DESC:F O R M 2
THE PATENTS ACT, 1970 (39 of
1970)
&
THE PATENTS
RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]

1. TITLE OF THE INVENTION: SYSTEM FOR IN-VITRO MODELLING OF NODAL METASTASIS IN ORAL SQUAMOUS CELL CARCINOMA

2. APPLICANT (A) NAME: MAZUMDAR SHAW MEDICAL
FOUNDATION
(B) ADDRESS: MAZUMDAR SHAW MEDICAL FOUNDATION, A-BLOCK, 8TH FLOOR, MAZUMDAR SHAW MEDICAL CENTRE, #258/A, NARAYANA HEALTH CITY, BOMMASANDRA, BANGALORE, KARNATAKA, INDIA - 560099

3. NATIONALITY (C) INDIAN

THE FOLLOWING SPECIFICATION DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED

PRIORITY CLAIM
[001] The instant patent application is related to and claims priority from the India provisional patent application entitled, “SYSTEM FOR IN-VITRO MODELLING OF NODAL METASTASIS IN ORAL SQUAMOUS CELL CARCINOMA”, Patent Application no.: 202341042308, Filed on: 23rd June 2023 and Post-dated Priority date: 23rd July 2023, which is incorporated in its entirety herewith.
BACKGROUND OF THE DISCLOSED EMBODIMENT
[002] Technical field
[003] The present invention is in technical field of a system and method for In-vitro modelling of nodal metastasis, one of the most significant prognosticators of oral cancer. The model consists of primary cell lines from tumor and more particularly from the nodal stroma patients of oral squamous cell carcinoma (OSCC) with and without nodal metastasis.
[004] Related art
[005] Oral cancer refers to the broad term for cancer that affects the inside of your mouth.
[006] The differentially expressed genes are genes that show significantly altered expression levels between different experimental conditions or groups. Differently engineered genes are identified through the comparison of gene expression profiles obtained from different samples or experimental conditions, such as comparing gene expression in cancer cells versus normal cells, treated versus untreated cells, or diseased tissue versus healthy tissue.
[007] Nodal metastasis is the most significant prognosticator in oral cancer. The management of nodal metastasis is a major challenge due to the delayed diagnosis, occult metastasis, and comparatively low sensitivity of pre-operative detection/prognostic methods.
[008] This is further accentuated due to mechanistic/conceptual gaps in knowledge attributed to the lack of comprehensive understanding of metastasis-induced changes in the nodal stroma and tumor-stroma interactions.
[009] Despite advancements and strategies in the preclinical In-vitro models, the lymph node stroma-tumor models are not explored much.
[0010] The stromal cells will prove to be an effective tool in studying the various aspects of pre-metastatic niche and the metastatic conditioning provided by the LNSCs to the tumor cells in the lymph node.
[0011] The indication of occult metastasis and over treatment is illustrated in FIG. 1(A) shows the occurrence of nodal metastasis is identified in ~50% of patients and ~30% among them have occult metastasis. The current treatment mandates neck dissection irrespective of the neck status.
[0012] Therefore, there is an urgent need for system and method for modelling nodal metastasis, one of the most significant prognosticators of oral cancer.
SUMMARY OF THE DISCLOSED EMMODIEMNT
[0013] According to an exemplary aspect of the present disclosure, an In-vitro system for modelling nodal metastasis, the system comprising of primary cultures from lymph nodes and tumor with and without nodal metastasis, one or more computer processors, one or more computer-readable storage media, and program instructions for execution by at least one of the one or more processors, wherein the system comprises of;
[0014] a) lymph node stromal cells (LNSCs) from N0nm, N0m, N+; epithelial primary cultures from tumors with nodal metastasis; CAF cultures from tumors with nodal metastasis;
co-culture combinations of lymph node stromal cells (LNSCs) from N0nm, N0m, N+; epithelial primary cultures from tumors with nodal metastasis; CAF cultures from tumors with nodal metastasis;
[0015] b) a model for characterizing nodal metastasis using co-culture combinations employing molecular-phenotyping, immune-phenotyping, STR profiling, cellular assays and molecular profiling;
[0016] c) a machine learning (ML) model, combining the molecular, cellular assay parameters of the different primary cultures and the co-cultures both pre and post treatment to assess the response to a drug or to assess the toxicity of a drug.
[0017] According to yet another aspect of the present disclosure, the In vitro system of the model is a regression or a non-regression model, wherein the model enables characterization of tumor-nodal cross talk by using multiple co-culture combinations.
[0018] According to one more aspect of the present disclosure, the In vitro system of the co-culture combinations are,
[0019] a. LNSCs N+ vs N0m; LNSCs N+ vs N0nm; LNSCs N0m vs N0nm,
[0020] b. Tumor cultures + N0nm/N0m/N+; Tumor cultures + CAF cultures +N0nm/N0m/N+.
[0021] According to another aspect of the present disclosure, the In vitro system for drug testing in nodal metastasis, system comprises of,
[0022] a. molecular profiling of cells comprising assaying for cellular invasion, migration, and spheroid assay;
[0023] b. characterizing cells;
[0024] c. cellular features of the cells;
[0025] d. modelling drug testing output such as evaluating drug efficacy and drug toxicity;
[0026] e. modelling research output such as characterizing molecular changes of cross-talk; analysing the effect of inhibiting the specific targets on the cross-talk.
[0027] According to another aspect of the present disclosure, the In vitro system cross talk is assessed co-culturing combinations of lymph node stromal cells (LNSCs) from N0nm, N0m, N+; epithelial primary cultures from tumors with nodal metastasis; CAF cultures from tumors with nodal metastasis.
[0028] According to an exemplary aspect of the present disclosure, a method for assessing/characterizing nodal metastasis, wherein the method comprises of;
[0029] d) establishing of primary lymph node stromal cells;
[0030] e) characterizing the primary cells morphology;
[0031] f) immunophenotyping and STR profiling;
[0032] g) establishing co-culture combinations of lymph node stromal cells (LNSCs) from N0nm, N0m, N+; epithelial primary cultures from tumors with nodal metastasis; CAF cultures from tumors with nodal metastasis;
[0033] h) profiling GP38 expression and growth kinetics by flow cytometry;
[0034] i) analyzing flow cytometry indicating heterogeneity between the cultures;
[0035] j) analyzing transcriptome of the stromal cultures from the metastatic and non-metastatic nodes;
[0036] k) employing transcriptomic profile of the pre metastatic nodes and from metastatic nodes;
[0037] l) profiling cytokine of the stromal cultures from the nodes; and
[0038] m) employing biomarkers panel for characterizing metastatic and non-metastatic nodes.
[0039] According to further aspect of the present disclosure, a method for evaluating drug efficacy/ toxicity, wherein the method comprises of;
[0040] a) establishing co-culture systems;
[0041] b) exposing co-cultures to a target drugs;
[0042] c) analysing cell death assays;
[0043] d) employing cellular assays;
[0044] e) quantifying molecular assays;
[0045] f) Profiling cytokines;
[0046] g) employing biomarkers panel for characterizing metastatic and non-metastatic nodes.
[0047] h) providing input data to a model; and
[0048] i) assessing efficacy of the drugs.
[0049] According to further aspect of the present disclosure, A method for characterizing nodal metastasis, wherein the method comprises of;
[0050] a) establishing co-culture systems;
[0051] b) exposing co-cultures to a target/ inhibitor;
[0052] c) analysing cell death assays;
[0053] d) employing cellular assays;
[0054] e) quantifying molecular assays;
[0055] f) Profiling cytokines;
[0056] g) providing input data to a model;
[0057] h) employing biomarkers/ inhibitors for characterizing metastatic and non-metastatic nodes;
[0058] i) assessing the mechanisms of the cross-talk between the cultures; and
[0059] j) identifying new targets by inhibitory studies.
[0060] According to further aspect of the present disclosure, the composition comprises of co-cultured combinations of lymph node stromal cells (LNSCs) from N0, N0m, Nm; epithelial primary cultures from tumors with nodal metastasis; CAF cultures from tumors with nodal metastasis; biomarkers from Table-3; a detection agent that binds to the biomarker products; and at least one reagent that inhibits the biomarker expression product in a biological sample.
[0061] According to further aspect of the present disclosure, primary cultures were labelled novel with a STR score filter of 80% for novelty; wherein the preferable biomarkers are CCL11, GPR4, EDNRB, CSF3, TRIM55, EYA4, ZP1, EPHB6, CABP1, TDRD9; wherein condition is lymph node metastasis; wherein the biological sample is a metastatic and non-metastatic primary lymph node stromal cell; wherein the composition can identify the differentially expressed genes in cell; wherein the composition can differentiate metastatic and non-metastatic lymph node with sensitivity and specificity.
[0062] According to further aspect of the present disclosure, the lymph nodes metastatic cells illustrated a doubling time at an average of 25.6 ± 1.0 hours, wherein the non-metastatic illustrated a doubling time at an average of 32.7 ± 6.4 hours.
