wherein said tetraspanin comprises tetraspanin 4 (TSPAN4) .

For example, the method for inhibiting the formation and/or function of a retractosome, and/or for regulating a retractosome mediated biological process, comprising knocking out or knocking down the expression of a gene encoding for said tetraspanin such as TSPAN4 in said cell.

For example, the method for inhibiting the formation and/or function of a retractosome, and/or for regulating a retractosome mediated biological process, comprising administering a cell migration inhibitor such as Blebbistatin to said cell.

In some embodiments, wherein said retractosome is an extracellular vesicle.

In some embodiments, wherein said retractosome is tetraspanin⁺ and/or PIGK⁺ vesicles.

In some embodiments, wherein said tetraspanin comprises tetraspanin 4.

In some embodiments, wherein said retractosome is CPQ^-, NDST1^-and/or PCCA^-.

In some embodiments, wherein said retractosome is Alix^-, Tsg101^-and/or CD63^-.

For example, wherein said retractosome may be:

i) tetraspanin 4⁺ and/or PIGK⁺;

ii) CPQ^-, NDST1^-and/or PCCA^-; and

iii) Alix^-, Tsg101^-and/or CD63^-.

In some embodiments, wherein said retractosome has a diameter of about 30 nm to about 400 nm.

For example, said retractosome may have a diameter of about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 110nm, about 120nm, about 130nm, about 140nm, about 150nm, about 160nm, about 170nm, about 180nm, about 190nm, about 200nm, about 210nm, about 220nm, about 230nm, about 240nm, about 250nm, about 260nm, about 270nm, about 280nm, about 290nm, about 300nm, about 310nm, about 320nm, about 330nm, about 340nm, about 350nm, about 360nm, about 370nm, about 380nm, about 390nm, or about 400nm.

In some embodiments, wherein said retractosome has a diameter of about 40 nm to about 350 nm.

In some embodiments, wherein said retractosome has a diameter of about 50 nm to about 250 nm.

providing a cell culture from which cells and cell debris have been removed;

isolating said retractosome from the filtrate.

For example, said isolating the characterized retractosome may comprises:

ii) removing the culture medium and washing cells with PBS to remove small EVs;

ii) collecting the cells and performing two steps of low-speed centrifugation to remove cell

bodies and large debris;

iii) passing the sample through a 0.22 μm filter to remove migrasomes; and

In some embodiments, wherein said retractosome is not a migrasome.

Engineered cell

change the amount and/or function of a tetraspanin therein;

change the amount and/or function of cholesterol therein;

change the migration ability thereof; and/or

change its ability to form and/or degrade a retraction fiber (RF) .

In some embodiments, wherein said tetraspanin comprises tetraspanin 4.

In one aspect, the present disclosure provides a method for preparing an engineered cell with altered ability for generating a retractosome comparing to an unmodified corresponding cell, comprising modifying said cell to:

change the amount and/or function of a tetraspanin therein;

change the amount and/or function of cholesterol therein;

change the migration ability thereof; and/or

change its ability to form and/or degrade a retraction fiber (RF) .

In one aspect, the present disclosure provides a use of an agent in the preparation of an engineered cell with altered ability for generating a retractosome comparing to an unmodified corresponding cell, wherein said agent may modify said cell to:

change the amount and/or function of a tetraspanin therein;

change the amount and/or function of cholesterol therein;

change the migration ability thereof; and/or

change its ability to form and/or degrade a retraction fiber (RF) .

A “corresponding unmodified cell” or “unmodified corresponding cell” generally refers to a cell that has not been modified to alter the amount and/or function of the sphingomyelin therein, while with all the other features substantially the same as the engineered cell. In some cases, the corresponding unmodified cell is a wildtype cell (e.g., of the same cell type as the engineered cell) . In some cases, the corresponding unmodified cell may comprise one or more modifications, but the modification may be for other purposes.

A cell may be modified by any approach applicable for the purpose of the present disclosure. For example, the modification may be a genetic modification. In some cases, the modification may comprise treating the cell with one or more agent causing the desired change or effect. The modification may be temporary, transient or may be stable or permanent. In some cases, the engineered cell may be a progeny of a parent cell that has been modified.

Agent

For example, said agent is capable of specifically identifying tetraspanin such as TSPAN4.

For example, said agent is capable of specifically identifying PIGK.

For example, said agent is capable of specifically identifying CPQ.

For example, said agent is capable of specifically identifying NDST1.

For example, said agent is capable of specifically identifying PCCA.

For example, said agent is capable of specifically identifying Alix.

For example, said agent is capable of specifically identifying Tsg101.

For example, said agent is capable of specifically identifying CD63.

For example, said agent is capable of specifically identifying tetraspanin and PIGK.

For example, said agent is capable of specifically identifying tetraspanin, PIGK and CPQ.

For example, said agent is capable of specifically identifying tetraspanin, PIGK, CPQ and NDST1.

For example, said agent is capable of specifically identifying tetraspanin, PIGK, CPQ, NDST1 and PCCA.

For example, said agent is capable of specifically identifying tetraspanin, PIGK, CPQ, NDST1, PCCA and Alix.

For example, said agent is capable of specifically identifying tetraspanin, PIGK, CPQ, NDST1, PCCA, Alix and Tsg101.

