Metastasis biomarkers

Abstract

The present invention relates to a method for determining whether a subject diagnosed with cancer is likely to develop or has developed metastasis, comprising detecting the level of membrane-bound actin in a sample obtained from the subject. The present invention also relates to methods and markers for diagnosing, prognosing, monitoring and / or staging cancer, particularly metastatic cancer.

Description

[Technical Field]

[0001] The present invention relates to biomarkers and their uses. In particular, the present invention relates to their use in predicting the likelihood of cancer metastasis or in diagnosing and / or monitoring cancer metastasis. [Background technology]

[0002] introduction Epithelial cell cancers, also known as carcinomas, account for 80 to 90 percent of all cancer cases. One such carcinoma, breast cancer, is the second most common type of cancer worldwide, accounting for more than 10% of all cancers. Breast cancer is the most common cancer in women, with more than 2 million women diagnosed with breast cancer annually and more than 500,000 patients dying from the disease annually.

[0003] Metastatic dissemination of primary tumors is the leading cause of cancer-related deaths, accounting for up to 90% of solid tumor cancer deaths. Furthermore, patients diagnosed with metastatic tumors have a 5-year survival rate of only 22%. Metastasis occurs when cancer cells acquire a migratory epithelial-mesenchymal transition (EMT) phenotype, which begins with the aggregation of cells that have disseminated from the primary tumor. The invasive phenotype of such cells is a fundamental characteristic that correlates with their invasion of the endothelial vascular layer during the early stages of metastasis.

[0004] Assessing the stage of cancer is crucial for determining any prognosis and appropriate treatment regimens. Current prognosis is often based on assessment of lymph node status, which requires invasive procedures such as lymph node biopsy, which can result in severe side effects such as lymphedema. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need in the art for the development of new and improved methods and markers for diagnosing, prognosing, monitoring and / or staging cancer. The present invention fulfills these needs and further provides other related advantages. [Means for solving the problem]

[0006] Summary of the Invention In a first aspect, the present invention provides a method for determining whether a subject diagnosed with cancer is likely to develop or has developed metastasis, the method comprising detecting membrane-bound actin in a sample obtained from the subject, and determining that the subject is likely to develop or has developed metastasis if membrane-bound actin is detected in the sample. The detected actin may be one or more of alpha, beta, and / or gamma actin. In one embodiment, the membrane-bound actin is beta actin.

[0007] In a second aspect, there is provided a method for diagnosing metastatic cancer in a subject, the method comprising detecting membrane-bound actin in a sample obtained from the subject, wherein the subject is determined to have metastatic cancer if membrane-bound actin is detected in the sample. In one embodiment, the membrane-bound actin is beta-actin.

[0008] In either the first or second embodiment, the detection of any level of membrane-bound actin in a sample obtained from a subject may enable the method to be performed.

[0009] In a third aspect, there is provided a method of determining whether a subject diagnosed with cancer is likely to develop or has developed metastasis, comprising: a. measuring the level of membrane-bound actin in a sample obtained from a subject; b. comparing the level of membrane-bound actin measured in the sample obtained from the subject with the level of membrane-bound actin in a standard sample; and c. determining that the subject is likely to develop or has developed metastasis if the level of membrane-bound actin in the sample obtained from the subject is approximately equal to or higher than the level of membrane-bound actin in the standard sample; A method is provided which includes:

[0010] In one embodiment, the membrane-bound actin is beta-actin.

[0011] In a fourth aspect, there is provided a method of diagnosing metastatic cancer in a subject, comprising: a. measuring the level of membrane-bound actin in a sample obtained from a subject; b. comparing the level of membrane-bound actin measured in the sample obtained from the subject with the level of membrane-bound actin in a standard sample; and c. Diagnosing a subject with metastatic cancer if the level of membrane-bound actin in the sample obtained from the subject is approximately equal to or higher than the level of membrane-bound actin in the standard sample. A method is provided which includes:

[0012] In one embodiment, the membrane-bound actin is beta-actin.

[0013] In a fifth aspect, there is provided a method of prognosing a subject diagnosed with cancer, comprising: a. measuring the level of membrane-bound actin in a sample obtained from a subject; b. comparing the level of membrane-bound actin measured in the sample with the level of membrane-bound actin in a standard sample; c. Determining that the patient has a poor prognosis if the level of membrane-bound actin in the sample obtained from the subject is approximately equal to or higher than the level of membrane-bound actin in the standard sample. A method is provided which includes:

[0014] In one embodiment, the membrane-bound actin is beta-actin.

[0015] A poor prognosis can refer to an increased likelihood of developing metastases and / or a decreased chance of survival of the subject.

[0016] In a sixth aspect, there is provided a method of identifying a patient diagnosed with cancer who may benefit from treatment with a known treatment for metastatic cancer, comprising: a. measuring the level of membrane-bound actin in a sample obtained from a subject; b. comparing the level of membrane-bound actin measured in the sample with the level of membrane-bound actin in a standard sample; c. determining that the patient is likely to benefit from treatment with a known treatment for metastatic cancer if the level of membrane-bound actin in the sample obtained from the subject is approximately equal to or higher than the level of membrane-bound actin in the standard sample; A method is provided which includes:

[0017] In one embodiment, the membrane-bound actin is beta-actin.

[0018] Those skilled in the art will be able to identify suitable known treatments for metastatic cancer, and will understand that known treatments for metastatic cancer vary depending on how widespread and advanced the metastatic disease may be and the molecular profile of the cancer.For example, suitable treatments may be adjuvant chemotherapy, and the specific treatment and / or regimen will depend on the characteristics of the particular tumor of the subject.

[0019] By way of example, the known treatment for metastatic cancer may be a treatment known for use in treating metastatic breast cancer, such as Herceptin™ or another Her2-blocking monoclonal antibody treatment.

[0020] In any embodiment, membrane-bound actin can be detected or measured in the membrane of a cell, such as an epithelial cancer cell.Membrane-bound actin can be detected or measured in the membrane of an extracellular vesicle (EV).Membrane-bound actin can be detected in both the membrane of a cell and the membrane of an extracellular vesicle.

[0021] In any embodiment, EVs may include, but are not limited to, exosomes, exomers, microvesicles, and apoptotic bodies.

[0022] In any embodiment, the sample obtained from the subject or used as a standard may be a biological fluid sample, such as a blood sample, a saliva sample, a urine sample, or the sample may be a tissue biopsy sample, such as a breast tissue biopsy sample.

[0023] In any embodiment, membrane-bound actin can be detected or measured from the plasma membrane of breast cancer epithelial cells from a solid breast tissue biopsy obtained from a subject.

[0024] In any embodiment, membrane-bound actin can be detected or measured from the membrane of extracellular vesicles isolated from a blood sample obtained from a subject.

[0025] In any of the third to sixth aspects, the standard sample may be a positive standard sample.

[0026] In any of the third to sixth aspects, the standard sample may be a negative standard sample.

[0027] In any embodiment, the membrane-bound actin may be beta-actin. In any embodiment, the membrane-bound actin may be alpha-actin. In any embodiment, the membrane-bound actin may be gamma-actin. In any embodiment, the membrane-bound actin may be one or more, or all, of alpha-actin, beta-actin, and gamma-actin.

[0028] If the standard sample is a positive standard sample, the determination in step c of any of the third to sixth aspects of the present invention is made if the level of membrane-bound actin detected or measured in the sample is equal to or higher than the level of membrane-bound actin detected or measured in the standard sample.

[0029] If the standard sample is a negative standard sample, the determination in step c of any of the third to sixth aspects of the present invention is made if the level of membrane-bound actin detected or measured in the sample is higher than the level of membrane-bound actin detected or measured in the standard sample. The magnitude of the difference may depend on the relationship between the sample obtained from the subject and the standard sample, and the characteristics of each sample. For example, a determination can be made if the level of membrane-bound actin detected or measured in a sample obtained from a subject is at least about 5% greater, at least about 10% greater, at least about 20% greater, at least about 50% greater, at least about 100% greater, at least about 200% greater, at least about 300% greater, at least about 400% greater, at least about 500% greater, at least about 1000% greater, at least about 2000% greater, at least about 5000% greater, at least about 10,000% greater, at least 20,000% or more, at least 50,000% greater, at least 100,000% greater, at least 500,000% greater, or at least 1,000,000% greater than the level of membrane-bound actin detected or measured in a standard sample. Alternatively, or in addition, a determination can be made if the level of membrane-bound actin detected or measured in a sample obtained from a subject is at least about 1-fold or more, at least about 2-fold or more, at least about 3-fold or more, at least about 4-fold or more, at least about 5-fold or more, at least about 10-fold or more, at least about 20-fold or more, at least about 50-fold or more, at least about 100-fold or more, at least about 250-fold or more, at least about 500-fold or more, at least about 1000-fold or more, at least about 5000-fold or more, at least about 10,000-fold or more, at least about 20,000-fold or more, at least about 50,000-fold or more, at least about 100,000-fold or more, or at least about 500,000-fold or more than the level of membrane-bound actin detected or measured in a standard sample.