[0063] According to further aspect of the present disclosure, the composition can identify differentially expressed genes (DEG) in N+/N0m, N+/N0nm and N0m/N0nm accordingly to Table-3; wherein the top 20 DEGs that differentiated the N+ from N0nm groups included C2orf72, KRT6A, HAPLN1 as top three upregulated genes and USP9Y, NLGN4Y, & CXCL10 as downregulated genes; wherein 7 genes, INHBE, ZP1, PCDH10, HOXA10, TSHZ2, CPM, and HAPLN1 were deregulated in the N+ nodes (N0m& N+) in comparison with the negative nodes; wherein the top 20 DEGs differentiated the two groups included HAPLN1, NPY4R, FOXG1 and top three upregulated genes and USP9Y, KDM5D, & DDX3Y as top 3 downregulated genes.
[0064] Several aspects of the disclosed embodiment are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the disclosed embodiment 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 disclosed embodiment. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] Example embodiments of the disclosed embodiment will be described with reference to the accompanying drawings briefly described below.
[0066] FIG. 1(A) illustrates the indication of occult metastasis and over treatment, according to aspect of present invention. Occurrence of nodal metastasis is identified in ~50% of patients and ~30% among them have occult metastasis. The current treatment mandates neck dissection irrespective of the neck status.
[0067] FIG. 1(B) illustrates the schematic representation of workflow of establishment and isolation of patient-derived oral cancer cells using explant based, and clonal ring extraction techniques, according to the aspects of present disclosure.
[0068] FIG. 2(A) illustrates the characteristics of the primary cells, morphology - The primary LNSCs from the N0nm groups, according to the aspects of present disclosure.
[0069] FIG. 2(B) illustrates the characteristics of the primary cells, morphology - The primary LNSCs from the N0m groups, according to the aspects of present disclosure.
[0070] FIG. 2(C) illustrates the characteristics of the primary cells, morphology - The primary LNSCs from the N+ groups, according to aspect of the present disclosure.
[0071] FIG. 2(D) illustrates the cells grouped into N0nm& N0m show morphology close to fibroblasts with stellate, reticular and fusiform shapes, according to aspect of the present disclosure.
[0072] FIG. 3(A) illustrates the molecular phenotyping by immune cytochemistry. These six primary cell lines, grouped into N0nm (Ln2a, Ln6a), N0m (Ln5b, Ln9b) and N+ (Ln5a, Ln9a), showed positive staining for Vimentin and Gp38, according to aspect of the present disclosure.
[0073] FIG. 3(B) illustrates the immune cytochemistry shows lack of CD31, panCK and CD45 expression in N0nm (Ln2a, Ln6a), N0m (Ln5b, Ln9b) and N+ (Ln5a, Ln9a) cells, according to aspect of the present disclosure.
[0074] FIG. 4 illustrates schematic representation of characterization of the patient-derived oral cancer epithelial/ fibroblast cells by flow cytometer-based experiments, according to aspect of present disclosure.
[0075] FIG. 5 illustrates characterization of primary OSCC cells epithelial and fibroblast phenotypes by immunocytochemistry, and flow cytometer analysis: (A) Flow cytometry analysis of established patient-derived cells. Epithelial cells display positivity for PanCK, and fibroblast cells display positivity for FSP1. (B) Tabulation of percentage positivity of patient-derived epithelial and fibroblast cells. (C) Epithelial patient-derived cancer cells of MhCB01, MhCB03, and MhCB04 samples stained with PanCK with bright field and secondary controls. (D) Epithelial patient-derived cancer cells of MhCB01, MhCB03, and MhCB04 samples stained with aSMA with bright field and secondary controls, according to aspect of the present disclosure.
[0076] FIG. 6(A) illustrates the growth kinetics. The growth kinetics for the six cells lines grouped into N0nm (Ln2a, Ln6a), N0m (Ln5b, Ln9b) and N+ (Ln5a, Ln9a) showed an initial lag phase followed by exponential phase, according to aspect of the present disclosure.
[0077] FIG. 6(B) illustrates the growth kinetics. The doubling time was calculated for the N0nm, N0m& N+ group of cells for every 24 hours and the mean doubling time was plotted on the graph, according to aspect of the present disclosure.
[0078] FIG. 6(C) illustrates the Gp38 expression profiling by flow cytometry. The flow cytometry analysis was done for detecting the Gp38 expression for the six primary cells grouped into N0nm (Ln2a, Ln6a), N0m (Ln5b, Ln9b) and N+ (Ln5a, Ln9a) and percentage positivity was determined against the unstained control, according to aspect of the present disclosure.
[0079] FIG. 6(D) illustrates the Gp38 expressions profiling by flow cytometry. The experiments were done in triplicates and the mean percentage positivity was estimated, according to aspect of the present disclosure.
[0080] FIG. 7(A) illustrates the molecular alteration in the N0nm, N0m& N+ groups. The expression heatmap was plotted using the differentially expressed genes (DEGs) identified by the deseq2 package in R, according to aspect of the present disclosure.
[0081] FIG. 7(B) illustrates the molecular alteration in the N0nm, N0m& N+ groups. The enriched pathways were identified using cluster profiler and represented with dot plots for N+ vs N0nm, according to aspect of the present disclosure.
[0082] FIG. 7(C) illustrates the molecular alteration in the N0nm, N0m& N+ groups - N+ vs N0nm. The cnetplots depicts the biological complexity of the downregulated molecules for N+ vs N0nm, according to aspect of the present disclosure.
[0083] FIG. 7(D) illustrates the molecular alteration in the N0nm, N0m& N+ groups. The enriched pathways were identified using cluster profiler and represented with dotplots for N+ vs N0m, according to aspect of the present disclosure.
[0084] FIG. 7(E) illustrates the molecular alteration in the N0nm, N0m& N+ groups. The cnetplots depicts the biological complexity of the downregulated molecules for N+ vs N0m, according to aspect of the present disclosure.
[0085] FIG. 7(F) illustrates the molecular alteration in the N0nm, N0m& N+ groups. The enriched pathways were identified using cluster profiler and represented with dotplots for N0m vs N0nm, according to aspect of the present disclosure.
[0086] FIG. 7(G) illustrates the molecular alteration in the N0nm, N0m& N+ groups. The cnetplots depicts the biological complexity of the upregulated molecules for N0m vs N0nm, according to aspect of the present disclosure.
[0087] FIG. 8(A) illustrates cytokines secreted by the cells grouped into N0nm (Ln2a, Ln6a), N0m (Ln5b, Ln9b) and N+ (Ln5a, Ln9a) are indicated by the dot blot of the cytokine array. Each blot contains three reference spots and a negative control in duplicates, according to aspect of the present disclosure.
[0088] FIG. 8(B) illustrates the heatmap that is generated for the cytokines with differential profile across the three groups N0nm, N0m& N+, according to aspect of the present disclosure.
[0089] FIG. 8(C) illustrates the bar graphs which represent the fold difference of the significant cytokines over expressed by the metastatic nodes in comparison to the non-metastatic nodes from the N0nm, N0m groups include MIF, IGFBP-3 & Cystatin C, according to aspect of the present disclosure.
[0090] FIG. 8(D) illustrates the bar graphs represents the fold difference of the significant cytokines by the metastatic nodes where pentraxin 3 and angiogenin showed significant expression difference in the N0m and N+ groups, according to aspect of the present disclosure.
[0091] FIG. 8(E) illustrates the bar graphs represents the fold difference of the significant cytokines by the metastatic nodes where IGFBP2 and leptin showed significant difference in expression in comparison to the N0m and N+ groups, according to aspect of the present disclosure.
[0092] FIG. 8(F) illustrates the bar graphs represents the fold difference of the significant cytokines by the metastatic nodes where DKK1 and CD26 expression show significant difference between the N0nm and N0m groups whereas IL-8 expression is significantly downregulated in the N+ group when compared to N0nm, according to aspect of the present disclosure.
[0093] FIG. 9 illustrates the design for the tumor-LNSC cross talk, according to aspect of the present disclosure.
[0094] FIG. 10(A), 10(B), 10(C) illustrates the Tumor-LN co-culture. This shows the effect of change in proliferation of HSC3 with 24, 48, 72 hrs of treatment with conditioned media from Ln9a, Ln9b, Ln6a with the different ratios (1:1, 3:7 and neat), according to aspect of the present disclosure.
[0095] FIG. 10(D), 10(E), 10(F) illustrates the Tumor-LN co-culture. This shows the effect of change in proliferation of CAL27with 24, 48, and 72 hrs of treatment with conditioned media from Ln9a, Ln9b, Ln6a with the different ratios (1:1, 3:7 and neat), according to aspect of the present disclosure.
[0096] FIG. 11(A), 11(B), 11(C) illustrates the proliferation assay of HSC3 treatment with conditioned media from Ln5a, Ln5b, Ln2a with the different ratios (1:1, 3:7 and neat), according to aspect of the present disclosure.
[0097] FIG. 11(D), 11(E), 11(F) illustrates the proliferation assay of CAL27treatment with conditioned media from Ln5a, Ln5b, Ln2awith the different ratios (1:1, 3:7 and neat), according to aspect of the present disclosure.
[0098] FIG. 12 illustrates the GO terms mapped to reactome. The upregulated genes in the CAL27 treated non-metastatic LNSC mapped to IL-10 signaling, ECM organization and other interleukin signaling among others, according to aspect of the present disclosure.
