Culture methods, a system and a kit for early-stage prediction of cancer metastasis

Abstract

The present invention is directed to a method for identifying i) metastatic potential of a cancer cell sample from a non-metastatic cancer or ii) potential for malignant alterations in a healthy cell, the method comprising the steps of: a) culturing a cancer cell sample from a non- metastatic cancer or a healthy cell sample in a composition comprising cancer-associated fibroblasts, CAFs, preferably in two-dimensional or three-dimensional cell culturing conditions; and b) monitoring morphological alterations in the sample cells during and / or after the culturing step a), and / or determining the expression level of biomarkers relating to metastasis in the sample cells during and / or after the culturing step a); wherein the sample cells which show morphological alterations relating to metastasis and / or changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measured from corresponding samples cultured in a composition comprising normal fibroblasts, NFs, are identified as having metastatic potential or potential for malignant alterations. The present invention is also directed to a system for culturing eukaryotic cells comprising a solid surface comprising at least one partition suitable for culturing eukaryotic cells, wherein said at least one partition contains a combined cell culture comprising i) cancer cells from a non- metastatic cancer or healthy cells and ii) cancer-associated fibroblasts, CAFs, wherein said partition is a well or chamber.

Description

Culture methods, a system and a kit for early-stage prediction of cancer metastasisFIELD

[0001] The present invention relates to the characterization of non-metastatic cancersand / or corresponding healthy cells, in particular non-metastatic breast cancer cells and / orhealthy breast cells. The present invention also relates to biomarkers and their use. Inparticular, their use in predicting the likelihood of a cancer metastasis, its initiation or indiagnosing and / or monitoring a cancer metastasis. Methods, kits and a system useful in theinvention are described. BACKGROUND

[0002] Cancer metastasis is almost always incurable and responsible for 90% of thecancer-related deaths.1 The lack of in vitro or in vivo models that can faithfully recapitulate theearly stages of cancer metastasis has hampered the progress in diagnosis, treatment andtherapeutic outcomes. Existing models utilize metastatic cell lines or tissues, which cannotprovide the key players responsible for the initiation and progression of metastasis of non-metastatic cancer and even healthy cells. Studies have shown that in patients with cancermetastasis, the stroma contains a significant number of activated fibroblasts, known as cancer-associated fibroblasts (CAFs).2CAFs promote cancer progression and metastasis and are responsible for extracellular matrix (ECM) remodeling through matrix remodeling and / ordegradation. CAFs facilitate the movement of metastatic cancer cells within their primary sitesor toward their distant target organs.3 However, the roles of CAFs in initiating a cancer innormal cells (i.e. converting healthy cells into non-metastatic cancer cells) and early-stage metastasis of non-metastatic cancer cells are not yet investigated.

[0003] Breast cancer is the number one cancer type in incidence and is the second leadingcause of cancer-related deaths worldwide.4It is estimated that around 2.3 million new breastcancer cases were diagnosed, including 685000 deaths in 2020 alone.5 About 90% of cancer-related deaths are due to its metastasis. In the case of breast cancer, a significant number of patients have already developed the advanced stage of breast cancer during their first diagnosis. More importantly, around 50% of the patients first diagnosed with primary breast cancer eventually develop metastasis within months to years after first diagnosis.6Breast cancer is intrinsically complex and molecularly and clinically heterogeneous, with a wide window of relapse.6

[0004] Therefore, more advanced methods focusing on early cancer diagnosis are neededto have information about the organ-specific metastasis probability of primary non-metastatic cells. This then will lead to the development of patient-specific treatments and offer directed attention to the clinical approaches in preventing metastasis.

[0005] In the present disclosure, we describe the roles of CAFs and the composition ofECM on the invasiveness of healthy and non-metastatic cancerous cells in the primary tumormicroenvironment and their dissemination to the secondary target organs through ECM remodeling and genomic regulation. We demonstrate the impacts of CAFs and ECM on thetransition of non-metastatic cancer cells and even healthy cells into a more aggressivemetastatic state by monitoring the expression levels of cancer-related biomarkers. For the first time, we show that changing the culturing method of healthy breast cells induces their conversion into non-metastatic and subsequently to more aggressive metastatic cancer cells. Inthe present disclosure, we describe a novel 3D cell culturing method that allows a cellulartransition of healthy breast cells into non-metastatic breast cancer cells and subsequently tometastatic breast cancer. Through this approach we identified metastasis-related biomarkersspecific to each transition stage. More importantly, we have identified the potential metastatic targets of different breast cancer cell populations expressing combinations of identifiedbiomarkers. Therefore, by monitoring the expression patterns of these biomarkers, we are ableto predict the potential of breast cancer to metastasize and / or where to metastasize even at veryearly stages of the disease. This allows us to understand the early driving elements for cancerinitiation and its progression into the more aggressive profile.

[0006] In addition, the present disclosure provides an in vitro diagnostic kit for use inrapid detection of the identified biomarkers in patient samples, e.g., solid biopsies, liquidbiopsies, or blood samples both at genomic and protein levels to predict the potential metastaticoutcome at very early stages of the disease. In a preferred embodiment, the kit includes twoseparate detection platforms which preferably are RT-qPCR-on-chip for gene expression detection and lateral flow test for protein-based expression detection for CYR61, Vimentin,Snail and E-cadherin. Some of the details of the kit are explained in Figures 7 and 8.SUMMARY OF THE INVENTION

[0007] The present invention provides methods for the detection of novel biomarkersrelating to the metastasis of non-metastatic cancer cells or promoting malignant alterations inhealthy cells. The present invention thus enables an early-stage detection of i) metastasis orpotential for metastasis from a non-metastatic cancer sample, or ii) malignant alterations or arisk thereof in healthy cells.

[0008] The invention is defined by the features of the independent claims. Some specificembodiments are defined in the dependent claims.

[0009] According to a first aspect of the present invention, there is provided an in vitromethod for identifying the metastatic potential of a cancer cell sample from a non-metastaticcancer, the method comprising the steps of: a) culturing said cancer cell sample from a non-metastatic cancer in a composition comprising cancer-associated fibroblasts, CAFs, preferably in two-dimensional or three-dimensional cell culturing conditions; and b) monitoring morphological alterations in the cells of said cancer cell sample during and / orafter the culturing step a), and / or determining the expression level of biomarkers relating tometastatic potential in the cells of said cancer cell sample during and / or after the culturing stepa); wherein the cells of said cancer cell sample from a non-metastatic cancer which show morphological alterations relating to metastatic potential and / or changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measured fromcorresponding non-metastatic cancer samples cultured in a composition comprising normalfibroblasts, NFs, are identified as having metastatic potential, and optionally the method further comprises steps of c) separately culturing a healthy cell sample in a composition comprising cancer-associated fibroblasts, CAFs, preferably in two- dimensional or three-dimensional cell culturing conditions, wherein said healthy cell sample istaken from the same patient and from the same tissue type as said cancer cell sample, and d)monitoring morphological alterations in the cells of said healthy cell sample during and / or afterthe culturing step c), and / or determining the expression level of biomarkers relating tometastatic potential in the cells of said healthy cell sample during and / or after the culturing stepc), wherein the cells of said healthy cell sample which show morphological alterations relatingto malignant alterations, metastatic alterations or increased potential for malignant alterations or metastatic alterations and / or changes in the expression levels of said biomarkers incomparison to the levels of said biomarkers measured from corresponding healthy cell samplescultured in the presence of normal fibroblasts, NFs, identify or confirm that said non-metastaticcancer has metastatic potential.

[0010] According to a second aspect of the invention, there is provided a kit comprisingmeans to detect at least three of the biomarkers selected from the group consisting of: vimentin,E-cadherin, Snail, and CYR61, wherein said kit comprises means to assay the level of geneexpression of said biomarkers.

[0011] According to a third aspect of the invention, there is provided a use of said kit foridentifying the metastatic potential of a cancer cell sample.