[0030] In one embodiment of any aspect, the sample obtained from the subject may contain epithelial cells, and / or EVs, and / or circulating tumor cells. The circulating tumor cells may be epithelial cells.

[0031] If the sample contains epithelial cells, detection of membrane-bound actin can be performed on these cells, which can optionally be first isolated, identified, or separated from the remainder of the sample.

[0032] If the sample contains EVs, detection or measurement of membrane-bound actin can be performed on these EVs, which can then be isolated, identified, or separated from the rest of the sample as needed.

[0033] If the sample contains circulating tumor cells, detection or measurement of membrane-bound actin can be performed on these cells, which can be isolated, identified, or separated from the remainder of the sample, if desired.

[0034] When cells and / or EVs are isolated, identified, or separated from the remainder of the sample, one or more appropriate biomarkers may be used.

[0035] For example, epithelial anti-EpCAM antibodies may be used in any manner deemed appropriate by one skilled in the art, such as FACS, to isolate, identify, or separate epithelial cells from a sample.

[0036] To isolate or separate EVs from a sample, one or more of the antibodies CD9, anti-CD63, and / or anti-CD81 may be used in any manner deemed appropriate by one of skill in the art, e.g., FACS. Similarly, an anti-EpCAM antibody may be used in combination with one or more EV-specific biomarkers to isolate, identify, or separate EVs derived from epithelial cells from the remainder of the sample.

[0037] Anti-EpCAM antibody or DNA or RNA that binds to EpCAM can be used to isolate, identify or separate circulating tumor cells from sample.This is because epithelial cells are not usually found in circulation, and their presence indicates circulating tumor cells.To isolate, identify or separate circulating tumor cells from sample, the means for detecting one or more of ER, PR, EGFR, HER2, TOP2A can be used (Nadal et al. Breast Cancer Research 2012, 14:R71).The means can be the antibody, DNA or RNA molecule that specifically binds to ER, PR, EGFR, HER2 or TOP2A.

[0038] In another aspect, a method for monitoring a subject's response to a treatment for metastatic cancer is provided, comprising measuring the level of membrane-bound actin in a sample obtained from the subject, wherein the subject is determined to be responding to the treatment for metastatic cancer if the measured level of membrane-bound actin in the sample is lower than the level of membrane-bound actin in a corresponding previous sample obtained from the subject. Alternatively, the subject may be determined to be not responding to the treatment for metastatic cancer if the measured level of membrane-bound actin in the sample is equal to or greater than the level of membrane-bound actin in the corresponding previous sample obtained from the subject. The corresponding previous sample may be obtained before or at the same time as the subject is administered the treatment. Preferably, the level of membrane-bound actin in the corresponding previous sample is compared with the level in one or more samples obtained after the administration of the treatment, and these samples may be obtained at one or more time points after the administration of the treatment. By monitoring the level of membrane-bound actin during and / or after the course of treatment, the subject's response to the treatment can be determined. It will be understood that a skilled physician can use the level of membrane-bound actin observed in the sample during treatment to adjust the treatment as necessary.

[0039] In another aspect, there is provided a method for monitoring disease progression in a subject diagnosed with metastatic cancer, comprising measuring the level of membrane-bound actin in a sample obtained from the subject, wherein the metastatic cancer is determined to be not progressing if the level of membrane-bound actin measured in the sample is lower than the level of membrane-bound actin in a corresponding previous sample obtained from the subject. Alternatively, the metastatic cancer can be determined to be progressing if the level of membrane-bound actin measured in the sample is equal to or greater than the level of membrane-bound actin in a corresponding previous sample obtained from the subject.

[0040] In another aspect, a method for monitoring the recurrence of metastatic cancer in a subject who has been successfully treated for the disease is provided, comprising measuring the level of membrane-bound actin in a sample obtained from the subject, wherein the metastatic cancer is determined not to have recurred if the level of membrane-bound actin measured in the sample is lower than the level of membrane-bound actin measured in a corresponding sample obtained from the subject earlier. Alternatively, the metastatic cancer can be determined to have recurred if the level of membrane-bound actin measured in the sample is equal to or greater than the level of membrane-bound actin measured in a corresponding sample obtained from the subject earlier. The corresponding sample may have been collected at the time of determining the treatment success, or within one week, two weeks, or four weeks of determining the treatment success.

[0041] Reference herein to a "corresponding sample" may refer to a sample of the same type, e.g., the same tissue type, taken from the same subject. The sample is preferably taken at an earlier time point.

[0042] The method of any aspect of the present invention may be carried out in vivo, ex vivo or in vitro. Preferably, the method is an in vitro method.

[0043] In a further aspect, a kit for (a) determining whether a subject diagnosed with cancer is likely to develop or has developed metastases, (b) prognosing a subject diagnosed with cancer, (c) diagnosing a subject with metastatic cancer, (d) monitoring a subject's response to treatment for metastatic cancer, or (e) monitoring disease progression in a subject diagnosed with metastatic cancer, comprising: means for detecting membrane-bound actin; i. a means of identifying the cell type of interest, and / or ii. Reagents for isolating EVs Kits are provided which may further comprise one or more of:

[0044] In one embodiment, the membrane-bound actin is beta-actin.

[0045] The means for detecting membrane-bound actin may be an actin-binding polypeptide such as an antibody. For example, the means for detecting membrane-bound actin may be an anti-actin antibody, such as an anti-beta-actin antibody. The means for detecting membrane-bound actin may be a DNA or RNA molecule, such as an aptamer, that can bind to actin.

[0046] The cell type of interest may be an epithelial cell or a circulating tumor cell. The circulating tumor cell may be an epithelial tumor cell.

[0047] The means for identifying a cell type of interest may be an EpCAM-binding polypeptide, such as an antibody. The means for identifying a cell type of interest may be an RNA or DNA molecule, such as an aptamer, that can bind to EpCAM.

[0048] In one embodiment, the kit may further comprise a reagent for isolating EVs from a sample. A person skilled in the art may select any set of reagents available in the art for isolating EVs from a sample. The exact reagents selected may depend on the nature of the sample; for example, a specific reagent that is particularly compatible with a blood sample may be selected.

[0049] In one embodiment, the kit may further comprise a set of instructions.

[0050] In certain non-limiting embodiments, the means for detecting membrane-bound actin and / or the means for identifying a cell type of interest may be provided attached to a solid support such as a column matrix, an array, or the well of a microtiter plate. Alternatively, the support can be provided as a separate component of the kit.

[0051] As used herein, the terms "actin-binding polypeptide" and "EpCAM-binding polypeptide" may refer to any polypeptide that binds to actin or EpCAM, respectively. Such polypeptides may be T-cell receptors or antibodies or antigen-binding fragments thereof, including, but not limited to, Fv, Fab, Fab', and F(ab')2 fragments, and single-domain antibodies, such as camelid single-domain antibodies. Antibodies or antigen-binding fragments, whether polyclonal or monoclonal, specific for actin, such as beta-actin, and / or specific for EpCAM can generally be prepared using conventional immunization techniques, as will be known to those skilled in the art.

[0052] Means for identifying a cell type of interest may also include DNA or RNA molecules capable of binding to EpCAM, such as aptamers.

[0053] The antibodies, polypeptides, or DNA or RNA molecules referred to herein may contain detectable labels that are bound to or linked to the given antibodies, polypeptides, or DNA or RNA molecules. Such detectable labels include, for example, chemiluminescent or fluorescent molecules (e.g., rhodamine, fluorescein, green fluorescent protein, luciferase, Cy3, Cy5, or ROX), radiolabels (e.g., H, S, P, C, I), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), or peptide / amino acid sequences (e.g., His-Tag, FLAG, Myc).

[0054] In any aspect or embodiment, the cancer may be or may originate from an epithelial cell cancer, such as breast cancer, colorectal cancer, lung cancer, or pancreatic cancer.

[0055] The metastatic cancer may be metastatic breast cancer, metastatic colorectal cancer, metastatic lung cancer or metastatic pancreatic cancer.

[0056] HPA or Helix pomatia agglutinin is a lectin derived from the Helix pomatia apple snail that can be used to detect glycans with terminal N-acetylgalactosamine (GalNAc). As used herein, "HPA positive" and "HPA positivity" refer to cells or EVs that display significant levels of GalNAc on their surface, and therefore are recognized by and bind to high levels of HPA. HPA-negative cells or EVs are defined as cells that do not display GalNAc or display low levels of GalNAc on their surface, and therefore are not recognized by HPA and do not bind to HPA or only bind to low levels of HPA.

[0057] A sample containing cells and / or EVs may be identified as HPA positive if more than about 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, or more than 99% of the cells and / or EVs in the sample are HPA positive.

[0058] A sample containing cells and / or EVs may be identified as HPA negative if more than about 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, or more than 99% of the cells and / or EVs in the sample are HPA negative.

[0059] In any embodiment, the present invention may further comprise detecting or measuring the level of HPA positivity in cells and / or EVs.