[0099] FIG. 13 illustrates the GO terms mapped to reactome. The upregulated genes in the HSC3 treated non-metastatic LNSC mapped to collagen formation, ECM organization and interleukin signaling, according to aspect of the present disclosure.
[00100] FIG. 14 illustrates the significant enrichment plots from the GSEA analysis. The corresponding NES score and the FDR q values are also indicated. N0m/N0nm in FIG. 14(A), N+/N0m FIG. 14(B), N+/N0nm, FIG. 14(C), according to aspect of the present disclosure.
[00101] FIG. 15 illustrates Oral cancer patient-derived cells cultured in 2D platform display differential response to chemotherapeutic drugs. The chemo-response of patient-derived MhCB03-E cells incubated with various concentrations of chemotherapeutic drugs such as cisplatin (A), and 5-FU (B). The percentage cell viability was monitored using MTT assays after a 72 hours’ incubation period. The percentage cell viability was represented by heat-maps at various drug concentrations, according to aspect of the present according to aspect of the present disclosure.
[00102] FIG. 16 illustrates Patient-derived OSCC cells cultured on 2D platform display differential response to different small molecule inhibitors. The response of patient-derived cells (MhCB03-E) incubated with various concentrations of SMIs such as ALDH1A1 inhibitor (A), Notch1 inhibitor (B), PLD2 inhibitor (C), and mTOR inhibitor (D). The percentage cell viability was monitored using MTT assays after a 72 hours’ incubation period. The percentage cell viability was represented by heat-maps at various drug concentrations, according to aspect of the present disclosure.
[00103] FIG. 17 illustrates MhCb03-E patient-derived OSCC cells cultured on 2D platform display differential response to combinatorial therapy. The response of P03 patient-derived cells incubated with SMIs and various concentrations of cisplatin resulted in increased efficacy of chemotherapeutic agent (A, B, C, D). The response of P03 patient-derived cells incubated with PLD2 inhibitor and various concentrations of mTOR inhibitor resulted in increased efficacy of chemotherapeutic agent (E, F). The percentage cell viability was monitored using MTT assays after a 72 hours incubation period. The percentage cell viability was represented by heat-maps at various drug concentrations, according to aspect of the present disclosure.
[00104] FIG. 18 is a block diagram illustrating the details of a digital processing system in which various aspects of the present invention are operative by execution of appropriate execution modules.
[00105] 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.
DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENT
[00106] 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.
[00107] 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 items. 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.
[00108] 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.
[00109] 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.
[00110] 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.
[00111] Example embodiments of the disclosed embodiment are described with reference to the accompanying figures.
[00112] 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.
[00113] DEFINITION
[00114] The term “Explant based” refers to techniques or studies that involve the use of explants.
[00115] The term “Clonal ring extraction technique” refers to a technique used in cell culture to isolate and propagate individual clones of cells.
[00116] The term “Gene ontology (GO)” refers to a framework for the model of biology that represents gene functions and their relationships across different species.
[00117] The term “Reactome” refers to a free, open-source, curated database of biological pathways and reactions.
[00118] The term “SMIS” stands for self-micro emulsifying drug delivery system.
[00119] The term “ALDH 1A1” refers to aldehyde dehydrogenase 1A1 enzyme that plays a crucial role in the oxidation of aldehydes to their corresponding carboxylic acids.
[00120] The term “LNSC” stands for lymph node stromal cells.
[00121] The term “Metastatic” refers to a cancer that has spread from its original site to other parts of the body.
[00122] The term “non-metastatic” refers to cancer that has not spread beyond its original (primary) site.
[00123] The term “Primary tumor tissue” refers to the original tissue from which a cancer originates.
[00124] The term “Lymph node” refers to a small, bean-shaped structures that are part of the lymphatic system.
[00125] The term “Immune phenotypes” refers to the process of identifying and characterizing the various types of immune cells present in a sample, typically using specific.
[00126] The term “Immunocytochemistry” refers to a laboratory technique used to visualize and analyze specific proteins or antigens within cells using antibodies tagged with detectable markers.
[00127] The term “N0nm” (N0 node from non-metastatic patients), “N0m” (N0 node from metastatic patients) and “N+” (Metastatic node from metastatic patients).
[00128] The term “Nodal metastasis” refers to the spread of cancer from its primary site to the lymph nodes. This can indicate a more advanced stage of cancer and may affect treatment options and prognosis.
[00129] The term “Short tandem repeat (STR) profiling” is a molecular biology method used to analyze specific regions of DNA that contain short, repeating sequence of base pairs.
[00130] The term “Sensitivity” refers to the ability of a test to correctly identify individuals with the disease.
[00131] The term “Specificity” refers to the ability of a test to correctly identify individuals who do not have the disease.
[00132] The term “Regression” refers to a reduction in the size of a tumor or the extent of cancer in the body.
[00133] The term “non-regression” refers to the situation where a tumor doe not decrease in size or the extent of cancer does not diminish despite treatment.
[00134] The term “Cross-talk” refers to the complex interactions between different signaling pathways, cells, and components within the tumor microenvironment that can influence cancer progression, treatment resistance, and metastasis.
[00135] The term “Cancer-associated fibroblasts (CAFs)” refers to a major component of the tumor microenvironment and -play a crucial role in cancer progression.
[00136] The term “Chemokine ligand 11 (CCL11)” refers to as eotaxin-1, belongs to the CC chemokine subfamily.
[00137] The term “G protein-coupled receptor 4 (GPR4)” refers to a receptor that plays a role in sensing changes in pH (acidosis) and is highly expressed in vascular endothelial cells.
[00138] The term “Endothelin Receptor Type B (EDNRB)” refers to a gene that encodes a protein that functions as a G protein-coupled receptor. Its ligand, endothelin, consists of a family of three potent vasoactive peptides: ET1, ET2, and ET3.
[00139] The term “Colony-Stimulating Factor 3 (CSF3)” refers to an important regulator in cancer, particularly colorectal cancer (CRC).
[00140] The term “Tripartite Motif Containing 55 (TRIM55)” refers to a gene that encodes a protein that contains a RING zinc finger motif, which is known to be involved in protein-protein interactions.
[00141] The term “Eyes Absent 4 (EAY4)” refers to a unique family of dual-functioning protein phosphatases (PP) known as the Eyes Absent (EYA) family. EYA proteins possess both transcriptional activation and phosphatase functions, with serine/threonine and tyrosine phosphatase domains.
[00142] The term “Zona Pellucida Glycoprotein 1 (ZP1)” refers to reproduction, and its expression patterns in cancer warrant investigation.
[00143] The term “Ephrin Type-B Receptor 6 (EPHB6)” refers to a gene that encodes a protein that belongs to the Eph receptor family. Eph receptors are the largest family of receptor tyrosine kinases (RTKs) and play diverse roles in various processes, including cancer development and progression.
[00144] The term “Calcium Binding Protein 1 (CABP1)” refers to a gene that encodes a protein that belongs to a subfamily of calcium binding proteins, sharing similarity to calmodulin.
[00145] The term “Tudor Domain Containing Protein 9 (TDRD9)” refers to a gene that encodes a protein that normally functions in the germline, where it is involved in the biosynthesis of PIWI-interacting RNAs (piRNAs).
[00146] The term machine learning (ML) model means A machine learning model is a program that can find patterns or make decisions from a previously unseen dataset.
[00147] A. EMBODIMENTS OF THE DISCLOSURE
[00148] The present invention relates to a system and method for In-vitro modelling of nodal metastasis, one of the most significant prognosticators of oral cancer. The model consists of primary cell lines from tumor and more particularly from the nodal stroma from patients of oral squamous cell carcinoma (OSCC) with and without nodal metastasis.
[00149] The present invention is a process to model nodal metastasis and to identify the differentially expressed genes that causes nodal metastasis in oral cancer. The invention also presents an invitro model combining lymph node stromal cells and primary tumor cells as a system to characterize nodal metastasis.
[00150] I. ESTABLISHMENT OF CULTURES
[00151] The samples were collected from eighteen patients and cultures were established and characterized from 11 patients which is roughly estimated to be 61% for reproducible isolation of cells from tumor/node tissues. Inventors have evaluated multiple methods for establishing the primary cells including explant culture and enzymatic digestion. Enzymatic digestion included cold trypsinization which yielded lower cell number after digestion leading to longer times for establishing primary cells. In contrast, explant culture was comparatively faster and reliable process to establish cultures.
[00152] ESTABLISHMENT OF PRIMARY LYMPH NODE STROMAL CELLS
[00153] In the present disclosure, the inventors established a reproducible protocol for the development of lymph node stromal cells (LNSCs) using the explant culture technique.
[00154] The metastatic status of the nodes was confirmed after histopathological evaluation.
[00155] Sixteen lymph node tissues from 9 OSCC patients including metastatic (n=3; P5, P8, P9) and non-metastatic (n=4; P1, P2, P6, P7), were collected and nodes from 2 patients were discarded due to necrosis.
[00156] The average age of the 7 patients included in the study was 57.5±2.8 years, 5 among them being males (Table 1) and 6/7 patients having risk habits.