For example, said agent is capable of specifically identifying tetraspanin, PIGK, CPQ, NDST1, PCCA, Alix, Tsg101 and CD63.

In the present disclosure, an agent may be a small molecule compound, an antibody, a nucleic acid molecule, a polypeptide, or fragments thereof. In some cases, the agent may comprise one or more active components, present in a single molecule or as separate molecules.

The agent may be provided in a non-active form and be converted into an active form in vitro or in vivo before, during or after administration.

The agent may be a pharmaceutical agent or an agent for non-pharmaceutical use.

The agent may exert the desired functions directly or indirectly via the function of additional agents, compositions or cells.

Isolated retractosome

In some embodiments, said retractosome is not a migrasome.

In some embodiments, said retractosome is CPQ^-, NDST1^-and/or PCCA^-.

In some embodiments, said retractosome is Alix^-, Tsg101^-and/or CD63^-.

In some embodiments, said retractosome is an extracellular vesicle.

In some embodiments, wherein said tetraspanin comprises tetraspanin 4.

Composition

The composition of the present disclosure may be a pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable excipient.

The composition may comprise an effective amount of the agent of the present disclosure. The effective amount may be an amount of the agent that when administered alone or in combination with another agent to a cell, tissue, or subject is effective to achieve the desired effect (e.g., regulating migrasome formation and/or in regulating a migrasome-mediated biological process) .

The compositions may further include pharmaceutically acceptable materials, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, i.e., carriers. These carriers are involved in transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier should be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.

The formulation and delivery methods will generally be adapted according to the site and the disease to be treated. Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intravenous, intra-arterial, intramuscular, or subcutaneous administration The dosage of the agents of the disclosure will vary according to the extent and severity of the need for regulation, the activity of the administered composition, the general health of the subject, and other considerations well known to the skilled artisan.

Agents as described herein can be incorporated into compositions suitable for administration. Such compositions typically comprise the agent and a pharmaceutically acceptable carrier. Supplementary active compounds can also be incorporated into the compositions. In yet other embodiments, the agents described herein are delivered locally. Localized delivery allows for the delivery of the agent non-systemically, for example, to the site of regulation in need.

Use

In some embodiments, said agent capable of specifically identifying tetraspanin, PIGK, CPQ, NDST1, PCCA, Alix, Tsg101 and/or CD63, for use in characterizing a retractosome.

In some embodiments, said agent is capable of regulating the amount and/or function of a tetraspanin in a cell generating said retractosome;

regulating the migration of a cell generating said retractosome; and/or

In some embodiments, said agent is capable of determining the presence, amount and/or function of a retractosome generated by said cell.

In some embodiments, said engineered cell has altered ability for generating a retractosome comparing to an unmodified corresponding cell.

In some embodiments, wherein said engineered cell has been modified to:

change the amount and/or function of a tetraspanin therein;

change the amount and/or function of cholesterol therein;

change the migration ability thereof; and/or

change its ability to form and/or degrade a retraction fiber (RF) .

Monitor cell migration and/or a cell migration related biological process

Cell migration and/or a cell migration related biological process may be monitored and/or determined by observation, e.g. using microscopy, such as confocal microscope, scanning electron microscope (SEM) and/or transmission electron microscope (TEM) . The migration of cultured cells attached to a surface or in 3D is commonly studied using microscopy. As cell movement is very slow, a few μm/minute, time-lapse microscopy videos are recorded of the migrating cells to speed up the movement.

In addition or alternatively, cell migration and/or a cell migration related biological process may be monitored and/or determined by detecting the expression and/or amount of a cell migration specific marker. Such detection may be at transcriptional level and/or at protein level. Such marker may include but not limited to Tetraspanin-4, integrin, pleckstrin homology (PH) domain, NDST1 (bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1) , PIGK (phosphatidylinositol glycan anchor biosynthesis, class K) , CPQ (carboxypeptidase Q) and/or EOGT (EGF domain-specific O-linked N-acetylglucosamine transferase) .

In some cases, cell migration and/or a cell migration related biological process may be monitored and/or determined by staining the cell or sample with a migrasome specific dye, for example, by using WGA (wheatgerm agglutinin, a sialic acid-and N-acetyl-D glucosamine-binding lectin) .

For example, TSPAN4-GFP or TSPAN4-mCherry can be analysed for the cell migration and/or a cell migration related biological process by confocal microscopy.

In some cases, cell migration and/or a cell migration related biological process may be monitored and/or determined by determining the presence, amount and/or function of structures depended on cell migration. For example, said structures may comprises retraction fibers, migrasomes, and/or retractosomes.

Cell migration related biological process

The cell migration related biological process includes all biological processes related to cell migration, such as cell migration-dependent biological processes.

The cell migration related biological process includes but not limit to cell-substrate adhesion, the production of migrasomes RFs, the production of migrasomes, the production of retractosomes, migracytosis.

In some embodiments, a cell migration related biological process comprises a cell migration-dependent mechanism for releasing cellular, contents, migrasomes and/or retractosomes.

Determining the presence, amount and/or function of a retractosome

Retractosome formation or a change thereof (e.g., an increase in retractosome formation or a decrease in retractosome formation) may be monitored and/or determined by observation, e.g. using microscopy, such as scanning electron microscope (SEM) and/or transmission electron microscope (TEM) . For example, the retractosome can be beads-on-a-string structure observed under cryo-TEM.