[0012] According to a fourth aspect of the present invention, there is provided an in vitromethod for identifying malignant alterations in a healthy cell, the method comprising the steps of:a) culturing said healthy cell in a composition comprising cancer-associated fibroblasts,CAFs, preferably in two-dimensional or three-dimensional cell culturing conditions; andb) monitoring morphological alterations in the cell during or after the culturing step a), and / ordetermining the expression level of biomarkers relating to metastatic potential in the cellduring and / or after the culturing step a);wherein the cell showing morphological alterations relating to metastasis and / or changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measuredfrom corresponding healthy cell samples cultured in a composition comprising normalfibroblasts, NFs, are identified as having malignant alterations.

[0013] According to a fifth aspect of the present invention, there is provided an in vitromethod for identifying metastatic potential of a cancer cell sample, the method comprising thesteps of:a) providing said cancer cell sample; andb) determining the expression level of biomarkers relating to metastatic potential in the cells of said cancer cell sample; wherein at least three of said biomarkers the expression level of which is determined in step b) are selected from the group consisting of: vimentin, E-cadherin, Snail, and CYR61; andwherein changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measured from corresponding cancer samples are identified as having metastatic potential.

[0014] According to a sixth aspect of the present invention, there is provided a systemfor in vitro culturing eukaryotic cells comprising a solid surface comprising at least one partitionsuitable for culturing eukaryotic cells, wherein said at least one partition contains a combined cell culture comprising i) cancer cells from a non-metastatic cancer or healthy cells and ii) cancer-associated fibroblasts, CAFs, wherein said partition is a well or chamber, and wherein said well or chamber is preferably interconnected via a channel or channels to other wells or chambers in said solid surface.

[0015] According to a seventh aspect of the present invention, there is provided an invitro method for screening drug candidates for preventing the metastatic spread of cancer cells,the method comprising the steps of:a) culturing a cancer cell sample from non-metastatic cancer cells or a sample of healthy cellsin a composition comprising cancer-associated fibroblasts, CAFs, and in the presence andabsence of a drug candidate compound, preferably in two-dimensional or three-dimensional cell culturing conditions; and b) monitoring morphological alterations in the cells of said cancer cell sample or of thesample of healthy cells during and / or after the culturing step a), and / or determining theexpression level of biomarkers relating to metastatic potential in the cells of said cancer cellsample or said sample of healthy cells during and / or after the culturing step a);wherein said drug candidate compound is having a preventing effect on the metastatic potential of said cancer or healthy cells if said cancer cells or healthy cells show less morphologicalalterations relating to metastatic potential and / or less changes in the expression levels of saidbiomarkers in comparison to the levels of said biomarkers measured from corresponding cancercells or healthy cells cultured in a composition comprising normal fibroblasts, NFs, in thepresence of said drug candidate compound than in the absence of said drug candidate compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGURE 1 illustrates the roles of both cellular and non-cellular components onbreast cancer initiation from healthy breast cells and their transition into a more aggressiveprofile. Breast CAFs (i.e., cancer-associated fibroblasts) in collagen I-based matrices promotethe cellular transition toward a more metastatic state by upregulating Vimentin, Snail and CYR61 genes while downregulating E-cadherin levels. The genomic regulation performed through the 3D culture model results in organ-specific metastasis of breast cancer cells.

[0017] FIGURE 2 illustrates a schematic diagram disclosing a preferred workflow forthe present method.

[0018] FIGURE 3 shows a) a schematic illustration of coculturing within the LOCplatform. The imaging was performed using FL microscope, and Z-stacks of each conditionwere obtained; b) phenotypic changes observed in MCF-7 and MDA-MB-231 cells when theyare cocultured with primary CAFs on Day 1 and Day 3. Scale bar: 100 μm.

[0019] FIGURE 4 shows scanning electron microscopy (SEM) images of 2D co-cultures.The SEM images showing the cellular interactions between healthy breast epithelial cells(bNEs) (a, d), non-metastatic breast cancer cells (b, e) and metastatic breast cancer cells (c, f)with primary normal fibroblasts (NFs) (d, e, f) or cancer-associated fibroblasts (CAFs) (a, b, c)in 2D cocultures after 7 days.

[0020] FIGURE 5 shows the effects of 2D- and 3D coculturing of healthy and cancerousbreast cells with primary NFs and CAFs on the expression of Snail gene. Herein are revealedthe mRNA expression levels of Snail observed from Day 1 to Day 7 in a) 2D and b) 3D co-culturing. These were measured in healthy breast epithelial cells (bNEs) and cancerous breastcells (MCF-7 and MDA-MB-231), whether cultured alone or alongside NFs or CAFs. Thevalues reflect the mean ± standard deviation from three independent experiments.

[0021] FIGURE 6 shows the effects of 2D- and 3D coculturing of healthy and cancerousbreast cells with primary NFs and CAFs on the expression of CYR61 gene from breast cells. Herein are revealed the mRNA expression levels of CYR61 observed from Day 1 to Day 7 ina) 2D and b) 3D co-culturing. These were measured in healthy breast epithelial cells (bNEs)and cancerous breast cells (MCF-7 and MDA-MB-231), whether cultured alone or alongsideNFs or CAFs. The values reflect the mean ± standard deviation from three independent experiments.

[0022] FIGURE 7 shows the effects of 2D- and 3D coculturing of healthy and cancerousbreast cells with primary NFs and CAFs on the expression of Vimentin gene from breast cells. Herein are revealed the mRNA expression levels of Vimentin observed from Day 1 to Day 7 ina) 2D and b) 3D co-culturing. These were measured in healthy breast epithelial cells (bNEs)and cancerous breast cells (MCF-7 and MDA-MB-231), whether cultured alone or alongsideNFs or CAFs. The values reflect the mean ± standard deviation from three independent experiments.

[0023] FIGURE 8 shows the effects of 2D- and 3D coculturing of healthy and cancerousbreast cells with primary NFs and CAFs on the expression of E-cadherin gene from breast cells.Herein are revealed the mRNA expression levels of Vimentin observed from Day 1 to Day 7 ina) 2D and b) 3D co-culturing. These were measured in healthy breast epithelial cells (bNEs)and cancerous breast cells (MCF-7 and MDA-MB-231), whether cultured alone or alongsideNFs or CAFs. The values reflect the mean ± standard deviation from three independent experiments.

[0024] FIGURE 9 shows the storage moduli (G´) of collagen I matrices with or withoutcells measured using in situ contactless rheology under cell culture conditions. The changes instorage modulus are shown from Day 0 to Day 7 in collagen I matrices (3 mg / mL) containinga) normal primary breast fibroblasts (NFs) individually or in combination with healthy breastepithelial cells (NEs) and breast cancer cells (MCF-7 and MDA-MB-231) or b) primary breastcancer-associated fibroblasts (CAFs) individually or in combination with NE, MCF-7 or MDA-MB-231 cells. The average storage moduli for each condition is shown (n=3). The rheologicaldata suggests nearly a two-fold increase in the stiffness of the cell-laden matrices from Day 0 to Day 7 compared to cell-free matrices.

[0025] FIGURE 10 illustrates a PDMS-based RT-qPCR on-chip platform that can beused in the present invention. The biological samples are loaded to the platform where their lysis is performed and the gene amplification for CYR61, Snail, Vimentin, and E-cadherin iscarried out within the microreactor port of the platform. “Loading Channel for biologicalsamples” is used for loading the biological samples to the platform. There will be 2 additionalinlets for loading the positive control and non-template control samples. “Lysis Part” is usedfor lysing the sample before PCR reaction within the platform. “Microreactor” contains specificprimer sets of genes of interest and is where amplification of the genes is performed. Preferably,there will be 9 microreactors – four of them for PCR detection of 4 biomarkers simultaneously,one of them for internal positive control, and four of them for non-template control reactions.“Heating element” is where localized thermal cycling is performed with the localized Peltierdevice at the bottom of the microreactors. “Air venting outlet” is where the air withinmicroreactors is taken through a port. “Absorbent Pad” is where the excess of the PCR mixtureis removed from the platform by capillary action flow to the pad.

