TECHNICAL FIELD

[001] The present disclosure generally relates to the field of cancer biology. More particularly, the present disclosure relates to products that serve as models for epithelial to mesenchymal transition, methods and uses thereof.

BACKGROUND

[002] Epithelial to Mesenchymal Transition (EMT) is a natural phenomenon that occurs during early development. This is required for cells to be mobile in the embryo during the developmental process and is generally lost in mature adults. In adults, EMT is reactivated in cancer where it is involved in the generation of cancer stem cells and also in the initial steps of metastasis (Ouyang G et al, 2010, Cell Mol Life Sci; 67 (15): 2605-18). EMT is required to confer the property of motility on the cancer epithelial cells, to enable metastasis. Frequently, internal organs including the lungs, liver and bone are the target sites for such metastases (Gupta PB et al, 2005, Cell 138 (4): 645-59). Establishment of tumour in vital internal organs interferes with their proper functioning leading to organ failure and death, thus decreasing the probability of successful treatment of the patient (Hanahan D et al, 2011, Cell 144: 646-674).

[003] The existing treatments are unable to successfully eradicate the disease due to higher rates of relapse primarily due to metastasis (Christofori G, 2006, Nature 441 : 444-450). Further the rate of discovery of newer and more effective therapeutic strategies for cancer has not been proportional to the amount of data available in literature about the development and behaviour of the disease. Therefore, there is still an unmet need for novel drugs that prevent cancer relapse and metastases.

[004] Studies on the mechanism of EMT through in vitro EMT models can throw light on many aspects of metastasis leading to novel cancer therapies and biomarkers to monitor metastasis (Lee JM et al, 2006, J Cell Biol, 172(7): 973-81). Therapeutics targeting EMT will target the crucial steps of metastasis and the formation of secondary sites of cancer. [005] Companies like Verastem Inc. and Boehringer Ingelheim have developed in vitro EMT platforms. The EMT model developed by Verastem Inc. uses SV40 viral oncogene for immortalising breast culture cells with overexpression of ras and inhibition of E-Cadherin. Boehringer Ingelheim has established EpH4 cell lines as an EMT platform by expressing ras/V12 ras, and bcl2 in a spontaneously immortalized breast cancer cell line. Use of viral regulators for immortalisation of cells frequently involves inactivation of tumor suppressor genes which might lead to transformation of cell lines. In such cases, there is a possibility that the basic properties of the cell lines might be altered which is not desirable.

[006] In the light of the aforementioned discussion, there exists a need for an in vitro EMT model that is derived from healthy and normal human tissue to mimic origin of cancer, to screen novel drugs which perturb and potentially inhibit EMT and also serve as a platform in which specific aspects of the mechanisms of EMT may be studied. It is an object of the present invention to develop an in vitro EMT model/platform based on the use of normal cells and thus be physiologically more relevant to human cancer. The present invention discloses products that serve as in vitro EMT models that can be used in the laboratory at several points in the drug discovery process such as target discovery, validation, screening for small and large molecule drugs alone or in combination with other drugs, or target deconvolution after identifying lead molecules in phenotypic screening in an unbiased manner. Cells demonstrating varying stages of EMT can be used to evaluate the modulatory effects of molecules with different mechanisms of action. Also, the products disclosed in the present invention would aid in identifying novel biomarkers related to cancer and to carry out basic mechanistic studies of generation of CSCs, cancer progression and metastasis.

[007] The EMT process has been shown to generate cancer stem cells (CSCs) that are more resistant to standard treatments such as chemo- or radio- therapies and are implicated in the relapse of cancer. Hence this platform can serve to create a process by which enriched population of stem cells or tumour-initiating cells can be obtained and be used as a screen for identification of new molecules or targets that can inhibit CSCs.

[008] The present invention also discloses a controlled generation of a platform of cell types representing EMT using specific engineered genes which are known to be involved in the process of EMT. In this invention, normal human mammary epithelial cells are used as a starting point and EMT is induced by over-expression of an EMT inducer. The present invention uses mammalian cellular regulators such as c-myc (Drissi R et al, 2001, J Biol Chem; 276 (32): 29994-30001) or hTERT (Dimri G et al, 2005; Breast Cancer Res: 7 (4): 171-9) to effect immortalisation instead of a viral oncogene. The epithelial to mesenchymal transition is effected by the over-expression of EMT inducers such as c-met which is a known initiator of EMT in several human epithelial cancers including liver, breast, gastric etc. and is reported to be a driver for the loss of epithelial phenotype and the acquisition of a migratory phenotype (Gaule PB et al, 2014; Expert Opin Ther Targets; 18 (9): 999-1009, Organ SL et al, 2011 ; Ther Adv Med Oncol; 3 (1 Suppl): S7-S19).

BRIEF SUMMARY

[009] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[010] Exemplary embodiments of the present disclosure are directed towards products that serve as EMT models. The product comprises a plurality of components, wherein the plurality of components are selected from one or more of the following groups: a primary epithelial cell population; a primary fibroblast cell population; a first immortalized stable cell line with epithelial or intermediate phenotype harbouring an engineered immortalizing gene construct; a second immortalized stable cell line with epithelial or intermediate phenotype harbouring an engineered immortalizing gene construct and an engineered assayable marker gene construct; a third immortalized stable cell line with mesenchymal-like or mesenchymal phenotype harbouring an engineered immortalizing gene construct, an engineered assayable marker gene construct and an engineered EMT inducing gene construct; an EMT modulating agent; and an enriched population of cancer stem cells derived from the third immortalized stable cell line.

[011] Other exemplary embodiments of the present subject matter are directed towards methods for making a product that serve as EMT models. The method starts with providing a normal epithelial cell population followed by introducing an engineered immortalizing gene construct into the normal epithelial cell population followed by culturing and selection of the transfected cell population through multiple passages to ensure stable expression of an immortalizing agent to yield a first immortalized stable cell line, wherein the first immortalized stable cell line harbours the engineered immortalizing gene construct and has an epithelial or intermediate phenotype. This is followed by introducing an engineered assayable marker gene construct into the first immortalized stable cell line to yield a second immortalized stable cell line, wherein the second immortalized stable ceil line harbours the engineered immortalizing gene construct and the engineered assayable marker gene construct and has an epithelial or intermediate phenotype. Then an engineered EMT inducing gene construct is introduced into the second immortalized stable cell line to yield a third immortalized stable cell line, wherein the third immortalized stable cell line has a mesenchymal-like or mesenchymal phenotype and harbours the engineered immortalizing gene construct, the engineered assayable marker gene construct and the engineered EMT inducing gene construct. The final step comprises of providing a product that serves as an EMT model by assembling a plurality of components, wherein the plurality of components comprise one or more of the following groups: a primary epithelial ceil population; a primary fibroblast cell population; the first immortalized stable cell line; the second immortalized stable cell line; the third immortalized stable cell line; an EMT modulating agent; and an enriched population of cancer stem cells derived from the third immortalized stable cell line.

[012] Other exemplary embodiments of the present subject matter are directed towards uses of products that serve as EMT models. The plurality of components disclosed herein can be used for evaluation of drug candidates that target EMT and/or cancer stem cells, evaluation of drug targets in EMT and/or cancer stem cells, evaluation of biomarkers in EMT and/or cancer stem cells, derivation of cancer stem cells and use as a disease model for studying underlying biology of EMT and stem cells.

[013] It is an object of the present invention to develop an in vitro EMT model/platform based on the use of normal cells and thus be physiologically more relevant to human cancer.

[014] It is yet another object of the present invention to create a novel in vitro EMT platform which can be used in the laboratory at several stages in the drug discovery process such as target discovery, validation, screening for small and large molecule drug candidates alone or in combination with other drugs, or target decon volution after identifying lead molecules in phenotypic screening in an unbiased manner. Cells demonstrating varying stages of EMT can be used to evaluate the modulatory effects of molecules with different mechanisms of action. [015] It is yet another object of the present invention to disclose products that would aid in identifying novel biomarkers related to cancer and to carry out basic mechanistic studies of cancer progression.

[016] It is yet another object of the present invention to disclose products that would create a process by which an enriched population of stem cells or tumour- initiating cells can be obtained and be used as a screen for identification of new molecules or targets that can inhibit CSCs.

BRIEF DESCRIPTION OF DRAWINGS

[017] Other objects and advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments, in conjunction with the accompanying drawings, wherein like reference numerals have been used to designate like elements, and wherein:

[018] Fig. 1 is a representative phase contrast microscopy image of normal human mammary epithelial cells (HMECs, Left panel) and normal human mammary fibroblasts (Right panel) grown in two dimensional culture at passage number 2. Objective magnification was 10X. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[019] Fig. 2 is an image representing expression of epithelial and mesenchymal markers in the isolated human mammary epithelial and fibroblast cells determined by PCR. PCR products denoting expression of the genes Mucin 1 (171 bp, Lanes 1,2), Desmoglein 2 (278 bp, Lanes 3,4), Slug (181 bp, Lanes 6,7), Snail (248 bp, Lanes 8,9), Twist 1 (200 bp, Lanes 11,12), and Fibronectin (382 bp, Lanes 13,14) are shown. GAPDH (115 bp, Lane 15) is used as a positive control for the PCR reactions. Lanes 5 and 10 represent the DNA ladder used to determine the size of the PCR products. The letter Έ' denotes epithelial cells and 'F' denotes fibroblasts as the source of the template. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[020] Fig. 3 represents immunofluorescence images of HMECs. Representative images are at passage 2 showing staining for epithelial markers EpCAM (Panel A) and Cytokeratin 8/18 (CK8/18, Panel B). The cells do not have any detectable staining for the mesenchymal marker Vimentin (Panel C). All cells were also stained for DAPI, a nuclear marker, to visualize the number of cells and nuclear vs. cytosolic localization. Objective magnification was 60X. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[021] Fig. 4 represents immunofluorescence staining of normal human mammary fibroblast cells. Representative images are at passage 2 demonstrating lack of staining with the epithelial marker EpCAM (Panel A). Cells stained for the mesenchymal markers, a-Smooth Muscle Actin (a-SMA, Panel B) and Vimentin (Panel C) show positive staining for both. All cells were also stained for DAPI, a nuclear marker, to visualize the number of cells and localize the nuclear vs cytosolic staining. Objective magnification was 60X. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[022] Fig. 5 is a graphic representation of expression of epithelial and mesenchymal markers in c-myc immortalized stable cell line as determined by Quantitative Real-Time PCR. Epithelial markers used were Mucin 1 and Desmoglein 2, while the mesenchymal markers used were Snail and Fibronectin. The open bars represent HMECs at passage 3, and the filled bars represent passage 10 of HMECs transfected with c-myc. Bars denote average and SD of the assay done in triplicate. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[023] Fig. 6 represents immunofluorescence images of HMECs transfected with c-myc. Representative images are at passage 10 showing cells stained with EpCAM (Left panel) and Vimentin (Right panel). All cells were stained with DAPI to visualize the number of cells and localization of cytosolic staining. Objective magnification was 20X. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[024] Fig. 7 is a graphic representation of expression of epithelial and mesenchymal markers in HMECs transfected with c-myc and E-Cadherin-GFP, as determined by Quantitative Real- Time PCR. Epithelial markers used were Mucin 1 and Desmoglein 2, while the mesenchymal markers used were Snail and Fibronectin. The open bars represent HMECs at passage 3, filled bars represent HMECs transfected with c-Myc and the grey shaded bars represent double transfected cells. Both the c-myc and double transfected cells were from passage 10. Bars denote average and SD of the assay done in triplicate. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[025] Fig. 8 represents immunofluorescence images of double transfected cells at passage 10. EpCAM (Panel A) was used as an epithelial marker and Vimentin (Panel B) was used as a mesenchymal marker. All cells were stained with DAPI to visualize the nucleus and hence the number of cells. Objective magnification was 20X. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[026] Fig. 9 is a representative phase contrast microscopy image of HMECs triple transfected with c-Myc, E-Cadherin-GFP, and c-Met-tomato maintained in two dimensional culture at passage number 10. Objective magnification is at 10X. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[027] Fig. 10 is a graphical representation of the expression of epithelial and mesenchymal markers in HMECs triple transfected with c-myc, E-Cadherin-GFP and c-Met-tomato, as determined by Quantitative Real-Time PCR. Epithelial markers used were Mucin 1 and Desmoglein 2, while the mesenchymal markers used were Snail and Fibronectin. The open bars represent HMECs at passage 3, filled bars represent HMECs transfected with c-myc alone and the grey shaded bars represent triple transfected cells. Both the c-myc and triple transfected cells were obtained from passage 10. Bars denote average and SD of the assay done in triplicate. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[028] Fig. 11 is an image showing immunofluorescence staining of HMECs triple transfected with c-Myc, E-Cadherin-GFP, and c-Met, at passage 10. Representative images showing staining for the epithelial marker EpCAM (Panel A) and for the mesenchymal marker Vimentin (Panel B). Cells were also stained with DAPI to visualize the nucleus and cytosolic staining. Objective magnification was 20X. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[029] Fig. 12 represents a fluorescence microscopy image of triple transfected HMECs representing the expression of red coloured tomato reporter gene (right panel). Phase contrast microscopy image of the cells is depicted in the left panel. Images are taken with 20X objective. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[030] Fig. 13 represents images of β-galactosidase assay to determine senescence in the different cell types developed. HMECs at passage 6 (Panel A) show intense blue colour in groups of cells indicated by arrows. C-myc (Panel B), C-myc and E-Cadherin-GFP (Panel C), and C-Myc, E-Cadherin-GFP, and C-Met-tomato (Panel D) transfected cells were negative for the β-galactosidase assay. All the three transfected cell types used in this assay were from passage 10. Objective magnification was 10X. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[031] Fig. 14 is a graphical representation of Real-Time quantitative PCR to determine the expression levels of hTERT. HMECs at passage 3 were used in this assay while the three transfected cell lines were from passage 10. Relative fluorescence units were normalized to HMECs set at 100%. Graph denotes the average and SD of the expression levels from an assay done in triplicate. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[032] Fig. 15 shows images representing sphere forming assay in non-adherent plates and serum-free culture conditions. 100,000 cells were seeded and allowed to form spheres for 7 days. HMECs (Panel A) failed to form spheres and remained dispersed and slowly died, while cells transfected with c-myc (Panel B), c-myc + E-Cadherin-GFP (Panel C), and with c-myc + E-Cadherin-GFP + c-met-tomato (Panel D) formed spheres, albeit at different rates and sizes. HMECs were at passage 3 while the other three cell lines were at passage 10. Images were taken with 10X objective. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[033] Fig. 16 shows images representing colony forming assay in soft agar. Cells were seeded and allowed to form colonies for 40 days. Primary epithelial cells (Panel A) failed to form colonies, while cells transfected with c-myc + E-Cadherin-GFP (Panel B), and with c-myc + E-Cadherin-GFP + c-met-tomato (Panel C) formed colonies of different sizes. Images were taken with 10X objective of phase contrast microscope. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure. [034] Fig. 17 is a graphical representation of the number of colonies formed in soft agar by the different cell types, based on the size of the colonies enumerated on Day 40. The graphs represent the average and SD from duplicate wells of a representative experiment. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[035] Fig. 18 represents a flow cytometric detection of cancer stem cells (CSCs) by CD44 and CD24 antibody staining. HMECs (Panel A) were grown in 2 dimensional culture and then stained by the antibodies, c-myc single transfected cells (Panel B) and triple transfected cells (Panel C) were grown as mammospheres and then used for the staining. Stained cells were gated for CD44+ and CD24- population and sorted in a FACS Canto II (BD Biosciences). The bottom right quadrant represents the CD44+/CD24- cells reported to be cancer stem cells. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

[036] Fig. 19 is a graphical representation of migration potential of the different cell types as determined by wound healing assay. The panels correspond to A) HMECs, B) HMECs transfected with c-Myc, C) HMECs transfected with c-Myc and E-Cadherin-GFP, and D) HMECs transfected with c-Myc, E-Cadherin-GFP, and c-Met-Tomato. The percentage of open wound area was calculated based on the density of the cells present in the scratch, when compared to the 0 hour of HMECs which was set at 100%. The graphs are average and SD of triplicate wells from a representative experiment of two independent experiments. This figure is in accordance with a particular non limiting exemplary embodiment of the present disclosure.

DETAILED DESCRIPTION

[037] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. [038] The use of "including", "comprising" or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms "a" and "an" herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms "first", "second", and "third", and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[039] According to non limiting exemplary embodiments of the present disclosure, products that serve as EMT models are disclosed.

[040] In accordance with non limiting exemplary embodiments of the present subject matter, methods for making products that serve as EMT models are disclosed.

[041] According to non limiting exemplary embodiments of the present disclosure, use of products that serve as an EMT model for different purposes is disclosed.

[042] Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts.

[043] The term "normal" used with reference to epithelial cells in the present disclosure refers to non-cancerous healthy tissue. The tissues used in these studies were obtained from young healthy females who did not have cancer. The individuals elected to undergo breast reduction surgery (reduction mammoplasty) for cosmetic reasons and voluntarily donated the tissue for research purposes. The epithelial and fibroblast cells isolated from such non-diseased healthy tissue samples are considered normal.

[044] The term "tissue" or "sample" used in the present disclosure refers to any biological fluid or tissue. In certain embodiments, a sample may be a tissue containing epithelial cells, either non-diseased or malignant cells. Useful biological samples include, without limitation, surgical material from solid tumours, biopsy, bone marrow, blood, saliva, sputum, urine, cerebrospinal fluid, cervical smear and other cellular exudates from a person. Such samples may further be diluted with sterile saline, PBS, cell culture medium, or a pH-balanced physiologically acceptable solution prior to grossing and processing. [045] The term "Immortalized stable cell line" used in the present disclosure encompasses primary cells modified either through introduction of a gene such as the catalytic subunit of the human telomerase enzyme (hTERT) encoded in a vector, or spontaneously due to acquisition of genetic changes, to become a continuously growing or 'immortalized cell line's. Unlike normal cells, immortalized cells do not undergo senescence typically observed at passage 5-7. Such immortalized cells, able to be passaged beyond passage 7 are considered a stable cell line. Examples of immortalizing genes include hTERT, myc, bmi-1 and those described in international patent publication numbers EP2611902 and references incorporated therein.

[046] The term "engineered immortalizing gene construct" used in the present disclosure refers to gene(s) cloned into vectors using standard recombinant DNA techniques to create plasmids that can be introduced into cells to immortalize them and generate stable cell lines. Examples of engineered immortalizing gene constructs include a vector (viral or others) coding for any of hTERT, bmi-1, myc genes and those described in international patent publication numbers EP2611902, US8642834, US7998688 and the references incorporated therein.

[047] The term "engineered assayable marker gene construct" refers to genes/promoters that can serve as reporters of a change in phenotype. These genes/promoters are engineered or cloned into vectors using standard recombinant DNA techniques to create gene constructs or plasmids expressing the markers fused to a fluorescent gene that when introduced into cells can be used for a quantitative assessment of change in phenotype. Examples of such assayable markers include but are not limited to E-cadherin, cytokeratin 8, cytokeratin 18, as epithelial markers and N-cadherin, vimentin, twist, snail, fibronectin, zeb as mesenchymal markers and those cited in international patent publication numbers incorporated herein by reference (US7998688, WO2010011642, US7951549) and the references incorporated therein.

[048] Reporter genes that can be fused with such assayable markers include but are not limited to fluorescent GFP, RFP, YFP, BFP, luminescent markers such as luciferase, colorimetric and others included herein by reference from international patent publication number, US7951549 and the references incorporated therein.

[049] The term 'engineered EMT inducing gene construct' used in the present disclosure refers to genes or promoters whose over- or under-expression can lead to EMT, that are cloned in vectors using standard recombinant DNA techniques. These vectors can be introduced into cells to effect EMT in those cells. Examples of genes and promoters include c-Met, Twist, Snail, Slug, CDH1 promoter, Src kinase and those described in International Patent Publication Numbers incorporated herein by reference (US7951549, US20110191868, WO2010011642)

[050] The term 'cancer stem cells' includes cells that have the property of self-renewal as well as initiating, progressing and maintaining tumours. These cells are generated during the process of EMT and may also be termed stem cells or progenitor cells, incorporated by references herein (CA2656548, WO2013131000). CSCs or SCs have been shown to be more resistant to chemo- and radiation therapies (Kwiatkowska-Borowczyk et al Contemp Oncol (Pozn) 2015; 19: A52-A59, Fillmore and Kuperwasser, Breast Cancer Res, 2008, 10: R25).

[051] The term "epithelial cancer" used in the present disclosure covers an epithelial cell cancer selected from the group consisting of carcinomas that derive from epithelial cell types. Among such cancers are included breast, colorectal, ovarian, lung, pancreatic, kidney, prostate and gastric cancer, among others. Also included among these cancers are aggressive cancer subtypes, basal cancer or lobular cancer, e.g., certain aggressive breast cancers.

[052] The term 'mesenchymal cells or phenotype' encompasses cells that are loosely associated and that have lost the tight junctions observed in epithelial cells, express markers including FN, snail, slug, vimentin, aSMA, zebl, N-cadherin, CDH1 methylation, FOXC2, twist demonstrate increased motility and invasiveness (Yausch et al Clin Cancer Res, 2005 11 ; 8686, Kim et al Experimental & Molecular Medicine (2015) 47, el37). Additional mesenchymal cell biomarkers include, fibrillin- 1, fibrillin-2, collagen alpha2(IV), collagen alpha2(V), LOXL1, nidogen, Cl l or f9, tenascin, tubulin alpha-3, and epimorphin. Basal-type breast cancers that are known to be the most aggressive with high rate of recurrence and poor overall survival display more mesenchymal phenotype compared with the luminal cancers (Kim et al Experimental & Molecular Medicine (2015) 47, el37).