The retractosome may be connected to or closely associated with retraction fibers (RFs) . Before breaking, the RFs goes through a stage of morphological transformation, with sections of the RF bulging into small vesicles, which likely form retractosomes.

In some embodiments, the presence, amount and/or function of a retractosome may be monitored and/or determined by detecting the expression and/or amount of a retractosome specific marker. Such detection may be at transcriptional level and/or at protein level. Such marker may include but not limited to Tetraspanin-4.

In some embodiments, the presence, amount and/or function of a retractosome may be monitored and/or determined by detecting the expression and/or amount of a retractosome specific cargo. Such detection may be at transcriptional level and/or at protein level. Such cargo may include but not limited to chemokines (such as CCL2, CCL9) , cytokines, and/or RNA (such as mRNA) .

The diameter of a retractosome

A retractosome may be oval shaped, with diameters from e.g. about 50 nm to about 250 nm, the morphology and size of the isolated retractosomes are very similar to retractosomes observed in cultured cells.

For example, the diameter of a retractosome may be about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, or about 260 nm.

Cell generating retractosome

A migratory cell generates retraction fibers (RFs) during migration. After the cell migrates away, large amounts of RFs break off from the cell and are left behind. When RFs break up, they form a type of extracellular vesicle named retractosomes. The retractosomes are cell migration dependent and have a continuous membrane, in many cases these vesicles are linked together, like a string of beads.

The cell generating retractosome may be any cell capable of migrating and/or producing retractosomes during and/or after the migration. For example, said cell may comprise cells of human or animal origin, such as rat, mouse, chicken, zebrafish. For example, said cell may comprise but not limited to L929 cells, D2SC cells, mouse neutrophils, zebrafish embryonic cells.

Regulating cell migration and/or a cell migration related biological process

For example, regulating cell migration and/or a cell migration related biological process may comprise promoting cell migration and/or a cell migration related biological process or inhibiting cell migration and/or a cell migration related biological process. For example, cell migration related biological process may comprise cell-substrate adhesion, the production of migrasomes RFs, the production of migrasomes, the production of retractosomes, migracytosis. For example, the degree of cell migration and/or a cell migration related biological process may be analyzed by detecting the expression and/or amount of a cell migration specific marker such as TSPAN4. For example, the degree of cell migration and/or a cell migration related biological process may be analyzed by analyzing the presence, amount and/or function of a migrasome and/or a retractosome.

For example, when treating the cell according to the method of the present application, the degree of cell migration and/or a cell migration related biological process may be lower than the untreated cell or the cell before treating. For example, the degree of cell migration and/or a cell migration related biological process may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower than the untreated cell or the cell before treating.

For example, when treating the cell according to the method of the present application, the degree of cell migration and/or a cell migration related biological process may be higher than the untreated cell or the cell before treating. For example, the degree of cell migration and/or a cell migration related biological process may be 0.1%higher, 1%higher, 10%higher, 20%higher, 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, 95%higher, 99%higher, 100%higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 10 times higher, 50 times higher, 100 times higher, 1000 times higher, or 10000 times higher than the untreated cell or the cell before treating.

Regulating the formation and/or degradation of a retraction fiber (RF) by a cell generating said retractosome

In some embodiments, the formation of contractile substrates can be regulated by regulating the formation and/or degradation of a retraction fiber (RF) .

For example, regulating the formation and/or degradation of a retraction fiber (RF) by a cell generating said retractosome may comprise promoting the formation a retraction fiber (RF) by a cell generating said retractosome.

For example, regulating the formation and/or degradation of a retraction fiber (RF) by a cell generating said retractosome may comprise promoting the degradation of a retraction fiber (RF) by a cell generating said retractosome.

For example, regulating the formation and/or degradation of a retraction fiber (RF) by a cell generating said retractosome may comprise inhibiting the formation degradation of a retraction fiber (RF) by a cell generating said retractosome.

For example, regulating the formation and/or degradation of a retraction fiber (RF) by a cell generating said retractosome may comprise inhibiting the degradation of a retraction fiber (RF) by a cell generating said retractosome.

For example, regulating the formation and/or degradation of a retraction fiber (RF) by a cell generating said retractosome may comprise promoting the formation and degradation of a retraction fiber (RF) by a cell generating said retractosome.

For example, regulating the formation and/or degradation of a retraction fiber (RF) by a cell generating said retractosome may comprise inhibiting the formation and degradation of a retraction fiber (RF) by a cell generating said retractosome.

Reducing the amount and/or function of the cholesterol

For example, the present application provides a method for reducing the amount and/or function of the cholesterol. For example, reducing the amount and/or function of the cholesterol may comprise lowering and/or function intracellular cellular cholesterol levels.

For example, when treating the cell according to the method of the present application, the amount and/or function of the cholesterol may be lower than the untreated cell or the cell before treating. For example, the amount and/or function of the cholesterol may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower than the untreated cell or the cell before treating.

For example, reducing the amount and/or function of the cholesterol may comprise decreasing cellular synthesis of cholesterol, increasing cellular degradation of cholesterol, decreasing cellular uptake of cholesterol, or culturing cells in a cholesterol depletion environment.

For example, reducing the amount and/or function of the cholesterol may comprise administering to cell a cholesterol synthesis inhibitor (such as pravastatin) and/or culturing cells in a cholesterol depletion environment.