[00157] Among the metastatic subset, positive and negative nodes were collected from two of them (P5 & P9), while both the nodes were non-metastatic in P8.
[00158] Two nodes each were collected from the 4 non-metastatic patients (P1, P2, P6 & P7).
[00159] ISOLATION OF PATIENT-DERIVED PRIMARY EPITHELIAL AND FIBROBLAST CELLS:
[00160] We’ve collected nine primary tumor tissue from OSCC patients, of which four patient samples have been successfully established.
[00161] The primary oral cancer samples were collected with the informed consent form from patients at MSMC. All the procedures in accordance with recommended guidelines and approval of the NHMEC, Bengaluru, India. Tumor samples were collected in sterile DMEM-F12 supplemented with 3X Penicillin-Streptomycin solution, and 20%FBS. The clinical details of the patients are mentioned in Table 1.
[00162] Primary cultures were established from primary oral cancer patient samples using explant culture method (FIG. 1B). The epithelial and fibroblast cells grew as a monolayer, identified based on their morphology (FIG. 1B). The epithelial and fibroblast cultures were isolated from the explant plate by using the clonal rings extraction method and were cultured separately thereafter (FIG. 1B).
[00163] TABLE 1: Patient demographics, pathologic diagnosis and cell line nomenclature. Abbreviations: BM- Buccal Mucosa, T – Tongue, SCC – Squamous cell carcinoma, ECS- Extracapsular Spread. Chewing indicates tobacco usage. Tumor Grade: ‘G1’ – Well differentiated, ‘G2’ – Moderately differentiated, ‘G3’ – Poorly differentiated.
[00164] Primary cultures were established from all the nodes using explant culture and cells grew as a monolayer with heterogeneous morphology including reticular-shaped, fusiform structures containing long extensions and stellate structures (FIG. 2(A), 2(B), 2(C), 2(D)).
[00165] The cells were cultured to 90% confluence, were expanded, and sub-cultured till passages of 10-20.
[00166] In an embodiment, the official nomenclatures for these cells were devised as per the institutional guidelines where the letters signify the following: ‘M’ – Mazumdar Shaw Medical Foundation, ‘h’ – human, ‘C’ - Cancer, ‘Ln’ – Lymph node (Table 1) ‘B’ – Buccal mucosa, ‘T’ - Tongue.
[00167] TABLE 1: Patient demographics, pathologic diagnosis and cell line nomenclature.
S. No Cell line code Cell culture identifier Patient Code Tissue type Age/ Gender Risk habits Previous cancer history Tumor site Histologic type of tumor Pathologic stage Tumor Grade WPOI LVI PNI No of lymph nodes examined Metastatic status of the node in culture ECS status of the node in culture ECS status of patient
1 Ln1a MhCLn1a P1 Lymph node 62/M Chewing, Alcohol No Buccal Mucosa SCC T2N0 G1 1-4 No No 0/32 NM Negative Negative
2 Ln1b MhCLn1b NM
3 Ln2a MhCLn2a P2 Lymph node 61/M Smoking, Chewing No Buccal Mucosa SCC T3N0 G2 NA No No 0/52 NM Negative Negative
4 Ln2b MhCLn2b NM
5 Ln3* MhCLn3 P3 Lymph node 53/M Smoking No Tongue SCC T4aN3b G2/3 5 No No 15/62 M Positive Positive
6 Ln4* MhCLn4 P4 Lymph node 38/F No habits No Buccal Mucosa SCC T2N2a G2/3 5 No No 1/42 NM Positive Positive
7 Ln5a MhCLn5a P5 Lymph node 56/F Chewing Carcinoma of Buccal mucosa pT1N0 in 2017 Buccal Mucosa SCC T4bN3b G2 1-4 No No 2/36 M Positive Positive
8 Ln5b MhCLn5b NM Negative
9 Ln6a MhCLn6a P6 Lymph node 42/M Smoking No Tongue SCC T4aN0 G1/2 5 No Yes 0/96 NM Negative Negative
10 Ln6b MhCLn6b NM
11 Ln7a MhCLn7a P7 Lymph node 56/M Chewing, Alcohol No Buccal Mucosa Verrucous carcinoma T2N0 G1 NA No No 0/32 NM Negative Negative
12 Ln7b MhCLn7b NM
13 Ln8a MhCLn8a P8 Lymph node 63/M Chewing No Buccal Mucosa SCC T2N1 G2 1-4 Yes No 1/25 NM Negative Negative
14 Ln8b MhCLn8b NM
15 Ln9a MhCLn9a P9 Lymph node 63/F No habits No Tongue SCC T3N3b G2 5 No Yes 2/47 M Positive Positive
16 Ln9b MhCLn9b NM Negative
17 P01Epi MhCB01E P10 Tumor 54/F Tobacco Chewing No Right BM SCC pT2pN3b G2 5 No Yes 5/55 - - Positive
18 P01Fibro MhCB01F - -
19 P02Epi MhCT02E P11 Tumor 40/M Smoking No Tongue SCC pT3pN3b G2/3 5 No Yes 5/85 - - Positive
20 P02Fibro MhCT02F - -
21 P03Epi MhCB03E P12 Tumor 53/M Smoking No Right lower alveolus SCC pT3pN3b G2 4 No No 5/50 Positive
22 P03Fibro MhCB03F
23 P04Epi MhCB04E P13 Tumor 39/F Areca nut chewing No Right BM SCC pT4apN0 G1 3 No No 0/48 Negative
24 P04Fibro MhCB04F

[00168] II CHARACTERISTICS OF THE PRIMARY CELLS – MORPHOLOGY
[00169] The FIG 2(A), 2(B), 2(C) illustrate characteristics of the primary cells – morphology.
[00170] The characteristics of the primary cell’s morphology in the primary LNSCs from the N0nm groups of cell line code Ln2a (202), and in the primary LNSCs from the N0nm groups of cell line code Ln6a (204). FIG. 2(A).
[00171] The characteristics of the primary cell’s morphology & growth kinetics in the primary LNSCs from the N0m groups of cell line code Ln5b (206) and in the primary LNSCs from the N0m groups of cell line code Ln9b (208). FIG. 2(B).
[00172] The characteristics of the primary cells – morphology & growth kinetics in the primary LNSCs from the N+ groups of cell line code Ln5a (210) and in the primary LNSCs from the N+ groups of cell line code Ln9a (212). FIG. 2(C).
[00173] The cells grouped into N0nm& N0m show morphology close to fibroblasts with stellate, reticular and fusiform shapes. FIG. 2(D).
[00174] The cells grouped into N0nm show morphology close to fibroblasts with stellate, reticular and fusiform shapes of cell line code Ln1a (214), Ln1b (216), Ln2b (218), Ln6b (220), Ln7a (222), and Ln7b (224).
[00175] The cells grouped into N0m show morphology close to fibroblasts with stellate, reticular and fusiform shapes of cell line code Ln8a (226), and Ln8b (228). FIG. 2(D).
[00176] Negative node from non-metastatic patient and Negative node from metastatic patient, Ln1a, Ln1b, Ln2b, Ln6b, Ln7a, Ln7b, Ln8a, & Ln8b represents primary cell lines.
[00177] The lymph node explants were cultured for approximately 4-6 weeks, trypsin zed, expanded, and frozen at the early passages (passage 1-10).
[00178] The H&E staining was done on cells cultured on coverslips overnight. The cells showed varied morphology with stellate, reticular and fusiform shaped cells.
[00179] Cultures developed from these lymph nodes tissues were grouped into three: N0nm (N0 node from non-metastatic patients), N0m (N0 node from metastatic patients) & N+ (Metastatic node from metastatic patients). H&E staining of the cells indicated heterogeneous cell populations as well as increased dendritic morphology in the N0m and N+ groups (FIG. 2(A), 2(B), 2(C)).
[00180] III. IMMUNOPHENOTYPING BY ICC REVEALED A HETEROGENEOUS CELL POPULATION
[00181] In an embodiment, LNSCs are heterogeneous cells majorly containing four cell types with specific marker patterns: Fibroblastic reticular cells (FRCs, CD31-gp38+), double negative cells (DNCs, CD31-gp38-), lymphatic endothelial cells (LECs, CD31+gp38-) and blood endothelial cells (BECs, CD31+gp38+).
[00182] The cells were immune phenotyped by ICC for these markers. All cells in the cultures developed were panCK-/CD45-/Vimentin+ establishing that these cells were of mesenchymal origin and lack epithelial and immune cells (FIG. 3(A)&3(B)).
[00183] The cell types identified in the cultures include FRCs (gp38+/CD31-) and DNCs (gp38-/CD31-). None of the cultures were CD31+ indicating the absence of lymphatic as well as blood endothelial cell populations (FIG. 3(A) & 3(B)).
[00184] IV. MOLECULAR PHENOTYPING BY IMMUNOCYTOCHEMISTRY
[00185] The molecular phenotyping by immunocytochemistry, the six primary cell lines grouped into N0nm Ln2a (302), Ln6a (304), N0m Ln5b (306), Ln9b (308) and N+ Ln5a (310), Ln9a (312), showed positive staining for Vimentin and Gp38.
