WO2023067582A1 - Tumor microenvironment platform comprising oral carcinoma for predicting drug sensitivity, drug resistance and disease progression - Google Patents

Abstract

The present disclosure relates to a tumor microenvironment platform comprising oral carcinoma for predicting drug sensitivity, drug resistance and disease progression. More particularly, invention focuses on a method for obtaining a tumor microenvironment platform comprising oral carcinoma for predicting chemoresistance and dysplastic progression primary cell lines derived from oral cancer patients, their characterization and validation as a model in early carcinogenesis and chemoresistance. Furthermore, invention deals with autologous pair of cancer cell line and development of Cancer Associated Fibroblast (CAF) cell lines from patients. In further embodiment, invention deals with development of spontaneously transformed cell lines from patients with oral cancer. In addition, the invention also provides kits that are useful for the practice of the methods of the invention.

Description

TUMOR MICROENVIRONMENT PLATFORM COMPRISING ORAL CARCINOMA FOR PREDICTING DRUG SENSITIVITY, DRUG RESISTANCE AND DISEASE PROGRESSION

[001] PRIORITY PARAGRAPH

[002] This application claims priority to the Indian provisional patent application No. 202141048345, filed on October 23^rd, 2021, titled "TUMOR MICROENVIRONMENT PLATFORM COMPRISING ORAL CARCINOMA FOR PREDICTING DRUG SENSITIVITY, DRUG RESISTANCE AND DISEASE PROGRESSION and is incorporated herein by reference.

[003] TECHNICAL FIELD OF THE INVENTION

[004] The present invention is in the technical field of tumor microenvironment platform comprising oral carcinoma for predicting drug sensitivity, drug resistance and disease progression. More particularly, invention focuses on primary cell lines derived from oral cancer patients, their characterization and validation as a model in early carcinogenesis and chemoresistance.

[005] BACKGROUND OF THE INVENTION

[006] Epithelial cancers presently do not have cure in spite of advances in different screening and surgical treatment. Tumor regression activated by known anti-cancer therapies does not correlate with patient survival. At present there is no specific model for predicting drug sensitivity, drug resistance and disease progression in oral cancer.

[007] Head and neck squamous cell carcinomas (HNSCC) are one of the most prevalent cancers worldwide, with specifically high incidence in the Asian sub-continent. Oral cancers constitute one of the most common types of head and neck cancers and despite easy accessibility of oral cavity for visual examination; these cancers are typically detected in their advanced stages.

[008] In India, 60-80% of HNSCC present are with advanced disease and poor overall survival. These statistics point out to the need for an increased focus on early detection as a strategy for downstaging of the disease at presentation [1].

[009] In the present scenario, the additional challenge associated with oral cancer is accurate prognosis or diagnostic subsequent to treatment. In patients with oral cancer, surgery is the preferred mode of treatment. Despite great progress in chemotherapy, radiotherapy, and targeted therapy in the last three decades, the prognosis of OSCC remains poor due to drug resistance, aggressive local invasion and metastasis, leading to recurrence. 26-47% of patients are known to develop a recurrence within 2 years of surgical resection with an annual 5% chance of developing a second primary tumor [2] .

[010] Patient-derived cell line models are most significant to study the underlying mechanisms and to evaluate possible targets of cancer. As now it is well accepted that cancer is not formed by the tumor tissues alone, rather by the tumour-CAF interaction, availability of such platforms are of utmost importance. In oral cancer, these models can be essential towards understanding tumor-stroma interactions in the process of early carcinogenesis, chemoresistance and poor prognosis.

[Oi l] In oral cancer, patient-derived cell lines, especially, Cancer Associated Fibroblast (CAF) cells are not available in the market, thus platform for studying the mechanism of tumour-CAF interaction or interception of the interaction-pathways is not available either.

[012] Further, obtaining autologous pairs of cancer cells and CAFs from the same patient is an additional challenge.

[013] Therefore, there is an urgent need in the art to develop a highly specific compostion/platform technology that can provide tumor microenvironment for predicting drug sensitivity, drug resistance and disease progression in oral cancer.

[014] SUMMARY OF THE INVENTION

[015] The primary objective of the present invention is to provide a tumor microenvironment platform comprising oral carcinoma for predicting drug sensitivity, drug resistance and disease progression. More particularly, invention deals with primary cell lines derived from oral cancer patients, their characterization and validation as a model in early carcinogenesis and chemoresistance.

[016] In an embodiment a method for obtaining a tumor microenvironment platform comprising oral carcinoma for predicting chemoresistance and dysplastic progression, wherein the method comprising:

(i) layering a platform with oral cancer cell lines and/or dysplastic cell lines and cancer- associated fibroblast cells, wherein the oral cancer cell lines comprise MhCT12-E and MhCT08-E cells, wherein the cancer-associated fibroblasts cells comprise MhCT08-F, MhCL03-F, MhCA04-F, and MhCB05 -F, wherein the dysplastic cell lines comprise DOK, wherein the cell lines are cultured in RPMI medium with 20% Fetal bovine serum (FBS); and

(ii) adding culture medium comprising epithelial spent media and fibroblast spent media to the platform to obtain the tumor microenvironment platform. [017] In further embodiment, wherein the oral cancer cell lines are autologous pairs of cancer cell lines, wherein the platform can identify novel targets/biomarkers for chemoresistance and therapy.

[018] In further embodiment, a method of predicting dysplastic progression using the tumor microenvironment platform as claimed, wherein the method is to assess the effectiveness of novel chemopreventive drugs, wherein the method comprising the steps of: a. co-culturing non-neoplastic and dysplastic epithelial cells together with MhCT08-F, MhCL03-F, MhCA04-F comprised in the tumor microenvironment platform, wherein the coculturing is performed for 48 hours in DMEM-F12 in 5% CO2 at 37°C, wherein the MhCB05-F to neoplastic and dysplastic epithelial cells are comprised in a ratio of 5:1; b. treating the platform with candidate drugs; and c. performing expression profiling, flow cytometer-based profiling of cancer stem cell markers in the co-cultured non-neoplastic and dysplastic epithelial cells and assessing cytotoxicity.

[019] In further embodiment, wherein the non-neoplastic cell is a HaCaT cell line, wherein the dysplastic epithelial cell is a DOK cell line.

[020] In an embodiment, wherein a) the co-culture of the non-neoplastic cells with CAFs showed up-regulation of ALDH1A1 by about 1.3 folds, CD133 by about 8.4 folds, and NOTCH1 by about 10.9 folds, wherein the expression profiling of the dysplastic epithelial cells co-cultured with fibroblasts showed up-regulation of SOX-2 by about 24.5 folds as compared to their respective monolayer cells; and

[021] b) the flow cytometer-based profiling of the non-neoplastic cell showed up-regulation of CD44+ by 96.8% compared to an untreated non-neoplastic cell having 74.6%, wherein the flow cytometer-based profiling of the dysplastic epithelial cells showed up-regulation of CD44+ by 72.7% compared to untreated dysplastic epithelial cells having 41.1%.

[022] In further embodiment, wherein the oral carcinoma cell line is obtained from a cancer tissue obtained surgically or by biopsy or as a xenograft or any combination thereof.