[0060] In any embodiment, the present invention may further or alternatively comprise detecting or measuring the level of HPA positivity in addition to detecting or measuring actin, or alternatively detecting or measuring actin.HPA positivity may be determined by EV.For example, in the method for determining whether a subject diagnosed with cancer is likely to develop or has developed metastasis, the method may include: a. Measuring the level of membrane-bound actin and / or HPA-positive in a sample obtained from the subject; b. comparing the level of membrane-bound actin and / or HPA positivity measured in the sample obtained from the subject with the level of membrane-bound actin and / or HPA positivity in a standard sample; and c. determining that the subject is likely to develop or has developed metastasis if the level of membrane-bound actin and / or HPA positivity in the sample obtained from the subject is approximately equal to or higher than the level of membrane-bound actin and / or HPA positivity in the standard sample; In such methods, EVs may be isolated, identified, or separated from the remainder of the sample as desired. [Brief explanation of the drawings]

[0061] [Figure 1]Figure 1 shows that GalNAc binds to multiple proteins in endothelial cells using 1D Western blot. A. Total endothelial cell proteins were extracted, subjected to SDS-PAGE, and then Western blotted. The membrane on the left was probed with GalNAc-BSA-biotin and showed several bands. As a negative control, the membrane on the right was labeled with BSA-biotin and no bands were observed. [Figure 2] Figure 2 shows the bands excised for analysis by mass spectrometry. 1D SDS-PAGE and blot showing where gel fragments were excised for mass spectrometry and five fragments were analyzed. [Figure 3] Figure 3 shows that GalNAc binds to multiple proteins in endothelial cells using 2D PAGE. 2D SDS-PAGE and blot showing where gel fragments were excised for mass spectrometry (13 fragments total). [Figure 4] Figure 4 shows that EVs can increase cell adhesion. EVs were isolated from breast cancer cells prior to static adhesion assays and incubated with either breast cancer cells, endothelial cells, or both. Breast cancer cell adhesion to endothelial cells is significantly increased in the presence of EVs. The greatest increase is seen when both breast cancer cells and endothelial cells are pretreated with EVs. Data are from three replicates; error bars are standard error of the mean (SEM); statistical testing was performed by one-way ANOVA with Tukey's test. Asterisks indicate significance compared to untreated controls. [Figure 5] Figure 5 shows that knockdown of plectin reduces breast cancer cell adhesion to endothelial cells. MCF7 and / or HUVEC cells were treated with negative scrambled siRNA or siRNA targeting plectin. Cell adhesion is significantly reduced when plectin is knocked down in either or both cell lines. Data are from three replicates; error bars represent standard error of the mean; statistical testing was performed by one-way ANOVA with Tukey's test. Asterisks indicate statistical significance compared to untreated controls. [Figure 6]Figure 6 shows that treatment of breast cancer cells with recombinant plectin reduces cell adhesion to endothelial cells. MCF7 cells were either untreated or treated with 0.1 μg / ml recombinant plectin before performing the static adhesion assay. Treatment of MCF7 cells with recombinant plectin significantly reduces cell adhesion to endothelial cells. Data from three replicates; error bars represent standard error of the mean; statistical testing was performed by one-way ANOVA with Tukey's test. [Figure 7] Figure 7 shows that cell adhesion can be partially restored in cancer cells with plectin knockdown by EVs derived from "wild-type" cancer cells. Plectin was knocked down using siRNA in MCF7 cells (second column), demonstrating reduced adhesion to endothelial cells. Plectin knockdown MCF7 cells treated with EVs derived from wild-type MCF7 cells show partial restoration of adhesion to endothelial cells. Data from three replicates; error bars represent standard error of the mean; statistical test performed was one-way ANOVA with Tukey's test. [Figure 8] Figure 8 shows that MCF7 cells have more HPA-binding proteins than BT474 cells. Lysates from the two breast cancer cell lines were subjected to SDS-PAGE before Western blotting using biotinylated HPA as a probe and anti-GAPDH antibody as a loading control. MCF7 lysates contained more HPA-binding bands than lysates from BT474 cells, which only had one HPA-binding band at 72 kDa. [Figure 9] Figure 9 shows a positive correlation between the HPA-binding capacity of cells and the HPA-binding capacity of EVs produced by those cells. Nanoview analysis of MCF7 and BT474 cells and the EVs produced by these cells. EVs from an HPA-negative BC cell line (BT474, left) and an HPA-positive BC cell line (MCF7, right) were labeled with fluorescently conjugated antibodies (first column = CD81, second column = CD9, third column = HPA). BT474 EVs were found to be HPA-negative, while MCF7 EVs were HPA-positive. MigG refers to the negative control, indicating the level of nonspecific binding. [Figure 10] Figure 10 shows that blocking cell surface beta-actin reduced breast cancer cell adhesion to endothelial cells. MCF7 cells were untreated or treated with antibodies to beta-actin or GAPDH. GAPDH was used as a control. Compared to untreated MCF7 BC cells, cells incubated with anti-beta-actin antibody adhered significantly less to endothelial cells. Data from three replicates; error bars are SEM. [Figure 11] Figure 11 shows that cell surface beta-actin levels correlate with HPA-binding / metastatic potential. Flow cytometry was used to detect surface actin. (A) Surface beta-actin expression in highly metastatic and HPA-positive MCF7 BC cells is greater than that in non-metastatic and weakly HPA-positive BT474 cells and moderately invasive and moderately HPA-positive ZR751 BC cells. Surface beta-actin expression in highly metastatic and HPA-positive MCF7 BC cells is comparable to that in invasive and highly HPA-positive T47D BC cells. (B) Surface beta-actin expression in MCF7 and BT474 cells is much higher than that in HPA-negative, normal breast cells (HME). [Figure 12-1] Figure 12 shows that metastatic breast cancer cells (MCF7) have more actin on their surface than non-metastatic breast cancer cells (BT474). Data were collected by flow cytometry. Pa refers to an actin antibody targeting pan-actin. IgG refers to a negative control showing the level of non-specific binding. No antibody (no anti) refers to no antibody added to show background autofluorescence. [Figure 12-2] Same as above [Figure 13-1] Figure 13 shows that metastatic cancer cells have more HPA on their surface than non-metastatic cancer cells. Data were collected by flow cytometry for pairs of metastatic and non-metastatic cell types: breast cancer (MCF7 is metastatic, BT-474 is non-metastatic), colorectal cancer (SW480 is metastatic, HT-29 is non-metastatic), and lung cancer (A539 is metastatic, NCI-H322 is non-metastatic). [Figure 13-2] Same as above [Figure 14] Figure 14 shows that metastatic cell plasma membranes have more actin than non-metastatic cells. Data were obtained by dot blot from probed membrane extracts of cell lines. ACTB is an antibody that recognizes actin beta, and ACTG is an antibody that recognizes actin gamma. Results are expressed as the intensity of the signal adjusted for background and normalized to the signal from the total membrane. [Figure 15] Figure 15 shows that lung metastatic cancer microvesicles have more actin than those derived from non-metastatic cells. Data were obtained by dot blot. The data show the binding levels of various different antibodies to microvesicles derived from either metastatic lung cancer (A549) or non-metastatic lung cancer (NCI-H322). For each cancer type, bars 1, 5, and 6 represent the control (IgG), Gm130, and no antibody, respectively, while bars 2, 3, and 4 represent three different clones of beta-actin antibodies. The results shown clearly demonstrate that microvesicles derived from metastatic lung cancer cells have more membrane-bound actin. Results are expressed as the intensity of the signal adjusted for background and normalized to the signal without primary antibody. [Figure 16-1]Figure 16 shows that pancreatic metastatic cancer microvesicles have more actin than non-metastatic cells. Data were obtained by dot blot. Figure 16A—Data shows the binding levels of various different antibodies to microvesicles derived from either metastatic pancreatic cancer (MIAPaCa2) or non-metastatic pancreatic cancer (BXPC-3). For each cancer type, bars 1, 5, and 6 are control—IgG, Gm130, and no antibody, respectively, while bars 2, 3, and 4 represent three different beta-actin antibodies. The results shown clearly demonstrate that microvesicles derived from metastatic pancreatic cancer cells have more membrane-bound actin. Figure 16B—Data shows the binding levels of various different antibodies to microvesicles derived from either metastatic pancreatic cancer (MIAPaCa2) or non-metastatic pancreatic cancer (BXPC-3). For each cancer type, bars 1, 3, and 4 are control—IgG, Gm130, and no antibody, respectively, while bar 2 is a gamma-actin antibody. The results shown clearly demonstrate that microvesicles derived from metastatic pancreatic cancer cells contain abundant membrane-bound gamma-actin. Results are expressed as the intensity of the signal adjusted for background and normalized to that without primary antibody. [Figure 16-2] Same as above [Figure 17-1]Figure 17 shows that metastatic breast cancer microvesicles have more actin than non-metastatic cells. Data were obtained by dot blot. Figure 17A—Data shows the binding levels of various different antibodies to microvesicles derived from either metastatic breast cancer (MCF7) or non-metastatic breast cancer (BT474). For each cancer type, bars 1, 5, and 6 are control-IgG, Gm130, and no antibody, respectively, and bars 2, 3, and 4 represent three different beta-actin antibodies. The results shown clearly demonstrate that there is more membrane-bound actin in microvesicles derived from metastatic lung cancer cells. Figure 17B—Data shows the binding levels of various different antibodies to microvesicles derived from either metastatic breast cancer or non-metastatic breast cancer. For each cancer type, bars 1, 3, and 4 are control-IgG, Gm130, and no antibody, respectively, and bar 2 is a gamma-actin antibody. The results shown clearly demonstrate that there is more membrane-bound actin. Results are expressed as the intensity of the signal adjusted for background and normalized to no primary antibody. [Figure 17-2] Same as above [Figure 18] Figure 18 shows that exosomes derived from metastatic breast cancer cells contain more actin than exosomes derived from non-metastatic breast cancer cells. Data were obtained by dot blot. Results are expressed as signal intensity normalized to the IgG control. [Figure 19] Figure 19 shows that a much greater proportion of exosomes from metastatic breast cancer cells are actin-positive than exosomes from non-metastatic breast cancer cells. Data were obtained by NanoFCM, a single-particle detection method that allows direct percentage of population comparison and supports dot blot averaging of total signal. Samples were probed with a pan-actin antibody. [Figure 20]Figure 20 shows that a greater proportion of exosomes derived from metastatic colorectal cancer cells are actin positive than exosomes derived from non-metastatic colorectal cancer cells. Data were obtained by NanoFCM, a single particle detection method that allows direct comparison of population proportions and supports dot blot averaging of total signal. Samples were probed with a pan-actin antibody. The metastatic proportion of positive EVs is expressed as a fold change compared to the non-metastatic proportion. [Figure 21] Figure 21 shows that a greater proportion of exosomes derived from metastatic colorectal cancer cells are HPA positive than exosomes derived from non-metastatic colorectal cancer cells. The data were obtained by NanoFCM as a single particle analysis method independent of Nanoview and support the findings of Figure 9 in different tissue types. [Figure 22] Figure 22 shows that a greater proportion of exosomes derived from (A) metastatic breast cancer cells and (B) metastatic colorectal cancer cells are gamma-actin positive than exosomes derived from comparable non-metastatic cancer cells. Data obtained by NanoFCM. DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention is based in part on the discovery of a positive correlation between the presence and / or level of membrane-bound actin on the surface of epithelial cancer cells and / or extracellular vesicles derived therefrom, and the metastatic potential of these cancer cells.