[0026] FIGURE 11 illustrates schematically a) a lateral flow test to detect the protein-based expression of CYR61, Snail, Vimentin, or E-cadherin; b) the lysed biological sample isabsorbed from the sample loading pad and flows to the labeled antibody of interest (either CYR61, Snail, Vimentin, or E-cadherin) embedded in the conjugate pad and binds to theantibody there; c) the antigen-antibody complex then flows toward the test line where it canreact with the membrane-bound antibody and d) cause a color change in the test line. In the absence of the protein of interest within the sample, no color change is observed in the test line. The removal of excess of the reaction will be absorbed in an absorbent pad at the end of the test. EMBODIMENTS

[0027] In the present context, the term "metastasis" is defined as cancer recurrence ordisease progression which may occur locally, regionally (such as nodal micrometastasis ormacrometastasis), or distally (such as brain, lung, bone, liver and other tissues).

[0028] In the present context, the term "non-metastatic cancer" is defined as a cancer thathas not spread from the primary site of the cancer to other tissues or parts in the body. Non-metastatic cancer cells differ from metastatic cancer cells in their gene expression profile for some specific cancer-related biomarkers and genes, morphology, adhesion strength and ability to migrate to distant organs. Non-metastatic cancer cells can be identified by their epithelial- like morphology, gene expression patterns and behavior, i.e., ability to migrate and invade to the neighboring or distant organs.7-9

[0029] In the present context, the term “cancer-associated fibroblasts” refers to afibroblast cell type within the tumor microenvironment that promotes tumorigenic features byinitiating the re-modelling of the extracellular matrix or by secreting cytokines. Fibroblasts areactivated during inflammation and fibrosis in tumors and are thus called “cancer‐associatedfibroblasts”. Cancer associated fibroblasts, CAFs, can be isolated from various cancer typessuch as breast cancer, prostate cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, gastric cancer, and colorectal cancer.10In the present invention, the CAFs used in the culturingstep of the present invention are preferably associated with the cancer type from which the non-metastatic and metastatic cancer cells originate.

[0030] In the present context, the term “normal” or “healthy”, when referring herein tocells, refers to cells which are not known to have any malignant changes or properties.11-12Normal or healthy cell, as described herein, responds appropriately to the signals that controlnormal cell behavior, while malignant cancer cells grow and divide in an uncontrolled manner.

[0031] The phrase "determining the expression level of biomarkers " as used herein refersto determining or quantifying RNA or proteins expressed by the gene of each biomarker. The term "RNA" includes mRNA transcripts, and / or specific spliced variants of mRNA. In the case of "protein", it refers to proteins translated from the RNA transcripts transcribed from the gene of each biomarker. A number of methods can be used to detect or quantify the level of RNA products of the gene or genes within a sample, including microarrays, RT-PCR (includingquantitative RT-PCR), nuclease protection assays and Northern blot analyses. An example of asuitable RT-PCR platform for the present invention is shown in Figure 7.

[0032] A person skilled in the art will appreciate that a number of methods can be usedto determine the amount of a protein product of a gene / biomarker of the present disclosure,including immunoassays such as Western blots, ELISA, and immunoprecipitation followed bySDS-PAGE and immunocytochemistry. To detect protein products of the biomarkers, ligandsor antibodies that specifically bind to the protein products can be used. An example of a suitabledetection platform for the biomarkers of the present invention as proteins is shown in Figure 8.

[0033] A person skilled in the art will appreciate that a number of detection agents canbe used to determine the expression of the gene of each biomarker. To detect the presence orabsence of expression of the biomarkers, probes, primers, complementary nucleotide sequences or nucleotide sequences that hybridize to the mRNA products or cDNAs thereof can be used.For instance, the gene expression patterns of the biomarkers can be detected through RT-qPCRtechnique where the mRNA levels of each biomarker are determined with biomarker-specificprimers.

[0034] As used herein the expression “changes in the expression levels” refers tomeasured differences in the level of expression of the genes / biomarkers at separate time pointsor between separate sample groups. The changes in the expression levels can be assayed bymeasuring the level of expression of the products of the genes, such as the difference in the level of messenger RNA transcript expressed or proteins expressed by the genes. Preferably, the difference is statistically significant. The expression "difference in the level of expression" refers to an increase or decrease in the measurable expression level of a given gene as measuredby the amount of messenger RNA transcript and / or the amount of protein in a sample as compared with the measurable expression level of a given gene in a control. The differential expression can be compared using the ratio of the level of expression of a given gene or genes as compared with the expression level of the given gene or genes of a control. For example, an RNA or protein is differentially expressed if the level of expression in a first sample ascompared with a second sample has a level of expression which is greater than or less than thelevel of expression in the second sample. In an embodiment, the sample is evaluated for thelevel of biomarker expression by comparing to a reference standard prepared for the comparisonof the expression levels. In an embodiment, the level of the biomarker expression may be atleast a 1.5, 2, 5, 10, 15, 20, 25, 50, 75, or 100-fold increase or decrease in expression compared to the reference standard.

[0035] The present disclosure is particularly directed to the detection of changes in theexpression levels of the biomarkers vimentin, E-cadherin, Snail, and / or CYR61. Vimentin is astructural protein that in humans is encoded by the VIM gene (GenBank: NM_003380,NP_003371). Cadherin-1 or E-cadherin (epithelial cadherin) is a protein that in humans isencoded by the CDH1 gene (GenBank: NM_004360, NP_004351). Zinc finger protein SNAI1(Snail) is a protein that in humans is encoded by the SNAI1 gene (GenBank: NM005985,NP_005976). Cysteine-rich angiogenic inducer 61 (CYR61) also known as CCN familymember 1 (CCN1), is a matricellular protein that in humans is encoded by the CYR61 gene(GenBank: NM_001554, NP_001545).

[0036] The present invention relates to the characterization of cell alterations leading toearly-stage cancer metastasis. The present invention is associated with the identification of specific factors playing key roles in promoting the metastasis of non-metastatic cancer cells oreven healthy cells. Therefore, it would open new paths to prevent the formation of organ-specific cancer metastasis at the very early stages. The present invention focuses on how non-metastatic cancer cells can gain invasiveness and become more aggressive to develop site-specific spread. The invention provides early information about the metastatic probabilities andcapacities of specific cellular profiles which enables a person skilled in the art to understandthe potential target sites of cancer cells in the very early stages of the disease and it may provide early information about which organ site the non-metastatic cancer cells would spread.

[0037] Accordingly, the present invention provides an in vitro method for identifyingmetastatic potential of a cancer cell sample from non-metastatic cancer cells, the methodcomprising the steps of: a) culturing said cancer cell sample from a non-metastatic cancer in a composition comprising cancer-associated fibroblasts, CAFs, preferably in two-dimensional or three-dimensional cell culturing conditions; and b) monitoring morphological alterations in the cells of said cancer cell sample during and / orafter the culturing step a), and / or determining the expression level of biomarkers relating tometastatic potential in the cells of said cancer cell sample during and / or after the culturing step a); wherein the cells of said cancer cell sample from a non-metastatic cancer which show morphological alterations relating to metastatic potential and / or changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measured fromcorresponding non-metastatic cancer samples cultured in a composition comprising normalfibroblasts, NFs, are identified as having metastatic potential, and optionally the method further comprises steps of c) separately culturing a healthy cell sample in a composition comprising cancer-associated fibroblasts, CAFs, preferably in two- dimensional or three-dimensional cell culturing conditions, wherein said healthy cell sampleis taken from the same patient and from the same tissue type as said cancer cell sample, and d)monitoring morphological alterations in the cells of said healthy cell sample during and / orafter the culturing step c), and / or determining the expression level of biomarkers relating tometastatic potential in the cells of said healthy cell sample during and / or after the culturingstep c), wherein the cells of said healthy cell sample which show morphological alterationsrelating to malignant alterations, metastatic alterations or increased potential for malignantalterations or metastatic alterations and / or changes in the expression levels of said biomarkersin comparison to the levels of said biomarkers measured from corresponding healthy cellsamples cultured in the presence of normal fibroblasts, NFs, identify or confirm that said non-metastatic cancer has metastatic potential.