[00186] The negative control represents no antibody staining indicating lack of non-specific staining by the secondary detection system. The images were taken at 10X magnification and scale bar represents 100µm.
[00187] In an embodiment, the immune cytochemistry shows lack of CD31, panCK and CD45 expression in N0nm Ln2a (314), Ln6a (316), N0m Ln5b (318), Ln9b (320) and N+ Ln5a (322), Ln9a (324) cells in FIG. 3(B).
[00188] The primary OSCC cells isolated were characterized for their purity for epithelial and fibroblast lineage. Specific markers were used to define the lineage of the patient-derived oral cancer cells. Inventors performed immunocytochemistry, and flow cytometer analysis using Pan-Cytokeratin (PanCK) as a marker for epithelial cells, and alpha Smooth Muscle Actin (aSMA), or fibroblast specific Fibroblast specific protein-1/S100A4 (FSP-1) as a marker for fibroblast cells.
[00189] The patient-derived that were able to differentiate as epithelial and fibroblastic lineage based on the morphology were quantified using flow cytometer analysis (FIG. 4).
[00190] The patient-derived epithelial and fibroblast OSCC cells were probed with anti-PanCK-APC to track epithelial cells, and anti-FSP1-Alexa488 to track fibroblast cells. The antibodies were used as per the dilutions instructed by the manufacturer. Inventors observed that the majority of cultures were stained more than 90% positive for respective markers (MhCT02-E, MhCT02-F, MhCB03-E, and MhCB04-F cells, were stained with PanCK for epithelial, and FSP1 for fibroblasts) (FIG. 4A, 4B). MhCB03-F, and MhCB04-E showed purity with >80% positive staining for respective markers (FIG. 4A, 4B).
[00191] Further, the patient-derived epithelial and fibroblast OSCC cells were subjected to immunocytochemistry. The above cells were probed with anti-PanCK to track epithelial cells, and anti- aSMA to track fibroblast cells. Majority of MhCB01-E, MhCB03-E, MhCB04-E cells were stained positive for PanCK (FIG. 5C, 5D). Whereas, MhCB01-F, MhCB03-F, MhCB04-F cells were stained positive for aSMA (FIG. 5C, 5D).
[00192] V. STR PROFILING:
[00193] Short Tandem Repeat (STR) profiling is a technique used to authenticate and characterize human cell lines. It involves analyzing specific regions of DNA that contain repetitive sequences (short tandem repeats). STR profiling includes PCR-amplified variable microsatellite regions from a genomic DNA template, separating the PCR amplicons on a genetic analyzer, and analyze the resulting data and compare the data from one specimen to databases housing previously generated STR sets. Repetitive DNA sequences with varying numbers of repeats, referred to as STR loci, are amplified and they are unique to each individual.
[00194] This information evaluates if two given cell lines are annotated as related and are thus expected to have similar STR profiles. A similarity score less than 80% for the STR marker is indicative of the novelty of the new cells as compared against the existing cell lines in the database.
[00195] VI. CHARACTERIZATION OF THE CULTURES OF METASTATIC AND NON-METASTATIC ORIGIN
[00196] In an embodiment, six of these primary lymph node stromal cells grouped into N+ (Ln5a, Ln9a), N0m (Ln5b, Ln9b) & N0nm (Ln2a, Ln6a) were taken for further characterizations.
[00197] Growth curve analysis indicated that the six cultures showed an initial lag period followed by exponential growth.
[00198] The doubling time was calculated by evaluating the growth kinetics (FIG.6 (A), 6(B)).
[00199] The growth kinetics -The growth kinetics for the six cells lines grouped into N0nm (Ln2a, Ln6a), N0m (Ln5b, Ln9b) and N+ (Ln5a, Ln9a) showed an initial lag phase followed by exponential phase. The experiment was done in triplicates and the mean cell counts were used for plotting the curve is illustrated in FIG. 6(A).
[00200] The growth kinetics - The doubling time was calculated for the N0nm, N0m& N+ group of cells for every 24 hours and the mean doubling time was plotted on the graph is illustrated in FIG. 6(B).
[00201] The average doubling time of the metastatic cells (Ln5a, Ln9a) is 25.6 ± 1.0 hours whereas the cells from the four non-metastatic nodes double at an average of 32.7 ± 6.4 hours.
[00202] In an embodiment, STR profiling using 10 STR markers were carried out to establish a baseline profile for these six cells and to compare with the parent tumors (Table 2).
[00203] The STR profile for these cells were verified against the existing cell lines for minimum 8 markers with a score filter of 80% and was found to be novel identifiers.
[00204] TABLE 2: STR profile determined for these six cultures as well as the corresponding tumor tissues.
Cell line
nomenclature
or Patient
code TH01 D21S11 D5S818 D13S317 D7S820 D16S539 CSF1PO AMEL vWA TPOX
MhCLn2a 6,9 27,31.2 11,13 9,12 7,11 13 12 X,Y 18 8
P2 6,9 27,31.2 11,13 9,12 7,11 13 12 X,Y 18 8
MhCLn5a 6,7 29,32.2 11 8 9,10 10,14 12 X 16,17 8,11
MhCLn5b 6,7 29,32.2 11 8 9,10 10,14 12 X 16,17 8,11
P5 6,7 29,32.2 11 8 9,10 10,14 12 X 16,17 8,11
MhCLn9a 6,7 28,30 11,12 11,12 9,12 8,13 12,13 X 14,16 9,11
MhCLn9b 6,7 28,30 11,12 11,12 9,12 8,13 12,13 X 14,16 9,11
P9 6,7 28,30 11,12 11,12 9,12 8,13 12,13 X 14,16 9,11
MhCLn6a 7,9.3 31.2,33.2 11 8,10 11,12 10,13 11,12 X,Y 17,19 9,11
P6 7,9.3 31.2,33.2 11 8,10 11,12 10,13 11,12 X,Y 17,19 9,11

[00205] VII. GROWTH KINETICS AND GP38 EXPRESSION PROFILING BY FLOW CYTOMETRY
[00206] In an embodiment, FIG. 6(A), 6(B), 6(C), 6(D) illustrates the growth kinetics and gp38 expression profiling by flow cytometry.
[00207] VIII. FLOW CYTOMETRY ANALYSIS INDICATED DISTINCT HETEROGENEITY BETWEEN THE CULTURES
[00208] In embodiment, cultures were analyzed for Gp38 expression by flow cytometry to assess the proportion of FRCs and DNCs (FIG. 6(C)).
[00209] The Gp38 expression profiling by flow cytometry. The flow cytometry analysis was done for detecting the Gp38 expression for the six primary cells grouped into N0nm (Ln2a, Ln6a), N0m (Ln5b, Ln9b) and N+ (Ln5a, Ln9a) and percentage positivity was determined against the unstained control is illustrated in FIG. 6(C).
[00210] The Gp38 expression was varying among the cultures indicating heterogeneity.
[00211] Among the metastatic patients, the N+ and N0m groups showed an average Gp38 expression of 62.6±32.7% and 53.03±11.5% respectively.
[00212] The non-metastatic patients (N0nm group) showed 22±10.5% average Gp38 expression.
[00213] Among all these six cultures, Ln6a from the N0nm group showed the lowest Gp38 expression of 11.5±4.3% indicating a higher proportion of DNCs in it rather than FRCs (FIG. 6(D)). The Gp38 expressions profiling by flow cytometry. The experiments were done in triplicates and the mean percentage positivity was estimated was shown in FIG. 6(D).
[00214] EXAMPLE EMBODIMENT
[00215] 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.
[00216] EXAMPLE 1: TRANSCRIPTOME OF THE STROMAL CULTURES FROM THE METASTATIC/NON-METASTATIC NODES
[00217] In an embodiment the differential gene expression analysis was carried out between the three groups of cells from metastatic nodes (Group N+: Ln5a, Ln9a), non-metastatic nodes (Group N0m: Ln5b, Ln9b) from N+ patients & non-metastatic nodes from N0 patients (Group N0nm: Ln2a, Ln6a).
[00218] The heatmap shows distinct expression of the DEGs between N0nm and N0m group whereas the N+ group show mixed expressions compared to either group.
[00219] Clustering also showed the N0m and N+ groups were closer to each other in comparison to N0nm group.
[00220] In an embodiment FIG. 7(A), 7(B), 7(C), 7(D), 7(E), 7(F), 7(G) illustrate molecular alteration in the N0nm, N0m& N+ groups.
[00221] The molecular alteration in the N0nm, N0m & N+ groups- The expression heatmap was plotted using the frc identified by the deseq2 package in R was shown in FIG. 7(A).
[00222] Differential gene expression analysis was carried out between the three groups of cells. A cluster heatmap (FIG 5(A)) shows distinct expressions of the DEGs (N=155) leading to the nodes from metastatic patients (N0m, N+) forming a separate cluster as compared to the N0nm group. The major cluster in the heat map (n=124 genes) representing the gene subset downregulated in the stromal cultures from the nodes of the metastatic patients (N0m, N+) was enriched for interferon alpha/gamma response, KRAS signaling, EMT, complement system, inflammatory response and TNFa signaling via NF?B.