[023] In further embodiment, a kit for predicting chemoresistance of novel drugs to oral carcinoma, wherein the kit comprises a tumor microenvironment platform comprising oral cancer cell lines and cancer-associated fibroblast cells, wherein the oral cancer cell lines comprise MhCT12-E and MhCT08-E, wherein the cancer-associated fibroblasts cells comprise MhCT08-F, MhCL03-F, MhCA04-F, and MhCB05-F, wherein the cell lines are cultured in RPMI medium with 20% Fetal bovine serum (FBS), and (ii) culture medium comprising of epithelial spent media and fibroblast spent media. [024] In further embodiment, a kit for predicting dysplastic progression, for the efficacy of novel candidate chemopreventive drugs, wherein the kit comprises a tumor microenvironment platform comprising dysplastic cell line (DOK) or non-neoplastic cell line (HaCaT) and cancer- associated fibroblast cells, wherein the cancer-associated fibroblasts cells comprise MhCT08-F, MhCL03-F, MhCA04-F, and MhCB05-F, wherein the cell lines are cultured in RPMI medium with 20% Fetal bovine serum (FBS), and (ii) culture medium comprising epithelial spent media and fibroblast spent media.

[025] According to a further aspect of the present invention, the invention also provides kits that are useful for the practice of the methods of the invention.

[026] According to a further aspect of the present invention, it deals with development of spontaneously transformed cell lines from patients with oral cancer.

[027] In preferred embodiments, invention provides development of,

[028] Spontaneously transformed cell lines from patients with oral cancer.

[029] Development of 5 CAF cell lines from patients.

[030] Autologous pair of cancer cell line and CAF from the same patient that are completely profiled and characterized.

[031] Models can be used to explore the role of CAF-cancer cell interactions during dysplastic progression, drug resistance as well as during metastasis.

[032] The platform can be used to study sars-cov2 viral entry through oral route.

[033] The platform grown in high glucose can be used to study sars-cov2 viral pathogenesis in comorbid (hyperglycemia) condition.

[034] According to a further exemplary aspect of the present invention, a platform to study interaction between tumor and stroma in oral squamous cell carcinoma.

[035] According to a further exemplary aspect of the present invention, a platform to study interaction between tumor and stroma in oral squamous cell carcinoma including oral cancers, cancers of larynx , pharynx, primary or secondary cancers as a cost-effective method for screening on large scale, for prognosis, screening at various stages of cancers in the subjects during and post -treatment, in the form of kits, strips, swabs, sticks, discs, meters and such other easily usable, disposable, multi-utility kits for measuring the preferred biomarkers to detect head and neck cancers including oral cancers with specificity, sensitivity, accuracy, speed, reliability and reproducibility.

[036] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.

[037] BRIEF DESCRIPTION OF THE DRAWINGS

[038] The foregoing and other objects and features of the present invention will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only typical embodiments of the invention and are, therefore, not to be considered limiting of its scope, the invention will be described with additional specificity and detail through use of the accompanying drawings. Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below, according to the aspects of present invention.

[039] FIG. 1A illustrates the Co-culture models of oral cancer cell lines as well as cancer- associated fibroblasts that have been derived from patients diagnosed with oral cancer, according to the aspects of the invention.

[040] FIG. IB illustrates the FACS staining of established cell lines, according to the aspects of the invention.

[041] FIG. 2 illustrates the HPV typing of established cell lines, according to the aspects of the invention.

[042] FIG. 3 illustrates the DNA ploidy determination of established cell lines, according to the aspects of the invention.

[043] FIG. 4 illustrates the Doubling time calculation of established cell lines, according to the aspects of the invention.

[044] FIG. 5 illustrates the Proliferation potential of MhCT12-E cells, according to the aspects of the invention.

[045] FIG. 6 illustrates the wound healing ability of MhCT12-E cells under the effect of MhCT12-F conditioned medium, according to the aspects of the invention.

[046] FIG. 7 illustrates the invasive potential of MhCT12-E cells under the effect of MhCT12-F conditioned medium, according to the aspects of the invention.

[047] FIG. 8 illustrates the sphere formation ability of MhCT12-E cells, according to the aspects of the invention.

[048] FIG. 9 illustrates the proliferation graph of MhCT12-E (B12-E) and MhCT12-F (B 12- F) autologous pairs, according to the aspects of the invention. [049] FIG. 10 illustrates the A. Comparative graph of MhCT12-F with and without conditioned media. B. Comparative graph of MhCT12-E with and without conditioned media, according to the aspects of the invention.

[050] FIG. 11 illustrates the Morphology of MhCB05-Fblast cells, according to the aspects of the invention.

[051] FIG. 12 illustrates the FACS staining with a-SMA and pan-CK, according to the aspects of the invention.

[052] FIG. 13 illustrates the Immunocytochemistry with a-SMA, Vimentin and pan- Cytokeratin, according to the aspects of the invention.

[053] FIG. 14 illustrates the Generation of CAF-conditioned medium, according to the aspects of the invention.

[054] FIG. 15 illustrates the Transwell co-culture system, according to the aspects of the invention.

[055] FIG. 16 illustrates the Expression profiling of epithelial cells under the effect of CAF- conditioned medium and Transwell co-culture, according to the aspects of the invention.

[056] FIG. 17 illustrates the FACS of epithelial cells under the effect of CAF-conditioned medium, according to the aspects of the invention.

[057] FIG. 18 illustrates the Invasion of epithelial cells under the effect of CAF-conditioned medium and Transwell co-culture, according to the aspects of the invention.

[058] FIG. 19 illustrates the Migration/wound healing assay of epithelial cells under the effect of CAF-conditioned medium, according to the aspects of the invention.

[059] FIG. 20 illustrates the Spheroid formation in epithelial cells under the effect of CAF- conditioned medium, according to the aspects of the invention.

[060] FIG. 21 illustrates the Comparative graph of curcumin treatment in A. HaCaT and B. DOK cells under the effect of CAF-conditioned medium, according to the aspects of the invention.

[061] FIG. 22 illustrates the Effect of the conditioned media on the resistance of Cal-27 (A) and Cal27 CisR (B), according to the aspects of the invention.

[062] FIG. 23 illustrates the Expression profile of the cells under the effect of the fibroblast conditioned media, according to the aspects of the invention.

[063] FIG. 24 illustrates the Effect of the conditioned media on the functional properties of Cal-27 and Cal27 CisR Effect of the co-culture was evaluated using spheroid formation (A), colony formation (B) and scratch assay (C), according to the aspects of the invention. [064] FIG. 25 illustrates the efficacy of anti-SARS-COV2 agents to inhibit viral entry, according to the aspects of the invention.

[065] FIG. 26 illustrates the pathogenesis of SARS-COV2 infection in comorbid condition like hyperglycemia, according to the aspects of the invention.

[066] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[067] DETAILED DESCRIPTION OF THE INVENTION

[068] 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.

[069] 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.

[070] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.

[071] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

[072] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.

[073] Example embodiments of the present invention are described with reference to the accompanying figures.

[074] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number. [075] DEFINITIONS

[076] The following terms are used as defined below throughout this application unless otherwise indicated.

[077] The terms “tumour” or “tumour tissue” refer to an abnormal mass of tissue which results from uncontrolled cell division. A tumour or tumour tissue comprises “tumour cells” which are neoplastic cells with anomalous growth properties and no functional bodily function. Tumours, tumour cells and tumour tissue can be benign or malignant.

[078] The phrase "differentially present" refers to differences in the quantity of the marker present in a sample taken from patients as compared to a control subject. A biomarker can be differentially present in terms of frequency, quantity or both.

[079] "Diagnostic" means identifying a pathologic condition.