For example, reducing the amount and/or function of the cholesterol may comprise transforming a cell into a cell with reduced cholesterol synthesis or a cell that is unable to synthesis cholesterol.

Cholesterol synthesis inhibitor

Examples of suitable cholesterol lowering agents include cholesterol absorption inhibitor (e.g., ezetimibe) and/or cholesterol synthesis inhibitor.

According to a embodiment the cholesterol synthesis inhibitor is one of atorvastati, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin, or a pharmaceutically acceptable salt, solvate, ester, amide, clathrate, stereoisomer, enantiomer, prodrug or analog thereof.

Cholesterol depletion environment

Cholesterol level in a cholesterol depletion environment is about 5%lower, about 10%lower, about 15%lower, about 20%lower, about 25%lower, about 30%lower, about 35%lower, about 40%lower, about 45%lower, about 50%lower, about 55%lower, about 60%lower, about 65%lower, about 70%lower, about 75%lower, about 80%lower, about 85%lower, about 90%lower or about 95%lower compared to a normal environment.

In some embodiments, said cells are cultured in conditions of complete lack of cholesterol.

The environment can include in vivo environment or in vitro environment (e.g. culture medium) .

For example, said cholesterol depletion environment may comprises cholesterol depleted medium.

Migration inhibitor

The term "migration inhibito″ generally refers to a compound/substance or composition that is capable of completely or partially preventing or reducing the migration and/or a cell migration related biological process. Suitable inhibitor molecules may include antagonist antibodies or antibody fragments, fragments or derivatives of small molecules, peptides, antisense oligonucleotides, small organic molecules, etc. In some embodiments, said inhibitor is capable of blocking the activation of cellular signaling pathways.

For example, when administering the migration inhibitor to cells, the degree of cell migration and/or a cell migration related biological process may be lower than the untreated cell or the cell before treating. For example, the degree of cell migration and/or a cell migration related biological process may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower than the non-or pre-administered cell.

For example, migration inhibitor may comprise CRISPR Cas9 system and/or miRNA targeting cell migration-associated proteins such as TSPAN4.

For example, migration inhibitor may comprise dynamin inhibitor ‘Dynasore’a nd the myosin II inhibitor ‘Blebbistatin’ .

Examples

The following examples are set 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 Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i. m., intramuscular (ly) ; i. p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; r.t., room temperature; and the like.

MATERIALS AND METHODS

Reagents and antibodies. Fibronectin (PHE0023) was from Life Technologies. Blebbistatin (ab120425) was from Abcam. Pravastatin sodium (T0672) was from TargetMol. Anti-Cpq (HPA023235) was from Sigma. Anti-Pigk (ab201693) , anti-NDST1 (ab129248) , anti-PCCA (ab187686) , anti-Eogt (ab190693) , anti-ITGAV (ab179475) , anti-Tsg101 (ab125011) , anti-CD63 (ab217345) , anti-Alix (ab186728) , and anti-Histone H3 (ab176882) were from Abcam. Anti-Syntenin-1 (505360) was from Zen bioscience. Anti-Calnexin (610523) and anti-Tim23 (611222) were from BD. For western blotting, the antibodies described above were used at 1: 1000 except for anti-ITGAV which was used at 1: 5000.

Cell culture. L929, U2OS, NRK, MGC803, MEF, HT1080 and NMuMG cells were cultured in DMEM (WISENT) supplemented with 10%FBS (BI) . D2SC and BV2 cells were cultured in RPMI-1640 (WISENT) supplemented with 10%FBS and 0.1%2-mercaptoethanol (M8211, Solarbio) . All cells were cultured at 37℃ with 5%CO₂.

Live-cell imaging. Cells were seeded in 35 mm glass-bottom dishes coated with (L929) or without (D2SC) 10 μg/ml fibronectin and cultured at 37 ℃ with 5%CO₂ overnight. Images were acquired with a Nikon A1 or Olympus F3000 confocal microscope.

Dye staining. L929 cells were cultured in 10 μg/ml fibronectin-coated dishes (D2SC cells were cultured without fibronectin) overnight and fixed by 4%paraformaldehyde for 15 min. Fixed cells were washed with PBS twice and incubated with 1 μg/ml WGA488 or 2.5 μM SYTO14 (S7576, Invitrogen) for 10 min, or 10 μg/ml Filipin III (70440, Cayman) for 1 hr. Images were acquired with a Nikon A1 or Olympus Fv3000 confocal microscope.

Counting migrasomes or retractosomes in confocal images. Migrasomes and retractosomes are distinguished by size, morphology and location. Under a confocal microscope, migrasomes are large vesicular structure localized on the branch points or the ends of retraction fibers, while retractosomes are small dots which are always arranged in a string. Counting was carried out in a non-blinded manner.