[00223] The molecular alteration in the N0nm, N0m& N+ groups - The enriched pathways were identified using cluster profiler and represented with dot plots for N+ vs N0nm was shown in FIG. 7(B).
[00224] The molecular alteration in the N0nm, N0m& N+ groups - N+ vs N0nm. The cnetplots depicts the biological complexity of the downregulated molecules for N+ vs N0nmwas shown in FIG. 7(C).
[00225] The molecular alteration in the N0nm, N0m& N+ groups- The enriched pathways were identified using cluster profiler and represented with dot plots for N+ vs. N0m. FIG. 7(D).
[00226] The molecular alteration in the N0nm, N0m& N+ groups- The cnetplots depicts the biological complexity of the downregulated molecules for N+ vs N0m. FIG. 7(E).
[00227] The molecular alteration in the N0nm, N0m& N+ groups- The enriched pathways were identified using cluster profiler and represented with dot plots for N0m vs N0nm. FIG. 7(F).
[00228] The molecular alteration in the N0nm, N0m& N+ groups- The cnetplots depicts the biological complexity of the upregulated molecules for N0m vs N0nm.
[00229] EXAMPLE 2: TRANSCRIPTOMIC PROFILE OF THE STROMAL CULTURES FROM METASTATIC NODES (N+)
[00230] Enrichment analysis of LNSCs from the metastatic nodes (N+)
[00231] E2F targets and KRAS signaling were positively enriched whereas oxidative phosphorylation and protein secretion were negatively enriched between N+ and N0mgroups of LNSCs. GSEA in comparison with the completely tumor-naive cultures (N0nm) identified hypoxia, G2M checkpoint, glycolysis among others as positively enriched in the metastatic LNSCs whereas interferon alpha response, interferon gamma response, oxidative phosphorylation among others were negatively enriched.
[00232] In an embodiment, the profile of the cultures from the metastatic nodes (N+), when compared to the non-metastatic nodes of the metastatic patients (N0m), identified 136 differentially expressed genes (DEGs; 96 upregulated; 40 downregulated).
[00233] Annotation of the differentials identified multiple pathways including collagen formation, assembly of collagen fibrils, collagen biosynthesis & modifying enzymes, and ECM organization among others, indicating an extensive remodeling in metastatic nodes (FIG. 7(B), 7(C)). The primary Gene Ontology (GO) terms included extracellular matrix organization, growth factor binding and external encapsulating structure organization.

[00234] TABLE 3: The top 20 differentially expressed genes (10 upregulated & 10 downregulated) across the different comparisons
N+/N0m N+/N0nm N0m/N0nm
Up Down Up Down Up Down
CCL11 KPRP C2orf72 USP9Y HAPLN1 USP9Y
GPR4 HOXA10 KRT6A NLGN4Y NPY4R KDM5D
EDNRB LCE2A HAPLN1 CXCL10 FOXG1 DDX3Y
CSF3 ACTC1 ZP1 SAMD5 HOXD4 NLGN4Y
TRIM55 PCDH10 CES1 OAS1 SYT15B OASL
EYA4 HHIP PADI2 KCNA1 LEP UTY
ZP1 PODXL KRT5 DGKB SCUBE3 ZFY
EPHB6 C11orf87 KRT17 HOXA10 CDK18 EDNRB
CABP1 ANKRD1 DPYSL4 NTSR1 HHIP RSAD2
TDRD9 HAPLN1 NCAM1 SEMA3E VAMP8 ADH1B

[00235] The top 20 DEGs that differentiated the two groups included top three upregulated genes as CCL11, GPR4, EDNRB, and top three downregulated genes as KPRP, HOXA10 & LCE2A (Table 3).
[00236] The differential analysis of the N+ stromal cultures in comparison to the cultures from the nodes from non-metastatic patients (N0nm) identified 85 DEGs (22 upregulated; 63 downregulated).
[00237] The GO analysis identified collagen-containing extracellular matrix, glycosaminoglycan binding and response/defence to virus among others.
[00238] The downregulated DEGs also mapped to pathways for interferon alpha/beta signaling and interferon signaling indicating possibility of inadequate immune profile (FIG. 7(D), 7(E)).
[00239] The top 20 DEGs that differentiated the N+ from N0nm groups included C2orf72, KRT6A, HAPLN1 as top three upregulated genes and USP9Y, NLGN4Y, & CXCL10 as downregulated genes (Table 3).
[00240] On comparison of the DEGs, 7 genes (INHBE, ZP1, PCDH10, HOXA10, TSHZ2, CPM, and HAPLN1) were commonly deregulated in the N+ nodes (N0m& N+) in comparison with the negative nodes.
[00241] In contrast to the genes that had a common regulatory trend (Upregulated: INHBE & ZP1; Downregulated: PCDH10 & HOXA10), HAPLN1 was downregulated when compared to N0m and upregulated when compared to N0nm whereas TSHZ2 and CPM were upregulated when compared to N0m and downregulated when compared to N0nm, indicating differential patterns between the cultures of the positive and negative nodes of a metastatic patient.
[00242] EXAMPLE 3: TRANSCRIPTOMIC PROFILE OF THE PRE-METASTATIC NODES (N0M)
[00243] Enrichment analysis of LNSCs from pre-metastatic nodes (N0m)
[00244] Enrichment analysis between negative nodes from the two sets of patients (N0m and N0nm) group identified various hallmark gene sets including hypoxia, KRAS signaling, epithelial mesenchymal transition, hedgehog signaling, glycolysis and TGF beta signaling as positively enriched whereas, interferon alpha response and interferon gamma response gene sets were negatively enriched.
[00245] Given that one set of LNSCs (N0m) is exposed to tumor-cells due to the presence of other metastatic nodes, it is possible that these pathways have been enriched in LNSCs in response to tumor-derived. In an embodiment differential expression analysis identified 267 DEGs (38 upregulated; 229 downregulated) in the non-metastatic nodes of metastatic patients (the pre-metastatic nodes) as compared to the nodes from the non-metastatic patients (N0nm).
[00246] The GO analysis identified collagen containing extracellular matrix, glycosaminoglycan binding, MHC protein complex, collagen trimer and response/defense to virus among others. Pathways identified included interferon alpha/beta signaling and interferon signaling whereas N0m against N0nm identified additional pathways including interleukin signaling and collagen chain trimerization (FIG. 7(F), 7(G)).
[00247] The top 20 DEGs that differentiated the two groups included HAPLN1, NPY4R, FOXG1 and top three upregulated genes and USP9Y, KDM5D, & DDX3Y as top 3 downregulated genes (Table 3).
[00248] In order to identify the differentials specific to the metastatic patient groups, the common DEGs between the two comparisons (N+ vs N0nm; N0m vs N0nm) were investigated; 45 common DEGs were identified.
[00249] Among the genes, 4 (LZTS1, HAPLN1, CDCP1, DUSP8) were upregulated and 41 downregulated. These genes, common to the lymph nodes of the metastatic patients (N+ or N0m), were further evaluated by gene sets from the Molecular Signature Database (MSigDB) to assess the common pathways that are enriched in the metastatic patients irrespective of the positive/negative nodes.
[00250] The canonical pathway gene sets from MSigDB identified interferon signaling, interferon alpha beta signaling, matrisome, type II interferon signaling, cytokine signaling in immune systems and trafficking & processing of endosomal TLR as the significant pathways.
[00251] EXAMPLE 4: CYTOKINE PROFILE OF THE STROMAL CULTURES FROM THE NODES
[00252] The cross talk between tumor and LNSC cells were evaluated as schematically represented in FIG. 9.
[00253] Design of tumor – LNSC cross talk (902) is done to check the effect of tumor on LNSC (904) and effect of LNSCs on tumor (906).
[00254] To check the effect of tumor on LNSC, the 48 hrs tumor condition media (TCM) (908) were collected and “LNSC” (910) were grown in it for 48 hrs. Later after 48 hrs it is followed by RNA sequencing (918).
[00255] To check the effect of LNSCs on tumor, the “tumor cells” (916) were cultured in LNSC (914) condition media (LCM) for 48 hrs. Later it is followed by tumorigenicity assay (912) (proliferation / invasion). The tumor condition media (TCM) and LNSC condition media (LCM) are shown in 920.
[00256] EXAMPLE 5: CO-CULTURING OF TUMOR-LYMPH NODE CROSS TALK
[00257] Effect of LNSCs on the tumor
[00258] To assess the tumor-lymph node cross talk, the tumor cells (HSC3 & CAL27) were co-cultured with the conditioned media (CM) from the metastatic (Ln5a, Ln9a) and the non-metastatic (Ln5b, Ln9b, Ln2a, Ln6a) primary lymph node cells.
[00259] Effect on the metastatic cell line (HSC3)
[00260] A time-point wise assessment for the three LNSCs (Ln9a, Ln9b, Ln6a) indicated that a percentage proliferation increase was observed in HSC3 (metastatic) cells at 48 hrs with all ratios of CM: Media followed by a decline at 72 hrs (FIG. 10(A), 10(B), 10(C)). When the proliferation was assessed for the next three LNSCs (Ln5a, Ln5b, Ln2a), a decrease in HSC3 proliferation was seen across all time points (24,48,72 hrs) and ratios (1:1, 3:7, neat) (FIG. 11(A), 11(B), 11(C)).