[080] The terms "detection", "detecting" and the like, may be used in the context of detecting markers or biomarkers.

[081] A "test amount" of a marker refers to an amount of a marker present in a sample being tested. A test amount can be either in absolute amount (e.g., pg/ml) or a relative amount (e.g., relative intensity of signals).

[082] The term, “plurality of agents”, “compositional elements”, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. In addition, no individual member of such list should be construed as an equivalent of any other member of the same list solely based on their listing in a common group without indications to the contrary.

[083] The term “Concentrations”, “amounts”, and other “numerical data” may be expressed or presented herein in a range format. It is to be understood that such range format is used merely for convenience and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[084] The term “about” means that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximated and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like and other factors known to those of skill. In addition, unless otherwise stated, the term “about” shall expressly include “exactly,” consistent with the specification regarding ranges and numerical data. [085] The term, “effective amount” refers to an amount of an ingredient which, when included in a composition, is sufficient to achieve an intended compositional or physiological effect.

[086] The term, “administration,” or “administering” refer to the manner in which an active agent, or composition containing such, is presented or given to a subject.

[087] The term, “stem cells’ refers to special human cells that have the ability to develop into many different cell types, for example, from muscle cells to brain cells. In some cases, they also have the innate ability to repair damaged tissues.

[088] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. "Polypeptide," "peptide" and "protein” can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.

[089] "Detectable moiety" or a "label" refers to spectroscopic, photochemical, biochemical, immunochemical, or chemical means of detection of a composition. For example, labels may include ³²P, ³⁵S, fluorescent dyes, biotin-streptavidin, dioxigenin, haptens, electron-dense reagents, and enzymes. The detectable moiety generates a measurable signal that can quantify the amount of bound detectable moiety in a sample. Quantitation of the signal is done by scintillation counting, densitometry, or flow cytometry.

[090] "Antibody” refers to a polypeptide ligand encoded by an immunoglobulin gene(s), which specifically binds and recognizes an epitope.

[091] The terms "subject", "patient" or "individual" generally refer to a human or mammals. [058] "Sample" refers to a polynucleotides, antibodies fragments, polypeptides, peptides, genomic DNA, RNA, or cDNA, polypeptides, a cell, a tissue, and derivatives thereof may comprise a bodily fluid or a soluble cell preparation, or culture media, a chromosome, an organelle, or membrane isolated or extracted from a cell.

[092] Subject refers to a subject or patient can include, but is not limited to, mammals such as bovine, avian, ovine, porcine, canine, equine, feline, or primate animals (including humans and non-human primates).

[093] The subject can have a pre-existing disease or condition, such as cancer. Furthermore, the subject may not have any known pre-existing condition. In addition, the subject may also be non-responsive to an existing or past treatment, such as a treatment for cancer.

[094] “Body fluid” refers to, but is not limited to, plasma, serum, urine, peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood.

[095] The term “oral cancer” refers to a group of malignant or neoplastic cancers originating in the oral cavity of an individual. Non-limiting examples of oral cancers include cancers of the buccal vestibule, hard or soft palate, tongue, gums (including gingival and alveolar carcinomas), lingual cancer, buccal mucosa carcinoma, and the like.

[096] “Head and neck squamous cell carcinoma” refers to group of cancers of epithelial cell origin originating in the head and neck, these tumors may arise from diverse locations, including the oral cavity, oropharynx, hypopharynx, larynx, and nasopharynx. The oral cavity includes the buccal mucosa, upper and lower alveolar ridges, floor of the mouth, retromolar trigone, hard palate, and anterior two thirds of the tongue.

[097] “Periodontal disease” refers diseases affecting the gums of an individual, including gingivitis, periodontitis, and the like.

[098] “Therapeutically effective amount or dose” refers to a dose that produces effects for which it is administered. The exact dose depends on the purpose of the treatment.

[099] Metastasis” refers to spread of a cancer from the primary origin to other tissues and parts of the body, such as the lymph nodes.

[0100] “Prognosis” refers to prediction of the likelihood of metastasis, predictions of disease free and overall survival, the probable course and outcome of cancer therapy, or the likelihood of recovery from the cancer, in a subject.

[0101] “Diagnosis” refers to identification of a disease state, such as cancer in a subject. The methods of diagnosis provided by the present invention can be combined with other methods of diagnosis well known in the art. Non-limiting examples of other methods of diagnosis include, detection of known disease biomarkers in saliva samples, co-axial tomography (CAT) scans, positron emission tomography (PET), oral radiography, oral biopsy, radionuclide scanning, and the like.

[0102] The term “xenografts” means the transfer of tissues or cells between two mammals of different species.

[0103] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.

[0104] The term “CSC” means Cancer Stem cells.

[0105] The term “ALDH1A” means Aldehyde dehydrogenase 1 family, member Al. [0106] The term” NOTCH1” means Neurogenic locus notch homolog protein 1 is a protein encoded in humans by the NOTCH1 gene. Notch 1 is a single-pass transmembrane receptor.

[0107] A particular nucleic acid sequence may also implicitly encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, in addition to the sequence explicitly indicated. Furthermore, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.

[0108] The cancer characterized by the methods of the invention can comprise, without limitation, a carcinoma, a germ cell tumor, a blastoma, a sarcoma, a lymphoma or leukemia, or other cancers. Carcinomas include without limitation transitional cell papillomas and carcinomas, adenomas and adenocarcinomas (glands), adenoma, adenocarcinoma, linitis plastica insulinoma, glucagonoma, gastrinoma, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, carcinoid tumor of appendix, epithelial neoplasms, squamous cell neoplasms squamous cell carcinoma, basal cell neoplasms basal cell carcinoma, prolactinoma, oncocytoma, hurthle cell adenoma, renal cell carcinoma, ductal, lobular and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, warthin's tumor, thymoma, specialized gonadal neoplasms, sex cord stromal tumor, thecoma, granulosa cell tumor, arrhenoblastoma, Sertoli leydig cell tumor, glomus tumors, paraganglioma, pheochromocytoma, glomus tumor, nevi and melanomas, melanocytic nevus, malignant melanoma, melanoma, nodular melanoma, dysplastic nevus, lentigo maligna melanoma, superficial spreading melanoma, and malignant acral lentiginous melanoma. Sarcoma includes without limitation Askin's tumor, botryodies, grawitz tumor, multiple endocrine adenomas, endometrioid adenoma, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic, mucinous and serous neoplasms, cystadenoma, pseudomyxoma peritonei, chondrosarcoma, Ewing's sarcoma, malignant hemangio endothelioma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, malignant schwannoma, osteosarcoma, soft tissue sarcomas including: alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and synovialsarcoma. Lymphoma and leukemia include without limitation chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases, extranodal marginal zone B cell lymphoma, also called malt lymphoma, nodal marginal zone B cell lymphoma (nmzl), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, T cell prolymphocytic leukemia, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides / sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified, anaplastic large cell lymphoma, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, classical hodgkin lymphomas (nodular sclerosis, mixed cellularity, lymphocyte -rich, lymphocyte depleted or not depleted), and nodular lymphocyte -predominant hodgkin lymphoma. Germ cell tumors include without limitation germinoma, dysgerminoma, seminoma, polyembryoma, and gonadoblastoma. Blastoma includes without limitation nephroblastoma, medulloblastoma, nongerminomatous germ cell tumor, embryonal carcinoma, endodermal sinus turmor, choriocarcinoma, teratoma, and retinoblastoma. Other cancers include without limitation labial carcinoma, adenocarcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervix carcinoma, uterine corpus carcinoma, meningioma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, medulloblastoma and peripheral neuroectodermal tumors, gall bladder carcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma, osteosarcoma, chondrosarcoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, fibrosarcoma, Ewing sarcoma, myosarcoma, liposarcoma, and plasmocytoma.