[053] The term "Intermediate phenotype" used in the present disclosure refers to the following: The EMT process is a continuum comprising many steps and cellular phenotypes that capture multiple discrete stages along the way of epithelial cells becoming mesenchymal cells. These phenotypes may be designated as 'intermediate or mixed phenotypes' since they exhibit some characteristics of epithelial cells and some of mesenchymal cells. Examples of such 'intermediate phenotypes' may range from mostly epithelial-like cells that demonstrate expression of some epithelial markers but not all, little to no expression of mesenchymal proteins but show altered morphology compared to normal epithelial cells. These cells may be at an early stage of transition towards mesenchymal cells. Intermediate phenotypes may also include mesenchymal-like cells that have acquired the expression of mesenchymal proteins whilst also expressing low levels of epithelial proteins, exhibit increased motility in migration and invasion assays, but not be able to form colonies in soft agar. Such intermediate phenotypes are valuable in identifying drug targets or molecules that can modulate early stages of EMT and cancer-promoting activities of mesenchymal cells. In one embodiment, co-expression of CK8/18 and EpCAM with Vimentin and aSMA may be termed an intermediate phenotype.

[054] The term "evaluation" used in the present disclosure includes any screening activity to identify, validate, repurpose, prioritize, rank order a 'drug candidate', a drug target, a biomarker in a comparative manner to select promising candidates or optimize leads as examplified incorporated herein by reference (US8642834, US7998688, US7951549, US20110191868)

[055] The term 'drug candidate' encompasses a compound, an antibody, a peptide, a protein or a RNA that can modulate the expression and/or activity of a molecule or complex of interest (the "target") with potential to be used as a therapeutic. For example, a compound that inhibits one or more activities of a target or a cell in the presence of the compound, or as a consequence of its use, as compared with in the absence of the compound, and/or if the level or amount of the target or cell function is modulated in the presence of the compound, or as a consequence of its use, as compared with in the absence of the compound may be considered as a drug candidate. In certain embodiments, drug candidate molecules act directly on their target in that they physically interact with it. In other embodiments, compounds act indirectly, e.g., by inhibiting a second molecule that is needed for synthesis or activity of the target or cells.

[056] The term "biomarker" is defined as a laboratory measurement that reflects the activity of a disease process as incorporated herein by reference (Katz NeuroRx. 2004; 1 : 189-195). Examples of such markers include positron emission tomographic (PET) scanning of cancers wherein markers quantitatively correlate (either directly or inversely) with disease progression. In one embodiment, a gene signature of EMT or FOXC2 may be used to diagnose, monitor and/or tailor treatment as shown in international patents incorporated herein by reference (US20120302572, US8088590, CA2601157, US20120159655, WO2012116248). [057] The term "disease model" used in the present disclosure refers to an in vitro or in vivo model system that is experimentally created or induced with pathological processes sufficiently similar to those in human disease that is used to understand the origin and progression of the disease, to monitor and treat it. In one embodiment, the panel of epithelial and mesenchymal cells isolated from normal breast tissue and the ones created herein using engineered constructs can be used as model to understand the biology of cancer as a disease and EMT as a process leading up to the disease. The insights gained from such fundamental understanding would be useful in blocking or reversing the EMT process in the lab resulting in more effective treatments in the clinic.

[058] In accordance with a non limiting exemplary embodiment of the disclosure, a product that serves as an EMT model is disclosed. The product comprises a plurality of components, wherein the plurality of components are selected from one or more of the following groups: a primary epithelial cell population; a primary fibroblast cell population; a first immortalized stable cell line with epithelial or intermediate phenotype harbouring an engineered immortalizing gene construct; a second immortalized stable cell line with epithelial or intermediate phenotype harbouring an engineered immortalizing gene construct and an engineered assayable marker gene construct; a third immortalized stable cell line with mesenchymal-like or mesenchymal phenotype harbouring an engineered immortalizing gene construct, an engineered assayable marker gene construct and an engineered EMT inducing gene construct; an EMT modulating agent; and an enriched population of cancer stem cells derived from the third immortalized stable cell line.

[059] In accordance with a non limiting exemplary embodiment of the present disclosure, a method for making a product that serves as an EMT model is disclosed. The method starts with providing a normal epithelial cell population followed by introducing an engineered immortalizing gene construct into the normal epithelial cell population followed by culturing and selection of the transfected cell population through multiple passages to ensure stable expression of an immortalizing agent to yield a first immortalized stable cell line, wherein the first immortalized stable ceil line harbours the engineered immortalizing gene construct and has an epithelial or intermediate phenotype. This is followed by introducing an engineered assayable marker gene construct into the first immortalized stable cell line to yield a second immortalized stable cell line, wherein the second immortalized stable ceil line harbours the engineered immortalizing gene construct and the engineered assayable marker gene construct and has an epithelial or intermediate phenotype. Then an engineered EMT inducing gene construct was introduced into the second immortalized stable cell line to yield a third immortalized stable ceil line, wherein the third immortalized stable ceil line has a mesenchymal-like or mesenchymal phenotype and harbours the engineered immortalizing gene construct, the engineered assayable marker gene construct and the engineered EMT inducing gene construct. The final step comprises of providing a product that serves as an EMT model by assembling a plurality of components, wherein the plurality of components comprise one or more of the following groups: a primary epithelial cell population; a primary fibroblast cell population; the first immortalized stable cell line; the second immortalized stable cell line; the third immortalized stable cell line; an EMT modulating agent; and an enriched population of cancer stem cells derived from the third immortalized stable ceil line.

[060] According to non limiting exemplary embodiments of the present disclosure, different uses of products that serve as EMT model is disclosed. The plurality of components disclosed herein can be used for evaluation of drug candidates that target EMT and/or cancer stem cells, evaluation of drug targets in EMT and/or cancer stem cells, evaluation of a biomarkers in EMT and/or cancer stem cells, derivation of cancer stem cells and use as a disease model for studying underlying biology of EMT and CSCs. In a preferred embodiment, the plurality of components disclosed herein can be used for evaluation of biomarkers, targets and/or drug candidates for triple negative breast cancer embodied by the triple transfected cell line. It has been reported that EMT induced by c-Met causes triple negative breast cancer (Minuti and Landi , 2015).

[061] In accordance with a non limiting exemplary embodiment of the present disclosure, the immortalized stable cell lines are obtained from human epithelial cells. The immortalized stable cells can be obtained from the epithelial cells of other mammalian species also without limiting the scope of the present disclosure.

[062] According to a non limiting exemplary embodiment of the present disclosure, the immortalized stable cell lines are obtained from mammary epithelial cells. The immortalized stable cells can be obtained from the epithelial cells of other organs also without limiting the scope of the present disclosure.

[063] In accordance with a non limiting exemplary embodiment of the present disclosure, the immortalized stable cell lines are obtained from normal epithelial cells. The immortalized stable cells can also be obtained from benign tumour cells of epithelial phenotype without limiting the scope of the present disclosure.

[064] Epithelial cells (or other cells) for use in composition and methods of the invention and/or to which methods of the invention may be applied, can be obtained from any of a wide variety of sources or, in the case of certain in vivo applications, may be present in a variety of tissues or organs. The cells may be primary cells, cells of a cell line, untransformed cells, transformed cells, genetically modified cells, or non-genetically modified cells, in various embodiments. For example, cells can be obtained from a human or other mammalian subject, from discarded surgical or cellular samples from a subject, or from a propagated cell line.

[065] According to a non limiting exemplary embodiment of the present disclosure, the engineered immortalizing gene construct is designed to overexpress an immortalizing agent. In different embodiments, c-myc, h-TERT and/or Bmil are used as immortalizing agents though any other cellular regulator that is known in the art for immortalizing epithelial cells can be used without limiting the scope of the present disclosure. The present invention discloses use of mammalian cellular regulator genes for immortalization instead of viral oncogenes that are generally used for immortalisation. This prevents the undesirable effect of transformation of cell lines that often occur when viral oncogenes are used for immortalisation.

[066] In accordance with a non limiting exemplary embodiment of the present disclosure, the engineered assayable marker gene construct is designed to overexpress an assayable marker.

In a particular embodiment, the engineered assayable marker gene construct was designed to overexpress an assayable marker and a reporter. In different embodiments, E-Cadherin, EpCAM, cytokeratin, N-Cadherin and/or Vimentin are used as the assayable markers though any other assayable marker known in the art for detecting and characterising EMT can be used without limiting the scope of the present disclosure. In different embodiments, GFP, BFP, YFP and RFP are used as the reporters though any other fluorescent reporter or luminescent reporter such as iuciferase or colorimetric reporter that is known in the art for assaying the expression of the gene of interest can be used without limiting the scope of the present disclosure.

[067] According to a non limiting exemplary embodiment of the present disclosure, the engineered EMT inducing gene construct is designed to overexpress an EMT inducer. In a particular embodiment, the engineered EMT inducing gene construct was designed to overexpress an EMT inducer and a reporter. In different embodiments, c-Met, Twist, ras, slug, zeb and/or snail are used as EMT inducers though any other EMT inducer that is known in the art capable of inducing EMT can be used without limiting the scope of the present disclosure.

In different embodiments, GFP, BFP, OFP, YFP and RFP are used as the reporters though any other fluorescent reporter or luminescent reporter such as iuciferase or coiorimetric reporter that is known in the art for assaying the expression of the gene of interest can be used without limiting the scope of the present disclosure.

[068] In accordance with a non limiting exemplary embodiment of the present disclosure, the EMT modulating agent that either induces or inhibits the process of EMT is an engineered gene construct, RNA, a protein, a synthetic molecule and/or an antibody.

[069] In a particular embodiment, the engineered gene construct used as the EMT modulating agent is designed to overexpress an EMT inducing gene in a transient manner. In another embodiment, the engineered gene construct used as the EMT modulating agent is designed to overexpress an EMT inducing gene and a reporter in a transient manner. In a particular embodiment, the protein used as the EMT modulating agent is an EMT inducer. In different embodiments, c-Met, Twist, ras, slug, zeb and/or snail gene are used as the EMT inducers though any other EMT inducer that is known in the art to be capable of inducing EMT can be used without limiting the scope of the present disclosure. In different embodiments, GFP, BFP, YFP and RFP are used as reporters though any other fluorescent or luminescent reporter such as Iuciferase or coiorimetric reporter that is known in the art for assaying the expression of the gene of interest can be used without limiting the scope of the present disclosure.

