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: 3D MODEL FOR TUMOUR
MICROENVIRONMENT ANALYSIS

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) INDIA

THE FOLLOWING SPECIFICATION PARTICULARLY 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 copending India provisional patent application entitled, “3D MODEL FOR TUMOUR MICROENVIRONMENT ANALYSIS”, Patent Application no.: 202241074987, Original Filing Date: 23 December 2022, Priority Filing Date: 23 March 2023 which is incorporated in its entirety herewith.

BACKGROUND OF THE DSCLOSED EMBODIMENT
[002] Technical field
[003] The present disclosure is in the technical field of a system for a 3D model for tumour microenvironment analysis. More particularly, the system is for analysis and prediction assay of clinical response for a subject upon targeting with radiotherapy and chemotherapy.
[004] Related art
[005] Head and neck squamous cell carcinoma is the world's eighth most common cancer type, with a 5-year survival rate of 25-60% depending on anatomical site and stage. India accounts for 60% of the global head and neck cancer burden. Head and neck cancers account for nearly 30% of all cancers in India, with men having the highest prevalence and women having the second highest. Despite significant efforts in clinical care and research to increase the 5-year survival rate through the use of various therapy options such as surgical excision, radiotherapy, chemotherapy, immunotherapy, and so on, they are far from perfect. Head and neck squamous cell carcinoma (HNSCC) targeting is difficult, due in part to their anatomic locations, which complicate surgery, and in part to the highly variable treatment response. Advanced cases necessitate combinations of surgery, radiotherapy, and chemotherapy, which rely heavily on clinical experience, demanding the development of preclinical models to predict therapy outcomes and guide targeting decisions at the individual level.
[006] The intra-tumor microenvironment is a complex composition of cells, ECM molecules, and different anatomical locations which differ in the levels of oxygen, pH, and nutrient availability. In contrast, the 2D platforms are oversimplified and introduce parameters that are not encountered in the tumor tissue. For example, when cells are cultured on unnaturally stiff plastic, cellular heterogeneity is lost, and other physicochemical features such as uniform oxygen, pH, and glucose-rich media are introduced, which do not mimic the in-vivo scenario. We have animal models, on the other hand in which patient-derived tumor cells or tumor fragments are implanted into immune-deficient mice to create PDXs. Even though the PDX models can successfully retain intratumoral heterogeneity, they have significant flaws of their own. For example, tumor cells will likely integrate ECM and different cell types of non-human origin, influencing test results. They lack some immune cells that are essential in tumor biology. Furthermore, the success rate of implantation is very low (10-20%), they are very expensive, and the generation time is very long (2-12months).
[007] The tumor microenvironment (TME) plays an important role in tumor development, and it is well understood that stroma and cancer cells co-exist and co-evolve over time. This complex stroma-cancer cell interaction results in an active feed-forward/feed-back loop with various biochemical and biophysical alterations supplementing the neoplastic conversion of cancer cells1. Tumorigenesis is a multistep process, reflecting all the inter-and intra-cellular interactions and genetic modifications that influence the cumulative transformation of normal human cells into malignant phenotypes. Although biochemistry has long been studied for understanding cancer progression, biophysical signaling is emerging as a critical paradigm determining cancer metastasis. Biophysically, tumor stiffness that is significantly greater than normal tissue correlates with cancer progression and metastasis. Biophysical forces pertinent to the TME have been reported to aid in the secretion of various cytokines, which further stimulate cancer progression/invasion of cancer cells.
[008] The increased stiffness of TME is the result of continuous deposition and remodeling of the extracellular matrix (ECM) by stromal cells. However, the different solid tumors show an extremely tumor-specific biophysical characteristic. GBM tumors have a significantly higher biophysical stiffness (0.5-13kPa) than normal tissue (< 0.5kPa). In breast cancer, the invasive front of invasive ductal carcinoma (IDC) has stiffer regions (2kPa- 10kPa) than adjacent normal tissue (up to 800Pa). Similarly, the tumor stiffness of OSCC has been reported to be in the range of 1-8kPa.