[00261] Effect on the non-metastatic cell line (Cal-27)
[00262] A time-point wise assessment for the three LNSCs (Ln9a, Ln9b, Ln6a) indicated that the percentage proliferation increase was observed in CAL27 with all three ratios (1:1, 3:7 & neat) and time points (48 & 72 hrs) (FIG. 10(E), 10(F), 10(G)). When the proliferation was assessed for the next three LNSCs (Ln5a, Ln5b, Ln2a), an increased proliferation is seen in CAL27 (FIG. 11(E), 11(F), 11(G)).
[00263] These results shows that there is a change in proliferation of CAL27 cells when treated with the conditioned media from the LNSCs although the change in much more evident in these LNSCs - Ln9a, Ln9b, Ln6a. HSC3 cells show a mixed response to the conditioned media effects. The proliferation does increase at 48 hrs for Ln9a, Ln9b, Ln6a, whereas it decreases for Ln5a, Ln5b, and Ln2a.
[00264] The inherent difference between these cells is their tumor origin i.e., Ln9a, Ln9b, Ln6a are derived from the nodes of patients with tongue tumor whereas Ln5a, Ln5b, Ln2a are derived from patients with buccal mucosa tumors.
[00265] Effect of Tumor on the LNSCs
[00266] Non-metastatic cell line (CAL27)-driven transcriptomic changes in the non-metastatic node (Ln2a) from an N0 patient showed the upregulated genes identified indicate pro-tumor factors favoring tumor growth/development of pre-metastatic niche due to a cross talk between tumor and LNSC. Gene ontology analysis led to identification of pathways enriched for IL-17 signaling, TNF signaling, chemokine signaling, IL-10 signaling, ECM organization, collagen degradation, and transcriptional mis-regulation in cancer (FIG. 12).
[00267] The changes driven by the metastatic cell line (HSC-3), in the same cell line Ln2awas explored. DESeq2 identified 230 differential genes (FC Cut off ±2; up: 163, down: 67). The upregulated genes were involved in processes/functions mapping to chemotaxis of neutrophils and leukocytes, CXCR-chemokine receptor binding, interleukin signaling (IL-17, IL-10, IL-4, IL-3) and the downregulated genes are involved in nucleosome/chromatin assembly/organization, DNA methylation, deacetylation by HDAC, oxidative stress induced senescence and transcriptional mis-regulation in cancer (FIG. 13).
[00268] FIG. 14 shows the significant enrichment plots from the GSEA analysis. The corresponding NES score and the FDR q values are also indicated. N0m/N0nm in FIG. 14(A), N+/N0min FIG.14(B), N+/N0nmin FIG. 14(C). Abbreviations: N0nm: Negative node from non-metastatic patient, N0m: Negative node from metastatic patient, N+: Positive node from metastatic patient, NES: Normalized enrichment score, FDR: False discovery rate
[00269] These results indicate that tumor conditioned factors that modulate the molecular changes in the lymph nodes are similar with regard to both the cell lines; further analysis is ongoing.
[00270] EXAMPLE 5: ASSESSMENT OF CHEMORESPONSE OF PATIENT-DERIVED OSCC CELLS:
[00271] Further, the patient-derived epithelial OSCC cells (MhCB03-E) were treated with various chemotherapeutic agents and molecular inhibitor to better understand the role of chemotherapeutic agents and molecular inhibitors. The percentage cell viability was calculated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.
[00272] The results suggest that MhCB03-E cells demonstrated higher sensitivity to cisplatin with an only 44.1% of cells viable at 3.9 µM (FIG. 15A), whereas, 5FU was able to inhibit only 47.8% of cells at the highest concentration used i.e., 500 µM (FIG. 15B).
[00273] ROLE OF MOLECULE INHIBITORS
[00274] Inventors also assessed the role of novel molecule inhibitors for inhibiting the proliferation of patient-derived epithelial OSCC cells (MhCB03-E). Inventor used various small molecule inhibitors (SMIs) blocking specific pathways such as ALDH1A1 inhibitor (NCT 501)1, Notch1 inhibitor (Begacestat)2, PLD2 inhibitor (ML298), and mTOR inhibitor (sirolimus). The patient-derived cells were treated with various concentration of SMIs for 72 hrs and the percentage cell viability was calculated by MTT assay. Inventors observed 61.9%, and 62.4% inhibition to ALDH1, and mTOR inhibitors at 1100nM, and 5000nM respectively (FIG. 16A, 16D). Whereas, inventor observed only 39.7%, and 36.5% inhibition to Notch1, and PLD2 inhibitors at 5200nM, and 1510nM respectively (FIG. 16B, 16C). Suggesting, differential ability of MhCB03-E cells to respond to different SMIs.
[00275] EXAMPLE 6: SCOPE OF COMBINATION THERAPY
[00276] Further, inventor could also assess the scope of combination therapy on inhibiting the proliferation of patient-derived epithelial OSCC cells (MhCB03-E). Inventors observed an increase in cell death upon using the combination of Cisplatin with novel SMIs. The viability of MhCB03-E cells reduced to 34.1%, 34.8%, and 21.8% when treated with cisplatin in combination of ALDH1 inhibitor, Notch1 inhibitor, and PLD2 inhibitor respectively, in contrast to 44.1% inhibition in cisplatin alone at 3.9µM concentration (FIG. 17A, 17B, 17C, 17D). Inventors observed similar effect upon using a combination of mTOR inhibitor and PLD2 inhibitor. The viability of MhCB03-E cells reduced to 30.3% when treated with mTOR inhibitor in combination of PLD2 inhibitor, in contrast to 71.7% inhibition in mTOR inhibitor alone at 1250nM concentration (FIG. 17E, 17F).
[00277] HARDWARE
[00278] Digital Processing System
[00279] (FIG. 18) is a block diagram illustrating the details of a digital processing system
(1800) in which various aspects of the present disclosure are operative by execution of appropriate execution modules, firmware or hardware components.
[00280] Digital processing system (1800) may correspond to each of user system: local
system or remote and server noted above. Digital processing system may contain one or more processors (such as a central processing unit (CPU) (1802)), random access memory (RAM) (1804), secondary memory (1806), graphics controller (GPU) (1812), display unit (1814), network interfaces like (WLAN) (1816), and input interfaces (1818).
[00281] CPU (1802) executes instructions stored in RAM (1804) to provide several
features of the present disclosure. CPU (1802) may contain multiple processing units, with each processing unit potentially being designed for a specific task.
[00282] Alternatively, CPU (1802) may contain only a single general-purpose processing
unit. RAM (1804) may receive instructions from secondary/system memory (1810).
[00283] Graphics controller (GPU) (1812) generates display signals (e.g., in RGB format)
to primary display unit (1814) based on data/instructions received from CPU (1802). Primary display unit contains a display screen (1814) (e.g., monitor, touchscreen) to display the images defined by the display signals. Input interfaces (1818) may correspond to a keyboard, a pointing device (e.g., touch-pad, mouse), a touchscreen, etc. which enable the various inputs to be provided. Network interface (1816) provides connectivity to a network (e.g., using Internet Protocol), and may be used to communicate with other connected systems. Network interface (1816) may provide such connectivity over a wire (in the case of TCP/IP based communication) or wirelessly (in the case of WIFI, Bluetooth based communication).
[00284] Secondary memory (1806) may contain hard drive (mass storage) (1806a), flash
memory (1806b), and removable storage drive (1806c). Secondary memory (1806) may store the data (e.g., the specific requests sent, the responses received, etc.) and executable modules, which enable the digital processing system (1800) to provide several features in accordance with the present disclosure.
[00285] Some or all of the data and instructions may be provided on a removable storage
unit (SD card) (1808), and the data and instructions may be read and provided by removable storage drive (1806c) to CPU (1802). Floppy drive, magnetic tape drive, CD-ROM drive, DVD Drive, Flash memory, removable memory chip (PCMCIA Card, EPROM) are other examples of such removable storage drive. (1806c).
[00286] Removable storage unit (1808) may be implemented using storage format
compatible with removable storage drive (1806c) such that removable storage drive (1806c) can read the data and instructions. Thus, removable storage (1806c) unit includes a computer readable storage medium having stored therein executable modules and/or data. However, the computer (or machine, in general) readable storage medium can be in other forms (e.g., non-removable, random access, etc.). CPU (1802) may retrieve the executable modules, and execute them to provide various features of the present disclosure described above.
[00287] 2. ADVANTAGES OF THE PRESENT DISCLOSURE:
[00288] As per the inventor, primary cell culture models derived from the tumors/cancer-associated fibroblasts have been widely used to study the different aspects of carcinogenesis, tumor progression, and metastasis enabling accurate reflection of the tumor itself.
[00289] As per the inventor the preclinical In-vitro models, the lymph node stroma-tumor models remain a less explored territory.
[00290] According to the inventor these cells could prove to be an effective tool in modeling the various aspects of pre-metastatic niche and the metastatic conditioning provided by the LNSCs to the tumor cells in the lymph node.