[070] As mentioned above, examples of engineered gene constructs include cMet, zeb, snail, slug, twist, ras and those described in International Patent Publication Nos EP2611902, US20120302572 and publications (Minuti and Landi, Ann Transl Med. 2015: 3: 181, Kim et al Experimental & Molecular Medicine (2015) 47, el 37) incorporated herein by reference.

[071] RNA interference (RNAi) may be employed to inhibit expression in eukaryotic cells, e.g., vertebrate cells, in a variety of ways as known in the art. As used herein, the term "RNAi agent" encompasses nucleic acids that can be used to achieve RNAi in eukaryotic cells. Exemplary RNAi agents are short interfering RNA (siRNA) and short hairpin RNA (shRNA). As known in the art, siRNAs typically comprise two separate nucleic acid strands that are hybridized to each other to form a duplex. They can be synthesized in vitro, e.g., using standard nucleic acid synthesis techniques or by cleavage of a longer dsRNA, e.g., by an RNase III or RNase Ill-like enzyme such as Dicer. RNAi agents also include microRNA (miRNA) and miRNA precursors. As used herein, "miRNA" and "miRNA precursor" encompasses naturally occurring and artificially designed nucleic acids that function in an analogous manner to naturally occurring miRNAs. In certain embodiments an RNAi agent is a vector that comprises a template for transcription of an siRNA (e.g., as two separate strands that can hybridize to each other), shRNA, or microRNA precursor. Such vectors can be used to introduce the template into vertebrate cells, e.g., mammalian cells, and result in transient or stable expression of the siRNA, shRNA, or miRNA precursor. Examples of RNA include miRNA, siRNA, shRNA and those described in International Patent Publication Nos EP2611902 and publications that are incorporated herein by reference.

[072] In different embodiments, proteins such as EGF, TGFb, HGF, Wnt, MSP, Hedgehog, TNF-alpha, IL4, HB-EGF, amphiregulin, betacellulin, epiregulin, epigen, oncostatin-M, IL-13 were used as EMT modulating agents and those described in International Patent Publication Nos (EP2611902, US7998688, US7951549), and publications that are incorporated herein by reference. Proteins may be naturally occurring e..g TGFb polypeptide (TGFbl, TGFb2, or TGFb3) or be synthesized as peptides. Any other protein that is known in the art is which is capable of modulating EMT can be used without limiting the scope of the present disclosure.

[073] In different embodiments synthetic molecules such as TGF-beta inhibitors A83-01 and SB435142 were used as EMT modulating agents though any other synthetic molecule that are known in the art which can modulate EMT can be used without limiting the scope of the present disclosure. Also, the synthetic molecules described in International Patent Publication Nos EP2611902, US20140010789, and publications (Li et al, J Biomol Screen 2011, Minuti and Landi, 2015) are incorporated herein by reference. For example, PCT/US2008/011648 (WO/2009/051660) discloses small molecules reported to activate TGF beta signaling.

[074] The antibodies that are used as EMT modulating agents can originate from a mammalian or avian species, e.g., human, rodent (e.g., mouse, rabbit), goat, chicken, etc., or can be generated ex vivo using a technique such as phage display. Such antibodies include members of the various immunoglobulin classes, e.g., IgG, IgM, IgA, IgD, IgE, or subclasses thereof such as IgGl, IgG2, etc., and, in various embodiments, encompasses antibody fragments or molecules that retains an antigen binding site and encompasses recombinant molecules comprising one or more variable domains. Examples of antibody include OnartuzuMab and those described in International Patent Publication Nos EP2611902 and publications (Minuti and Landi, Ann Transl Med. 2015: 3: 181) that are incorporated herein by reference. Also any other antibody that is known in the art to modulate EMT can be used without limiting the scope of the present disclosure.

[075] It is also anticipated that inventive compositions and methods may be employed singly or together with modulators of multiple signaling pathways such as the Notch pathway, Hedgehog pathway, signaling via tyrosine kinase receptors such as Met, FGF, IGF, EGF, HGF, VEGF, and/or PDGF receptor family members, the NFkB pathway, hypoxia inducible factor (HIF) pathway, and/or microRNA regulatory pathways. The EMT modulating agents may modulate or perturb EMT, stimulate MET, or inhibit the growth of mesenchymal-like cells as shown in the reference US7951549 that is incorporated herein.

[076] In one embodiment, breast cancer stem cells may be defined as the subpopulation of breast cells staining positively with CD44 antigen but with low to no expression of CD24 in flow cytometry assays (Sheridan et al, Breast Cancer Research 2006, 8:R59 and references cited therein). Expression levels of proinvasive genes (IL-la, IL-6, IL-8, and urokinase plasminogen activator [UPA]) have been reported to be higher in cell lines with a significant CD44 ⁺ /CD24 ^" population than in other cell lines, and thus can be used to identify strategies to overcome invasive properties of CSCs.

[077] In an embodiment, the assayable marker gene construct may be a promoter-reporter construct used to measure promoter activity in cells. In one embodiment, the activity of an epithelial biomarker gene promoter is assessed by inclusion of an epithelial biomarker gene promoter-reporter gene construct into HMECs such that said promoter activity can be monitored by reporter gene expression level or activity. For example, the epithelial biomarker gene promoter-reporter gene construct may be an E-cadherin promoter-GFP construct. The promoter-reporter gene construct may be permanently incorporated into the HMECs as a stable engineered cell line, or may be transiently expressed. Multiple promoter-gene-reporter gene constructs may also be employed in order to monitor several biomarkers simultaneously, e.g. an E-cadherin promoter-GFP construct and a cMet-tdTomato construct, in order to, for example, monitor simultaneous repression of the E-cadherin gene and induction of the cMet gene during EMT (US7951549) and references incorporated therein). One example of an inducible promoter is a tetracycline (tet)-responsive promoter. Fluorescent or luminescent reporters can also be used in vivo to track tumour cell growth and metastasis (US7951549).

[078] In a particular embodiment, the engineered EMT inducing gene constructs may comprise constitutively active promoters and hence activity of the EMT inducing gene. In another embodiment, engineered EMT inducing gene constructs may comprise inducible promoters and hence inducible on-off activity of the EMT inducing gene (US7998688). In another embodiment, the EMT inducing gene constructs may comprise constitutively active promoter and EMT inducing gene but the construct may be used to induce EMT in a transient or metastable manner without deriving stable cell lines using antibiotic selection (US20120302572, US8642834, EP2611902).

[079] The plurality of components disclosed herein can be used for different purposes such as evaluation of a drug candidate targeting EMT and cancer stem cells, evaluation of a drug target targeting EMT and cancer stem cells, evaluation of biomarkers targeting EMT and cancer stem cells, derivation of cancer stem cells and use as a disease model for studying underlying biology of EMT, triple negative breast cancer and cancer stem cells.

[080] The term 'drug candidate' encompasses a compound, an antibody, a peptide, a protein or a RNA that can modulate the expression and/or activity of a molecule or complex of interest (the "target") with potential to be used as a therapeutic. For example, a compound that inhibits activity of a target or a cell in the presence of the compound, or as a consequence of its use, as compared with in the absence of the compound, and/or if the level or amount of the target or cell function is modulated in the presence of the compound, or as a consequence of its use, as compared with in the absence of the compound may be considered as a drug candidate. In certain embodiments, drug candidate molecules act directly on their target in that they physically interact with it. In other embodiments, compounds act indirectly. This platform is particularly suited for conducting phenotypic screening to identify drug candidates that demonstrate desired phenotype without knowing their target. The target may be deconvoluted later using mechanism-of-action studies. Drug candidate examples that are expected to be identified are incorporated herein by reference (EP2611902, US8642834, US7998688, US7951549, US20110191868, CA2656548, US20130158087, US20100088775, US20110033471, US20120156216, US20120220546, WO2013123588, EP2569335, Li et al / Biomol Screen 2011 16: 141)

Methods of inhibiting encompass methods that result in a decreased amount of a target or cellular function and methods that interfere with one or more functions of a target or cell. A variety of methods useful for inhibiting or interfering with expression can be applied in embodiments of the present invention.

[081] The present invention provides compositions and methods useful for modulating the epithelial-mesenchymal transition. In one aspect, the invention provides a composition comprising normal cells or engineered cell lines selected from each of at least two of the following groups: (a) normal human mammary epithelial or fibroblast cells that can serve as control; (b) engineered immortalized stable cell line derived from normal mammary epithelial cells; (c) engineered immortalized stable cell line expressing a gene that can serve as a marker of E to M transition; and (d) engineered immortalized stable cell line expressing a gene that can serve as a marker of E to M transition in which EMT has been induced by an oncogene relevant to breast cancer including triple negative breast cancer and many human cancers of epithelial origin.

[082] In some embodiments, the composition comprises at least one engineered cell line from each of these three groups: (a) engineered immortalized stable cell line derived from normal mammary epithelial cells; (b) engineered immortalized stable cell line expressing a gene that can serve as a marker of E to M transition; and (c) engineered immortalized stable cell line expressing a gene that can serve as a marker of E to M transition in which EMT has been further induced by an oncogene involved in a variety of human cancers of epithelial origin.

[083] In one aspect, the invention provides composition/methods comprising one control and one mesenchymal cell type to identify targets, drug candidates or markers to modulate EMT. Control could be primary epithelial cells as the normal unmanipulated non-cancerous cells compared to the triple transfected cells that have acquired migratory, sphere-formation and CSC properties. The triple transfected cells are triple negative breast cancer.

[084] In another embodiment, benign tumours of epithelial origin such as phylloids may be compared with triple transfected stable line to delineate changes resulting in uncontrolled proliferation to invasiveness to tumour initiation. [085] In another embodiment, the normal fibroblast cells can be used as a control for normal unmanipulated non-cancerous but mesenchymal cells but which don't demonstrate the migratory, anchorage independent phenotype of triple transfected cells.

[086] In another embodiment, hTERT immortalized cells can be used as a control to compare to triple transfected cells that have acquired migratory, sphere-formation and CSC properties.

[087] In another embodiment, the double transfected cells may be used as a control for triple transfected cells whereby the double transfected cells have intermediate phenotype of enhanced migration and sphere formation but are not able to form colonies in soft agar as the triple transfected are able to. This may identify the penultimate changes required for the mesenchymal-like double transfected cells to transform into completely mesenchymal cells generating CSCs.

[088] Identification of the changes between normal epithelial cells, hTERT immortalized cells, cMyc immortalized cells, double transfected cells and triple transfected cells may illustrate differences in the pathways and genes involved in the EMT.

[089] In accordance with a particular non-limiting exemplary embodiment of the present disclosure, normal human mammary epithelial cells are immortalized using cellular regulators such as c-myc or hTERT to effect immortalisation. The stable cell lines obtained after immortalization were transfected with E-Cadherin-GFP, wherein E-Cadherin served as the assayable marker and GFP served as the reporter. This is followed by further transfection of the immortalised stable cell lines with an EMT inducing gene construct. In a particular embodiment, c-Met-RFP was used, wherein c-Met served as the EMT inducer and GFP served as the reporter. In a particular embodiment tdTomato fluorescent protein was used as the reporter along with c-Met.