[009] Currently available in vitro and in vivo models do not accurately mimic the biological framework, limiting their ability to assess the therapeutic potential of anti-cancer drugs. This is primarily due to the oversimplification of 2D culture systems, which introduce variables (lack of cellular heterogeneity, uniform oxygen levels, excessive glucose, and uniform pH) that are not in sync with native tissue. In vivo mouse models, on the other hand, are prohibitively expensive, time-consuming, and inherently governed by multiple signals from surrounding non-human host cells.
[010] These inaccurate platforms are insufficient to cure all the patients, resulting in a high incidence of therapeutic resistance in patients, necessitating the need for accurate prediction of patients’ clinical response to therapeutic agents. Furthermore, the current chemotherapy management practice of “one treatment fits all” results in 40% non-responders in HNSCC, necessitating the development of better models capable of simulating therapeutic resistance to either chemotherapeutic drugs or radiotherapy.
[011] However, none of these models are currently in clinical practice. Other prediction models which have been developed to predict the clinical response of patients to chemotherapy or radiotherapy are primarily histocultures-based techniques. Histocultures are the cultures in which micro-sections of the tumor tissue are grown in-vitro and targeted with chemotherapeutic drugs or radiotherapy. In this case, they predict the patient’s clinical response based on the response of these tumor tissues cultured in-vitro. These methods also have the following drawbacks:
[012] a. Inadequate tissue samples to perform multiple assays.
[013] b. These micro-tissues have a limited duration of viability period of 7-21 days, which results in necrotic tissue.
[014] c. The histocultures lack biophysical properties that are present in the in-vivo tumor microenvironment.
[015] d. Because the tumor microenvironment is extremely heterogeneous, the micro tissues produced may not be similar replicates of each other and thus are not proper biological replicates.
[016] These drawbacks result in inappropriate outcomes, which jeopardize the patients’ treatment regimen. As a result, a model system is required to overcome all of these drawbacks and produce more accurate results, which will aid the patients’ treatment regimen.
[017] Therefore, there is an urgent need to develop a test/assay that allows for individual testing of a specific molecule during the patient's diagnosis and predicts the therapeutic effect of both chemotherapeutic drugs and radiotherapy. 3D scaffolds have emerged as a novel tool for revisiting various stages of cancer progression as well as overcoming the challenges posed by in vitro and in vivo models.
[018] These 3D culture techniques are one of such valuable models that eliminate these shortcomings and aid in achieving the goal. These models prove to be critical in personalized medicine because prediction models are scarce in the clinic.
SUMMARY OF THE DISCLOSED EMBODIMENT
[019] The primary objective of the present disclosure relates to a system for 3D model for tumour microenvironment analysis. More particularly, the system is for analysis and prediction assay of clinical response for a subject upon targeting with radiotherapy and chemotherapy.