1. EMBODIMENTS OF THE INVENTION

[0109] In an embodiment a method for obtaining a tumor microenvironment platform comprising oral carcinoma for predicting chemoresistance and dysplastic progression, wherein the method comprising:

(ii) layering a platform with oral cancer cell lines and/or dysplastic cell lines and cancer- associated fibroblast cells, wherein the oral cancer cell lines comprise MhCT12-E and MhCT08-E cells, wherein the cancer-associated fibroblasts cells comprise MhCT08-F, MhCL03-F, MhCA04-F, and MhCB05 -F, wherein the dysplastic cell lines comprise DOK, wherein the cell lines are cultured in RPMI medium with 20% Fetal bovine serum (FBS); and (ii) adding culture medium comprising epithelial spent media and fibroblast spent media to the platform to obtain the tumor microenvironment platform.

[0110] In further embodiment, wherein the oral cancer cell lines are autologous pairs of cancer cell lines, wherein the platform can identify novel targets/biomarkers for chemoresistance and therapy.

[0111] In further embodiment, wherein the cancer-associated fibroblasts are spontaneously transformed.

[0112] In an embodiment, a method of predicting chemoresistance using the tumor microenvironment platform as claimed, wherein the method comprises the steps of: a. subjecting the MhCT12-E cells and the cancer-associated fibroblast cells to a drug with a concentration ranging from 1.9 to 200uM in a ratio of 1:1; and b. validating proliferation of the MhCT12-E cells and the cancer-associated fibroblasts by calculating IC50 value.

[0113] In further embodiment, the method as claimed, wherein the IC50 value of MhCT12-E cell is in the range of 2.3151 uM and the IC50 value in the presence of the MhCT12-F cells is in the range of 6.4542 uM.

[0114] In further embodiment, wherein one of the drug is cisplatin.

[0115] In further embodiment, a method of predicting dysplastic progression using the tumor microenvironment platform as claimed, wherein the method is to assess the effectiveness of novel chemopreventive drugs, wherein the method comprising the steps of: a. co-culturing non-neoplastic and dysplastic epithelial cells together with MhCT08-F, MhCL03-F, MhCA04-F comprised in the tumor microenvironment platform, wherein the coculturing is performed for 48 hours in DMEM-F12 in 5% CO2 at 37°C, wherein the MhCB05-F to neoplastic and dysplastic epithelial cells are comprised in a ratio of 5:1; b. treating the platform with candidate drugs; and c. performing expression profiling, flow cytometer-based profiling of cancer stem cell markers in the co-cultured non-neoplastic and dysplastic epithelial cells and assessing cytotoxicity.

[0116] In further embodiment, wherein the non-neoplastic cell is a HaCaT cell line, wherein the dysplastic epithelial cell is a DOK cell line.

[0117] In an embodiment, wherein a) the co-culture of the non-neoplastic cells with CAFs showed up-regulation of ALDH1A1 by about 1.3 folds, CD133 by about 8.4 folds, and NOTCH1 by about 10.9 folds, wherein the expression profiling of the dysplastic epithelial cells co-cultured with fibroblasts showed up-regulation of SOX-2 by about 24.5 folds as compared to their respective monolayer cells; and

[0118] b) the flow cytometer-based profiling of the non-neoplastic cell showed up-regulation of CD44+ by 96.8% compared to an untreated non-neoplastic cell having 74.6%, wherein the flow cytometer-based profiling of the dysplastic epithelial cells showed up-regulation of CD44+ by 72.7% compared to untreated dysplastic epithelial cells having 41.1%.

[0119] In further embodiment, wherein the oral carcinoma cell line is obtained from a cancer tissue obtained surgically or by biopsy or as a xenograft or any combination thereof.

[0120] In an embodiment, wherein the tumor microenvironment platform is for maintaining an intact tissue microenvironment, cellular architecture, and integrity of tumor-stroma interaction.

[0121] In further embodiment, wherein the method includes producing a xenograft model by injecting a mixture of the epithelial and fibroblast cells into a mouse model. Experimental xenografts in animals are essential tool in cancer research and more particularly, in studying the efficacy of anti-cancer drugs or other therapeutic procedures. Experimental xenografts also have potential applications in the study of the mode of action of drugs or other chemical compounds or biological substances or agents in biological systems. Xenograft provide an improvement to the determination of efficacy of candidate therapeutic agents in xenograft animal models and their effects.

[0122] In further embodiment, a kit for predicting chemoresistance of novel drugs to oral carcinoma, wherein the kit comprises a tumor microenvironment platform comprising oral cancer cell lines and cancer-associated fibroblast cells, wherein the oral cancer cell lines comprise MhCT12-E and MhCT08-E, wherein the cancer-associated fibroblasts cells comprise MhCT08-F, MhCL03-F, MhCA04-F, and MhCB05-F, wherein the cell lines are cultured in RPMI medium with 20% Fetal bovine serum (FBS), and (ii) culture medium comprising of epithelial spent media and fibroblast spent media.

[0123] In further embodiment, a kit for predicting dysplastic progression, for the efficacy of novel candidate chemopreventive drugs, wherein the kit comprises a tumor microenvironment platform comprising dysplastic cell line (DOK) or non-neoplastic cell line (HaCaT) and cancer- associated fibroblast cells, wherein the cancer-associated fibroblasts cells comprise MhCT08-F, MhCE03-F, MhCA04-F, and MhCB05-F, wherein the cell lines are cultured in RPMI medium with 20% Fetal bovine serum (FBS), and (ii) culture medium comprising epithelial spent media and fibroblast spent media.

[0124] MATERIALS AND METHODS: [0125] Tumor specimen and establishment:

[0126] Tumor samples were collected after obtaining informed consent from patients. Tissues were collected aseptically in RPMI-1640 (cat. no. AT222A; Himedia Laboratories, LLC) with triple strength penicillin- streptomycin (cat. no. 15140122; Gibco; Thermo Fisher Scientific, Inc.) from two 65-year-old females with no risk habits, diagnosed with Oral squamous cell carcinoma. The tissue samples were thoroughly washed 3 times at 5 min interval with 3X penicillin- streptomycin followed by 10% povidone iodine solution (Win Medicare Pvt. Ltd.) and finally with complete growth medium. The tissue was cut into small sections and treated with 0.25% trypsin (cat. no. 25200056; Gibco; Thermo Fisher Scientific, Inc.) for 30 min at 37°C. The chopped and digested pieces were placed in a serrated 60-mm petri dishes and supplemented with 10 ng/pl each of human recombinant epidermal growth factor (hEGF; cat. no. E9644; Sigma- Aldrich; Merck KGaA), N2 suppliment-lX (cat. no. 17502048; Gibco; Thermo Fisher Scientific, Inc.), Epilife defined growth supplement (EDGS; cat. no. S0125; Gibco; Thermo Fisher Scientific, Inc.) along with 20% FBS (cat. no. RM10434; Himedia Laboratories, LLC), in RPMI-1640 media with IX penicillin- streptomycin. The media was changed every 48 h to remove the dead cells. The epithelial cells were enriched by differential trypsinization and further sub-cultured. Briefly, the cells were trypsinized for two different time points. After a min of trypsinization, floating cells (fibroblasts) were removed and seeded in a separate flask. Since fibroblasts can detach faster than epithelial cells, this differential trypsinization technique yielded two separate cellular populations. The separated cells were cultured in RPMI-1640 media with 20% FBS and no additional growth supplements. The cells were passaged for more than P50 and were characterized for cell-type specificity at both early and late passages. Later passages of the cells were maintained in RPMI-1640 medium, pH 7.2, supplemented with 20% FBS and IX penicillin-streptomycin solution. Both the epithelial and fibroblast cells originated from the oral tissues of the patient. The established cell types were stained with Pan-cytokeratin (PanCK; epithelial specific marker) and FSP-1 (fibroblast specific marker), to verify their identities after differential trypsinization. Isolated epithelial and fibroblast cells were denoted as mentioned in table I. The names have been arrived at through an acronym: M, Mazumdar shaw medical foundation; h, Human; C, Cancer of; T, Tongue; B, Buccal Mucosa; L , Larynx; A, Upper alveoulus; 03/04/05/08/12, Patient code; E, Epithelial; and F, Fibroblast.