Examples:

[090] The different methods that were used for carrying out the present invention are given below. The methods listed here are in accordance with different non limiting exemplary embodiments and they can be practiced or carried out in various ways. [091] Certain conventional techniques of ceil biology, cell culture, molecular biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, etc., which are within the skill of the art, may be of use in aspects of the invention. Non-limiting descriptions of certain of these techniques are found in the following publications: Ausubei, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., editions as of 2008; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001 ; Harlow, E. and Lane, D., Antibodies— A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Burns, R., Immunochemical Protocols (Methods in Molecular Biology) Humana Press; 3rd ed., 2005, Monoclonal antibodies: a practical approach (P. Shepherd and C Dean, eds., Oxford University Press, 2000); Freshney, R. L, "Culture of Animal Cells, A Manual of Basic Technique", 5th ed., John Wiley & Sons, Hoboken, N.J., 2005; Cancer: Principles and Practice of Oncology (V. T. De Vita et al., eds., J. B. Lippincott Company, 8th ed., 2008). Further information on cancer may be found in The Biology of Cancer, Weinberg, R A, et al, Garland Science, 2006. All patents, patent applications, websites, databases, scientific articles, and other publications mentioned herein are incorporated herein by reference in their entirety.

Human Mammary Epithelial cell culture

A) Tissue processing & Digestion:

[092] Human mammary tissue from normal breast reduction mammoplasty was processed after surgical procedures.

The tissue was placed in a sterile container containing DMEM media and transported to the lab. The epithelial tissue was collected by scraping off the stroma, surrounding connective tissue using sterile scalpel, forceps and blades. The epithelial tissue was collected into a sterile plastic 100mm culture dish and minced into finer pieces (l-3mm2) for proper digestion, collected into a 50ml tube test tube containing collagenase enzyme (100 U/ml) and DNase enzyme (2U/ml) in DMEM media. The tissue with the enzyme solution was incubated for overnight digestion (Day 1) in shaking water bath @ 370C/ 80rpm/10-12hrs. The following day of digestion the tubes were spun @ 600g (1800 rpm); 5 min; at room temperature. The supernatant fat and enzyme solution from the tubes were discarded. Two ml media was added to the tubes with pellet and triturated gently using 10 ml serological pipettes; 5ml and finally with pi 000 pipette not more than 10 times overall.

The tissue was observed for organoid like structures with a drop onto a sterile slide under microscope. Ensuring the organoids are free of any stromal tissue, the tissue was plated at a density of 40-80 organoids/100 mm dish. For complete assurance of stroma free ductal structures the tissue was digested one more time in a fresh digestion solution overnight at 370C. After the digestion was complete, the pellet containing 10-25 organoids was plated in 35mm dish in DMEM-F12(1 :1) media supplemented with 4% FCS, EGF (20ng/ml), Insulin (lOug/ml), Hydrocortisone (0. lug/ml), Bovine pituitary extract (35ug/ml) & N2 (IX).

B) Primary Culture Maintenance

After attachment of organoids for around 24 hours, the primary cultures were fed with Insulin and EGF every day and every third day complete media change was done with all the supplements. The cultures were monitored daily and photographed under light microscopy. Each epithelial colony with 80-100 cells approximately was considered ready for passaging.

Cell were trypsinized and tapped gently to lift off. Trypsin activity was inhibited by adding FBS and cells were collected into a 15 ml tube. The cells were centrifuged at 100 rpm for 15 min at room temperature. For sub culturing, cells were plated at a density of 1-2x105 cells/ 35mm dish with the same media composition. The doubling time for the cultures was calculated to be approximately 4 days. Cultures were fed daily with EGF and Insulin and media change every third day. Human mammary epithelial cultures (HMECs) were characterized by immunofluorescence and PCR for epithelial markers.

Normal human mammary fibroblast culture:

Normal breast tissue was dissected in a sterile manner as mentioned above. The stromal part was separated and digested with 100 U/ml Collagenase and lU/ml DNase I for overnight at 37°C water bath. Next day, cells were counted using haemocytometer and plated onto T-75 flask in DMEM/F12 containing 10% FBS medium. Confluent cells from T-75 flask were frozen and characterized for cell surface markers, such as Smooth muscle actin and Vimentin. Established fibroblast cultures were used for generating conditioned medium for culturing epithelial cells. For this, fibroblasts were grown to 90% confluency and fresh Serum-free media was added for 72 h. This was collected and concentrated using Amicon filters. Aliquots of 1 ml were stored at -20°C until further use. After several trials and conditions, 5-10% condition medium was optimized for culturing transfected cells.

Immunostaining with epithelial and mesenchymal markers

For immunofluorescence, the cells were plated onto sterile coverslip in 12 well dish. 5000 cells were added onto each coverslip and incubated at 37°C for 30min for cell attachment before lml media was added. Once the cells were ready for fixation, the spent media was removed and cells were fixed with 4% paraformaldehyde and incubated for 20min at room temperature.

Cells were permeabilized with 0.1% Triton x-100 for 15min, blocked with 1% BSA in PBS then washed 3 times with PBS at room temperature. The cells were incubated with primary antibody (dissolved in blocking buffer) anti-E-cadherin, anti-cytokeratin 8/18, anti-vimentin, anti-aSMA for overnight at 4°C in dark. Later, cells were washed 3 times with PBS and incubated with secondary antibody for lhr at RT or overnight at 4°C in dark. Cells were washed with PBS 3 times for 20 min each and mounted with vectashield mounting media with counterstaining with DAPI. The coverslips were imaged using confocal microscopy.

Plasmid Construction

a) cMyc plasmid

The plasmid pCMV-XL5 and cMyc cDNA were purchased from Origene. Both 1 μg pCMV- XL5 and 1 μg cMyc cDNA were digested using 1U EcoRI (New England Biologies) and 1U Notl (New England Biologies) enzyme at 37°C water bath for 2 h. Digested DNA was performed for gel electrophoresis for confirmation and isolation of the DNA from agarose gel.

Digested cMyc insert was cloned into multiple cloning site of the pCMV-XL5 vector. DNA extracted from agarose gels was used for ligation using DNA Ligase enzyme and incubated at 16°C for overnight. Ligation mixture was used for transforming competent E.coli DH 5a cells.Transformed DH 5a cells were plated onto LB amp plates and incubated at 37°C for overnight. After incubation, positive colonies were picked and plated into 5 ml LB broth medium. Incubation was performed at 37°C shaking incubator for overnight. Plasmid DNA was isolated from LB culture by Qiagen Mini prep. Restriction digestion using EcoRI and Notl was performed for all the isolated DNA. The E.coli with plasmid containing gene of interest was inoculated into 200 ml LB medium and incubated for overnight at 37°C in shaking incubator. Isolated endofree plasmid from LB culture by Qiagen Midiprep. The isolated plasmid was used for transfecting human mammary epithelial cells. b) pCMV-hTERT-BFP plasmid

The plasmid pCMV-BFP was purchased from Origene. hTERT gene was amplified from genomic DNA of normal epithelial cells using specific primers. Both 1 μg pCMV-BFP and 1 μg hTERT PCR product was digested using 1U Hind III (New England Biologies) and 1U Not I (New England Biologies) enzyme at 37°C water bath for 2 h. Digested DNA was run on gel electrophoresis for confirmation and isolation of the DNA from agarose gel. Digested hTERT insert was cloned in the multiple cloning site of the pCMV-AC-mBFP vector.

DNA extracted from agarose gels was ligated using DNA Ligase enzyme and incubated at 16°C for overnight. Ligation mixture was used to transform competent E.coli DH5a cells. Transformed DH5a cells were plated onto LB amp plates and incubated at 37°C for overnight. After incubation, positive colonies were picked and plated into 5 ml LB broth medium. Incubation was performed at 37°C shaking incubator for overnight. Plasmid DNA was isolated from LB culture by Qiagen Mini prep kit. For confirmation of the clone, restriction digestion using Hind III and Not I was performed for all the isolated DNA. The E.coli with plasmid containing gene of interest was inoculated into 200 ml LB medium and incubated for overnight at 37°C in shaking incubator. Isolated endofree plasmid from LB culture by Qiagen Midiprep kit. The isolated plasmid was used for transfecting human mammary epithelial cells. c) E-Cad GFP plasmid

The plasmid pCMV E-Cadherin-GFP-neo was purchased from Origene. E-Cadherin promoter was amplified from genomic DNA of normal epithelial cells. Both 1 μg pCMV-E-Cadherin- GFP-neo and 1 μg E-Cadherin promoter DNA was digested using 1U Spel (New England Biologies) and 1U Bgl II (New England Biologies) enzyme at 37°C water bath for 2 h. Digested DNA was run on gel electrophoresis for confirmation and isolation of the DNA from agarose gel. Digested E-Cadherin promoter insert was cloned upstream of the E-Cadherin gene of the pCMV-CDHl-GFP vector. DNA extracted from agarose gels was used for ligation using DNA Ligase enzyme and incubated at 16°C for overnight. Ligation mixture was used to transform competent E.coli DH 5a cells. Transformed DH 5a cells were plated onto LB amp plates and incubated at 37°C for overnight. After incubation, positive colonies were picked and plated into 5 ml LB broth medium. Incubation was performed at 37°C shaking incubator for overnight. Plasmid DNA was isolated from LB culture by Qiagen Mini prep kit. For confirmation of the clone, restriction digestion using Spe I and Bgl II was performed for all the isolated DNA. The E.coli with plasmid containing gene of interest was inoculated into 200 ml LB medium and incubated for overnight at 37°C in shaking incubator. Isolated endofree plasmid from LB culture by Qiagen Midiprep. The isolated plasmid was used for transfecting cMyc cells. d) cMet-tomato Plasmid

The plasmid pCMV tomato-neo was purchased from Origene. cMet was amplified from pCRXL TOPO cMet plasmid using specific primers. Both 1 μg pCMV-tomato-neo and 1 μg PCR product of cMet were digested using 1U Nhe I (New England Biologies) and 1U Sac I (New England Biologies) enzyme at 37°C water bath for 2 h. Digested DNA was run on gel electrophoresis for confirmation and isolation of the DNA from agarose gel. Digested cMet insert was cloned into multiple cloning site of the pCMV-tomato-neo vector. DNA extracted from agarose gels was ligated using DNA Ligase enzyme and incubated at 16°C for overnight. Ligation mixture was used to transform competent E.coli DH5a cells. Transformed DH 5a cells were plated onto LB kanamycin agar plates and incubated at 37°C for overnight.