[0063] Therefore, determining the presence of actin on cell surface or membrane of extracellular vesicle, or the increase in actin level, can allow clinicians to accurately identify the metastatic potential of cancer cells, and classify the stage of cancer and / or diagnose prognosis.This allows identifying breast cancer patients at a later stage, or those who may develop metastasis or may have already developed metastasis, so that they can receive earlier and more appropriate treatment.Method can also be used to monitor patient's response to treatment and / or monitor disease progression.

[0064] Furthermore, the present invention may allow patients to avoid unnecessary lymph node biopsies. For example, the present invention can be performed on cells derived from tissue obtained from or around a primary tumor, such as breast tissue from an initial biopsy used to diagnose breast cancer, or breast tissue after removal during treatment. Additionally or alternatively, when detecting or determining the level of actin in the membrane of extracellular vesicles, the present invention can be performed on readily available biological fluids without the need for a biopsy. The present invention may enable the diagnosis, prognosis, and / or identification of patients who require specific treatments quickly, minimally or non-invasively, and / or prior to surgical intervention, as well as the determination of the metastatic potential of cancer, disease staging.

[0065] Those skilled in the art will understand that several methods or techniques in the art can be used to detect or measure the level of membrane-bound actin. Suitable techniques include immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, lateral flow assays, Western blots after biochemical or mechanical separation / isolation of membranes, dot blots, slot blots, and mass spectrometry. In any embodiment, actin detection may be direct or indirect, for example, using a primary detection reagent that detects actin and a secondary detection reagent that specifically detects the primary detection reagent. The secondary detection reagent may be labeled. The present invention can therefore be widely adopted in many hospitals or laboratories around the world without requiring highly specialized equipment or training.

[0066] In one embodiment, the level of actin may be detected by forming a complex between actin and a detection agent, and then detecting the actin / detection agent complex.

[0067] Those skilled in the art will appreciate that several methods or techniques in the art can be used to isolate EVs from a sample (reviewed in Konoshenko et al., 2018, BioMed. Res. Intl.).

[0068] Actin referred to herein is a protein that is endogenous to the host species.For example, in humans, actin can refer to beta-actin, a protein encoded by the ACTB gene.Actin can refer to alpha-actin, a protein encoded by the ACTA1 or ACTA2 gene.Actin can refer to gamma-actin, a protein encoded by the ACTG1 or ACTG2 gene.

[0069] As used herein, "membrane-bound actin" refers to actin, such as beta-actin, present or detectable on the surface of a cell, preferably in / on the plasma membrane of a cell, or present or detectable on the surface of an extracellular vesicle. The actin may have post-translational modifications, such as glycosylation markers. The actin may be detectable without the need for chemical or mechanical disruption of the cells and / or EVs. The actin may be detectable in the membrane fraction of mechanically and / or chemically disrupted cells and / or EVs, where the fraction contains only the plasma membrane of the cells or EVs. The actin detected or measured may be an integral membrane protein or a peripheral membrane protein that can interact with an integral membrane protein.

[0070] "Likely to develop or have developed metastases," as used herein, refers to an increased likelihood of developing or already having developed metastases compared to a subject that does not have the required characteristics described in an aspect and / or embodiment of the present invention.

[0071] As used herein, the term "higher" can refer to a number that is at least about 5% or more, at least about 10% or more, at least about 20% or more, at least about 50% or more, at least about 100% or more, at least about 200% or more, at least about 300% or more, at least about 400% or more, at least about 500% or more, at least about 1000% or more, at least about 2000% or more, at least about 5000% or more, at least about 10000% or more, at least about 20000% or more, at least about 50000% or more, at least about 100000% or more, at least about 500000% or more, or at least about 1000000% or more higher than the level of membrane-bound actin in a standard sample. Alternatively, the term "higher" can refer to a number that is at least about 1-fold or more, at least about 2-fold or more, at least about 3-fold or more, at least about 4-fold or more, at least about 5-fold or more, at least about 10-fold or more, at least about 20-fold or more, at least about 50-fold or more, at least about 100-fold or more, at least about 250-fold or more, at least about 500-fold or more, at least about 1000-fold or more, at least about 5000-fold or more, at least about 10000-fold or more, at least about 20,000-fold or more, at least about 50,000-fold or more, at least about 100,000-fold or more, or at least about 500,000-fold or more higher than the level of membrane-bound actin in a standard sample.

[0072] As used herein, the terms "actin-binding polypeptide" and "EpCAM-binding polypeptide" can refer to any polypeptide that binds to actin or EpCAM, respectively. Such polypeptides may be T cell receptors, or antibodies or antigen-binding fragments thereof, including but not limited to Fv, Fab, Fab', and F(ab')2 fragments.

[0073] As used herein, the term "sample obtained from a subject" refers to a sample of biological material obtained from a subject, e.g., a human subject, including tissue, tissue samples, biopsies, cell samples, tumor samples, fecal samples, and samples of biological fluids, e.g., plasma, serum, blood, urine, lymph, ascites, saliva. In one embodiment, the sample is a tissue sample, e.g., from a biopsy of breast tissue from the subject. In one embodiment, the sample is a blood sample obtained from the subject.

[0074] The terms "patient" or "subject," as used interchangeably herein, refer to any mammal, e.g., a human. Non-limiting examples of non-human mammals include non-human primates, dogs, cats, mice, rats, guinea pigs, rabbits, poultry, pigs, horses, cows, goats, sheep, etc.

[0075] In this disclosure, samples referred to as "biopsies" can be obtained by conventional methods using methods well known to those skilled in the relevant medical arts. Methods for obtaining biopsy samples include dividing tumors into large masses, or microdissection, or other cell isolation methods known in the art. Tumor cells can also be obtained by cytology via aspiration using a small-gauge needle. To simplify sample storage and handling, samples can be fixed in formalin and immersed in paraffin, or first frozen and then immersed in a tissue-freezing medium such as OCT compound by immersion in a cryogenic medium that allows for rapid freezing. Other methods for storing and / or processing samples are known to those skilled in the art.

[0076] A sample may be manipulated to isolate a cell type of interest or to isolate EVs. For example, a marker for a cell type of interest in a tissue sample or biopsy may be used to isolate or separate such cells. One example is the use of EpCAM-binding polypeptides to identify and / or separate epithelial cells from biopsied tissue before membrane-bound actin, such as beta-actin, is detected or its level determined. Another example is the use of EpCAM-binding polypeptides to identify and / or separate EVs derived from epithelial cells from other EVs and / or biological materials in a sample.

[0077] A standard sample may refer to an equivalent sample, for example, an equivalent sample from the same tissue and / or cell type as the sample obtained from the subject or another subject, or a cell line that closely resembles the sample type obtained from the subject.