Isolation of migrasomes from cultured cells. The isolation procedure was based on a modification of previous protocols (Ma et al, 2015; Zhao et al, 2019) . Briefly, D2SC cells were grown in 150 mm dishes (NEST) coated with 1 μg/ml fibronectin in full RPMI (DMEM for L929 cells) medium for 16 hr. The cells and migrasomes on plates were digested with trypsin and collected in 50 ml tubes, and conditioned medium was harvested for small EV isolation. All subsequent manipulations were performed at 4℃. Cells and large debris were removed by centrifugation at 1000 g for 10 min followed by 4000 g for 20 min. Crude migrasomes were then collected as the pellet by centrifugation at 18,000 g for 60 min. Migrasome fractionation was performed by density gradient centrifugation, using Optiprep as the density medium (Sigma-Aldrich, D1556) . First, a step gradient was built starting with 30% (500 μl) , followed by 25% (500 μl) , crude migrasomes (19%, 800 μl) , 15% (500 μl) , 12% (500 μl) , 10% (500 μl) , 8% (500 μl) , 5% (500 μl) and 2% (500 μl) . The crude migrasome sample was prepared by resuspending the pellet with 137.5 μl dilution buffer and then mixing with 400 μl 1ⅹ extraction buffer and 252.5 μl 60%Optiprep. Second, the prepared gradient was centrifuged at 150 000× g for 4 hr at 4℃ in an MLS-50 rotor (Beckman) . Third, samples were collected from top to bottom (480 μl per fraction) . Each fraction was mixed with the same volume of PBS (480 μl) and centrifuged at 20,000g for 45 min to collect the pellet. The pellets were washed with PBS and centrifuged again at 20,000g for 45 min. The samples were compatible with western blot analysis, negative staining EM and mass spectrometry.

Isolation of small EVs from cultured cells. The isolation procedure was based on a modification of previous protocols (Clotilde et al, 2006; Zhao et al, 2019) . Briefly, D2SC cells were grown on 150 mm dishes (NEST) coated with 1 μg/ml fibronectin in RPMI medium (DMEM for L929 cells) with 10%FBS depleted of small EVs. After 16 hr, conditioned medium was harvested and at the same time cells were harvested for migrasome or retractosome isolation. All subsequent manipulations were performed at 4℃. Cells and large debris were removed by centrifugation at 300 g for 10 min followed by 2000g for 20 min and 10,000g for 30 min in 50 ml tubes. The supernatant was passed through a 0.22 μm filter (16541-k, Minisart) and then centrifuged at 120,000 g for 70 min at 4℃ in a Type 45 Ti rotor (Beckman) . The crude small EV pellet was washed with PBS, followed by a second step of ultracentrifugation at 120,000 g for 70 min at 4℃ in a TLA-55 rotor (Beckman) to collect the crude small EVs in the pellet. Sucrose density gradient centrifugation was performed to further purify small EVs. Briefly, crude small EVs were resuspended in 500 μl of HEPES/sucrose stock solution (2.25 M sucrose, 20 mM HEPES/NaOH solution, pH 7.4) . Crude small EVs were loaded in between the 2 M-2.5 M sucrose steps, and then 1.75, 1.5, 1.25, 1.0, 0.75, 0.5, 0.25 M sucrose (20 mM HEPES/NaOH, pH 7.4, 500 μl for each) were laid on top. The gradient was spun at 150,000g at 4℃ for 4 hr. Gradient fractions of 500 μl were collected from top to bottom and each fraction was mixed with the same volume of PBS and centrifuged at 120,000g for 70 min to collect the pellet. The samples were compatible with western blot analysis, negative staining EM and mass spectrometry.

Isolation of retractosomes from cultured cells. D2SC cells were grown in 150 mm dishes (NEST) coated with 1 μg/ml fibronectin in full RPMI medium (DMEM for L929 cells) for 16 hr. The cells and retractosomes on plates were digested with trypsin and collected in 50 ml tubes, and conditioned medium was harvested for small EV isolation. All subsequent manipulations were performed at 4℃. Cells and large debris were removed by centrifugation at 1000 g for 10 min followed by twice at 4000g for 10 min. The supernatant was passed through a 0.22 μm filter (16541-k, Minisart) and retractosomes were collected by centrifugation at 18,000 g for 60 min at 4℃. The crude retractosome pellet was washed with PBS, followed by a second centrifugation step at 18,000 g for 60 min at 4℃. The samples were compatible with western blot analysis, negative staining EM and mass spectrometry.

Field emission scanning electron microscopy. For the conventional fixation procedure, the culture cells were grown on coverslips, fixed with 2.5%glutaraldehyde in PB buffer for 2 h at room temperature, washed three times with PB buffer and then post fixed with 1%osmium containing 1.5%potassium ferrocyanide for 60 min at room temperature. All samples were then dehydrated with a graded series of ethanol (50%, 70%, 90%, 95%, and 100%) for 8 min each. After changing ethanol with tert-Butanol, samples were frozen at -20 ℃, then dried with a freeze drier. The dried samples were coated with an approximately 10-nm-thick gold film by sputter coating before examination with a field emission scanning electron microscope using an SE detector at an acceleration voltage of 3 kV.

Negative staining and TEM imaging. Purified migrasomes, retractosomes or small EVs were resuspended in 50-100 μl PBS, then a 5 μl sample of each was mixed with the same volume of 2.5%glutaraldehyde (PB buffer, pH 7.4) , and fixed for 30 min at room temperature. The sample was spread onto glow-discharged Formvar-coated copper mesh grids (Electron Microscopy Sciences, Hatfield) for about 5 min, then stained with 1%uranyl acetate for 30 s. Excess staining solution was blotted off with filter paper and the copper mesh grids were washed with water. After drying, grids were imaged at 10-100 kV using a transmission electron microscope H-7650.