After incubation, positive colonies were picked and plated into 5 ml LB broth medium. Incubation was performed at 37°C shaking incubator for overnight. Plasmid DNA was isolated from LB culture by Qiagen Mini prep kit. For confirmation of the clone, restriction digestion using Nhe I and Sac I was performed. The E.coli with plasmid containing gene of interest was inoculated into 200 ml LB medium and incubated for overnight at 37°C in shaking incubator. Isolated endofree plasmid from LB culture by Qiagen Midiprep. The isolated plasmid was used for transfecting cMyc + Ecad cells.

Transfection of HMECs

Before transfection, cells were plated at 0.2x106 epithelial cells/35 mm culture dish with media conditions as DMEM-F12 (1 :1) + 4% FBS + Antibiotics + Glutamax + EGF + Insulin + Hydrocortisone + N2. Cells were 70-80% confluent at the time of transfection. On the day of transfection, complete medium was removed 2 hrs before transfection and DMEM-F12 was added. DNA & Fugene HD complex (Total volume of complex; 500μ1 for a 35mm culture dish) was prepared with different concentrations of each plasmid in DMEM-F12 media without serum and antibiotics Different concentrations of DNA were used to transfect e.g., 0.01-0.5 μg c-Myc plasmid, 0.5 - 2.5 μg of hTERT-BFP, 0.5 - 2.5 μg Ecad-GFP, and 0.5 - 2.5 μg of cMet-tomato. DNA was added to the Fugene HD and incubated for 10 min at room temperature. Transfections were performed in duplicate. 250μ1 of the complex was added and incubated for lhr @ 37°C in a C02 incubator. After 1 hr incubation cells were washed gently with DMEM-F12. Fresh complete media with DMEM-F12 4% FBS + Antibiotics + Glutamax + EGF + Insulin + Hydrocortisone + N2 was added. Selection medium with the antibiotic was added 2-7 days after transfection, and continued for 2-3 weeks, with frequent changes of medium to eliminate dead cells and debris until distinct colonies were visualized. Individual colonies were trypsinized and transferred to culture dishes for further propagation in the presence of selective medium which was decreased in a step-wise manner over 2-3 months to obtain stable clones.

Transfectant cell culture conditions

Stable transfectant cMyc, cMyc + Ecad and cMyc + Ecad + cMet cells were regularly cultured in media containing Knockout DMEM, 15% FBS, 5% condition media, 20 ng/ml bFGF at 37°C/5%C02 incubator.

Cell Viability Assay

Viability assay was carried out using WST cell proliferation reagent (Takara, Cat # MK 400) as per the supplier's instructions. Normal HMEC cells and stable transfectant lines were seeded in triplicate in 96 well plates at a density of typically 5000 cells/well in culture medium and allowed to attach for 24 h at 37C/5%C02 incubator. After 24 hrs of attachment, test compounds were added and incubated for 3 to 6 days. 20 μΐ of WST reagent was added to each well and incubated for 3 h at 37°C in 5% C02 incubator. The plate was shaken gently before reading for homogenous distribution of the colour. The absorbance was read using a microplate reader at 450 nm and 630 nm.

Senescence Assay

HMECs and transfected cell lines were tested for senescence using beta galactosidase chromogenic assay. Cells were seeded on sterile coverslips at 5000 cells/well and allowed to attach for 24hrs. Fixation solution 4% paraformaldehyde was added to the cells & incubated for lOmin at room temp and then washed twice with PBS. Staining solution (K4 [Fe (CN) 6] 3H2 O and K3 [Fe (CN) 6]) was added to the cells and incubated at 37°C overnight in a humidifier in dark. After incubation, blue color can be seen visually. The staining solution was washed off, rinsed with PBS and then methanol. The cover slips were kept for drying and then viewed under phase microscope.

Mammosphere Assay

For 3D mammosphere formation, early passage monolayer HMECs and single, double and triple transfected cells were detached and plated in serum free media [DMEM-F12 (1: 1)] containing EGF, bFGF, B27 and N2 as supplements. Cells at 1-2x105 cells were plated in a 6 cm non-adherent culture dish and incubated for 3-7 days. Spheres were visible within 48hrs in triple transfected cells. Approximately 100-150 μπι size of spheres were formed by 5-7 days and were ready for passaging or drug treatment. The spheres were collected into 15ml falcon centrifuge tube, spun at lOOg for 5 min, followed by a PBS wash. The pellet was resuspended in 0.5ml of Accutase and incubated in a shaking water bath at 37°C for 10-15min for sphere dissociation. The pellet was triturated with a pi 000 pipette gently then spun down at 200g for 5min and the supernatant was discarded. The pellet was resuspend in 0.5ml of serum free media. Cell were counted and plated as needed for second generation of spheres.

Migration Assay

Cells were plated at a density of 0.75x106 cells/well in 6 well plates and allowed to become confluent. For wound creation, a scratch using a yellow tip was made. Floating cells were removed followed by a wash with PBS and replenishment of culture medium. For compound treatment, test compound are added in duplicate to the media, with 0.25% DMSO used as a vehicle control for compounds. The wells with the scratch were recorded at a uniform time up to 48hrs using a phase contrast microscopy. The percentage of wound healed was quantified using TScratch software (Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich, Zurich, Switzerland) and data was plotted as percentage of wound closure over time using HMECs migration at 0 hours as 100%.

Soft Agar Assay

Six-well plates were coated with 1 ml of 1% agarose as base agar. A 1 :1 mixture of 0.8% agarose at 40°C was prepared with warm 2X DMEM/F12, 20% FBS and 30,000 cells. 5,000 cells per well were plated in 6-well plates. After agarose solidified, 1 ml of IX DMEM/F12, 10% FBS containing media was added on top. Cells were cultured in soft agar medium for 30 - 40 days with media change twice a week. The number of colonies formed were counted visually. RNA Isolation

It was done as per standard protocol using Trizol reagent and RNA concentration and quality was assessed using Nano drop. cDNA Synthesis using High-Capacity cDNA Reverse transcription kit (Invitrogen)

Allowed the kit components to thaw on ice. Reverse transcription master mix was prepared as below,

Above 2X Reverse transcription master mix was placed on ice and mixed gently, lug of RNA was added into the master mix reaction and mixed gently. Tubes were placed in the thermal cycler. Program/Thermal cycling conditions as follows,

PCR (Emerald Amp GT PCR Master mix-Takara)

Master mix composed of a DNA polymerase, optimized reaction buffer, dNTPs, and a density reagent. Master mix was placed on ice and allowed to thaw. Reaction mix was prepared as below. Component Volume

Emerald Amp GT PCR Master mix (2X 25μΙ.

Premix)

Template/cDNA <500ng

Forward Primer 0.2μΜ (final concentration)

Reverse Primer 0.2μΜ (final concentration) dH ₂ 0 Make up the volume to 50μ1

A 'no template control' was included in every reaction. Program/Thermal cycling conditions were as below:

95°C for 2 min

95°C for 15 sec

50-60°C for 15sec 25 cycles

72°C for 30 sec

72°C for 1 min

Hold at 4°C.

The resulting products were analysed by 1.5% agarose gel electrophoresis (Reference - Matt Lewis, Department of Pathology, and University of Liverpool)

Cells isolated from normal breast tissue demonstrate morphological and gene expression characteristics of epithelial cells

[093] Human breast cells were isolated from organelles after enzymatic digestion of surgical specimens obtained from normal healthy females undergoing elective breast reduction surgeries (Stampfer M et al, 1980; In Vitro; 16 (5): 415-25). The samples were obtained with patient consent and due ethical clearances. Breast tissue was enzymatically digested, differentially processed and cultured to obtain fibroblasts and epithelial cells. The epithelial cells were cultured in the presence of growth medium supplemented with EGF and Insulin, while the fibroblasts were grown in DMEM-F12 (1 :1) supplemented with 10% FBS.

[094] Referring to Fig. 1 , it is a Representative phase contrast microscopy image of normal Human Mammary Epithelial cells (HMECs, Left panel) and normal human mammary fibroblasts (Right panel) grown in two dimensional culture at passage number 2. Objective magnification was 10X. Normal Human Mammary Epithelial cells (HMECs) showed a typical cuboidal morphology with a small nucleus as seen in the left panel of Fig. 1. Very few vacuoles were observed indicating healthy growth of isolated cells that grew in close apposition. Cells from the fibroblast culture showed mostly flattened, more elongated and dispersed cells with no consistent shape (Right panel).

[095] HMECs and fibroblast cells were characterized for the expression of epithelial and mesenchymal markers by the use of immunofluorescent antibodies and PCR (Prat A et al, 2013; Breast Cancer Res Treat; 142 (2): 237-55). Specific markers used for PCR included Mucin 1 and Desmoglein 2 (epithelial markers), and Slug, Snail, Twist 1, and Fibronectin (mesenchymal markers or fibroblast markers). Normal mammary epithelial and fibroblast cells at passage 2 were used for this characterization.

[096] Referring to Fig. 2, it is an image representing expression of epithelial and mesenchymal markers in the isolated human mammary epithelial and fibroblast cells determined by PCR. PCR products denoting expression of the genes Mucin 1 (171 bp, Lanes 1,2), Desmoglein 2 (278 bp, Lanes 3,4), Slug (181 bp, Lanes 6,7), Snail (248 bp, Lanes 8,9), Twist 1 (200 bp, Lanes 11,12), and Fibronectin (382 bp, Lanes 13,14) are shown. GAPDH (115 bp, Lane 15) is used as a positive control for the PCR reactions. Lanes 5 and 10 represent the DNA ladder used to determine the size of the PCR products. The letter Έ' denotes epithelial cells and 'F' denotes fibroblasts as the source of the template.Gene expression as determined by PCR (Fig. 2) showed that HMECs had a high expression of the epithelial markers Mucin 1 and Desmoglein 2. In contrast, normal human fibroblast cells did not have significant expression of these epithelial markers. Fibroblasts isolated from the same human mammary tissue showed high expression of Slug, Snail, Twist 1, and Fibronectin mesenchymal markers whose expression was absent in the HMECs.

[097] Referring to Fig. 3, they represent immunofluorescence images of HMECs. Representative images are at passage 2 showing staining for epithelial markers EpCAM (Panel A) and Cytokeratin 8/18 (CK8/18, Panel B). The cells do not have any detectable staining for the mesenchymal marker Vimentin (Panel C). All cells were also stained for DAPI, a nuclear marker, to visualize the number of cells and nuclear vs. cytosolic localization. Objective magnification was 60X. The epithelial nature of the cells isolated from normal breast epithelium was confirmed by immunofluorescence staining for antibodies to specific epithelial markers. Epithelial cells from passage 2 were stained for epithelial markers EpCAM and Cytokeratin 8/18, and mesenchymal marker Vimentin. Good staining for both epithelial markers was observed in HMECs (Fig. 3; Panels A and B), while the expression of the mesenchymal marker Vimentin was below the detection level (Panel C). More than 90% of the cells in culture were positive for epithelial markers pointing to a purified population of epithelial cells.