[0038] In a specific embodiment, the steps a) and b) of the present method are used forpredicting the risk of metastasis, overall survival, or both, in a patient, wherein the cells of thecancer cell sample from the non-metastatic cancer cells taken from said patient showmorphological alterations relating to metastatic potential and / or changes in the expressionlevels of said biomarkers in comparison to the levels of said biomarkers measured fromcorresponding non-metastatic cancer samples cultured in a composition comprising normalfibroblasts, NFs, are identified as having metastatic potential and thus increasing the risk ofcancer recurrence and cancer death as well as decreasing the probability of overall survival ofsaid patient.

[0039] In another specific embodiment, the steps a) and b) of the present method are usedfor diagnosing a patient with metastatic cancer or potentially metastatic cancer, wherein thecells of the cancer cell sample from non-metastatic cancer cells taken from the said patientwhich show morphological alterations relating to metastatic potential and / or changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measured from corresponding non-metastatic cancer samples cultured in a composition comprisingnormal fibroblasts, NFs, are identified as having metastatic potential and thus diagnosing thepatient to have metastatic cancer or potentially metastatic cancer when the level of measuredbiomarkers in the sample obtained from the patient are differentially expressed compared to areference sample of non-metastatic cancers as described in the present disclosure.

[0040] In another specific embodiment, the steps a) and b) of the present method are usedfor diagnosing patients with cancer who are likely to benefit from treatment with a knowntreatment for metastatic cancer, wherein the patient is likely to benefit from treatment with aknown treatment for metastatic cancer, if the cells of the cancer cell sample from the non-metastatic cancer cells taken from said patient show morphological alterations relating tometastatic potential and / or changes in the expression levels of said biomarkers relating to metastatic potential in comparison to the levels of said biomarkers measured fromcorresponding non-metastatic cancer samples cultured in a composition comprising normalfibroblasts, NFs, as described in the present disclosure.

[0041] The present invention also provides a kit comprising means to detect at least threeof the biomarkers selected from the group consisting of: vimentin, E-cadherin, Snail, andCYR61, wherein said kit comprises means to assay the level of gene expression of saidbiomarkers, preferably in a cancer sample from a non-metastatic cancer or a pre-treated samplethereof. The suitable samples include but are not limited to solid biopsies, liquid biopsies, orblood samples.

[0042] In one aspect, a kit or product comprising a means to assay the level of geneexpression of vimentin, E-cadherin, Snail, and CYR61 is provided. In some embodiment, thekit or product comprises an agent capable of interacting with a gene expression product of vimentin, E-cadherin, Snail, or CYR61. In some embodiment, the kit or product comprises an agent capable of interacting with a gene expression product of vimentin, E-cadherin, Snail, or CYR61. In some embodiments the agent is an antibody. In some embodiments the agent is anoligonucleotide. In some embodiments the gene expression product is an RNA molecule. Insome embodiment the gene expression product is a mRNA molecule. In some embodiment the gene expression product is a polypeptide or protein.

[0043] The kit can further optionally include reagents for performing the assaysdescribed herein. For example, the kit can include buffers, solvents, stabilizers, preservatives, purification columns, detection reagents, and enzymes, which may be necessary for isolating nucleic acids from a patient sample, amplifying the samples, e.g., by qRT-PCR, and applying the samples to the agent described above; or for isolating proteins from a subject sample, and applying the samples to the agent described above; or reagents for directly applying the subject sample to the agent described above. A kit can also include positive and negative control samples, e.g., control nucleic acid samples.

[0044] In a preferred embodiment, the kit comprises a RT-qPCR platform for detectingand quantifying the amount of mRNA of at least three of the biomarkers selected from the group consisting of: vimentin, E-cadherin, Snail, and CYR61.

[0045] In a preferred embodiment, the kit comprises a lateral flow test to detect theprotein-based expression of CYR61, Snail, Vimentin, or E-cadherin.

[0046] In another preferred embodiment, the kit comprises i) a RT-qPCR platform fordetecting and quantifying the amount of mRNA of at least three of the biomarkers selected fromthe group consisting of: vimentin, E-cadherin, Snail, and CYR61, preferably all four, and / or ii)a lateral flow test to detect the protein expression of at least one, preferably all four, of thebiomarkers CYR61, Snail, Vimentin, and E-cadherin.

[0047] Accordingly, in some embodiments, the expression level(s) of one or more of thebiomarkers described herein are detected using a lateral flow immunoassay test, also known asthe immunochromatographic assay, or strip test. A lateral flow test is a form of immunoassayin which the test sample flows along a solid substrate via capillary action. After the biological sample is applied to the test, it encounters a colored reagent which mixes with the sample and transits the substrate encountering the test lines or zones, which have been pretreated with anantibody or antigen. Depending upon the antigens present in the biological sample, the colored reagent can become bound at the test line or zone.

[0048] The sample for detecting the present biomarkers can be obtained from a cancercell culture as described in the present disclosure or directly from a cancer patient using routinetechniques known to those skilled in the art, and the sample may be used directly as obtained from the source or following a pretreatment to modify the character of the sample. Such pretreatment may include, for example, diluting viscous fluids, filtration, precipitation, dilution, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, extracting nucleic acids and the like.

[0049] After the sample is obtained and preferably pre-treated, the sample or the pre-treated sample or a fraction thereof can be contacted with the detection means of the kit.

[0050] The present invention is also directed to a use of the kit as disclosed above foridentifying the metastatic potential of a cancer cell sample, preferably from a non-metastaticcancer.

[0051] The present invention further provides an in vitro method for identifyingmalignant alterations in a healthy cell, the method comprising the steps of:a) culturing said healthy cell in a composition comprising cancer-associated fibroblasts,CAFs, preferably in two-dimensional or three-dimensional cell culturing conditions; andb) monitoring morphological alterations in the cell during or after the culturing step a), and / ordetermining the expression level of biomarkers relating to metastatic potential in the cellduring and / or after the culturing step a);wherein the cell showing morphological alterations relating to metastasis and / or changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measuredfrom corresponding healthy cell samples cultured in a composition comprising normalfibroblasts, NFs, are identified as having malignant alterations.

[0052] The present invention further provides an in vitro method for identifyingmetastatic potential of a cancer cell sample, the method comprising the steps of: a) providing said cancer cell sample, preferably from a non-metastatic cancer; andb) determining the expression level of biomarkers relating to metastatic potential in the cells of said cancer cell sample; wherein at least three of said biomarkers the expression level of which is determined in step b) are selected from the group consisting of: vimentin, E-cadherin, Snail, and CYR61; and wherein changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measured from corresponding cancer samples, preferably samples from non-metastatic cancer, or healthy cell samples are identified as having metastatic potential.

[0053] In a preferred embodiment, said biomarkers the expression level of which isdetermined in step b) comprise or consists of vimentin, E-cadherin, Snail, and CYR61.

[0054] In another preferred embodiment, the expression level of vimentin, Snail, and / orCYR61 is / are substantially upregulated in cells having metastatic potential or upon attaining metastatic potential in comparison to the levels in a reference standard prepared for the comparison of the expression levels.

[0055] In another preferred embodiment, the expression level of E-cadherin issubstantially downregulated in cells having metastatic potential or upon attaining metastatic potential in comparison to the levels in a reference standard prepared for the comparison of the expression levels.

[0056] The present invention also provides an in vitro method for screening drugcandidates for preventing the metastatic spread of cancer cells, the method comprising the stepsof: a) culturing a cancer cell sample from non-metastatic cancer cells or a sample of healthy cells in a composition comprising cancer-associated fibroblasts, CAFs, and in the presence and absence of a drug candidate compound, preferably in two-dimensional or three-dimensional cell culturing conditions; and b) monitoring morphological alterations in the cells of said cancer cell sample or of the sample of healthy cells during and / or after the culturing step a), and / or determining the expression level of biomarkers relating to metastatic potential in the cells of said cancer cell sample or said sample of healthy cells during and / or after the culturing step a); wherein said drug candidate compound is having a preventing effect on the metastatic potential of said cancer or healthy cells if said cancer cells or healthy cells show less morphologicalalterations relating to metastatic potential and / or less changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measured from corresponding cancercells or healthy cells cultured in a composition comprising normal fibroblasts, NFs, in thepresence of said drug candidate compound than in the absence of said drug candidate compound.