[00291] This co-culture using different combinations of the primary cultures will be valuable in studying the pre-metastatic niche and the molecular alterations undergone by the tumor cells once they inhabit the lymph nodes.
[00292] In addition, for a subset of the patients, inventor also have the entire model including primary culture from the tumor, corresponding cancer-associated fibroblasts (CAF) and the LNSC.
[00293] The primary cultures can be a model system for characterizing nodal metastasis, as a 2D co-culture model for drug testing for nodal metastasis and biomarkers identified in the study can be a differentiator of stromal cells from nodes with/without metastasis.
[00294] 3. USES, APPLICATIONS AND BENEFITS OF THE DISCLOSED EMBODIEMNT
[00295] The technology and process developed herein is novel in terms of its application and usage.
[00296] The invention can be used to analyse the alterations in the nodal stroma in response to tumor-derived cues (exosomes, micro vesicles, metabolites, proteins).
[00297] The invention can be used for assessing the possible alterations occurring in the incoming tumor cells in the lymph nodes and enabling them with “pro-metastatic” features.
[00298] The invention can be used to assess the metabolic changes undergone by a pre-metastatic nodal stroma.
[00299] The invention can be used to assess the metabolic profile of the tumor before and after inhabiting the node.
[00300] The invention can be used to carry out inhibition studies to confirm the role of “identified” biomarkers in the processes mentioned above.
[00301] The invention can also be used to carry out drug testing for therapeutics.
[00302] The invention uses in vitro cultures which can enable the development of in-vivo models mimicking the patient status; this can be invaluable in drug testing as well as mechanistic studies.
[00303] 4. BEST MODE TO PRACTICE
[00304] Best mode to practice the invention is as in vitro model systems.
[00305] A combination of metastatic and non-metastatic cultures from the metastatic patient in comparison to the nodes from non-metastatic nodes to delineate mechanistic differences in academic field
[00306] A combination of tumor-associated epithelial and fibroblast cultures along with the lymph node stromal cultures to understand tumor-node cross talk (academic research)
[00307] The models can be used for delineating mechanisms as well as for drug screening of novel molecules against nodal metastasis.
[00308] The invention enables other researchers who lack access to lymph node tissues or cells to conduct their cancer study, these cells could be made commercially available.
[00309] The same patient's metastatic and non-metastatic LNSC combinations can also be studied.
[00310] The kits comprising of cell combinations are useful for the practice of present disclosure.
[00311] Best mode to practice the above-mentioned disclosure is through commercialization of this invention through the distributors, and marketing to reach research organizations, private clinics and hospitals.
[00312] 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 sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.
[00313] While specific embodiments of the present disclosure have been shown and described in detail to illustrate the inventive principles, it was understood that the present disclosure may be embodied otherwise without departing from such principles.
[00314] 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 disclosure. 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.
[00315] 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 configurable, such that it may be utilized in ways other than that shown in the accompanying figures
[00316] 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 was 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.
,CLAIMS: I/WE Claim
1) An In-vitro system for modelling nodal metastasis, the system comprising of primary cultures from lymph nodes and tumor with and without nodal metastasis, one or more computer processors, one or more computer-readable storage media, and program instructions for execution by at least one of the one or more processors, wherein the system comprises of;
a) lymph node stromal cells (LNSCs) from N0nm, N0m, N+; epithelial primary cultures from tumors with nodal metastasis; CAF cultures from tumors with nodal metastasis;
co-culture combinations of lymph node stromal cells (LNSCs) from N0nm, N0m, N+; epithelial primary cultures from tumors with nodal metastasis; CAF cultures from tumors with nodal metastasis;
b) a model for characterizing nodal metastasis using co-culture combinations employing molecular-phenotyping, immune-phenotyping, STR profiling, cellular assays and molecular profiling;
c) a machine learning (ML) model, combining the molecular, cellular assay parameters of the different primary cultures and the co-cultures obtained both pre and post treatment to assess the response to a drug or to assess the toxicity of a drug.
2) The In vitro system as claimed in claim 1, wherein the model is a regression or a non-regression model, wherein the model enables characterization of tumor-nodal cross talk by using multiple co-culture combinations.
3) The In vitro system as claimed in claim 1, wherein the co-culture combinations are,
a. LNSCs N+ vs N0m; LNSCs N+ vs N0nm; LNSCs N0m vs N0nm;
b. Tumor cultures + N0nm/N0m/N+; Tumor cultures + CAF cultures +N0nm/N0m/N+.
4) The In vitro system as claimed in claim 1, wherein the system is for drug testing in nodal metastasis, system comprises of,
a. molecular profiling of cells comprising assaying for cellular invasion, migration, and spheroid assay pre and post adding of the drugs
b. characterizing cells;
c. cellular features of the cells;
d. modelling drug testing output such as evaluating drug efficacy and drug toxicity;
5) The In vitro system as claimed in claim 4, wherein cross talk is assessed by co-culturing combinations of lymph node stromal cells (LNSCs) from N0nm, N0m, N+; epithelial primary cultures from tumors with nodal metastasis; CAF cultures from tumors with nodal metastasis.
6) A method for assessing/characterizing nodal metastasis, wherein the method comprises of;
d) establishing of primary lymph node stromal cells;
e) characterizing the primary cells morphology;
f) immunophenotyping and STR profiling;
g) establishing co-culture combinations of lymph node stromal cells (LNSCs) from N0nm, N0m, N+; epithelial primary cultures from tumors with nodal metastasis; CAF cultures from tumors with nodal metastasis;
h) profiling GP38 expression and growth kinetics by flow cytometry;
i) analyzing flow cytometry indicating heterogeneity between the cultures;
j) analyzing transcriptome of the stromal cultures from the metastatic and non-metastatic nodes;
k) employing transcriptomic profile of the pre metastatic nodes and from metastatic nodes;
l) profiling cytokine of the stromal cultures from the nodes; and
m) employing biomarkers panel for characterizing metastatic and non-metastatic nodes.
7) modelling research output by assessing the above mentioned assays before and after inhibiting specific molecular targetsA method for evaluating drug efficacy/ toxicity, wherein the method comprises of;
a) establishing co-culture systems;
b) exposing co-cultures to a target drugs;
c) analysing cell death assays;
d) employing cellular assays;
e) quantifying molecular assays;
f) Profiling cytokines;
g) employing biomarkers panel for characterizing metastatic and non-metastatic nodes;
h) providing input data to a model; and
i) assessing efficacy of the drugs.
8) A method for characterizing nodal metastasis, wherein the method comprises of;
a) establishing co-culture systems;
b) exposing co-cultures to a target/ inhibitor;
c) analysing cell death assays;
d) employing cellular assays;
e) quantifying molecular assays;
f) Profiling cytokines;
g) providing input data to a model;
h) employing biomarkers/ inhibitors for characterizing metastatic and non-metastatic nodes;
i) assessing the mechanisms of the cross-talk between the cultures; and
j) identifying new targets by inhibitory studies and establishing their effect in nodal metastasis
9) A composition for modelling nodal metastasis, wherein the composition comprises of co-cultured combinations of lymph node stromal cells (LNSCs) from N0nm, N0m, N+; epithelial primary cultures from tumors with nodal metastasis; CAF cultures from tumors with nodal metastasis; biomarkers from Table-3; a detection agent that binds to the biomarker products; and at least one reagent that inhibits the biomarker expression product in a biological sample.
10) The composition as claimed in claim 9, wherein primary cultures were labelled novel with a STR score filter of 80% for novelty; wherein the preferable biomarkers are CCL11, GPR4, EDNRB, CSF3, TRIM55, EYA4, ZP1, EPHB6, CABP1, TDRD9; wherein condition is lymph node metastasis; wherein the biological sample is a metastatic and non-metastatic primary lymph node stromal cell; wherein the composition can identify the differentially expressed genes in cell; wherein the composition can differentiate metastatic and non-metastatic lymph node with sensitivity and specificity.
11) The composition as claimed in claim 10, wherein the lymph nodes metastatic cells illustrated a doubling time at an average of 25.6 ± 1.0 hours, wherein the non-metastatic illustrated a doubling time at an average of 32.7 ± 6.4 hours.
12) The composition as claimed in claim 1, wherein the composition can identify differentially expressed genes (DEG) in N+/N0m, N+/N0nm and N0m/N0nm accordingly to Table-3; wherein the top 20 DEGs that differentiated the N+ from N0nm groups included C2orf72, KRT6A, HAPLN1 as top three upregulated genes and USP9Y, NLGN4Y, & CXCL10 as downregulated genes; wherein 7 genes, INHBE, ZP1, PCDH10, HOXA10, TSHZ2, CPM, and HAPLN1 were deregulated in the N+ nodes (N0m& N+) in comparison with the negative nodes; wherein the top 20 DEGs differentiated the two groups included HAPLN1, NPY4R, FOXG1 and top three upregulated genes and USP9Y, KDM5D, & DDX3Y as top 3 downregulated genes.
Dated this 23th day of July, 2024
(LIPIKA SAHOO)
Registration Number: IN/PA-2467
Agent for Applicant
This document is signed with the digital signature of Patent Agent for the
Applicant LIPIKA SAHOO (IN/PA-2467)