[098] Referring to Fig. 4, they represent immunofluorescence staining of normal human mammary fibroblast cells. Representative images are at passage 2 demonstrating lack of staining with the epithelial marker EpCAM (Panel A). Cells stained for the mesenchymal markers, a-Smooth Muscle Actin (a-SMA, Panel B) and Vimentin (Panel C) showed positive staining for both. All cells were also stained for DAPI, a nuclear marker, to visualize the number of cells and localize the nuclear vs cytosolic staining. Objective magnification was 60X. Fibroblast cells isolated from the normal breast tissue were also stained for the presence or absence of epithelial and mesenchymal markers by immunofluorescence. Fibroblast cells from passage 2 did not stain for the epithelial marker EpCAM (Figure 4, Panel A) but showed positive staining for the mesenchymal markers a-Smooth Muscle Actin (a-SMA, Panel B) and Vimentin (Panel C). Most of the cells (more than 90%) showed positive staining for the presence of fibroblast cells, suggesting that the culture methods produced a pure population of fibroblast cells.

Over-expression of c-myc gene in HMECs increased the life-span of cells causing immortalization

[099] Normal HMECs were transfected with a plasmid designed to over-express c-myc (Drissi et al, 2001 ; J Biol Chem; 276 (32): 29994-30001) or hTERT (telomerase) genes. Over- expression of these genes may be used to immortalize the epithelial cells, extending their life span to beyond that of the primary cells.

[0100] Transfected cells were slowly weaned off the selection antibiotic (neomycin) in a stepwise manner over multiple passages, and allowed to grow in antibiotic free medium after passage 6. These cells, henceforth called singly transfected cells, showed epithelial characteristics such as cuboidal morphology with little vacuolization in phase contrast microscopy, c-myc transformed cells were grown for over 10 passages without significant loss of cell proliferation and viability unlike the untransfected HMECs which did not grow beyond passage 6. c-myc transfected cells from passage 10 were used for characterization for the expression of epithelial and mesenchymal markers using both PCR and immunofluorescence.

[0101] Referring to Fig. 5, it is a graphic representation of expression of epithelial and mesenchymal markers in c-myc immortalized stable cell line as determined by Quantitative Real-Time PCR. Epithelial markers used were Mucin 1 and Desmoglein 2, while the mesenchymal markers used were Snail and Fibronectin. The open bars represent HMECs at passage 3, and the filled bars represent passage 10 of HMECs transfected with c-myc. Bars denote average and SD of the assay done in triplicate. Results of quantitative real-time PCR (Fig. 5) showed little change in the expression levels of the epithelial markers (Mucin 1 and Desmoglein 2) compared to HMECs. Some expression of mesenchymal markers (Snail and Fibronectin) was also seen compared to HMECs. These cells may represent an intermediate phenotype to epithelial and mesenchymal cells.

[0102] Referring to Figure 6, they represent immunofluorescence images of HMECs transfected with c-myc. Representative images are at passage 10 showing cells stained with EpCAM (Left panel) and Vimentin (Right panel). All cells were stained with DAPI to visualize the number of cells and localization of cytosolic staining. Objective magnification was 20X. HMECs transfected with c-myc were stained with fluorescent antibodies for markers of epithelial and mesenchymal cells. EpCAM was used as epithelial markers while Vimentin was used as mesenchymal marker, c-myc transfected cells showed intense positive staining for EpCAM (Left Panel). The staining is seen all over the cells and is shown as bright even distribution in the image. Cells stained for Vimentin did not show any detectable levels of positive staining (Panel B). Only the nucleus stained with DAPI can be observed. As can be clearly seen from the image on the right, the size of the bright spots are smaller than seen in the left panel. These bright spots on the right panel only represent the nucleus unlike the left panel that shows both nuclear and cytoplasmic staining.

[0103] Together with the results obtained from gene expression analysis using PCR, normal HMECs transfected with c-myc show some changes in gene expression wherein a low expression of mesenchymal markers was observed in these cells without any significant decrease in the expression of epithelial markers. Over-expression of E-Cadherin gene tagged with Green Fluorescence Protein (GFP) as a reporter for EMT

[0104] Immortalized human mammary cells (c-myc transfected primary cells) were further transfected with a plasmid to over-express E-cadherin gene under the regulation of its own promoter. This was tagged with a Green Fluorescence Protein (GFP) gene serving as an inbuilt reporter. During the process of Epithelial to Mesenchymal transition, E-cadherin is downregulated and this can be followed by the reduced expression of GFP in a phenotypic screen.

[0105] Referring to Fig. 7, it is a graphic representation of expression of epithelial and mesenchymal markers in HMECs transfected with c-myc and E-Cadherin-GFP, as determined by Quantitative Real-Time PCR. Epithelial markers used were Mucin 1 and Desmoglein 2, while the mesenchymal markers used were Snail and Fibronectin. The open bars represent HMECs at passage 3, filled bars represent HMECs transfected with c-Myc and the grey shaded bars represent double transfected cells. Both the c-myc and double transfected cells were from passage 10. Bars denote average and SD of the assay done in triplicate. Stable doubly transfected (c-myc and E-cadherin-GFP) cells were selected over multiple passages and characterized by both PCR and immunofluorescence staining to determine the expression levels of epithelial (Mucin 1 and Desmoglein 2), and mesenchymal markers (Snail and Fibronectin) at passage 10. The double transfected cells express both the epithelial and mesenchymal markers (Fig 7) suggesting that these cells may also represent an intermediate stage of the EMT process. Both Mucin 1 and Desmoglein 2 showed only a small decrease in their expression levels compared to the HMECs. However, the expression levels of both mesenchymal markers was higher in double transfected cells compared to both the HMECs and the cells transfected with c-myc alone.

[0106] Referring to Fig. 8, they are representative immunofluorescence images of double transfected cells at passage 10. EpCAM (Panel A) was used as an epithelial marker and Vimentin (Panel B) was used as a mesenchymal marker. All cells were stained with DAPI to visualize the nucleus and hence the number of cells. Objective magnification was 20X. Double transfected cells were stained with fluorescent labelled antibodies for detecting the expression of epithelial (EpCAM) and mesenchymal (Vimentin) markers. The double transfected cells showed staining for both these markers. However, as shown in Fig. 8, EpCAM showed a very intense positive staining and was seen all over cells with an even distribution (panel A). Cells stained for Vimentin show a moderate signal that was not distributed throughout the cells but was seen in limited areas of the cell cytoplasm (panel B). The images show both the cytoplasmic staining with the antibodies and the nuclear staining with DAPI.

Over-expression of c-Met in the double transfected cells transforms the intermediate phenotype cells into mesenchymal cells

[0107] Referring to Fig. 9, it is a representative phase contrast microscopy image of HMECs triple transfected with c-Myc, E-Cadherin-GFP, and c-Met-tomato maintained in two dimensional culture at passage number 10. Objective magnification is at 10X. Cells isolated from breast epithelium and stably transfected with c-myc and E-Cadherin-GFP were subsequently transfected with a c-Met-tomato reporter plasmid to evaluate the induction of EMT. Upon successful transfection, antibiotic-selected cells were maintained in a culture medium that was optimized to support mesenchymal cell growth. Cell growth in this specialized medium containing conditioned medium from normal fibroblast cultures was much higher compared to their growth in commonly used medium designed to support epithelial cells. These triple transfected cells morphologically appeared similar to mesenchymal cells with spindle-shaped structure and long filament like extensions on either side (Fig. 9), resembling fibroblasts.

[0108] Referring to Fig. 10, it is a graphical representation of the expression of epithelial and mesenchymal markers in HMECs triple transfected with c-myc, E-Cadherin-GFP and c-Met- tomato, as determined by Quantitative Real-Time PCR. Epithelial markers used were Mucin 1 and Desmoglein 2, while the mesenchymal markers used were Snail and Fibronectin. The open bars represent HMECs at passage 3, filled bars represent HMECs transfected with c-myc alone and the grey shaded bars represent triple transfected cells. Both the c-myc and triple transfected cells were obtained from passage 10. Bars denote average and SD of the assay done in triplicate. Analysis for gene expression by quantitative real time PCR for epithelial and mesenchymal markers in the triple transfected cells, at passage 10, showed even higher expression of mesenchymal markers compared to either HMECs or c-myc immortalized cells (Fig. 10). Moreover, the expression levels of the epithelial markers was observed to be much lower in these cells, -29% in the case of Desmoglein 2 and -41% for Mucin 1 compared to HMECs, and about half that of single transfected cells. [0109] Referring to Fig. 11, it is an image showing immunofluorescence staining of HMECs triple transfected with c-Myc, E-Cadherin-GFP, and c-Met, at passage 10. Representative images showing staining for the epithelial marker EpCAM (Panel A) and for the mesenchymal marker Vimentin (Panel B). Cells were also stained with DAPI to visualize the nucleus and cytosolic staining. Objective magnification was 20X. Staining the triple transfected cells with immunofluorescence for the expression of epithelial (EpCAM) and mesenchymal (Vimentin) markers showed a significant loss of expression of the epithelial markers. As shown in Fig. 11, the triple transfected cells showed staining for both the markers, however, EpCAM (Panel A) showed a moderate signal that was localized to only certain areas of the cytoplasm. In contrast, positive staining for Vimentin (Panel B), was represented as a bright signal evenly distributed throughout the cell. The differences in the sizes of the distribution of the positive staining was also evident. The staining for EpCAM is visible as an area that is only slightly larger than the nucleus.

[0110] Taken together with the results of the real time PCR, these results suggest that the transfection of HMECs with a combination of c-myc, E-cadherin-GFP, and c-Met- tomato pushes the cells towards a dominant mesenchymal nature through the process of EMT.

[0111] Referring to Fig. 12, it represents a fluorescence microscopy image of triple transfected HMECs representing the expression of red coloured tomato reporter gene (right panel). Phase contrast microscopy image of the cells is depicted in the left panel. Images are taken with 20X objective. Expression of the reporter protein tomato (red fluorescence) was detected in the triple transfected cells by immunofluorescence as shown in Fig. 12. In the right panel, the bright dots indicated by arrows, are groups of cells that show intense red fluorescence and are the ones expressing c-Met-tomato and they range between 30 to 50% of the cells in culture.