[0057] In a preferred embodiment, in culturing step a) of any of the above methods, saidcancer cell sample from non-metastatic cancer cells and said cancer-associated fibroblasts,CAFs, are cultured in a composition comprising one or more extracellular matrix, ECM,component, preferably selected from the group consisting of: collagens, elastin, fibronectin and laminins.

[0058] In another preferred embodiment, in culturing step a) of any of the above methods,said cancer cell sample from non-metastatic cancer cells and said cancer-associated fibroblasts,CAFs, are cultured in a composition comprising one or more of the following ingredients:agarose, alginate, fibrin, chitosan, and albumin.

[0059] In another preferred embodiment, said extracellular matrix, ECM, component iscollagen-1.

[0060] In another preferred embodiment, said CAFs are from breast tissue.

[0061] In another preferred embodiment, said non-metastatic cancer is breast cancer.

[0062] In another preferred embodiment, said morphological alterations monitored instep b) of any of the above methods are related to the form of the cells, cell-cell interactions,and motility of the cells.

[0063] In another preferred embodiment, one or more of said biomarkers the expressionlevel of which is determined in step b) of any of the above methods are selected from the groupconsisting of: vimentin, E-cadherin, Snail, and CYR61.

[0064] In a more preferred embodiment, one or more of said biomarkers the expressionlevel of which is determined in step b) of any of the above methods are selected from the groupconsisting of: i) vimentin, E-cadherin, and Snail; ii) vimentin, Snail, and CYR61; iii) vimentin,E-cadherin, and CYR61; iv) E-cadherin, Snail, and CYR61; v) vimentin and E-cadherin; vi) vimentin and Snail; vii) vimentin and CYR61; viii) E-cadherin and Snail; ix) E-cadherin andCYR61; or x) Snail and CYR61. In another more preferred embodiment, said biomarkers theexpression level of which is determined in step b) of any of the above methods comprise at leastthe biomarkers listed in any of the groups i) - x).

[0065] In another preferred embodiment, the expression level of vimentin, Snail, and / orCYR61 is / are substantially upregulated in cancer cells having metastatic potential or upon attaining metastatic potential, when cultured in a composition comprising CAFs, in comparison to the levels of said biomarkers measured from corresponding non-metastatic cancer samplescultured in a composition comprising normal fibroblasts.

[0066] In another preferred embodiment, the expression level of E-cadherin issubstantially downregulated in cancer cells having metastatic potential or upon attainingmetastatic potential, when cultured in a composition comprising CAFs, in comparison to thelevel of said biomarker measured from corresponding non-metastatic cancer samples culturedin a composition comprising normal fibroblasts.

[0067] The present invention further provides a system for in vitro culturing eukaryoticcells comprising a solid surface comprising at least one partition suitable for culturing eukaryotic cells, wherein said at least one partition contains a combined cell culture comprising i) cancer cells from a non-metastatic cancer or healthy cells and ii) cancer-associated fibroblasts, CAFs, wherein said partition is a well or chamber, and wherein said well or chamber is preferably interconnected via a channel or channels to other wells or chambers in said solid surface.

[0068] In a preferred embodiment, said at least one partition in said system furthercomprises one or more extra cellular matrix, ECM, component, preferably selected from the group consisting of: collagens, elastin, and fibronectin.

[0069] In another preferred embodiment, said at least one partition in said system furthercomprises one or more of the following ingredients: agarose, alginate, fibrin, chitosan, and albumin.

[0070] In another preferred embodiment, said extracellular matrix, ECM, component iscollagen-1.

[0071] In another preferred embodiment, said cancer cells are from a non-metastaticbreast cancer.

[0072] In another preferred embodiment, said healthy cells are breast tissue cells.

[0073] In another preferred embodiment, said system comprises a parallel control cellculture on the same or separate solid surface, wherein said control cell culture comprises a combined cell culture comprising i) cancer cells from non-metastatic cancer or healthy cells and ii) normal fibroblasts, NFs.

[0074] It is to be understood that the embodiments of the invention disclosed are notlimited to particular structures, process steps, or materials disclosed herein, but are extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0075] Reference throughout this specification to “one embodiment”, “preferredembodiment” or “an embodiment” means that a particular feature, structure, or characteristicdescribed in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in a preferredembodiment” or “in an embodiment” in various places throughout this specification are notnecessarily all referring to the same embodiment.

[0076] As used herein, a plurality of items, structural elements, compositional elements,and / or materials may be presented in a common list for convenience. However, these listsshould be construed as though each member of the list is individually identified as a separateand unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. In addition, various embodiments and example of the present invention may be referred to herein along with alternatives for the various components thereof. It is understood that such embodiments, examples, and alternatives are not to be construed as de facto equivalents of one another, but are to be considered as separate and autonomous representations of the present invention.

[0077] Furthermore, the described features, structures, or characteristics may becombined in any suitable manner in one or more embodiments. In the following description,numerous specific details are provided, such as examples of lengths, widths, shapes, etc., toprovide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well- known structures, materials, or operations are not shown or described in detail to avoidobscuring aspects of the invention.

[0078] While the forgoing examples are illustrative of the principles of the presentinvention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the claims set forth below.