[020] According to the aspects of the disclosure, a system for predicting a subject-specific response of progression, recurrence or complete remission of a condition using biomimetic 3D models, the system comprises:
a. the biomimetic 3D models to culture cells, the biomimetic 3D models comprise (i) an organoid culture, the cells are grown in a biomimetic basal membrane extract, (ii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 0.3kPa to 0.6kPa forming soft hydrogels, (iii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 1.0kPa to 5.0kPa forming stiff hydrogels, (iv) a spheroid cultures, the cells are cultured with a serum-free medium under ultra-low attachment conditions, the cultured cells comprises in-vitro formed micro-tumors, the biomimetic 3D models comprising the in-vitro formed micro-tumors are subjected to a radiation ranging from 0 to 8gy in a single-dose and a chemotherapeutic agent for about 72 hours,
b. a server, the server stores instructions in a database to perform the following:
c. obtaining from an external device, data related to an ability of the cells to form the spheroids, an extent of the DNA damage, a resistance index, a proliferative index, and an apoptosis rate of the cells on the biomimetic 3D models after the treatment with the radiation and the chemotherapeutic agent,
d. the ability of the cells to form spheroids in the hydrogels of the biomimetic 3D models is determined by a morphological analysis, the ability of the cells to form the spheroids is determined by measuring a time taken to form the spheroids, the time taken to form spheroids is inversely proportional to an aggressiveness of the cells, the aggressive cells form spheroids within 5-days from the day of seeding, the less aggressive cells take about 7 days to form the spheroids,
e. the extent of DNA damage of the cells is determined by performing a TUNEL assay,
f. the resistance index of the cells is determined by performing a cytotoxicity assay, the cytotoxicity assay comprises MTT or CCK-8 assay, an inhibitory concentration 50 for the chemotherapeutic agent is calculated against a 2D model, the resistance index of greater than or equal to 3 is considered a non-responder;
g. the proliferative index of the cells is determined using Ki-67 stain, the proliferative index is measured by stained the cells with Ki-67, a value of 30% or above is considered as no response to the chemotherapeutic agent, the value of lesser than 30% score is considered a response to the chemotherapeutic agent,
h. the cellular apoptosis rate is determined by performing anti-cleaved caspace-3 assay, the cellular apoptosis is generated by measuring the cleaved caspase-3 before and after the treatment with the chemotherapeutic agent, cellular apoptosis rate is determined based on percentage of cells having the expression for cleaved caspase-3,
i. generating a response score of the cells based on the data related to the ability to form the spheroids, the extent of the DNA damage, the resistance index, the proliferative index, and the apoptosis rate; and
j. generating a nomogram based on the generated response score of the cells and predicting the subject-specific response of progression, recurrence or complete remission of the condition, the subject-specific response is predicted based on the response score of the cells along with habit history, clinic-pathological staging of the subject.
[021] According to the one aspect of the disclosure, the condition is head and neck squamous cell carcinoma.
[022] According to an aspect of the disclosure, the cells comprise neoplastic cells, fibroblasts, immune cells and endothelial cells, the cells are obtained from the subject with head and neck squamous cell carcinoma, the cells comprise Cal27, HSC3 or other HNSCC cell lines.
[023] According to another aspect of the disclosure, the chemotherapeutic agent comprises a concentration ranging from 1nM to 1mM, the chemotherapeutic agent is selected from Cisplatin (platinum-based drugs), 5-fluro uracil, and Taxol.
[024] According to the further aspect the present disclosure, the biomimetic 3D models are subjected to the chemotherapeutic agent along with one or more therapeutic drugs selected from ALDH1 inhibitor/NCT-501, Notch1 inhibitor/begacestat, PLD2 inhibitor/ML298, mTOR inhibitor/rapamycin, celecoxib, methotrexate, immunotherapy drugs (anti-PD1, anti-PDL1, anti-CTLA4), Celecoxib, targeted therapies (anti-EGFR, anti-mTOR, anti-VEGF, anti-PI3K, anti-CDKs inhibitors of the PI3-AKT, JAK-STAT, RAS-RAF, anti-NOTCH, anti-FGFR, anti-MET, anti-RET pathways) or combinations thereof.
[025] According to the aspect of the present disclosure, the biomimetic 3D model is prepared by,
a. dissolving gelatin in PBS comprising a pH of 7.4 and stirring at 50°C for 1 hour to obtain a first mixture;
b. adding 0.06 to 0.6g per 1g of (w/v) of methacrylic anhydride dropwise to the first mixture;
c. adding the PBS to the first mixture comprising the methacrylic anhydride to stop the reaction and dialyzing for 3 days with distilled water at 37 °C to obtain a second mixture;
d. freeze-drying the second mixture in a lyophilizer to obtain a methacrylamide-modified gelatin as a dry white powder and storing at -20°C;
e. dissolving the methacrylamide-modified gelatin in 1X PBS at a concentration of 5% - 10% along with a photo-initiator comprising 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone of 0.5 mg/mL to obtain a third mixture; and
f. placing the third mixture in culture vessels followed by 10 minutes of UV-365 exposure to obtain a microporous 3D hydrogel of the methacrylamide-modified gelatin forming the biomimetic 3D model.