Mass spectrometric analysis. The proteins were lysed with 8 M urea and the protein concentration was determined by the BCA method. After reductive alkylation of the proteins, the reaction was carried out overnight at 37℃ according to a trypsin: sample mass ratio of 1: 50. After enzymatic digestion, the samples were desalted using a C18 desalting cartridge and put on the machine. The mass spectrometry assay was performed using a tims pro mass spectrometer with data acquisition (DDA) in data dependent mode.

Statistical analysis. Statistical analysis was performed in Graphpad Prism. Different experimental groups were compared with two-tailed t-tests (Fig. 1i, j, l, m, o; Supplementary information, Fig. S1b, c, e, and S3b, d) . All data were obtained from independent experiments.

Example 1

Tspan4-overexpressing L929 cells were observed with live-cell imaging, it was found that during migration, retraction fibers (RFs) were pulled out at the trailing edge of cells, and migrasomes soon formed on the tips or branch points of the RFs. At this stage, the Tspan4 signal was evenly distributed along the RFs (FIG. 1A) . Eventually, the RFs started to break. In most cases, the sections of RF far away from the cells broke first, after the RFs broke up, the Tspan4 signal appeared as a large number of small puncta along the paths which were occupied by the RFs (FIG. 1A) .

Scanning electron microscopy (SEM) analysis revealed that on the far end of RFs, large numbers of round, small extracellular vesicles were left behind; in many cases these vesicles are linked together, like a string of beads (FIG. 1B) .

Transmission electron microscopy (TEM) analysis revealed that these vesicles did indeed have a continuous membrane (FIG. 1C) , and thus they were true vesicles instead of broken membrane fragments.

Cryo-TEM analysis confirmed that the RF puncta were small extracellular vesicles (FIG. 1D) . The size of these vesicles varies considerably, from 50 nm to 250 nm, which was much smaller than migrasomes (FIG. 1E) .

These data suggested that when RFs broke up, they may form a type of previously unreported extracellular vesicle. Similar to migrasomes, these small extracellular vesicles were migration dependent: treating cells with blebbistatin blocked their formation (FIG. 2A-2C) . These vesicles were named “retractosomes” , to reflect their retraction fiber and plasma membrane origin.

The beads-on-a-string structure was frequently observed under cryo-TEM (FIG. 1F) . This indicated that, before breaking, the RF went through a stage of morphological transformation, with sections of the RF bulging into small vesicles, which likely formed retractosomes.

Wheat germ agglutinin (WGA) effectively labels retraction fibers and migrasomes³. WGA staining revealed that retractosomes were formed by all the migratory cells tested, none of which overexpress Tspan4 (FIG. 2D) .

Moreover, the formation of retractosomes in various in vivo settings, including circulating neutrophils in mice, and embryonic cells during zebrafish gastrulation was observed (FIG. 3A-3B) .

Example 2

Live-cell imaging using resonant scanning mode revealed that Tspan4 started to condense and became enriched in puncta along the RF before the RF breaks; these puncta eventually became retractosomes (FIG. 1G) . Moreover, it was found that overexpression of Tspan4 significantly enhanced both migrasome and retractosome formation (FIG. 1H-1J) . This effect on retractosomes was largely due to enhanced RF formation. In Tspan4-overexpressing cells, there was a 2.68-fold increase in the total length of RFs, which then broke into more retractosomes (FIG. 2E) .

It was found that cholesterol was hardly detected on retractosomes (FIG. 4A) . Detailed analysis showed that cholesterol was much less enriched on retractosomes than on migrasomes (FIG. 4B) .

This suggested that retractosomes may form from mechanisms other than assembly of tetraspanin-enriched macrodomains.

Cells were cultured in cholesterol depletion medium; it was found that cholesterol depletion promoted retractosome formation (FIG. 1K-1M; FIG. 4-4D) . Taken together, these data suggested that retractosome formation depended on Tspan4 but not cholesterol, which was different from migrasome formation.

Example 3

First, the culture medium was removed and the cells were washed with PBS, which removed the majority of small extracellular vesicles (small EVs) . Next, the cells were collected and performed two steps of low-speed centrifugation to remove cell bodies and large debris. Migrasomes were removed by passing the sample through a 0.22 μm filter. Finally, to collect retractosomes, the flow-through were subjected to high-speed centrifugation (FIG. 5A) ⁵.

The sample was checked by electron microscopy, which revealed vesicles with sizes ranging from 50 nm to 250 nm (FIG. 1) . The morphology and size of the isolated retractosomes were very similar to retractosomes observed in cultured cells. The isolated retractosomes were smaller than migrasomes but slightly larger than small EVs (FIG. 1N, FIG. 1O) .

To further check the purity of retractosomes, western blot analysis using antibodies against various markers of migrasomes and small EVs was carried out (FIG. 1P, FIG. 6) . It was found that the isolated retractosomes contained very little Tsg101, CD63 and Syntenin-1, three well-established markers for small EVs. ⁶ Thus, the isolated retractosomes were not contaminated with significant amounts of small EVs. The isolated retractosomes were positive for the migrasome marker PIGK, but not for migrasome markers CPQ, NDST1 and PCCA. This suggested that the retractosome preparation was not contaminated by large amounts of migrasomes.