[0079] The verbs “to comprise” and “to include” are used in this document as openlimitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", i.e. a singular form, throughout this document does not exclude a plurality. EXPERIMENTAL SECTION Materials and Methods Cell Lines Human breast cancer cell lines, basal type triple negative breast cancer cell line (MDA-MB- 231) and luminal type breast cancer cell line (MCF-7), primary normal breast epithelial cells(NEs) were obtained from ATCC, primary breast cancer-associated fibroblasts (CAFs) weretaken from BIOIVT. All cell lines were cultured in DMEM high glucose (11965092, Gibco) with Fetal Bovine Serum (FBS, A3840001, Gibco, 10%) and Penicillin / Streptomycin (15070063, Gibco, 1%). All cell lines were cultured at 37°C in a humidified incubator with 5% CO2. 2D and 3D Cell Culture The impacts of primary breast CAFs on healthy epithelial cells, non-metastatic luminal MCF- 7 and TNBC MDA-MB-231 cells were investigated in 2D and 3D cell culture conditions. In2D cell culture experiments, MCF-7 and MDA-MB-231 cells were labeled with CellTrackerGreen to monitor their phenotypic changes, while breast CAFs remained unlabeled. Cells weredetached from their culture plates and mixed at a 1:1 ratio (MCF-7 + breast CAFs or MDA-MB-231 + breast CAFs) in DMEM-1X complete cell culture media. For each cell line, a density of 5x105cells / mL was used. In each study,10 μL of cell suspension was loaded to the mediumchannels of the LOC platform. The low-density-cell mixture was essential to properly monitor the interactions and morphological changes among individual cells. The same conditions were performed with normal breast fibroblasts (NFs). The morphological changes of each cell were monitored using a fluorescence microscopy imaging for 3 days. In 3D cell culture, the effects of primary CAFs on breast cells were determined within collagen- I-based matrices in LOC platforms. Collagen-I working solution was prepared according to Table 1. Collagen-I stock solution was added to a mixture containing known volume of 10X PBS, 1N NaOH, and ultrapure H2O in an Eppendorf tube on ice bath. Cells were prepared at two times the final concentration in serum-free media to have a final cellular density of 5x105cells / mL. The cells were mixed with collagen-I working solution in a 1:1 ratio to obtain the required concentration of cells in 3 mg / mL final collagen-I concentration. The cell-laden collagen matrices were loaded into the MC channels of the LOC platform and the polymerization of collagen-I was performed for 30 minutes at RT or 37 °C in a humidified incubator with 5% CO2. Following the polymerization, DMEM 1X culture media was added to the HMC of LOC. The cells were visualized using 3D imaging by fluorescence microscopy for 3 days. Z-stack images of the cells within 3D matrices for each condition were obtained each day from Day 0 to Day 3 with a 10X objective and by scanning throughout the hydrogel for 500 μm with a Z-step size of 7.52 µm using a fluorescence microscope. The sum projection of z-stacks of all conditions for each day was prepared and then the same threshold value was applied to all images.Table 1. The required volumes of collagen type I, 10X PBS or medium, 1NaOH, and dH2O forneutralization of collagen type I. (V: total volume, S: stock concentration of collagen type I, F: final concentration of collagen type I)Total RNA Isolation and Semi-quantitative Real-time PCR For the recovery from collagen-I matrices, the media was removed, and the matrix was detached from the plate by a scraper. The gel was transferred to an Eppendorf tube and centrifuged at 1000xg for 1 minute at 4oC to spin down the collagen I matrix. The supernatant was carefully removed and lysis buffer which was prepared by adding 1 µL β-mercaptoethanol to every 100µL lysis buffer of PureLink RNA Mini Kit was added. The gel was then homogenized on iceto prevent the heating of the samples and denaturation of RNA. The process was performed for a minute with a break after 30 seconds. The homogenized gel was then passed through an insulinneedle several times. Following a quick centrifugation at 1000xg and 4 oC for 30 seconds, thesupernatant was collected. The RNA isolation process was performed by following the protocol of PureLink RNA Mini Kit. The samples were stored at -80oC before the homogenization step if needed. cDNAs were synthesized from 1 μg total RNAs by RevertAid First Strand cDNA Synthesis Kit(K1622, ThermoFisher Scientific, USA). mRNA levels were analyzed with semi-quantitativereal time reverse transcriptase PCR (RT-qPCR) using FastStart Essential DNA Green Master Kit (06402712001, Roche) on Light Cycler® 96 Instrument. The relative expression levels of each gene were calculated by ∆∆Ct method using human or rat TATA-box binding (TBP) protein as the housekeeping gene. Non-template controls were included in each condition and p-values were calculated using two-tailed t-test. Rheological Measurements The storage moduli (G´) of collagen-I matrices with or without primary fibroblasts was measured using the nondestructive in situ contactless ElastosensTMBio (Rheolution, Canada, Montreal) rheometer. In a typical experiment, 250 μL of collagen-I containing cell solution was transferred to a microvolume sample holder, and the collagen was polymerized at 37oC. For Day 0, the elastic modulus of the samples was measured immediately after forming the gelwithin the sample holder. The samples together with the holder were placed in a 12-well platecontaining complete cell culture media. The incubation of the samples was carried out for 5days and each day the elastic moduli of the samples was measured. The measurements wereperformed in triplicates for each condition. ResultsCoculturing with CAFs promotes the invasion of healthy and cancerous breast cellsTo investigate the effects of primary breast CAFs on cell migration and invasion, we coculturedprimary breast CAFs with MCF-7 and MDA-MB-231 breast cancer cells within collagen-Imatrices. The culture system includes a multichannel lab-on-a-chip (LOC) platform, allowing simulated primary and secondary tumor microenvironments (Figure 3A). MCF-7 cell After 3days of coculturing, MCF-7 cells revealed phenotypic changes, they started to form coloniesrather than being single (Figure 3B). The coculturing of MCF-7 cells with breast CAFs increasetheir ability to migrate as they seemed more elongated and mesenchymal-like compared to their original phenotype, as supported using fluorescence microscope imaging. The phenotypicchanges observed, especially in MCF-7 cells, directed us to consider if any change in the geneexpression patterns plays a role in the epithelial-to-mesenchymal transition (EMT). EMT is implemented in cancer metastasis by increasing the mobility and invasion capacities of cancer cells. Therefore, the expression levels of E-cadherin and Vimentin genes, which are epithelial and mesenchymal markers, were checked using RT-qPCR analysis. Specifically, we have checked the gene expression profiles of metastatic (MDA-MB-231) and non-metastatic (MCF- 7) breast cancer cells cultured alone, coculture with breast CAFs, and cocultured with normalfibroblasts (Figures 5, 6, 7, and 8)A significantly increased expression of Vimentin and a decreased expression of E-cadheringenes in MCF-7 cells cocultured with breast CAFs were observed which is relevant to theirphenotypic changes upon coculturing with CAFs (**p-value <0.01). There was no significant change in the expression levels in MDA-MB-231 cells. Besides, similar expression patterns of the genes were observed when the cancer cells were cocultured with normal fibroblasts (NFs).This reveals that the presence of breast CAFs leads to the transition of MCF-7 cells fromepithelial to mesenchymal phenotype through upregulating the EMT-related genes.In addition to EMT-related genes, we have also checked the expression levels of genes corresponding to Snail-a zinc-finger transcriptional repressor controlling EMT during tumorprogression and CYR61-cysteine-rich protein 61, which regulate the adhesion, migration,proliferation, and extracellular matrix remodeling. Snail plays an important role in ECM degradation and invasion, and it suppresses the expression of cell adhesion molecules such as E-cadherin. In clinical samples, it has been shown that patients with high snail expression have poor prognosis. Therefore, it is an important prognostic biomarker in breast cancer patients. CYR61 genes are known to have low expression in normal healthy cells. However, it is strongly upregulated in mechanically challenged organs or tissues. Therefore, mechanical stiffness might be one of the primary inducers of the CYR61 expression in a pathological state. RT-qPCR analysis revealed a significant increase in the expression of Snail and CYR61 genes whenhealthy breast cells and non-metastatic MCF-7 cells were cocultured with breast CAFs withincollagen I matrices (Figures 5, 6, 7, and 8). However, the expression level of Snail and CYR61was not changed when MCF-7 cells were cultured alone or cocultured with normal fibroblasts.On the other hand, for MDA-MB-231 cells, the expression levels remained unchanged when cultured alone, or cocultured with CAFs or normal fibroblasts. The above results offer numerous insights on how CAFs can have significant effects on themorphology and even genotype of healthy breast cells and non-metastatic breast cancer cellsfor their transition into a more metastatic profile. The presence of primary breast CAFs increases the elastic moduli of collagen-based 3D matrices In addition to biochemical cues and adhesion factors, mechanical stiffness is an important parameter in cancer biology. The increased expression level of CYR61 indicates altered mechanical properties of the culture system in the presence of CAFs. To determine the impacts of breast CAFs on the mechanical properties of the matrices, we cultured CAFs within collagen I-based matrices. The stiffness of the matrices was determined non-destructively using a contactless rheological measurement from Day 0 to Day 7. The rheological data suggest a two-fold increase in the stiffness of the matrices was observed from Day 0 to Day 7, indicating theroles of CAFs in remodeling the collagen I matrices (Figure 9).ACRONYMS LISTCAF cancer-associated fibroblastECM extracellular matrixNE normal epithelialNF normal fibroblastRT-qPCR quantitative reverse transcription polymerase chain reactionCITATION LIST1. D. Wirtz, K. Konstantopoulos and P. C. Searson. Nat. Rev. Cancer, 2011, 11, 512 —5222. S. Koustoulidou, M. W. H. Hoorens, S. U. Dalm, S. Mahajan, R. Debets, Y. Seimbille andM. de Jong. Cancers, 2021, 13(5), 1100.3. J. Winkler, A. Abisoye-Ogunniyan, K. J. Metcalf and Z. Werb. Nat. Commun., 2020, 11,5120. 4. N. Harbeck, F. Penault-Llorca, J. Cortes, M. Gnant, N. Houssami, P. Poortmans, K. Ruddy,J. Tsang and F. Cardoso. Nat. Rev. Dis. Primers, 2019, 5, 665. M. Arnold, E. Morgan, H. Rumgay, A. Mafra, D. Singh, M. Laversanne, J. Vignat, J. R.Gralow, F. Cardoso, S. Siesling and I. Soerjomataram. Breast (Edinburgh, Scotland), 2022,66, 15–23. 6. A. I. Riggio, K. E. Varley and A. L. Welm, Br. J. Cancer, 2021, 124, 13-26.7. J. Fares, M. Y. Fares, H. H. Khachfe, H. A. Salhab and Y. Fares. Molecular principles ofmetastasis: a hallmark of cancer revisited. Sig. Transduct. Target Ther. 2020, 5, 28.8. X. Jin, Z. Demere, K. Nair, A. Ali, G. B. Ferraro, T. Natoli, A. Deik, L. Petronio, A. A.Tang, C. Zhu, L. Wang, D. Rosenberg, V. Mangena, J. Roth, K. Chung, R. K. Jain, C. B.Clish, M. G. V. Heiden and T. R. Golub. A metastasis map of human cancer cell lines. Nature2020, 588, 331–336.9. A. Fuhrmann, A. Banisadr, P. Beri, T. D. Tlsty and A. J. Engler. Metastatic state of cancercells may be indicated by adhesion strength. Biophys. J. 2017, 112, 736-745.10. K. Shiga, M. Hara, T. Nagasaki, T. Sato, H. Takahashi and H. Takeyama. Cancer-Associated Fibroblasts: Their Characteristics and Their Roles in Tumor Growth. Cancers,2015, 7, 2443–2458.11. F. Rossi, H. Noren, R. Jove, V. Beljanski and K. H. Grinnemo. Differences andsimilarities between cancer and somatic stem cells: therapeutic implications. Stem Cell Res. Ther.2020, 11, 489. 12. C. Alibert, B. Goud and J. -B. Manneville. Are cancer cells really softer than normal cells? Biol. Cell.2017, 109, 167-189.