[0078] A "negative standard sample" may refer to a standard sample that is known to be non-cancerous or cancerous / immortalized but has not or will not develop metastasis. For example, the negative standard sample may be a corresponding sample from the area surrounding the subject's tumor, such as a biopsy sample, or corresponding tissue from one or more healthy subjects. The negative standard sample may also be a cell line known to be non-metastatic in nature and with the same or similar properties as the sample obtained from the subject, such as a breast epithelial cell line known to have low invasiveness as a negative standard sample for breast tissue samples obtained from the subject, such as BT474 cells [Lasfargues et al., 1978, J. Nat. Cancer Institute, 61(4): 967-978], or HMT3522 human breast epithelial cells isolated from benign fibrocystic breast tissue [Briand et al., 1987, In Vitro Cell and Dev. Biol., 23(3): 181-188].

[0079] When the presence or level of membrane-bound actin is detected or measured in EVs, a negative standard sample may refer to EVs isolated from one or more subjects who do not have cancer, or one or more subjects who have / had cancer and who have not or have not developed metastasis, or from a comparison cell line known to have low invasiveness as described above.

[0080] Thus, the level of membrane-bound actin detected or measured in such a negative standard sample serves as a negative control.

[0081] A "positive standard sample" may refer to a standard sample that is known to be cancerous and / or has or may develop metastasis. For example, the positive standard sample may be a corresponding sample, such as a biopsy sample, from one or more subjects known to have metastasis. The positive standard sample may also be a cell line known to be invasive and / or metastatic in nature and having the same or similar properties as the sample obtained from the subject, for example, a breast epithelial cell line known to be highly invasive as a positive standard sample for breast tissue samples obtained from the subject, such as MCF7 cells [Soule et al., 1973, J. Nat. Cancer Institute, 51(5): 1409-14-16].

[0082] When the presence or level of membrane-bound actin is detected or measured in EVs, a positive standard sample may refer to EVs isolated from one or more subjects who have / had cancer and who have developed metastasis, or from a comparison cell line known to be highly invasive, as described above.

[0083] Thus, the level of membrane-bound actin detectable or measured in such a positive standard sample serves as a positive control.

[0084] The sample that is derived from the subject diagnosed with cancer and that measures a higher level than the level of membrane-bound beta actin in the standard sample can be identified as being more likely to develop or develop metastasis, or being more likely to be diagnosed with metastatic cancer.The appropriate treatment regimen for the patient can then be identified and provided.

[0085] One skilled in the art can consult databases that record levels of membrane-bound actin in a given sample type from subjects with confirmed or documented no metastases, or from cell lines known to be metastatic or non-metastatic in nature, and one skilled in the art can readily identify such metastatic or non-metastatic cell lines as needed.

[0086] Those skilled in the art will understand that preferred features of any one embodiment and / or aspect of the invention may be applied to all other embodiments and / or aspects of the invention.

[0087] material and method

[0088] [Table 1]

[0089] [Table 2]

[0090] Protein extraction and quantification from endothelial cells HUVEC cells were grown in T175 flasks to confluence and then treated with 10µg / ml TNFα in complete growth medium in a humidified atmosphere at 37°C for 2 hours. Cells were washed with ice-cold PBS. The cells were pelleted by brief centrifugation, the supernatant discarded, and the pellet resuspended in 1x RIPA buffer supplemented with 10µl / ml of protease inhibitor cocktail and placed in an end-over-end mixer at 4°C for 30 minutes. After this time, the tube was centrifuged at 13,000 RPM for 20 minutes at 4°C. The supernatant was transferred to a new 1.5ml tube and the pellet discarded. Extracted protein was quantified using a BCA assay.

[0091] SDS-PAGE and Western blot 10 μg of protein sample was used for SDS-PAGE using 3 μl of Laemmli sample buffer and 1 μl of 1M DTT. This was mixed, heated to 100°C for 10 minutes, and then placed on ice. The sample, along with the Precision Plus Protein Dual Color Standard, was run on a mini PROTEAN TGX stain-free precast gel (12%) in 1x TGS buffer. The gel was run at 100 volts for 2 hours. Protein was transferred using a Trans-Blot Turbo Midi PVDF transfer pack. The gel was placed on top of the membrane, sandwiched between the layers, and then placed in a Transblot Turbo machine at the high MW setting. The membrane was washed three times with TBST (0.05% Tween) for 5 minutes on a rocking platform. The membrane was then blocked by incubation with 5% BSA / TBST on a rocking platform at RT for 2 hours. The blocked membrane was incubated overnight at 4°C with 10 ml of 1 μg / ml GalNAc-BSA-biotin in 5% BSA / TBST or 10 ml of 1 μg / ml BSA-biotin in 5% BSA / TBST. After washing the membrane three times for 5 minutes with 5% BSA / TBST, the membrane was incubated for 1 hour at room temperature with 10 ml of 5 μg / ml streptavidin-HRP in 5% BSA-TBST. The membrane was washed twice for 5 minutes with 5% BSA-TBST, then once for 15 minutes. HRP substrate was prepared in a 1:1 ratio (Clarity+Clarity Max Western ECL Substrate, BioRad, 170-5060) and mixed. This was added to the membrane and imaged using a Chemidoc® transilluminator.

[0092] 2D PAGE Total activated HUVEC protein at a concentration of 1 μg / µL in 1x RIPA buffer supplemented with protease inhibitors was purified for use in 2D PAGE using the GE Healthcare 2D Clean-up Kit according to the manufacturer's instructions (80-6484-51). Samples were prepared for 2D PAGE using the BioRad ReadyPrep 2-D Starter Kit according to the manufacturer's instructions (163-2105). This allowed samples to be absorbed onto 11 cm IPG strips and then subsequently separated by isoelectric focusing with Ettan IPGphor 3 for 16 hours. The IPG strips were washed, rehydrated, and then placed into a Criterion™ XT Bis-Tris precast gel. The 12% gel was loaded into the tank, standards were added, and the gel was sealed with agarose. The agarose was fixed with 1x MOPS buffer before loading into the tank. 250 μl of NuPage Antioxidant (Thermo, NP0005) was added per gel. Gels were run at 200 volts for 70 minutes. The blotting gel was removed from its cassette and washed twice with dH2O. The remaining steps were carried out at room temperature. The gel was placed in Invitrogen™ Novex™ SimplyBlue™ SafeStain (Thermo, LC6065) and placed on a rocking platform for 1 hour. The gel was then destained with dH2O overnight on a rocking platform. The gel was blotted using the iBlot™ 2 Dry Blotting System. The membrane was washed briefly with dH2O and then blocked with 5% Marvel / TBST on a rocking platform for 1 hour. The membrane was incubated with 1 μg / ml GalNAc-BSA-biotin in 5% BSA / TBST on a rocking platform for 2 hours, then washed with TBST three times for 10 minutes each on a rocking platform. The membrane was incubated with 5 μg / ml streptavidin-peroxidase in 5% BSA / TBST for 1 hour on a rocking platform, then washed three times with TBST for 10 minutes each on a rocking platform. ECL HRP substrate was prepared and added to the membrane, which was then imaged using a Chemidoc® transilluminator.

[0093] Preparation of gels for mass spectrometry The 1D gel was stained with Coomassie blue and the 2D gel with silver stain. Bands and dots of interest were then excised from the 1D and 2D gels using the blots as references. The gels were then sent for mass spectrometry at Porton Biopharma Ltd.

[0094] EV extraction from cancer cells Cancer cells were grown in T175 flasks to 70% confluency and fed with 25 ml / flask of EV-depleted medium (DMEM containing 10% FBS, previously spun at 120,000 g for 16 hours to remove any EVs present) and allowed to condition for 48 hours. The medium was removed and spun at 300 x g for 5 minutes, then at 16,000 x g for 20 minutes at 4°C. The medium was removed from the flasks into "normal" 50 ml tubes (Greiner Bio-One, 227285) and spun at 300 x g for 5 minutes. For microvesicles, the supernatant was transferred to a high-speed (green or orange top) 50 ml tube (Alpha Laboratories, CT1120), the pellet was discarded, and the tube was centrifuged at 16,000 g for 20 minutes. The supernatant was transferred to a normal 50 ml tube—this is the EV portion. The pellet contains the microvesicles. The microvesicles were pooled and resuspended in 20 ml of PBS. This is then spun again at 16,000 g for 20 minutes. The supernatant is poured off from the tube and the pellet is resuspended in a volume of PBS leaving approximately 300 μl. A 0.1% BSA (Sigma, A7906-100G) solution in PBS (40 μl of 10% BSA in 40 ml PBS) is used to block a 0.22 micron (Fisher, fdr-050-071n) filter and the medium is filtered through this filter. The medium is added to a Vivaspin 20 100 kDa concentrator (Fisher, 10774797). The medium is concentrated by spinning at 3,000 g (swinging bucket - 25 min) or 5,000 g (fixed angle rotor - 20 min step).

[0095] For exosomes, the supernatant was filtered through a 0.22 μm filter blocked with 0.1% BSA. The supernatant was concentrated to 500 μl using a Vivaspin 20, 100 kDa concentrator. An SEC column (BioRad, 7321010) was prepared by adding 14 ml of Sepharose and 10 ml of PBS, which was allowed to precipitate for 2 hours, followed by the addition of a column support and PBS flowing through. The column was then washed three times with 10 ml of PBS. 500 μl of sample was added to the column support, allowed to adsorb, and then 10 ml of PBS was added. 2.5 ml of flow-through was collected and discarded, and then 2 ml of flow-through (containing EVs) was collected. EVs were quantified using a Particlematrix™ particle analyzer. EVs may then be stored in a refrigerator for short-term use.