[0127] Diagnostic and Prognostic Methods

[0128] The current invention focuses on primary cell lines derived from oral cancer patients, their characterization as well as validation of the use of the model in early carcinogenesis as well as chemoresistance.

[0129] Table 1: Names of Cell line. Cell lines used in the invention have been renamed to provide uniformity.

Oral cancer cell lines (104) as well as cancer-associated fibroblasts (106) have been derived from patients diagnosed with oral cancer via explant culture (102). Co-culture models (108) were tested for their relevance in early carcinogenesis (112) and drug resistance (114).

[0130] The profiling/characterization of the cell lines were made (110). (FIG 1A)

[0131] Five fibroblasts (MhCT12-F, M08 fibre, MhCL03-F, MhCA03-F, and MhCB05-F) and two epithelial cell lines (MhCT12-E and MhCT08-E) were established from patient tumor samples and the following experiments were performed.

[0132] Table 2 shows Clinical and pathological details of established cell lines

[0133]

[0134] The present invention is further elaborated with the help of the following examples.

However, these examples should not be construed to limit the scope of the present invention.

[0135] EXAMPLE 1: CHARACTERIZATION OF THE PATIENT-DERIVED CELL

LINES

[0136] The established cell lines (MhCT12-F, M08 fibre, MhCT08-E and MhCT12-E) were characterized using various techniques as detailed below:

1. FACS

[0137] The epithelial and cancer associated fibroblast nature of the cell lines was confirmed by the presence of surface markers like Pancytokeratin and Fibroblast surface protein 1 (FSP-1) respectively, using FACS (FIG IB). The removal of fibroblasts from epithelial population was confirmed by negative staining with fibroblast surface protein specific antibody. Similarly, a pure population of fibroblast was confirmed by negative staining with anti-Pan Cytokeratin antibody. As depicted, the cell lines were characterized at both an early (p8) and late passage (p43). As shown, the respective epithelial and fibroblast cell lines were enriched in their lineage in the later passage as compared to the early passage. (FIG IB) [0138] 2. HPV Infection Status

[0139] High risk of HPV 16 and 18 infections have been associated with oral cancer. More than 25% of all the oral cancers are associated with HPV 16 infection, while a lower percentage of about 1-3% is attributed to HPV 18 infection. Factually, HPV positive oral cancers are associated with a favourable prognosis when compared to the HPV negative oral cancers. The established cell lines were examined for the presence of HPV infection status by staining with pl6 and E7 antibody via immunocytochemistry. HeLa cells, a cervical cancer cell line was used as a positive control for the experiment. As depicted in FIG 2, both the epithelial and fibroblast cells were lightly stained for p 16 and E7.

[0140] 3. DNA ploidy

[0141] DNA content was determined in the established cell lines by performing ploidy analysis. Normal human lymphocytes from a healthy individual was used as a control for diploid DNA content. Dividing the mean channel of the cells in the Go phase by that of the Go mean channel of diploid lymphocytes gave the DNA index number. The DNA indices of M08 and B12 cells were 1.1 and 0.8 respectively. M08 cell line had slightly higher than diploid DNA content, while B 12 was towards the lower DNA content. The results therefore indicate that both the patient samples have abnormal DNA content which might be responsible for the cancerous transformation of these cells. (FIG 3)

[0142] 4. STR profiling

[0143] STR profiling of 10 loci was performed to establish the genomic identity, cell line identity and exclude any cross -contamination (Table 3 and 4). The STR profile of the established cell lines was distinct from that of any other cell lines deposited in ATCC and Expasy Cellosaurus STR (CLASTR) database. These results thus indicated that both the M08 and B12 lines were novel. The similar genotypic identity between MhCT12-E and fibroblast cells from the same patient distinctly proved the autologous nature of the cells (Table 4). [0144] Table 3: Shows important attributes of established cell lines

E, Epithelia! ; F. Fibroblast,

[0145]

[0146] Table 4: Shows STR profiling

5 [0147] 5. Doubling time determination

[0148] The cell lines exhibited different doubling times as depicted in the FIG. As depicted from FIG 4 doubling time of approximately 25 hours was observed for MhCT12-E cell line. Both the fibroblast cell lines depicted higher doubling times than epithelial cells as depicted in FIG 4.

10 [0149] EXAMPLE 2: ANALYSIS OF TUMORIGENIC PROPERTIES

[0150] The primary epithelial cells were also tested for their tumorigenic properties as detailed below: [0151] 1. Proliferation

[0152] Treatment with CAF conditioned medium (neat as well as mixed in different ratios with fresh medium) increased the proliferative potential MhCT12-E cells (FIG 5). However, due to over-confluency, the proliferative potential of MhCT12-E cells decreased at 72 hours.

[0153] 2. Wound Healing

[0154] Established primary epithelial cells were treated with conditioned media from its respective primary fibroblast pair. Treatment with conditioned media from the autologous fibroblast pair significantly increased the migration potential MhCT12-E cell type (p-value < 0.05) as depicted in FIG 6.

[0155] 3. Invasion

[0156] Established primary epithelial cell was treated with conditioned media from its autologous primary fibroblast pair. Treatment with conditioned media from the autologous fibroblast pair significantly increased the invasive potential MhCT12-E cell types (p-value < 0.05) as depicted in FIG 7.

[0157] 4. Sphere formation

[0158] Established primary MhCT12-E cell was treated with conditioned media from its autologous primary fibroblast pairs. Treatment with conditioned media from the autologous fibroblast pair significantly increased the sphere formation potential in GelMA hydrogels as depicted in FIG 8a. Not only the number, but the size of the spheres was also observed to be increased upon treatment with conditioned medium (FIG 8b).

[0159] FIG. 8a is MhCT12-E cells with no treatment and Neat CM.

[0160] FIG. 8b is MhCT12-E cells with no treatment and CM treatment.

[0161] FIG. 8c depicts results of no treatment and neat concentrated medium.