Immortalized HMECs showed a longer life-span without senescence and expressed hTERT mRNA

[0112] Primary human cells have a limited life-span mainly due to the lack of hTERT (human Telomerase Reverse Transcriptase) expression leading to progressive shortening of the telomeres. When the telomeres reach a critical length, the cells undergo senescence and ultimately die. [0113] Referring to Fig. 13, they are representative images of β-galactosidase assay to determine senescence in the different cell types developed. HMECs at passage 6 (Panel A) show intense blue colour in groups of cells indicated by arrows. C-myc (Panel B), C-myc and E-Cadherin-GFP (Panel C), and C-Myc, E-Cadherin-GFP, and C-Met-tomato (Panel D) transfected cells were negative for the β-galactosidase assay. All the three transfected cell types used in this assay were from passage 10. Objective magnification was 10X. HMECs transfected with c-myc proliferate for a much longer time compared to the primary epithelial cells. These transfected cells do not show senescence even after passage number 10. As shown in Fig. 13, the primary epithelial cells, at passage 6, used as positive control cells take up blue colour of the beta-galactosidase stain (depicted by darker colour in panel A) in the cytoplasm indicating their being senescent. More than 50% of the cells in culture show senescence at this passage. These cells do not proliferate further and eventually die in culture. Cells that have been transfected with c-myc overcome this crisis and continue to proliferate. Lack of colour and thereby absence of sensescence was observed for the c-myc single transfected (panel B), c-myc and E-Cadherin-GFP double transfected cells (panel C), and the triple transfected cells (panel D). None of the cells in the three transfected cultures showed blue colouration in the cytoplasm and therefore demonstrated lack of senescence at passage 10.

[0114] Referring to Fig. 14, it is a graphical representation of Real-Time quantitative PCR to determine the expression levels of hTERT. HMECs at passage 3 were used in this assay while the three transfected cell lines were from passage 10. Relative fluorescence units were normalized to HMECs set at 100%. Graph denotes the average and SD of the expression levels from an assay done in triplicate. In addition to the β-galactosidase assay, expression of hTERT mRNA is also used as an indicator of cellular immortalization. Expression of this gene was determined by quantitative real time PCR. When compared to the HMECs at passage 3, cells transfected with c-myc showed high hTERT expression (Fig. 14). When compared between the different groups of immortalized cells, the triple transfected cells had the highest expression levels of hTERT.

Cells transfected with c-myc, E-Cadherin-GFP, and c-Met-tomato gain the mesenchymal cell characteristic of anchorage independent growth in serum free medium and the ability to form colonies in soft agar

[0115] Normal breast epithelial cells stably transfected with c-myc alone, c-myc and E- Cadherin-GFP, and triple transfected c-myc, E-Cadherin-GFP and c-Met-tomato were grown in non-adherent culture plates under serum-free conditions to test for their ability to form 3- dimensional colonies, which is a feature of cancer cells.

[0116] Referring to Fig. 15, they are images representing sphere forming assay in non-adherent plates and serum-free culture conditions. 100,000 cells were seeded and allowed to form spheres for 7 days. HMECs (Panel A) failed to form spheres and remained dispersed and slowly died, while cells transfected with c-myc (Panel B), c-myc + E-Cadherin-GFP (Panel C), and with c-myc + E-Cadherin-GFP + c-met-tomato (Panel D) formed spheres, albeit at different rates and sizes. HMECs were at passage 3 while the other three cell lines were at passage 10. Images were taken with 10X objective. These cells did form 3D spheres, also called mammospheres, over several days in serum-free medium. The absence of serum selects for cells, namely cancer stem cells (CSCs), which are able to not only tolerate lack of nutrients but also have the ability to regenerate cancer cells. After seven days of culture, the number of mammospheres and the size of the spheres was determined. The results of the sphere forming assay, as represented in the Fig. 15, showed that the primary epithelial cells (Panel A) do not have the ability to form spheres but the transfected cells do. However, the size and number of spheres was significantly higher in single (Panel B) and triple transfected cells (Panel D) compared to double transfected cells (Panel C). In addition, the single and triple transfected cells started to form spheres by day 3 while the double transfected cells started to form small spheres as late as day 7. The triple transfected cells were embedded in albumin and then in paraffin as a block. The block was sectioned and immunostained for ER, PR and Her2 markers and shown to be triple negative breast cancer.

[0117] Referring to Fig. 16, they are images representing colony forming assay in soft agar. Cells were seeded and allowed to form colonies for 40 days. Primary epithelial cells (Panel A) failed to form colonies, while cells transfected with c-myc + E-Cadherin-GFP (Panel B), and with c-myc + E-Cadherin-GFP + c-met-tomato (Panel C) formed colonies of different sizes. Images were taken with 10X objective of phase contrast microscope. (Change images to passage 10. In EMT PCT folder). Colony formation in soft agar, a hallmark of cancer cells, was assessed for 20-40 days in the presence of serum. As shown in the Fig. 16, HMECs (Panel A) did not form any colonies as expected from literature and remained dispersed as single cells that did not survive past 20 days. The single, double and triple transfected cells did have the ability to form colonies. However, the colonies in the double transfected cells (Panel B) were significantly smaller than in the triple transfected cells (Panel C) at 30 days. It took 40 days for the double transfected cells to form any colonies of about ΙΟΟμπι size compared to only 30 days for the triple transfected cells. Subsequent experiments with c-myc transformed cells at passage 10, showed colony formation similar to the triple transfected cells.

[0118] Referring to Fig. 17, it is a graphical representation of the number of colonies formed in soft agar by the different cell types, based on the size of the colonies enumerated on Day 40. The graphs represent the average and SD from duplicate wells of a representative experiment. Representative data of the colony formation assay using cells from passage 10 is shown in Fig. 17. Though the double transfected cells do form a few colonies in soft agar, they are primarily below 50μπι in diameter. Colonies that are more than ΙΟΟμπι are present only in the triple transfected cells indicating the presence of many tumour initiating cells able to form independent colonies, renew themselves and proliferate in the hypoxic conditions of soft agar that mimic tumour environment.

[0119] Taken together, the results from the mammosphere and colony formation assays suggest that the epithelial cells transfected with the three plasmids have attained the characteristics of mesenchymal cells and also indicate the presence of cancer stem cells. Moreover, the triple transfected cells formed multiple new colonies rapidly in soft agar that suggests their ability to metastasize and form new cancers compared to HMECs.

Triple transfected cells grown as 3D mammosphere cultures are enriched for cancer stem cells

[0120] Triple transfected cells grown in 2D culture at passage 9 were trypsinized and seeded in non-adherent culture plates under serum-free condition to generate mammospheres. Spheres that were more than 100 μπι after day 7 were collected and were dispersed into single cells using accutase. In order to detect cancer stem cells, two antibodies to CD44 antigen (Biolegend) and CD24 antigen (BD Biosciences) were used to stain the cells and analysed by FACS. Unstained cells were used to gate for the size by forward and side scatter while single stained cells were used to determine the gating strategy for the final sorting. Cell population obtained by gating for CD44+ and CD24- belong to the cancer stem cell group.

[0121] Referring to Fig. 18, it represents a flow cytometric detection of cancer stem cells (CSCs) by CD44 and CD24 antibody staining. HMECs (Panel A) were grown in 2 dimensional culture and then stained by the antibodies, c-myc single transfected cells (Panel B) and triple transfected cells (Panel C) were grown as mammospheres and then used for the staining. Stained cells were gated for CD44+ and CD24- population and sorted in a FACS Canto II (BD Biosciences). The bottom right quadrant represents the CD44+/CD24- cells reported to be cancer stem cells. As shown in Fig. 18, 9.3% of cells in the 2 dimensional HMEC culture are CD44 ⁺ and CD24 ^" and therefore may potentially be cancer stem cells (Panel A). This result is corroborated from other groups where in the stem cell population in isolated HMECs has been reported to be around 10% (Fillmore and Kuperwasser, Breast Cancer Research 2008, 10:R25).

[0122] Single and triple transfected HMECs when grown in 3 dimensional mammosphere culture showed a higher population of the CD44 ⁺ and CD24 ^" cells. C-myc transfected cells (Figure 18, Panel B) showed about 47% of cells in the quadrant which represents the cancer stem cell population (Quadrant 4). This population is further increased in the triple transfected cells (Panel C), wherein about 58% of the cells may belong to the cancer stem cell group. These cells can be enriched further through sequential culture as mammospheres.

Mesenchymal cells derived in this study have a high migratory potential and the migration is modulated by small molecules

[0123] Referring to Fig. 19, it is a graphical representation of migration potential of the different cell types as determined by wound healing assay. The panels correspond to A) HMECs, B) HMECs transfected with c-Myc, C) HMECs transfected with c-Myc and E- Cadherin-GFP, and D) HMECs transfected with c-Myc, E-Cadherin-GFP, and c-Met-Tomato. The percentage of open wound area was calculated based on the density of the cells present in the scratch, when compared to the 0 hour of HMECs which was set at 100%. The graphs are average and SD of triplicate wells from a representative experiment of two independent experiments. The increased ability of cells to migrate from their point of origin is a hallmark of metastatic cancer and also of mesenchymal cells. In order to evaluate the migratory potential of cells, a uniform scratch is made in a confluent area of cell growth and all the cells in that area are removed; this absence of cells represents a wound. The migration potential is measured by the ability of neighbouring cells to migrate into the scratched area to fill in the wound. As shown in the Fig. 19, the area of the open wound remained much higher in the HMECs (designated A) compared to that in cells which were transfected with either c-myc alone (B), c-myc and E-Cadherin-GFP (C), or with c-myc, E-Cadherin-GFP and c-met-tomato (D). At 48 hours, the area of the open wound is the least in the triple transfected cells compared to either the singly or doubly transfected cells suggesting that the triple transfected cells have the highest migratory potential. Consistent with published literature, a gain of migratory phenotype is observed even for the c-myc single transfected cells.

Use of the differently transfected cells, demonstrating different stages of EMT, for evaluating the anti-cancer activity of drug candidates in different assays

[0124] Cell growth inhibitory effects of several anti-cancer agents were tested on the different cell types in both 2D and3D formats. IC50 values of each of the compound was calculated using the WST cell proliferation assay in the absence or presence of the compound for 72 hours. Cells were cultured in a standard 2D format in 96-well plates in the presence of serum, treated with various concentrations of the compounds for 3 to 6 days. The triple transfected cells showed differential sensitivity to some anti-cancer agents relative to the double transfected cells and normal epithelial and fibroblast cells.

Conclusion:

[0125] We have created an in vitro platform with a panel of cells that span epithelial to mesenchymal phenotype with varying degrees of expression of specific markers and functionality, including but not limited to immortalization, migration, invasion, extravasation, formation of 3D spheres and colonies that no longer require serum or adherence, generation of CSCs and with differential response to standard of care chemotherapy drugs. We have generated triple cancer breast cancer cells in our EMT model from normal epithelial cells in a stepwise manner.

[0126] We propose that this EMT model can be used for discovering and/or validating novel biomarkers, new drug candidates or repurposing of known drugs for use in treating cancer e.g. triple negative breast cancer singly or in combination with other drugs.

[0127] Although the present disclosure has been described in terms of certain preferred embodiments and illustrations thereof, other embodiments and modifications to preferred embodiments may be possible that are within the principles and spirit of the invention. The above descriptions and figures are therefore to be regarded as illustrative and not restrictive.

[014] Thus the scope of the present disclosure is defined by the appended claims and includes both combinations and sub combinations of the various features described herein above as well as variations and modifications thereof, which would occur to persons skilled in the art upon reading the foregoing description.

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