[0096] Endothelial static adhesion assay with or without EVs Coverslips containing activated endothelial cells were prepared as described above. MCF7 breast cancer cells were grown to 70% confluency in T75 flasks and treated with 10 mg / ml 8-hydroxypyrenetrisulfonic acid (HTPS) in complete medium for 2 hours at 37°C and 5% CO2. After this time, the HTPS was removed, and the cells were washed five times with 10 ml of PBS until the washes ran clear. Cells were scraped and counted. A 20,000 cell / ml solution was prepared. MCF7 cells were treated with either 500 μl of EV or 500 μl of PBS and mixed for 10 minutes at room temperature. HUVEC cells were treated with either 500 μl of EV or 500 μl of PBS for 30 minutes at 37°C and 5% CO2. EV or PBS was removed from the cells, and MCF7 cells were added at 20,000 cells / ml per well. The cells were then incubated for 10 minutes. The cells were then removed, and the wells were gently washed with warm PBS to remove any unbound cells. The wells were then fixed with 4% PFA for 10 minutes at room temperature. The fixative was removed, the wells were washed three times with PBS, and then coverslips were mounted using Fluoromount™. The total adherent cells per coverslip were counted.

[0097] Coverslips containing activated endothelial cells were prepared as described above. MCF7 breast cancer cells were grown in T75 flasks to 70% confluency and treated with 10 mg / ml HTPS in complete medium for 2 hours at 37°C and 5% CO2. The HTPS was removed, and the cells were washed five times with 10 ml of PBS until the wash solution ran clear. The cells were scraped and counted. A concentration of 20,000 cells / ml was prepared. MCF7 cells were treated with either 0.1 μg / ml recombinant plectin (2b Scientific SKU RPC754Hu01 (residues Asp 175 to Pro 400 of human plectin Uniprot ID Q15149, corresponding to the actin-binding domain) in PBS or PBS for 10 minutes at room temperature. The recombinant plectin or PBS was removed from the cells, and 20,000 cells / ml MCF7 cells were added per well and incubated for 10 minutes. The cells were then removed, and the wells were gently washed with warm PBS to remove any unbound cells. The wells were then fixed with 4% PFA for 10 minutes at room temperature. The fixative was removed, and the wells were washed three times with PBS and then coverslips were mounted using Fluoromount. The total adherent cells per coverslip were counted.

[0098] Endothelial static adhesion assay with or without plectin silencing and EV rescue MCF7 and HUVEC cells were grown in 24-well cell culture plates to 70% confluency and treated with Dharmafect™ transfection reagent and 5 μM plectin siRNA or scrambled negative control for 24 hours. Some HUVEC and MCF7 cells were untreated. HUVEC cells were activated by treatment with 10 μg / ml TNFα in complete medium for 2 hours at 37°C and 5% CO2. MCF7 breast cancer cells were grown in T75 flasks to 70% confluency and treated with 10 mg / ml HTPS in complete medium for 2 hours at 37°C and 5% CO2. The HTPS was removed, and the cells were washed five times with 10 ml of PBS until the wash flowed clear. Cells were scraped and counted. A concentration of 20,000 cells / ml was prepared. MCF7 cells were treated with either 500 μl of EV isolated using the extraction method outlined above or 500 μl of PBS for 10 minutes at RT. EV or PBS was removed, and 1 ml of MCF7 cells was added to the HUVEC cells. After incubation at 37°C and 5% CO2 for 10 minutes, the cells were removed and the wells were gently washed with warmed PBS to remove any unbound cells. The wells were then fixed with 4% PFA for 10 minutes at room temperature. The fixative was removed, and the wells were washed three times with PBS, followed by mounting a coverslip using Fluoromount. The total adherent cells per coverslip were counted.

[0099] Dot blot assay Membrane isolation was performed using the Plasma Membrane Protein Extraction Kit (Abcam, ab56400) as described in the exosome / MV binding protocol. Using a narrow-nosed pipette tip, 2 μl of sample or buffer solution was spotted onto a nitrocellulose membrane. The solution was applied slowly to minimize the area penetrated by the solution (typically 3-4 mm). The membrane was allowed to dry for approximately 30 minutes. Each membrane was placed inside a 6-well plate. The membrane was blocked by immersion in Carbo-free blocking solution (2BScientific, SP-5040-125) diluted in dH2O with 5% marvel in TBS-T, or 0.1% Tween 20 for HPA (1 hour at room temperature). Biotinylated HPA in Carbo-Block-T (10 μg / ml, or 1:1,000 for all antibodies) and 5% marvel in TBS-T were incubated at 37°C for 2 hours or at 4°C overnight. Wash the membrane three times with TBS-T (3 x 5 min). Incubate with biotinylated anti-Ms (Vector Labs, BA-2000-1.5) or biotinylated anti-Rb (Vector Labs, BA-1000-1.5) in 5% marvel TBST at 37°C for 1 hour. Wash the membrane three times with TBS-T (3 x 5 min). Incubate with 1 μg / ml streptavidin-peroxidase in 5% marvel TBS-T, or Carbo-Block-T for HPA. Wash three times with TBS (3 x 10 min). Mix ECL substrates A and B (BioRad Clarity kit). Incubate each membrane with ECL reagent for 1 minute, remove excess solution from the surface, and image as a chemi-high sensitivity blot.

[0100] HPA probing after Western blot of MCF7 and BT474 breast cancer cells MCF7 and BT474 cell lines were grown to 70% confluence in T75 flasks as previously described, and their proteins were extracted, quantified, subjected to SDS-PAGE, and then blotted. Membranes were probed with biotinylated HPA or anti-GAPDH antibodies.

[0101] Nanoview EVs were extracted from MCF7 and BT474 breast cancer cells using SEC (previously described) and labeled with fluorescently conjugated antibodies (CD81 and CD9) and HPA lectin according to standard protocols for R100 and analysis by Nanoviewer.

[0102] Preparation of activated endothelial cells on coverslips Sterile 13mm D glass coverslips were placed in the wells of a 24-well plate and seeded with 100,000 HUVEC cells. The cells were incubated at 37°C in a 5% CO2 atmosphere until a monolayer was formed. HUVEC cells were activated by treatment with 10µg / ml TNFα in complete medium at 37°C in 5% CO2 for 2 hours.

[0103] Endothelial adhesion assay with or without actin antibody inhibition Coverslips containing activated endothelial cells were prepared as described above. MCF7 breast cancer cells were grown in T75 flasks to 70% confluency and treated with 10 mg / ml HTPS in complete medium for 2 hours at 37°C and 5% CO2. After this time, the HTPS was removed, and the cells were washed five times with 10 ml of PBS until the wash solution ran clear. The cells were scraped and counted. A 20,000 cell / ml solution was prepared. MCF7 cells were treated with either 1:1,000 actin IgG (Abcam ab8227), 1:1,000 GAPDH IgG, or PBS for 30 minutes at room temperature. The antibody or PBS was removed from the cells, and 20,000 cells / ml of MCF7 cells were added per well and incubated for 10 minutes. The cells were then removed, and the wells were gently washed with warm PBS to remove any unbound cells. The wells were then fixed with 4% PFA for 10 minutes at room temperature. The fixative was removed, the wells were washed three times with PBS, and then coverslips were mounted using Fluoromount. The total adherent cells per coverslip were counted.

[0104] Flow cytometry For viability testing, cultured cells were harvested at approximately 70% confluence and detached from the bottom of the flask using Accutase. Cells were collected by centrifugation (1400 rpm, 5 min). For analysis, 0.5 × 10 6 Cells were used at a concentration of 1 / ml. Cells were washed with PBS and incubated with HPA antibody (7.5 μg / ml) in 3% BSA / TBS in the dark at RT for 10 minutes to detect the HPA GalNAc epitope on the cell surface or 1 / 50 actin (clone SP124). Cells were treated with propidium iodide (10 μg / ml) to gate the live population. The wavelengths of the 647 conjugate were quantitatively recorded in the APC-A channel using a 13-color, 4-laser CytoFLEX S NVBR flow cytometer equipped with 375 nm, 405 nm, 488 nm, and 638 nm lasers and operated using CytExpert software.

[0105] Each sample was analyzed in triplicate, and the determination of the surface GalNAc epitope level of the analyzed cells was based on the average fluorescence signal from the APC detector. Gating: 1) FSC-H vs. FSC-A - doublet elimination; clumped cells will have twice the area and height. 2) Pi viability gate - distinguish live cells from dead cells. Dead cells become permeable, and therefore PI binds to the nucleus and therefore has more fluorescence (we also included our own unstained Pi control to demonstrate differences). 3) antibody / lectin channel of interest to determine peak shifts between different conditions.

[0106] For fixed cells, cells were harvested and resuspended in cold PBS. Cells were incubated with anti-actin Ab (Proteintech catalog: 60008-1-Ig) for 1 hour at 4°C. Cells were then washed and resuspended in PBS before flow cytometry.