[0162] 5. Chemoresistance

[0163] The Proliferation assay was performed to check the intrinsic Cisplatin resistance of MhCT12-E and fibroblast cells. The cells seeded, (10000 cells per well) were treated with cisplatin concentration ranging from 1.9 to 200 uM in a ratio of 1:1. The proliferation trend in both cells is almost same, but the difference can be validated by calculating the IC50 value, which was determined to be 2.3151 uM for MhCT12-E and 6.4542 uM for fibroblast. As a result, epithelial cells are more susceptible to the cisplatin medication than fibroblast cells. The same can be observed in the following proliferation graph of MhCT12-F vs Epithelial cells (FIG. 9). [0164] Furthermore, to simulate the actual circumstance in the human body, the cells were treated with the respective 100% conditioned medium for five passages and the proliferation was monitored.

[0165] The above comparative graphs of with and without condition media for MhCT12-F and epithelial cells, respectively, shows the proliferation rate of each cell type (FIG 10). Thus, the IC50 for cells in neat and conditioned media were calculated using these values. For epithelial cells, the IC50 for neat media was 2.3151uM, whereas for conditioned media it slightly increased to 3.6205 uM, while for fibroblast, the IC50 for neat was calculated as 6.4542 uM and for conditioned media it was 4.0938 uM. However, for the fibroblast in conditioned media the cell shows 60% proliferation at 100 uM concentration.

[0166] FIG. 10a illustrates Comparative graph of MhCT12-F with and without conditioned media and FIG. 10b illustrates comparative graph of MhCT12-E with and without conditioned media.

[0167] EXAMPLE 3: Patient-derived cells as a model for early carcinogenesis

[0168] The feasibility of using the fibroblasts cells along with the dysplastic and normal keratinocytes as a model for early carcinogenesis and dysplastic progression was assessed.

[0169] The morphology of the MhCB05-Fblast cells was initially checked through phase contrast microscope (FIG 11).

[0170] The established cell line (MhCB05-F) was characterized using various techniques as detailed below,

1. Flow cytometer: The cancer associated fibroblast nature of the cell line was confirmed by the presence of alpha-Smooth Muscle Actin (a-SMA) using FACS. A pure population of fibroblast was confirmed by negative staining with pan-Cytokeratin (pan-CK) antibody. The MhCB05-Fblast cells showed 89.5%±3.18 positivity for a-SMA, while pan-CK showed 0.815%±0.36 indicating absence of epithelial cells (FIG 12).

2. Immunocytochemistry: The morphology and nature of the fibroblast cells were also confirmed by immunocytochemistry with a-SMA, Vimentin and pan-CK. The fibroblast specific markers, a-SMA and Vimentin showed >80% positivity, while pan-CK was negative (FIG 13).

[0171] EXAMPLE 4: POTENTIAL TO INDUCE TUMORIGENICITY

[0172] The MhCB05-Fblast cells were tested for their potential to induce tumorigenicity in non-neoplastic (HaCaT) and dysplastic (DOK) epithelial cells. The various experiments performed are as detailed below:

1. Generation of CAE-conditioned medium (1402) and transwell co-culture assay [0173] The fibroblast medium for conditioned medium assay was obtained from 60-70% confluent CAF (1404) monolayer culture with DMEM: F12 (1:1) medium without FBS for a period of 48 hours. The culture was changed to SFM (1406). The medium was then collected and spun at l lOOrpm for 3 minutes to pellet down any floating cells or debris (1408). The supernatant was collected in a fresh tube and stored for further use (1412). Indirect Co-culture (1410) was thus achieved by changing the supernatant to CAF-CM (1414) (FIG 14).

[0174] The transwell co-culture was performed in human epithelial (HaCaT and DOK) and fibroblast (h-CAF) cells using transwell inserts (1502). The epithelial and fibroblast cells were trypsinized, counted (50,000 cells/well) and plated in complete medium (1506) onto the 24- well plate and the trans well insert respectively. The medium was changed to serum-free medium after 24 hours and the cells were then allowed to grow for 48 hours. Post 48 hours, the epithelial cells were trypsinized (1504) and used for further characterization (FIG 15).

[0175] 2. Expression profiling

[0176] Transcriptomic profiling of CSC markers indicated that HaCaT cells showed upregulation of CD133 (5.3fold. p=0.0007) and NOTCH1 (10.6 fold, p=0.0001) at higher [5:1 (5 parts of conditioned medium)] dilution of the conditioned media, along with up-regulation of CD44 (1.2 fold). DOK cells showed up-regulation in both CD44 (1.1 fold) and NOTCH1 (1.2 fold, p=0.0462) at 5:1 dilution along with up-regulation of SOX-2 (24.7 fold, p=0.0001) as compared to untreated controls (FIG 16). All further co-culture experiments were performed with a 5:1 concentration of the conditioned medium in both HaCaT and DOK cells. Transwell co-culture with epithelial and fibroblast cells was also carried out in human cell lines. Profiling of these transwell co-cultured cells showed significant up-regulation of ALDH1A1 (1.3fold), CD133 (8.4 fold), and NOTCH1 (10.9 fold) in HaCaT cells, while DOK cells showed upregulation of SOX-2 (24.5 fold) alone as compared to their respective monolayer cells (FIG 16).

[0177] 3. Flow cytometer-based profiling

[0178] Protein profiling of CD44 under the effect of CAF-conditioned medium showed enrichment of CSCs. In human cell lines, HaCaT cells showed significantly up-regulated expression of CD44⁺ in CAF-conditioned medium treated cells (96.8%±1.06, p=0.0042) as compared to untreated cells (74.6%±0.99), while in DOK cells, CAF-conditioned medium treated cells showed 72.7%±0.21 (p=0.0007) as compared to untreated cells (41.15%±0.81) (FIG 17).

[0179] 4. Invasion

[0180] The cells treated with CAF-conditioned medium and co-cultured with trans well inserts were also assessed for their invasiveness as compared to their respective monolayer cells. HaCaT cells showed a higher percentage of invasiveness in both CAF-conditioned media treated (5.4+0.22) and transwell co-cultured (5.5+0.39) cells as compared to control (3+0.67) cells and a comparison between CAF-conditioned medium treated cells and transwell cocultured cells did not show any difference in invasiveness (FIG 18). DOK cells also showed similar results, wherein both CAF-conditioned media treated (7+0.05, p=.O496') and transwell co-cultured (7.2+0.21, p=0.0527) cells were significantly invasive as compared to their control (5.6+0.32) cells and a comparison of CAF-conditioned medium treated cells with transwell co- cultured cells did not show any difference (FIG 18). A comparison of invasiveness between the cell lines showed significantly high invasiveness in DOK cells (7+0.05, p=0.01 3) treated with CAF-conditioned medium as compared to HaCaT cells (5.4+0.22) treated with CAF- conditioned medium (FIG 18).

[0181] 5. Migration/wound healing assay

[0182] Evaluation of the migratory capacity under the effect of the conditioned media showed that at 6th hour, HaCaT cells showed 52% wound closure, while DOK showed 74% wound closure as compared to untreated cells (HaCaT: 37%; DOK: 49%). At 12th hour, HaCaT (p=0.0001) and DOK (p=0.0102) cells showed complete wound closure as compared to their control cells (HaCaT: 49% and DOK: 87%) (FIG 19).

[0183] 6. Spheroid formation

[0184] All the cell lines were assessed for the CSC enrichment by the spheroid formation in the presence of the CAF-conditioned media. It was observed that the cells that did not show any potential previously, formed spheroids under the effect of CAF-conditioned medium (5:1). The total number of spheroids formed was significantly high in DOK (22+0.75, p=().()OI8) as compared to HaCaT (16.5+1.07) cells. The size of the spheres was also significantly high in DOK (0.28+0.013, p=0.0028) as compared to that of HaCaT (0.23+0.012) spheres (FIG 20).