[026] According to the aspect of the present disclosure, the microporous 3D hydrogel of the methacrylamide-modified gelatin is equilibrated with 500µL of a culture medium for 24 hours before performing seeding of the cells.
[027] According to another aspect of the present disclosure, a method for predicting a subject-specific response of progression, recurrence or complete remission of a condition using biomimetic 3D models, the method comprises:
a. generating the biomimetic 3D models to culture cells, the biomimetic 3D models comprise (i) an organoid culture, the cells are grown in a biomimetic basal membrane extract, (ii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 0.3kPa to 0.6kPa forming soft hydrogels, (iii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 1.0kPa to 5.0kPa forming stiff hydrogels, (iv) a spheroid cultures, the cells are cultured with a serum-free medium under ultra-low attachment conditions, the cultured cells comprises in-vitro formed micro-tumors;
b. subjecting the biomimetic 3D models with the in-vitro formed micro-tumors to radiation ranging from 0 to 8gy in a single dose and a chemotherapeutic agent for about 72 hours,
c. determining (i) an ability of the cells to form spheroids in the hydrogels of the biomimetic 3D models by performing a morphological analysis, (ii) an extent of DNA damage of the cells by performing a TUNEL assay; (iii) a resistance index of the cells by performing a cytotoxicity assay, the cytotoxicity assay comprises MTT or CCK-8 assay, (iv) a proliferative index of the cells, the proliferative index of the cells are determined using Ki-67 stain, (v) an apoptosis rate by performing anti-cleaved caspace-3 assay;
d. the ability of the cells to form spheroids in the hydrogels of the biomimetic 3D models are determined by a morphological analysis, the ability of the cells to form the spheroids is determined by measuring a time taken to form the spheroids, the time taken to form spheroids is inversely proportional to an aggressiveness of the cells, the aggressive cells form spheroids within 5-days from the day of seeding, the less aggressive cells take about 7 days to form the spheroids,
e. the extent of DNA damage of the cells is determined by performing a TUNEL assay,
f. the resistance index of the cells is determined by performing a cytotoxicity assay, the cytotoxicity assay comprises MTT or CCK-8 assay, wherein an inhibitory concentration 50 for the chemotherapeutic agent is calculated against a 2D model stored in the database, the resistance index of greater than or equal to 3 is considered a non-responder;
g. the proliferative index of the cells is determined using Ki-67 stain, the proliferative index is measured by stained the cells with Ki-67, a value of 30% or above is considered as no response to the chemotherapeutic agent, the value of greater than 30% score is considered a response to the chemotherapeutic agent,
h. the cellular apoptosis rate is determined by performing anti-cleaved caspace-3 assay, the cellular apoptosis is generated by measuring the cleaved caspase-3 before and after the treatment with the chemotherapeutic agent, cellular apoptosis rate is determined based on percentage of cells having the expression for cleaved caspase-3,
i. generating a response score of the cells based on the data related to the ability to form the spheroids, the extent of the DNA damage, the resistance index, the proliferative index, and the apoptosis rate; and
j. generating a nomogram based on the generated response score of the cells and predicting the subject-specific response of progression, recurrence or complete remission of the condition, the subject-specific response is predicted based on the response score of the cells along with habit history, clinic-pathological staging of the subject.
[028] According to one another aspect of the present disclosure, the condition is head and neck squamous cell carcinoma.
[029] According to the yet another aspect of the present disclosure, the cells comprise neoplastic cells, fibroblasts, immune cells and endothelial cells, the cells are obtained from the subject with head and neck squamous cell carcinoma, the cells comprise Cal27, HSC3 or other HNSCC cell lines.