Claims

CLAIMS1. An in vitro method for identifying metastatic potential of a cancer cell sample from a non-metastatic cancer, the method comprising the steps of:a) culturing said cancer cell sample from a non-metastatic cancer in a composition comprising cancer-associated fibroblasts, CAFs, preferably in two-dimensional or three-dimensional cell culturing conditions; and b) monitoring morphological alterations in the cells of said cancer cell sample during and / orafter the culturing step a), and / or determining the expression level of biomarkers relating tometastatic potential in the cells of said cancer cell sample during and / or after the culturing stepa); wherein the cells of said cancer cell sample from a non-metastatic cancer which show morphological alterations relating to metastatic potential and / or changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measured from corresponding non-metastatic cancer samples cultured in the presence of normal fibroblasts, NFs, are identified as having metastatic potential.

2. The method according to claim 1 further comprising steps of c) separately culturing ahealthy cell sample in a composition comprising cancer-associated fibroblasts, CAFs, preferably in two-dimensional or three-dimensional cell culturing conditions, wherein saidhealthy cell sample is taken from the same patient and from the same tissue type as saidcancer cell sample, and d) monitoring morphological alterations in the cells of said healthycell sample during and / or after the culturing step c), and / or determining the expression levelof biomarkers relating to metastatic potential in the cells of said healthy cell sample duringand / or after the culturing step c), wherein the cells of said healthy cell sample which showmorphological alterations relating to malignant alterations, metastatic alterations or increasedpotential for malignant alterations or metastatic alterations and / or changes in the expressionlevels of said biomarkers in comparison to the levels of said biomarkers measured fromcorresponding healthy cell samples cultured in the presence of normal fibroblasts, NFs,identify or confirm that said non-metastatic cancer has metastatic potential.

3. The method according to claim 1 or 2, wherein, in culturing step a) or c), the cell sampleand said cancer-associated fibroblasts, CAFs, are cultured in a composition comprising one ormore extracellular matrix, ECM, component, preferably selected from the group consisting of:collagens, elastin, fibronectin and laminins.

4. The method according to any one of claims 1-3, wherein, in culturing step a) or c), the cellsample and said cancer-associated fibroblasts, CAFs, are cultured in a compositioncomprising one or more of the following ingredients: agarose, alginate, fibrin, chitosan, andalbumin.

5. The method according to claim 3 or 4, wherein said extracellular matrix, ECM, componentis collagen-1.

6. The method according to any one of claims 1-5, wherein said non-metastatic cancer is breast cancer.

7. The method according to any one of claims 1-6, wherein said morphological alterationsmonitored in step b) or d) are related to the form of the cells, cell-cell interactions, andmotility of the cells.

8. The method according to any one of claims 1-7, wherein one or more of said biomarkers the expression level of which is determined in step b) or d) are selected from the group consisting of: vimentin, E-cadherin, Snail, and CYR61.

9. The method according to claim 8, wherein the expression level of vimentin, Snail, and / orCYR61 is / are substantially upregulated in cancer cells having metastatic potential or uponattaining metastatic potential, when cultured in a composition comprising CAFs, incomparison to the levels of said biomarkers measured from corresponding non-metastatic cancer samples cultured in the presence of normal fibroblasts.

10. The method according to claim 8, wherein the expression level of E-cadherin issubstantially downregulated in cancer cells having metastatic potential or upon attainingmetastatic potential, when cultured in a composition comprising CAFs, in comparison to thelevel of said biomarker measured from corresponding non-metastatic cancer samples cultured in the presence of normal fibroblasts.

11. The method according to claim 2, wherein the expression level of vimentin, Snail, and / orCYR61 is / are substantially upregulated in healthy cells having malignant alterations orincreased potential for malignant alterations, when cultured in a composition comprising CAFs, in comparison to the levels of said biomarkers measured from corresponding healthy cells cultured in a composition comprising normal fibroblasts.

12. The method according to claim 2, wherein the expression level of E-cadherin is substantially downregulated in healthy cells having malignant alterations or increased potential for malignant alterations, when cultured in a composition comprising CAFs, in comparison to the levels of said biomarker measured from corresponding healthy cells cultured in a composition comprising normal fibroblasts.

13. A kit comprising means to detect at least three of the biomarkers selected from the group consisting of: vimentin, E-cadherin, Snail, and CYR61, or expression level thereof, whereinsaid kit comprises means to assay presence and / or the level of gene expression of saidbiomarkers, preferably in a cancer sample from a non-metastatic cancer or a pre-treated sample thereof.

14. The kit according to claim 13, wherein said kit comprises an RT-qPCR platform fordetecting and quantifying the amount of mRNA of at least three of the biomarkers selected from the group consisting of: vimentin, E-cadherin, Snail, and CYR61.

15. The kit according to claim 13, wherein said kit comprises a lateral flow test to detect theprotein expression of at least one of the biomarkers CYR61, Snail, Vimentin, and E-cadherin.

16. The kit according to claim 13, wherein said kit comprises i) an RT-qPCR platform fordetecting and quantifying the amount of mRNA of at least three of the biomarkers selectedfrom the group consisting of: vimentin, E-cadherin, Snail, and CYR61, and ii) a lateral flowtest to detect the protein expression of at least one of the biomarkers CYR61, Snail, Vimentin,and E-cadherin.

17. The kit according to any one of claims 13-16, wherein the kit consists of means to detectthe biomarkers vimentin, E-cadherin, Snail, and CYR61, or expression level thereof.

18. Use of the kit according to any one of claims 13-17 for identifying the metastatic potentialof a cancer cell sample.

19. The use according to claim 18, wherein said cancer cell sample is taken from a cancer patient having a non-metastatic cancer.

20. The use according to claim 18 or 19, wherein said cancer cell sample is a solid biopsy, a liquid biopsy, or a blood sample.

21. The use according to claim 18, wherein said cancer cell sample is taken from a cellculture, wherein the cancer cells from a non-metastatic cancer have been cultured in acomposition comprising cancer-associated fibroblasts, CAFs.