[0107] NanoFCM The NanoFCM is a 40 nm to 1 micron flow cytometer focused on exosome analysis, independently reviewed at https: / / onlinelibrary.wiley.com / doi / epdf / 10.1002 / jev2.12044 and https: / / doi.org / 10.1002 / jev2.12079. All of these results were performed by a company based in Nottingham, UK. Exosomes were extracted as previously described. The instrument was configured with the following settings: SN-N30E, V1.11 software, sample pressure - 1 Kpa, laser - 10 / 50 mW 488 @ 20 / 100 mW 640, SS attenuation - 10%, and minimum width - 0.3 ms. Calibration was performed using silica nanoparticles, and EV concentration was quantified. PBS, antibody controls, and EV unstained and stained samples were optimized. For actin antibodies, approximately 2e8-2e10 particles were incubated with the antibody / stain at 1 / 50-1 / 500 for 30 minutes at room temperature and then diluted 1 / 5-1 / 50 in PBS. For HPA antibodies, approximately 2e8 particles were incubated with the antibody / stain at 1 / 500 for 30 minutes at room temperature and then diluted 1 / 20-1 / 50 in PBS. For high background, samples were ultracentrifuged at 110k x g for 1 hour and resuspended in 50 μl. The supernatant was removed and the sample resuspended in 50 μl PBS. Events gated between approximately 40-199 nm were recorded for 1 minute and reported as the number of exosomes with a positive signal as a percentage of the total exosomes gated at that size.

[0108] [Table 3] [Example]

[0109] Example 1 - Identification of membrane-bound actin as a biomarker of metastatic potential Binding of the snail-derived lectin, Hemicentrotus pallidus agglutinin (HPA), to breast cancer (BC) and other epithelial cancers is associated with metastatic potential and poor patient prognosis [Brooks S., 2000, Histology and Histopathology, 15(1): 143-158]. Although the specific binding site of HPA is unknown, it has been shown to bind to a series of glycoproteins bearing terminal α-GalNAc, the monosaccharide for which HPA has the highest affinity. One of these glycoproteins is the cancer-associated Tn antigen (Ser / Thr-GalNAc). This initial structure, typically produced by O-linked glycosylation and always further elaborated, is frequently present in cancer (Brooks SA & Leathem AJC, 1995, British J. Cancer, 71: 1033-1038).

[0110] Highly HPA-positive BC cells adhere to endothelial cells significantly more than weakly HPA-positive or HPA-negative BC cells in static adhesion assays. This assay mimics metastatic cancer cell arrest in narrow capillaries. For cancer cells to successfully metastasize, they must bind to and extravasate into blood vessels. Incubation of cells with HPA or BSA-GalNAc inhibits cell binding, indicating that binding is mediated through this glycosylation marker.

[0111] Identification of a receptor on endothelial cells that mediates HPA-positive cell adhesion. To identify putative receptors on endothelial cells that recognize HPA-linked glycans on BC cells, total protein was extracted from endothelial cells activated with TNFα (a proinflammatory cytokine known to upregulate endothelial receptors). Proteins were subjected to SDS-PAGE and then blotted. GalNAc-BSA-biotin (GalNAc is the sugar for which HPA has nominal maximal specificity) was used as a probe to identify the endothelial protein of interest. As a negative control, membranes were also probed with BSA-biotin. As illustrated in Figure 1, several bands were observed when the membrane was probed with GalNAc-BSA-biotin, but no bands were observed when the membrane was probed with BSA-biotin.

[0112] Using GalNAc-BSA-biotin as a probe, several bands were consistently identified. From these, five gel fragments were selected and excised for mass spectrometry (Figure 2). Because the separation was only by size, the bands observed in the blot from the 1D gel could represent multiple proteins. To better resolve the bands, 2D-PAGE was used to separate the proteins by both size and pH. The 2D gel was blotted and probed with GalNAc-BSA-biotin. Thirteen spots were identified as spots of interest and excised from the 2D PAGE gel (Figure 3).

[0113] Mass spectrometry data revealed several proteins in the gel fragments from both 1D- and 2D-PAGE, some of which overlapped between the two. One of the identified proteins was plectin, which is normally an intracellular scaffolding protein. However, Shin et al. (2013) showed that plectin can be localized to the cell membrane through exosome secretion. To investigate this, EVs were isolated from BC cells and incubated with either BC cells or endothelial cells, or both, before static adhesion assays. Incubation of both BC cells and endothelial cells with EVs was found to significantly increase BC cell binding to endothelial cells compared with no treatment, BC cell treatment alone, or endothelial cell treatment alone (Figure 4). This finding suggests that EVs may play an active role in supporting BC cell adhesion to endothelial cells.

[0114] To further investigate this, we used siRNA to knock down plectin in either BC cells, endothelial cells, or both. In all cases, binding of BC cells to endothelial cells was significantly reduced compared to untreated and scrambled siRNA-treated cells (referred to as negative in the graphs), suggesting that plectin is required for BC cell binding to endothelial cells (Figure 5).

[0115] Confirmation that receptors on endothelial cells are specific for glycosylated markers on BC cells and mediate cell binding To confirm that the receptor on endothelial cells is specific for glycosylated markers on BC cells, we used a recombinant plectin fragment (containing the actin-binding domain of plectin, which can bind to all actin isoforms (Fontao et al., 2001, J. Cell. Sci., 114: 2065-2076)) to block BC cells by incubation for 10 minutes before the addition of endothelial cells. Cell adhesion to endothelial cells was significantly reduced compared with untreated control cells (Figure 6). We hypothesized that the reduced binding observed when BC cells were incubated with recombinant plectin was the result of plectin binding to HPA-positive glycosylated markers, thereby "capping" the BC cells and preventing their binding to endothelial cells.

[0116] Investigating how BC cell-derived EVs increase adhesion to endothelial cells The findings of plectin knockdown in BC cells, combined with the observation that BC cell-derived EVs can increase BC cell adhesion to endothelial cells, led to the hypothesis that EVs may deliver plectin from BC cells to the surface of endothelial cells, thereby increasing the effective "landing platform" for BC cell binding. To begin testing this hypothesis, we performed a "rescue" experiment. Knockdown of plectin in BC cells reduced BC cell binding to endothelial cells (Figure 7). Interestingly, treating endothelial cells with EVs derived from control BC cells partially rescued this reduced binding. This is consistent with the hypothesis that EVs derived from normal cells (which contain plectin) provide a "landing platform" that allows binding to occur.

[0117] We tested the above hypothesis using two previously reported model BC cell lines: one highly HPA-positive (MCF7) and one weakly HPA-positive (BT474). Proteins were extracted from the two cell lines, subjected to SDS-PAGE, and then blotted and probed with HPA. The HPA-positive BC cell line (MCF7) had a broader range of HPA-positive glycoproteins than the weakly HPA-positive BC cell line (BT474), which only had a single band at 75 kDa, thereby confirming the use of this model system (Figure 8).

[0118] Using NanoView, we demonstrated that HPA-binding positive cells produce HPA-binding positive EVs, and similarly, HPA-binding negative cells produce HPA-binding negative EVs. To do this, we isolated EVs from two BC cell lines (one HPA-binding negative, the other HPA-binding positive) by labeling two EV populations with two commonly used EV markers and a fluorescently conjugated antibody specific for HPA, allowing us to quantify the amount of EVs that possess "HPA-binding positivity." EVs derived from HPA-binding negative BC cells (BT474 left, Figure 9) were found to be HPA-binding negative, while EVs derived from HPA-binding positive BC cells (MCF7 right, Figure 9) were HPA-binding positive. Notably, HPA predominantly colocalized with CD9 in vesicles—up to 45% of CD9-positive vesicles are HPA-positive, compared to 13% of CD81-positive vesicles and 9% of CD63-positive vesicles. Due to overlap with the APC emission spectrum, the fluorescently conjugated antibody CD63 was not included in this experiment.

[0119] Identification of glycoproteins in BC cells that mediate binding to receptors on endothelial cells Cytoskeletal plectin has several binding partners that interact with its actin-binding domain (ABD), including beta-actin. To investigate whether glycosylated beta-actin is a potential binding partner of cell surface plectin, we performed blocking experiments using anti-beta-actin IgG (Abcam ab8227) to mask beta-actin for 30 min before incubation with endothelial cells. Compared to untreated control cells, blocking beta-actin significantly reduced cell adhesion, therefore suggesting that beta-actin is a binding partner of plectin and that this interaction contributes to BC cell attachment to endothelial cells (Figure 10).