[030] According to the one aspect of the present disclosure, the chemotherapeutic agent comprises a concentration ranging from nano molar concentrations to micro molar, wherein the chemotherapeutic agent is selected from Cisplatin (platinum-based drugs), 5-fluro uracil, and Taxol.
[031] According to the further aspect the present disclosure, the biomimetic 3D models are subjected to the chemotherapeutic agent along with one or more therapeutic drugs selected from ALDH1 inhibitor/NCT-501, Notch1 inhibitor/begacestat, PLD2 inhibitor/ML298, mTOR inhibitor/rapamycin, methotrexate, celecoxib, immunotherapy drugs (anti-PD1, anti-PDL1, anti-CTLA4), Celecoxib, targeted therapies (anti-EGFR, anti-mTOR, anti-VEGF, anti-PI3K, anti-CDKs inhibitors of the PI3-AKT, JAK-STAT, RAS-RAF, anti-NOTCH, anti-FGFR, anti-MET, anti-RET pathways) or combinations thereof.
[032] According to the one aspect of the present disclosure, the biomimetic 3D model is prepared by,
a. dissolving gelatin in PBS comprising a pH of 7.4 and stirring at 50°C for 1 hour to obtain a first mixture;
b. adding 0.06 to 0.6g per 1g of (w/v) of methacrylic anhydride dropwise to the first mixture;
c. adding the PBS to the first mixture comprising the methacrylic anhydride to stop the reaction and dialyzing for 3 days with distilled water at 37 °C to obtain a second mixture;
d. freeze-drying the second mixture in a lyophilizer to obtain a methacrylamide-modified gelatin as a dry white powder and storing at -20°C;
e. dissolving the methacrylamide-modified gelatin in 1X PBS at a concentration of 5% - 10% along with a photo-initiator comprising 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone of 0.5 mg/mL to obtain a third mixture; and
f. placing the third mixture in culture vessels followed by 10 minutes of UV-365 exposure to obtain a microporous 3D hydrogel of the methacrylamide-modified gelatin forming the biomimetic 3D model.
[033] According to the one aspect of the present disclosure, the microporous 3D hydrogel of the methacrylamide-modified gelatin is equilibrated with 500µL of a culture medium for 24 hours before performing seeding of the cells.
[034] 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 30 combinations are described herein for conciseness.

BRIEF DESCRIPTION OF THE DRAWING
[035] Example embodiments of the disclosed embodiment will be described with reference to the accompanying drawings briefly described below.
[036] FIG. 1 illustrates approach of drug discovery in relevance to personalized medicine, according to the aspects of the disclosed embodiment.
[037] FIG. 2 illustrates schematic representation of the work flow for correlation with patients’ clinical outcome, according to the aspects of the disclosed embodiment.
[038] FIG. 3 illustrates critical steps involved in the preparation of 3D GelMA hydrogels, according to the aspects of the disclosed embodiment.
[039] FIG. 4 illustrates characterization of the physical properties of GelMA, according to the aspects of the disclosed embodiment.
[040] FIG. 5 illustrates phenotypic properties of OSCC cell lines cultured on 2D TCPS and GelMA Hydrogels of varying mechanical stiffness, according to the aspects of the disclosed embodiment.
[041] FIG. 6 illustrates OSCC cell lines Cal27 and HSC3 behave differently upon varying the mechanical property of 3D GelMA, according to the aspects of the disclosed embodiment.
[042] FIG. 7 illustrates radio-response of patient-derived oral cancer cells cultured on 2D platform, according to the aspect of the disclosed embodiment.
[043] FIG. 8 illustrates chemo-response of patient-derived OSCC cells cultured upon 2D platform, according to the aspect of the disclosed embodiment.