Next, retractosomes were subjected to mass spectrometry analysis. It was found that the protein profile of retractosomes was significantly different from cell bodies: 1107 proteins were enriched and 1309 proteins were depleted in retractosomes compared to cell bodies (FIG. 5B) . Quantitative mass spectrometry analysis on isolated small EVs and migrasomes were carried out, and it was found that the protein composition of retractosomes shared significant similarity with migrasomes, but is very different from small EVs (FIG. 1Q) .

During zebrafish embryonic development, migrasomes modulate organogenesis by enriching and releasing chemokines at spatially restricted locations. ⁷ To test whether retractosomes also contain chemokines, DS2C cells were used, a dendritic cell line in which the chemokines CCL2 and CCL9 are expressed. It was found that both CCL2 and CCL9 were enriched in migrasomes; in contrast, some but not all retractosomes contained these chemokines (FIG. 1R, FIG. 5C) .

mRNA can be stained by SYTO14, a dye for nucleic acids. In contrast to migrasomes, there was no SYTO14 signal in retractosomes (FIG. 1S) . Thus, retractosomes may have a different cargo composition from migrasomes. ⁸

A migrating cell can generate both retractosomes and small EVs. The relative amount of retractosomes and small EVs generated by the same cultured cells was investigated.

First, the culture medium was taken and used high-speed centrifugation to isolate the crude small EV fraction. Then, the plate was washed and retractosomes were collected. Finally, the isolated crude small EV and retractosome fractions were diluted with an equal volume of buffer and the relative amount of small EVs and retractosomes were checked based on the total protein concentration. The purity of the isolated small EVs and retractosomes was checked by western blot (FIG. 1T) . It was found that the total amount of protein in small EVs was roughly 3-fold more than in retractosomes (FIG. 1U) .

While exemplary embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

REFERENCES

1 Rilla, K. Diverse plasma membrane protrusions act as platforms for extracellular vesicle shedding. J Extracell Vesicles 10, doi: ARTN e1214810.1002/jev2.12148 (2021) .

2 Ma, l. et al. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration. Cell Res 25, 24-38 (2015) .

3 Chen, L., Ma, L. &Yu, L. WGA is a probe for migrasomes. Cell Discov 5, 13 (2019) .

4 Huang, Y. W. et al. Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains. Nat Cell Biol 21, 991-1002 (2019) .

5 Zhao, X. X. et al. Identification of markers for migrasome detection. Cell Discov 5, doi: ARTN 2710.1038/s41421-019-0093-y (2019) .

6 Thery, C., Clayton, A., Amigorena, S. &Raposo, G. Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids. Current Protocols in Cell Biology, 3.22.21-23.22.29 (2006) .

7 Jiang, D. et al. Migrasomes provide regional cues for organ morphogenesis during zebrafish gastrulation. Nat Cell Biol 21, 966-977 (2019) .

8 Zhu, M. L. et al. Lateral transfer of mRNA and protein by migrasomes modifies the recipient cells. Cell Res 31, 237-240, doi: 10.1038/s41422-020-00415-3 (2021) .

Claims

A method for monitoring cell migration and/or a cell migration related biological process, comprising determining the presence, amount and/or function of a retractosome generated by said cell.
The method of claim 1, wherein determining the presence, amount and/or function of said retractosome comprises determining the presence and/or amount of tetraspanin⁺ and/or PIGK⁺ vesicles.
The method of any one of claims 1-2, wherein said vesicle is CPQ^-, NDST1^-and/or PCCA^-.
The method of any one of claims 1-3, wherein said vesicle is Alix^-, Tsg101^-and/or CD63^-.
The method of any one of claims 1-4, wherein said vesicle is an extracellular vesicle.
The method of any one of claims 1-5, wherein said vesicle has a diameter of about 30 nm to about 400 nm.
The method of any one of claims 1-6, wherein said cell comprises an epithelial cell, a cancer cell, a fibroblast, an embryonic cell, a neural cell and/or an immune cell.
A method for regulating cell migration and/or a cell migration related biological process, comprising:

i) monitoring said cell migration and/or said cell migration related biological process according to any one of claims 1-7; and

ii) administering an appropriate regulator according to the result of step i) .
A method for characterizing a retractosome, comprising:

determining the presence and/or amount of tetraspanin and/or PIGK in a vesicle to be characterized.
The method of claim 9, further comprising determining the presence and/or amount of CPQ, NDST1 and/or PCCA in said vesicle.
The method of any one of claims 9-10, further comprising determining the presence and/or amount of Alix, Tsg101 and/or CD63 in said vesicle.
The method of any one of claims 9-11, wherein said vesicle is an extracellular vesicle.
The method of any one of claims 9-12, wherein said vesicle has a diameter of about 30 nm to about 400 nm.
The method of any one of claims 9-13, wherein the vesicle that is:

i) tetraspanin⁺ and/or PIGK⁺;

ii) CPQ^-, NDST1^-and/or PCCA^-; and

iii) Alix^-, Tsg101^-and/or CD63^-

is characterized as a retractosome.
A method for isolating and/or regulating a retractosome, comprising:

i) characterizing the retractosome according to the method of any one of claims 9-14; and

ii) isolating the characterized retractosome, and/or administering a regulating agent to said charactrerized retractosome.
A method for regulating the formation and/or function of a retractosome, and/or for regulating a retractosome mediated biological process, comprising:

regulating the amount and/or function of a tetraspanin in a cell generating said retractosome;

regulating the migration of a cell generating said retractosome; and/or

regulating the formation and/or degradation of a retraction fiber (RF) by a cell generating said retractosome.
The method of claim 16, which promotes the formation of the retractosome, and comprises:

increasing the amount and/or function of the tetraspanin in the cell generating said retractosome;

reducing the amount and/or function of cholesterol in the cell generating said retractosome;

promoting the migration of the cell generating said retractosome; and/or

promoting the formation and/or inhibiting the degradation of the retraction fiber (RF) by the cell generating said retractosome.
The method of claim 17, wherein increasing the amount and/or function of the tetraspanin comprises overexpressing said tetraspanin in said cell.
The method of any one of claims 17-18, wherein reducing the amount and/or function of the cholesterol comprises inhibiting the synthesis and/or uptake of cholesterol by said cell.
The method of claim 19, wherein inhibiting the synthesis and/or uptake of cholesterol by said cell comprises administering to said cell a cholesterol lowering agent.
The method of claim 20, wherein said cholesterol lowering agent comprises cholesterol absorption inhibitor and/or cholesterol synthesis inhibitor.
The method of any one of claims 19-21, wherein inhibiting the uptake of cholesterol comprises culturing said cells in a cholesterol depletion environment.
The method of claim 16, which inhibits the formation of the retractosome, and comprises:

decreasing the amount and/or function of the tetraspanin in the cell generating said retractosome;

inhibiting the migration of the cell generating said retractosome; and/or

inhibiting the formation and/or promoting the degradation of the retraction fiber (RF) by the cell generating said retractosome.
The method of claim 23, wherein decreasing the amount and/or function of the tetraspanin comprises knocking out or knocking down the expression of a gene encoding for said tetraspanin in said cell.
The method of any one of claims 23-24, wherein inhibiting the migration of said cell comprises administering a cell migration inhibitor to said cell.
The method of claim 25, wherein said cell migration inhibitor comprises Blebbistatin.
The method of any one of claims 2-26, wherein said tetraspanin comprises tetraspanin 4.
A method for regulating a cell migration mediated biological process, comprising regulating the formation and/or function of the retractosome according to any one of claims 16-27.
A method for isolating a retractosome, comprising:

providing a cell culture from which cells and cell debris have been removed;

passing said cell culture through a filter with a pore size not exceeding about 0.25 μm to obtain a filtrate containing said retractosome; and

isolating said retractosome from the filtrate.
The method of any one of claims 1-29, wherein said retractosome is not a migrasome.
An engineered cell with altered ability for generating a retractosome comparing to an unmodified corresponding cell, wherein said engineered cell has been modified to:

change the amount and/or function of a tetraspanin therein;

change the amount and/or function of cholesterol therein;

change the migration ability thereof; and/or

change its ability to form and/or degrade a retraction fiber (RF) .
The engineered cell of claim 31, wherein said tetraspanin comprises tetraspanin 4.
The engineered cell of any one of claims 31-32, which comprises an epithelial cell, a cancer cell, a fibroblast, an embryonic cell, a neural cell and/or an immune cell.
An agent capable of determining the presence, amount and/or function of a retractosome generated by a cell, for use in monitoring the migration of said cell and/or a biological process related to the migration of said cell.
An agent capable of regulating the formation and/or function of a retractosome generated by a cell, for use in regulating a biological process mediated by the migration of said cell.
An agent capable of regulating the amount and/or function of a tetraspanin in a cell, regulating the amount and/or function of cholesterol in a cell, regulating the migration ability of a cell, and/or regulating the ability of a cell to form and/or degrade a retraction fiber (RF) , for use in regulating the formation and/or function of a retractosome.
An agent capable of specifically identifying tetraspanin, PIGK, CPQ, NDST1, PCCA, Alix, Tsg101 and/or CD63, for use in characterizing a retractosome.
An isolated retractosome, which is a tetraspanin⁺ and/or PIGK⁺ vesicle.
The isolated retractosome of claim 38, which is not a migrasome.
The isolated retractosome of any one of claims 38-39, which is CPQ^-, NDST1^-and/or PCCA^-.
The isolated retractosome of any one of claims 38-40, which is Alix^-, Tsg101^-and/or CD63^-.
The isolated retractosome of any one of claims 38-41, which is an extracellular vesicle.
The isolated retractosome of any one of claims 38-42, which has a diameter of about 30 nm to about 400 nm.
The isolated retractosome of any one of claims 38-43, wherein said tetraspanin comprises tetraspanin 4.
A composition, comprising the engineered cell according to any one of claims 31-33, the agent according to any one of claims 34-37, and/or the isolated retractosome according to any one of claims 38-44.
The composition of claim 45, which is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable excipient.
Use of the agent according to any one of claims 34-37, in the preparation of a reagent for characterizing the retractosome.
Use of the agent according to any one of claims 34-37, in the preparation of a reagent for regulating the formation and/or function of a retractosome.
Use of the agent according to any one of claims 34-37 and/or the engineered cell according to any one of claims 31-33, in the preparation of a reagent for regulating a biological process mediated by the migration of said cell.
Use of an agent according to any one of claims 34-37, in the preparation of a reagent for monitoring the migration of said cell and/or a biological process related to the migration of said cell.