22. An in vitro method for identifying malignant alterations in a healthy cell, the methodcomprising the steps of:a) culturing said healthy cell in a composition comprising cancer-associated fibroblasts,CAFs, preferably in two-dimensional or three-dimensional cell culturing conditions; andb) monitoring morphological alterations in the cell during or after the culturing step a), and / ordetermining the expression level of biomarkers relating to metastatic potential in the cellduring and / or after the culturing step a);wherein the cell showing morphological alterations relating to metastasis and / or changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measured from corresponding healthy cell samples cultured in a composition comprising normal fibroblasts, NFs, are identified as having malignant alterations.

23. The method according to claim 22, wherein, in culturing step a), said healthy cell and saidcancer-associated fibroblasts, CAFs, are cultured in a composition comprising one or moreextra cellular matrix, ECM, component, preferably selected from the group consisting of: collagens, elastin, and fibronectin.

24. The method according to claim 22 or 23, wherein, in culturing step a), said cancer cellsample from a non-metastatic cancer and said cancer-associated fibroblasts, CAFs, arecultured in a composition comprising one or more of the following ingredients: agarose,alginate, fibrin, chitosan, and albumin.

25. The method according to claim 23 or 24, wherein said extra cellular matrix, ECM, component is collagen-1.

26. The method according to any one of claims 22-25, wherein said healthy cell is a breast tissue cell.

27. The method according to any one of claims 22-26, wherein said morphological alterations monitored in step b) are related to the form of the cells, cell-cell interactions, and motility of the cells.

28. The method according to any one of claims 22-27, wherein one or more of said biomarkers the expression level of which is determined in step b) are selected from the group consisting of: vimentin, E-cadherin, Snail, and CYR61.

29. The method according to claim 28, wherein the expression level of vimentin, Snail, and / orCYR61 is / are substantially upregulated in cells having metastatic potential or upon attainingmetastatic potential, when cultured in a composition comprising CAFs, in comparison to thelevels of said biomarkers measured from corresponding cells cultured in a compositioncomprising normal fibroblasts.

30. The method according to claim 28, wherein the expression level of E-cadherin issubstantially downregulated in cells having metastatic potential or upon attaining metastaticpotential, when cultured in a composition comprising CAFs, in comparison to the levels ofsaid biomarker measured from corresponding cells cultured in a composition comprising normal fibroblasts.

31. A system for in vitro culturing eukaryotic cells comprising a solid surface comprising atleast one partition suitable for culturing eukaryotic cells, wherein said at least one partition contains a combined cell culture comprising i) cancer cells from a non-metastatic cancer or healthy cells and ii) cancer-associated fibroblasts, CAFs, wherein said partition is a well orchamber, and wherein said well or chamber is preferably interconnected via a channel orchannels to other wells or chambers in said solid surface.

32. The system according to claim 31, wherein said at least one partition further comprises one or more extra cellular matrix, ECM, component, preferably selected from the group consisting of: collagens, elastin, and fibronectin.

33. The system according to claim 31 or 32, wherein said at least one partition further comprises one or more of the following ingredients: agarose, alginate, fibrin, chitosan, and albumin.

34. The system according to claim 32 or 33, wherein said extracellular matrix, ECM,component is collagen-1.

35. The system according to any one of claims 31-34, wherein said cancer cells are from anon-metastatic breast cancer.

36. The system according to any one of claims 31-34, wherein said healthy cells are breast tissue cells.

37. The system according to any one of claims 31-36, wherein said system comprises a parallel control cell culture on the same or separate solid surface, wherein said control cell culture comprises a combined cell culture comprising i) cancer cells from non-metastaticcancer or healthy cells and ii) normal fibroblasts, NFs.

38. The system according to any one of claims 31-36, wherein said system comprises a firstpartition and a second partition, wherein said first partition contains a combined cell culturecomprising cancer cells from a non-metastatic cancer and cancer-associated fibroblasts,CAFs, and wherein said second partition contains a combined cell culture comprising healthycells and cancer-associated fibroblasts, CAFs, wherein said healthy cells are taken from thesame patient and from the same tissue type as said cancer cells.

39. An in vitro method for identifying metastatic potential of a cancer cell sample, the methodcomprising the steps of:a) providing said cancer cell sample; andb) determining the expression level of biomarkers relating to metastatic potential in the cells of said cancer cell sample; wherein at least three of said biomarkers the expression level of which is determined in step b) are selected from the group consisting of: vimentin, E-cadherin, Snail, and CYR61; and wherein changes in the expression levels of said biomarkers in comparison to the levels of said biomarkers measured from corresponding cancer samples are identified as having metastatic potential.

40. The method according to claim 39, wherein said biomarkers the expression level of whichis determined in step b) comprise or consists of vimentin, E-cadherin, Snail, and CYR61.

41. The method according to claim 39 or 40, wherein the expression level of vimentin, Snail,and / or CYR61 is / are substantially upregulated in cells having metastatic potential or uponattaining metastatic potential in comparison to the levels in a reference standard prepared forthe comparison of the expression levels.

42. The method according to any one of claims 39-41, wherein the expression level of E-cadherin is substantially downregulated in cells having metastatic potential or upon attainingmetastatic potential in comparison to the levels in a reference standard prepared for thecomparison of the expression levels.

43. An in vitro method for screening drug candidates for preventing the metastatic spread ofcancer cells, the method comprising the steps of:a) culturing a cancer cell sample from a non-metastatic cancer or a sample of healthy cells in acomposition comprising cancer-associated fibroblasts, CAFs, and in the presence and absenceof a drug candidate compound, preferably in two-dimensional or three-dimensional cell culturing conditions; and b) monitoring morphological alterations in the cells of said cancer cell sample or of the sample of healthy cells during and / or after the culturing step a), and / or determining theexpression level of biomarkers relating to metastatic potential in the cells of said cancer cellsample or said sample of healthy cells during and / or after the culturing step a); wherein said drug candidate compound is having a preventing effect on the metastatic potential of said cancer or healthy cells if said cancer cells or healthy cells show lessmorphological alterations relating to metastatic potential and / or less changes in the expressionlevels of said biomarkers in comparison to the levels of said biomarkers measured fromcorresponding cancer cells or healthy cells cultured in a composition comprising normalfibroblasts, NFs, in the presence of said drug candidate compound than in the absence of said drug candidate compound.

44. The method according to claim 43, wherein, in culturing step a), i) said cancer cell sample from a non-metastatic cancer and said cancer-associated fibroblasts, CAFs, or ii) said sample of healthy cells and said cancer-associated fibroblasts, CAFs, are cultured in a compositioncomprising one or more extra cellular matrix, ECM, component, preferably selected from thegroup consisting of: collagens, elastin, and fibronectin.

45. The method according to claim 43 or 44, wherein, in culturing step a), said cancer cell sample from a non-metastatic cancer and said cancer-associated fibroblasts, CAFs, arecultured in a composition comprising one or more of the following ingredients: agarose,alginate, fibrin, chitosan, and albumin.

46. The method according to claim 44 or 45, wherein said extra cellular matrix, ECM, component is collagen-1.

47. The method according to any one of claims 43-46, wherein said non-metastatic cancer is breast cancer, or said healthy cell is a breast tissue cell.

48. The method according to any one of claims 43-47, wherein said morphological alterations monitored in step b) are related to the form of the cells, cell-cell interactions, and motility of the cells.

49. The method according to any one of claims 43-48, wherein one or more of said biomarkers the expression level of which is determined in step b) are selected from the group consisting of: vimentin, E-cadherin, Snail, and CYR61.

50. The method according to claim 49, wherein the expression level of vimentin, Snail, and / orCYR61 is / are substantially upregulated in cells having metastatic potential or upon attainingmetastatic potential, when cultured in a composition comprising CAFs, in comparison to thelevels of said biomarkers measured from corresponding cells cultured in a compositioncomprising normal fibroblasts.

51. The method according to claim 49, wherein the expression level of E-cadherin issubstantially downregulated in cells having metastatic potential or upon attaining metastaticpotential, when cultured in a composition comprising CAFs, in comparison to the level of saidbiomarker measured from corresponding cells cultured in a composition comprising normalfibroblasts.

Images