[044] FIG. 9 illustrates phenotypic properties of patient-derived OSCC cells cultured on 2D TCPS and GelMA Hydrogels of varying mechanical stiffness, according to the aspect of the disclosed embodiment.
[045] FIG. 10 illustrates chemo-response of patient-derived OSCC cells cultured upon 2D, 3D GelMA hydrogels of varying the mechanical property, according to the aspect of the disclosed embodiment.
[046] FIG. 11 illustrates chemo-response in terms of the Resistance Index of patient-derived OSCC cells cultured upon 2D, 3D GelMA hydrogels of varying the mechanical property, according to the aspect of the disclosed embodiment.
[047] FIG. 12 illustrates chemo-response of patient-derived OSCC cells cultured upon 2D, 3D GelMA hydrogels of varying the mechanical property to combination therapy, according to the aspect of the disclosed embodiment.
[048] FIG. 13 illustrates schematic representation of the work flow for testing novel chemotherapeutic drugs, according to the aspect of the disclosed embodiment.
[049] FIG. 14 illustrates a block diagram illustrating the details of a digital processing system in which various aspects of the disclosed embodiment are operative by execution of appropriate execution modules, firmware, or hardware components, according to the aspects of the disclosed embodiment.
[050] 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
[051] 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.
[052] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in disclosed embodiment. 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.
[053] 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.
[054] 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.
[055] 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.
[056] 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.
[057] 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.
[058] Example embodiments of the present disclosure are described with reference to the accompanying figures.
[059] DEFINITIONS
[060] The term ‘cancer’ refers for a disease in which abnormal cells divide without control and can invade nearby tissues.
[061] The term ‘remission’ means that cancer treatment reduced or eliminated the symptoms and signs of cancer. Remission may last for months, years or the rest of your life. Remission may not mean you're free of cancer (cured), but it's an important turning point for you and your cancer care team.
[062] The term ‘3D models’ refers to the process of creating three-dimensional representations of an object or a surface. 3D models are made within computer-based 3D modelling software.
[063] The term ‘Tumour microenvironment’ refers to the normal cells, molecules, and blood vessels that surround and feed a tumor cell. A tumor can change its microenvironment, and the microenvironment can affect how a tumor grows and spreads.
[064] The term ‘stroma’ refers to the cells and tissues that support and give structure to organs, glands, or other tissues in the body.
[065] The term ‘prediction assay’ refers to utilize specialized assay/instrumentation, computational and mathematical tools (algorithms), and/or biochemical information and techniques to aid in the prediction of the response of the model to the cytotoxic effects of natural or synthetic compounds on the functioning of biological systems.
[066] The term ‘metastasis’ refers to the spread of cancer cells from the place where they first formed to another part of the body.
[067] The term ‘Progressive disease’ refers to a disease or physical ailment whose course in most cases is the worsening, growth, or spread of the disease.
[068] The term ‘gene-signature based’ refers to the information about the activity of a specific group of genes in a cell or tissue.
[069] The term ‘Tumorigenesis’ means a multistep process, reflecting all the inter-and intra-cellular interactions and genetic modifications that influence the cumulative transformation of normal human cells into malignant phenotype.
[070] The term ‘Histocultures’ means the cultures in which micro-sections of the tumor tissue are grown in-vitro and treated with chemotherapeutic drugs or radiotherapy.
[071] The term ‘Matrigel’ means a solubilized extract derived from mouse tail that contains ECM components and growth factors.
[072] The term ‘Organoid’ means tiny, self-organized three-dimensional tissue cultures that are derived from stem cells.
[073] The term ‘Radiotherapy’ means the treatment of disease, especially cancer, using X-rays or similar forms of radiation.
[074] The term ‘Chemotherapy’ means a type of cancer treatment that uses one or more anti-cancer drugs as part of a standardized chemotherapy regimen.
[075] The term ‘Tumor’ means an abnormal mass of tissue that forms when cells grow and divide more than they should or do not die when they should. Tumors may be benign (not cancer) or malignant (cancer). Benign tumors may grow large but do not spread into, or invade, nearby tissues or other parts of the body.