[0185] 7. Chemo-resistance

[0186] Cytotoxic assays were carried out to evaluate the CAF-conditioned medium effect on Curcumin sensitivity indicated that HaCaT cells showed a 20% increase in cell viability; the IC50 increasing by 2.1fold (266pM vs 563pM) (FIG 21A & C). In DOK, comparatively, a more sensitive cell line, a 30-50% increase in cell viability (FIG 2 IB & C) was observed with the IC50 increasing by 4.2fold (18pM vs 75pM, p=0.0101

[0187] EXAMPLE 5: CANCER CELL-CAF MODEL FOR RESISTANCE TO CHEMOTHERAPY

[0188] Both BMS12 (primary tumor derived fibroblast) and M05 (Recurrent patient derived Fibroblast) were also assessed for their effect on (i) resistance towards cisplatin drug and (ii) cancer stem cell enrichment potential, on oral cancer HNSCC cell lines Cal-27 and its cisplatin resistant counter-part Cal-27CisR cell lines. a) Resistance towards Cisplatin on HNSCC (Cal-27 and Cal-27 CisR cell lines)

[0189] Cal-27 and Cal-27CisR cells were assessed for their IC50 and resistance index (RI) for cisplatin. Cal-27 cells showed an increased IC50 (44.05 pM, p=0.001; RI >2 and IC50 (28.4 pM, p=0.001; RI >1.2 under the effect of BMS12 and MhCB05-Fblast conditioned media respectively as compared to parental IC50 (26.25 pM) (FIG. 22 A). However, Cal-27 CisR cells showed an increased IC50 (47.82 pM, p=0.001; RI >1.5) as compared to its parental IC50 (31.5 pM) under the effect of BMS12 fibroblast conditioned media. A similar effect was observed under MhCB05-Fblast conditioned media (ICso:49.95pM, p=0.001; RI =1.59) (FIG. 22 B).

[0190] (b) Expression profile: Multi-drug (MDR) resistance genes such as MDR1, MRP2, ABCG2, Survivin, ERCC1, showed up-regulation under M05 conditioned media treatment. MDRl(2.95fold, P<0.03), TS (1.52fold), ABCG2 (4.3fold, P<0.001) Survivin (1.3fold, P<0.0003), ERCC1 (5.56fold, P<0.05) showed a significant increase in expression. The cancer stem cell regulatory genes, NANOG (1.68-fold, P<0.0054), OCT4 (2.45-fold, P<0.001) were also upregulated along with Notchl (1.16fold, P<0.0051), CXCR4 (1.19-fold, P<0.045) and SDFlalpha (2-fold, p<0.04).

[0191] Similarly, Cal-27CisR showed an up-regulation MDRl(1.39fold, P<0.03), TS (1.69fold), ABCG1/2 (1.39fold, P<0.001) Survivin (1.75fold, P<0.0003 and ERCC1 (1.23fold, P<0.05) as compared to Cal-27CisR untreated cells. The cancer stem cell regulatory genes, SOX2 (4.27-fold, P<0.0001), NANOG (1.87-fold, P<0.0054), OCT4 (1.38-fold, P<0.001) also showed an increase along with Notchl (1.16fold, P<0.0051), CXCR4 (1.16-fold, P<0.02) and SDFlalpha (1.5-fold, p<0.04) (FIG 23).

[0192] c) Functional Assays:

[0193] Spheroid assay showed that the Cal-27 cells showed an 8-fold increase (n=8, P=0.0001) in Cal-27M05 cells and 3-fold increase (n=3, P=0.0001) in Cal-27BMS12 when treated M05 and BMS12 conditioned media as compared to the untreated Cal-27 cells (n=l). Cal-27 CisR cell line in presence of M05 conditioned media generated 3 -fold increase (n=25, P=0.0001) in comparison with the untreated plain media Cal-27 CisR cells (n=8).

[0194] On the other hand, multicellular spheroids (epithelial + fibroblast) generated using M05 and BMS12 fibroblast with Cal-27 and Cal-27CisR cells showed difference in size and morphology of spheroids. Both Cal-27 and Cal-27 CisR cells in presence of BMS12/MhCB05- Fblast cells showed to develop (n=3) spheroids as compared to the Cal-27(n=l) and cal-27cisR (n=8) cells alone. [0195] Similar results were observed in the anchorage dependent clone formation capacity of Cal-27 cells, with a significant 3.5fold increase in colony formation capacity in Cal-27cM (n=56, P=0.0001) cells as compare to the Cal-27 (n=15) cells maintained in basal growth media in 15 days interval (FIG. 24 A&B). In the wound closure assay, Cal-27 cells, under the effect of M05 conditioned media treatment (Cal-27cM) lead to a significantly increased migratory potential. Cal-27cM cells showed complete wound closure (100%, p=0.0001) at 24^th h, as compared to wound closure of 55% in Cal-27 cell at the same time period. Cal-27 cells completed wound closure only at 36 h after the scratch was made (FIG 24. C).

[0196] d) Primary Cell line model to study the efficacy of anti-SARS-COV2 agents to inhibit viral entry:

[0197] MO8 oral epithelial cells were plated in a 96-well plate at 3.0x104 cells per well and incubated overnight. Different concentration of the anti-SARS-COV2 agent at (500, 250,125 & 62.5, 31.25 and 15.625 pg/ml) was preincubated with SARS-CoV-2 virus at an MOI of 0.01 at 37 oC for 1.5 hours and then added to the seeded cells and incubated for 1 h at 37 °C with 5% CO2. The infection media was removed and fresh growth media was added onto the cells and incubated for 48 hours. After the completion of 48 hours the supernatant was collected into viral transport media. The virus was inactivated and the viral load was checked by q-RT-PCR assay with Nucleocapsid-N-gene, Envelop-E- gene and RdRp (RNA dependent RNA polymerase) gene primers. As observed in FIG 25, there was a dose dependent decrease in viral load with an increasing concentration of SolAce2 protein.

[0198] e) Cell line model to study the pathogenesis of SARS-COV2 infection in comorbid condition like hyperglycemia

[0199] M08 and B12 were cultured in both high and low glucose media conditions. SARS- COV2 viral particles at an MOI of 0.1 was used to infect the cells by incubating for 1 hour at 37°C. The media was changed into fresh media and incubated for 48 hours. The cells were lysed with Trizol and RNA isolated. The viral load was detected in the cells by qRT PCR by amplifying the Nucleocapsid (N), and the RNA dependent RNA polymerase (RdRp) gene of SARS-COV2. The differential CT values between the cells grown at Low and high glucose conditions was calculated and plotted to see the difference in the viral entry into the cells. As seen in FIG 26, the viral infectivity was higher when cells were subjected to high glucose conditions.

[0200] 2. ADVANTAGES OF THE INVENTION

[0201] Development of spontaneously transformed cell lines from patients with oral cancer. These are not mere adaption of cells outside the human body. The fibroblast and epithelial cells have been separated out and are growing as pure culture.

[0202] Development of 5 CAF cell lines from patients.

[0203] Autologous pair of cancer cell line and CAF from the same patient that are completely profiled and characterized. One of the pairs MhCT12-E and MhCT12-Fs are derived from the same patient ie they are autologous pairs

[0204] The CAF cell lines derived are also from two cohorts of patients, treatment naive and recurrent. These cell lines are generated from oral cancer patients. However only cancer cell lines have been generated.