[0120] Levels of surface actin in different cell lines Using a series of cells with known invasiveness and HPA-binding profiles, we determined the levels of surface actin by flow cytometry in different cell types, ranging from normal / HPA-negative to non-metastatic / weakly HPA-positive to highly metastatic / highly HPA-positive (Figure 11). HME cells are normal breast cells and are HPA-negative; BT474 are derived from a primary invasive ductal carcinoma without evidence of metastasis [Lasfargues et al. 1978, J. Nat. Institute, 61(4): 967-978] and are weakly labeled with HPA; ZR751 are derived from malignant ascites from an invasive primary ductal carcinoma—i.e., cells that had already metastasized (Engel et al. 1978, Canc. Res., 38, 3352-3364) and are moderately labeled with HPA; and T47D are derived from malignant pleural effusion from an invasive primary ductal carcinoma—i.e., cells that had already metastasized [Keydar et al. 1979, Eur J Cancer, 15 (5):65 9-70] and are highly labeled in the HPA; MCF7 are derived from malignant pleural effusions from invasive primary ductal carcinoma of the breast—i.e., cells that have already metastasized [Soule et al. 1973, J. Nat. Cancer Institute, 5 1(5): 1409-14-16] and are highly labeled in the HPA. Actin labeling appears to follow a similar trend as HPA labeling, in that increased surface actin is measured in more invasive / metastatic cells.

[0121] Figure 12 further demonstrates using flow cytometry that actin levels are higher on the surface of metastatic breast cancer cells (MCF7) than non-metastatic breast cancer cells (BT474). This data was generated using antibodies capable of detecting both beta and gamma actin. Figure 13 further demonstrates that metastatic cancers, including breast cancer MCF7, colorectal cancer HT-29, and lung cancer A539, ​​also exhibit elevated levels of HPA on their surface. These results further demonstrate that surface levels of both actin and HPA are increased in metastatic cancer cells, thus demonstrating their diagnostic value.

[0122] Figure 14 further supports increased surface levels of actin in metastatic cancer cells. The data compare non-metastatic and metastatic breast cancers and use antibodies to either beta-actin or gamma-actin, both of which show increased actin levels in metastatic cancer cells.

[0123] Levels of surface actin in microvesicles obtained from different cell lines Figures 15-17 compare actin levels on the surface of microvesicles obtained from metastatic and non-metastatic breast, lung, and pancreatic cancer cells. The results clearly show elevated levels of actin in microvesicles obtained from metastatic cells.

[0124] Levels of surface actin in exosomes obtained from different cell lines Figures 18-20 compare actin levels on the surface of exosomes obtained from metastatic and non-metastatic breast and colorectal cancer cells. The results clearly demonstrate elevated levels of actin in exosomes obtained from metastatic cells compared to non-metastatic cells. Figure 21 further demonstrates that exosomes obtained from metastatic cancer, specifically colorectal HT-29 cancer cells, also exhibit elevated levels of HPA on their surface. Figure 22 demonstrates that metastatic breast and colorectal cancer cells exhibit elevated levels of gamma-actin on their surface.

[0125] Consideration In conclusion, the data presented herein demonstrate that actin on the surface (membrane-bound) of epithelial cancer cells and / or extracellular vesicles derived therefrom can serve as a diagnostic or prognostic marker for the metastatic potential of cancer cells. Actin levels can also be used to monitor the response to specific cancer treatments, disease progression, and / or disease recurrence, and HPA levels can also be used as biomarkers, either alone or in combination with actin levels.

Claims

1. 1. A method for assisting in determining whether a subject diagnosed with cancer is likely to develop or has developed metastasis, comprising: a. detecting the level of membrane-bound actin in a sample obtained from a subject; b. comparing the level of membrane-bound actin in the sample obtained from the subject with the level of membrane-bound actin in a standard sample; and c. i) the standard sample is a positive standard sample and the level of membrane-bound actin in the sample obtained from the subject is approximately equal to or higher than the level of membrane-bound actin in the standard sample; or ii) if the standard sample is a negative standard sample and the level of membrane-bound actin in the sample obtained from the subject is higher than the level of membrane-bound actin detected or measured in the standard sample; Determining that a subject is likely to develop or has developed metastasis A method comprising:

2. 1. A method for aiding in diagnosing metastatic cancer in a subject, comprising: a. measuring the level of membrane-bound actin in a sample obtained from a subject; b. comparing the level of membrane-bound actin in the sample obtained from the subject with the level of membrane-bound actin in a standard sample; and c. i) the standard sample is a positive standard sample and the level of membrane-bound actin in the sample obtained from the subject is approximately equal to or higher than the level of membrane-bound actin in the standard sample; or ii) if the standard sample is a negative standard sample and the level of membrane-bound actin in the sample obtained from the subject is higher than the level of membrane-bound actin detected or measured in the standard sample; The subject is determined to have cancer metastasis. A method comprising:

3. 1. A method for aiding in prognosis in a subject diagnosed with cancer, comprising: a. measuring the level of membrane-bound actin in a sample obtained from a subject; b. comparing the level of membrane-bound actin measured in the sample with the level of membrane-bound actin in a standard sample; c. i) the standard sample is a positive standard sample and the level of membrane-bound actin in the sample obtained from the subject is approximately equal to or higher than the level of membrane-bound actin in the standard sample; or ii) if the standard sample is a negative standard sample and the level of membrane-bound actin in the sample obtained from the subject is higher than the level of membrane-bound actin detected or measured in the standard sample; Identifying patients as having a poor prognosis A method comprising:

4. 4. The method of claim 3, wherein the poor prognosis is associated with an increased likelihood of developing metastases and / or a decreased chance of survival.

5. 1. A method for assisting in identifying a patient diagnosed with cancer who may benefit from treatment with a known treatment for metastatic cancer, comprising: a. measuring the level of membrane-bound actin in a sample obtained from a subject; b. comparing the level of membrane-bound actin measured in the sample with the level of membrane-bound actin in a standard sample; c. i) the standard sample is a positive standard sample and the level of membrane-bound actin in the sample obtained from the subject is approximately equal to or higher than the level of membrane-bound actin in the standard sample; or ii) if the standard sample is a negative standard sample and the level of membrane-bound actin in the sample obtained from the subject is higher than the level of membrane-bound actin detected or measured in the standard sample; To determine whether a patient is likely to benefit from treatment with a known treatment for metastatic cancer A method comprising:

6. The method of any one of claims 3 to 5, wherein the level of membrane-bound actin in the sample obtained from the subject is determined to be higher than the level of membrane-bound actin in the standard sample when the level of membrane-bound actin in the sample obtained from the subject is at least about 50%, 75%, 100%, 150%, 200%, 300%, 500%, 1000%, 5000%, or 10000% higher than the level of membrane-bound actin in the standard sample.

7. The method of any one of claims 3 to 6, wherein the standard sample is an equivalent sample from a healthy patient who has not been diagnosed with cancer, or an equivalent sample known to be metastatic.

8. The method according to any one of claims 1 to 7, wherein the method is an ex vivo method or an in vitro method.

9. The method according to any one of claims 1 to 8, wherein the sample is a tissue biopsy and / or a blood sample and / or a saliva sample.

10. 10. The method of claim 9, wherein the tissue biopsy is a breast tissue biopsy.

11. The method according to any one of claims 1 to 10, wherein the detection or measurement of the level of membrane-bound actin is carried out by flow cytometry, immunohistochemistry, immunocytochemistry, Western blot, and / or ELISA.

12. A method for assisting in the decision of whether to treat a subject for metastatic cancer, comprising: a. The subject is determined to be likely to develop or have developed metastasis, or b. The subject is diagnosed with metastatic cancer, or c. The subject is assessed as having a poor prognosis, or d. The subject is determined to be likely to benefit from treatment for metastatic cancer. If so, the subject is determined to be a candidate for treatment of metastatic cancer.

13. 1. A kit for (a) determining whether a subject diagnosed with cancer is likely to develop or has developed metastasis, or (b) prognosing a subject diagnosed with cancer, or (c) diagnosing metastatic cancer in a subject, comprising: A kit comprising a means for detecting membrane-bound actin.

14. 14. The kit of claim 13, wherein the means for detecting membrane-bound actin is an actin-binding polypeptide.

15. 15. The kit of claim 13 or 14, wherein the cell type of interest is an epithelial cell and the means for identifying an epithelial cell is an EpCAM-binding polypeptide.

16. The method according to any one of claims 1 to 12, wherein the membrane-bound actin is detected in the membrane of a cell and / or the membrane of an extracellular vesicle.

17. 17. The method of claim 16, wherein the cell is an epithelial cell or a circulating tumor cell.

18. 18. The method of claim 17, wherein the epithelial cells are derived from breast tissue.

19. The method of any one of claims 1 to 12 or 15 to 18, wherein the cancer is an epithelial cell cancer.

20. 20. The method of claim 19, wherein the epithelial cell cancer is breast cancer.

21. The method of any one of claims 1 to 12 or 15 to 20, wherein the actin is beta-actin.

22. A kit described in any of claims 15 to 17, wherein membrane-bound actin is detected in the membrane of a cell and / or the membrane of an extracellular vesicle.

23. The kit of claim 22, wherein the cells are epithelial cells or circulating tumor cells.

24. The kit described in claim 23, wherein the epithelial cells are derived from breast tissue.

25. A kit described in any one of claims 13 to 15 or 22 to 24, wherein the cancer is epithelial cell cancer.

26. The method or kit described in claim 25, wherein the epithelial cell cancer is breast cancer.

27. ​​A kit described in any one of claims 13 to 15 or 22 to 26, wherein the actin is beta-actin.

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