[0205] These cell lines are spontaneously transformed.

[0206] Models can be used to explore the role of CAF-cancer cell interactions during dysplastic progression, drug resistance as well as during metastasis.

[0207] The platform can be used to study SARS-CoV2 viral entry through oral route

[0208] The platform grown in high glucose can be used to study SARS-CoV2 viral pathogenesis in comorbid (hyperglycemia) condition.

[0209] 3. NOVELTY AND BENEFITS OF THE INVENTION.

[0210] The platform can be used in,

[0211] Discovery of markers affecting tumor and stromal microenvironment interaction: Can be done by co-culture experiments in 2D and 3D cell culture systems.

[0212] Validation of markers affecting tumor and stromal microenvironment interaction by co-culture experiments in 2D and 3D cell culture systems.

[0213] Co-culture model for understanding early carcinogenesis, identifying markers of chemoprevention .

[0214] Co-culture model for evaluating the role of fibroblast niche in chemoresistance of oral cancer cells.

[0215] Validating novel targets for chemoprevention and resistance reversal.

[0216] Identifying and validating markers of early diagnosis and resistance of oral cancer.

[0217] 4. BEST MODE TO PRACTICE THE INVENTION

[0218] The possible uses of this invention include but not limited to,

[0219] Provide service to pharmaceutical industries through an accredited laboratory,

[0220] Provide service to academia through an accredited laboratory.

[0221] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and subblock diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed. [0222] According to a non limiting exemplary aspect of the present invention, the markers can be used for the development of kits that enable saliva collection, processing, and marker detection. These diagnostic kits developed can then be utilized by hospitals/private clinic s/dental doctors or the public as such to screen/diagnose oral cancer.

[0223] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.

[0224] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0225] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configureurable, such that it may be utilized in ways other than that shown in the accompanying figures.

[0226] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[000] References

1) Coelho, K.R., Challenges of the oral cancer burden in India. J Cancer Epidemiol, 2012. 2012: p. 701932.

2) Loree, T.R. and E.W. Strong, Significance of positive margins in oral cavity squamous carcinoma. Am J Surg, 1990. 160(4): p. 410-4.

Claims

27

CLAIMS:

We Claim,

1) A method for obtaining a tumor microenvironment platform comprising oral carcinoma for predicting chemoresistance and dysplastic progression, wherein the method comprising:

(i) layering a platform with oral cancer cell lines and/or dysplastic cell lines and cancer-associated fibroblast cells, wherein the oral cancer cell lines comprise MhCT12-E and MhCT08-E cells, wherein the cancer-associated fibroblasts cells comprise MhCT08-F, MhCL03-F, MhCA04-F, and MhCB05 -F, wherein the dysplastic cell lines comprise DOK, wherein the cell lines are cultured in RPMI medium with 20% Fetal bovine serum (FBS); and (ii) adding culture medium comprising epithelial spent media and fibroblast spent media to the platform to obtain the tumor microenvironment platform.

2) The method as claimed in claim 1, wherein the oral cancer cell lines are autologous pairs of cancer cell lines, wherein the platform can identify novel targets/biomarkers for chemoresistance and therapy.

3) The method as claimed in claim 1, wherein the cancer-associated fibroblasts are spontaneously transformed.

4) A method of predicting chemoresistance using the tumor microenvironment platform as claimed in claim 1, wherein the method comprises the steps of: a. subjecting the MhCT12-E cells and the cancer-associated fibroblast cells to a drug with a concentration ranging from 1.9 to 200uM in a ratio of 1 : 1 ; and b. validating proliferation of the MhCT12-E cells and the cancer-associated fibroblasts by calculating IC50 value.

5) The method as claimed in claim 4, wherein the IC50 value of MhCT12-E cell is in the range of 2.3151 uM and the IC50 value in the presence of the MhCT12-F cells is in the range of 6.4542 uM.

6) The method as claimed in claim 4, wherein one of the drug is cisplatin.

7) A method of predicting dysplastic progression using the tumor microenvironment platform as claimed in claim 1, wherein the method is to assess the effectiveness of novel chemopreventive drugs, wherein the method comprising the steps of: a. co-culturing non-neoplastic and dysplastic epithelial cells together with MhCT08-F, MhCE03- F, MhCA04-F comprised in the tumor microenvironment platform, wherein the co-culturing is performed for 48 hours in DMEM-F12 in 5% CO2 at 37°C, wherein the MhCB05-F to neoplastic and dysplastic epithelial cells are comprised in a ratio of 5: 1 ; b. treating the platform with candidate drags; and c. performing expression profiling, flow cytometer-based profiling of cancer stem cell markers in the co-cultured non-neoplastic and dysplastic epithelial cells and assessing cytotoxicity.

8) The method as claimed in claim 7, wherein the non-neoplastic cell is a HaCaT cell line, wherein the dysplastic epithelial cell is a DOK cell line.

9) The method as claimed in claim 7, wherein a) the co-culture of the non -neoplastic cells with CAFs showed up-regulation of ALDH1A1 by about 1.3 folds, CD133 by about 8.4 folds, and NOTCH1 by about 10.9 folds, wherein the expression profiling of the dysplastic epithelial cells co-cultured with fibroblasts showed up-regulation of SOX-2 by about 24.5 folds as compared to their respective monolayer cells; and b) the flow cytometer-based profiling of the non-neoplastic cell showed up-regulation of CD44+ by 96.8% compared to an untreated non-neoplastic cell having 74.6%, wherein the flow cytometerbased profiling of the dysplastic epithelial cells showed up-regulation of CD44+ by 72.7% compared to untreated dysplastic epithelial cells having 41.1%.

10) The method as claimed in claim 1, wherein the oral carcinoma cell line is obtained from a cancer tissue obtained surgically or by biopsy or as a xenograft or any combination thereof.

11) The method as claimed in claim 1, wherein the tumor microenvironment platform is for maintaining an intact tissue microenvironment, cellular architecture, and integrity of tumor-stroma interaction.

12). The method as claimed in claim 1, wherein the method includes producing a xenograft model by injecting a mixture of the epithelial and fibroblast cells into a mouse model.

13). A kit for predicting chemoresistance of novel drags to oral carcinoma, wherein the kit comprises a tumor microenvironment platform comprising oral cancer cell lines and cancer- associated fibroblast cells, wherein the oral cancer cell lines comprise MhCT12-E and MhCT08- E, wherein the cancer-associated fibroblasts cells comprise MhCT08-F, MhCL03-F, MhCA04-F, and MhCB05-F, wherein the cell lines are cultured in RPMI medium with 20% Fetal bovine serum (FBS), and (ii) culture medium comprising of epithelial spent media and fibroblast spent media.

14) A kit for predicting dysplastic progression, for the efficacy of novel candidate chemopreventive drags, wherein the kit comprises a tumor microenvironment platform comprising dysplastic cell line (DOK) or non-neoplastic cell line (HaCaT) and cancer-associated fibroblast cells, wherein the cancer-associated fibroblasts cells comprise MhCT08-F, MhCL03-F, MhCA04- F, and MhCB05-F, wherein the cell lines are cultured in RPMI medium with 20% Fetal bovine serum (FBS), and (ii) culture medium comprising epithelial spent media and fibroblast spent media.