[076] The term ‘Disease progression’ means a term used to characterize the course of an illness, including pain and targeting response biomarkers like blood pressure. Over time, a drug's action for example, blocking an enzyme or activating a receptor—causes the state of the disease to alter.
[077] The term ‘Cancer recurrence’ (come back), usually after a period of time during which the cancer could not be detected. The cancer may come back to the same place as the original (primary) tumor or to another place in the body. Also called recurrent cancer.
[078] The term ‘Gelatin methacryloyl (GelMA)’, a photocurable hydrogel, is widely used in 3D culture, particularly in 3D bioprinting, due to its high biocompatibility, tunable physicochemical properties, and excellent formability.
[079] The term ‘spheroid cultures’ refers to a cell aggregates, self-assembling in an environment that prevents attachment to a flat surface.
[080] The term ‘Cancer Stem Cells (CSCs)’ refers to are a small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity when transplanted into an animal host.
[081] The term ‘Cytotoxicity assays’ refers to measure loss of some cellular or intercellular structure and/or functions, including lethal cytotoxicity. They thus give an indication of the potential to cause cell and tissue injury and as such have been used by some investigators to predict tissue injury, including eye injury.
[082] The term ‘hydrogel’ is a network of polymer chains with great water absorbance ability.
[083] The term ‘polymers’ are large molecules made by bonding (chemically linking) a series of building blocks.
[084] The term ‘glioblastoma’ is caused by DNA mutations that result in uncontrolled cell growth. The underlying causes for these genetic cell mutations are largely unknown.
[085] The term ‘neurospheres’ refers to a small cluster of nerve stem cells that is grown in the laboratory. Neurospheres can be grown that are similar to normal human nerve tissue or to a specific type of tumor.
[086] The term ‘Oral squamous cell carcinoma (OSCC)’ is the most common oral malignancy, representing up to 80-90% of all malignant neoplasms of the oral cavity.
[087] The term ‘Transcriptomics’, also commonly known as expression profiling, is the study of transcriptome of organisms resulting from the expression of genes under specified conditions.
[088] The term ‘Chemoresistance’ refers to the ability of cancer cells to evade or to cope with the presence of therapeutics, is a key challenge that oncology research seeks to understand and overcome.
[089] 1. EMBODIMENTS OF THE DISCLOSURE
[090] The present disclosure relates to a system for 3D model for tumour microenvironment analysis. More particularly, the system is for analysis and as a prediction assay of clinical response for a subject upon targeting with radiotherapy and chemotherapy. The system also enables assessing the efficacy of novel drugs across different HNSCC patient-derived 3D models.
[091] The present disclosure focuses on prediction of patient specific response of disease progression, recurrence or complete remission of cancer disease. The prediction models developed will include the 3D models also profiled for their er gene-signature based or marker-based (ct-DNA in blood, proteins in saliva etc.). These models propose to predict the survival benefit and aggressiveness of the disease on treatment with established drug in addition to assessing the efficacy of novel drugs.
[092] Approach of drug discovery in relevance to personalised medicine- The current treatment regimen includes treating the patient with both radiation and chemotherapy based on the bare minimum of information available, namely the stage and grade of the disease. This usually results in a chance-based effect in which some patients show complete regression with improved survival, or no effect (i.e., progressive disease), or it can result in harmful side-effects. Consequently, a proper analysis is required beforehand, and the patient is then treated based on the information available as to which drug or radiation treatment is better suited for the patient. (FIG. 1). Further this also enables testing of novel drugs for their response/efficacy in the 3D models for preclinical validation.
[093] 2. SYSTEM FOR 3D MODEL FOR TUMOUR MICROENVIRONMENT
[094] According to the aspects of the disclosure, a system for predicting a subject-specific response of progression, recurrence or complete remission of a condition using biomimetic 3D models, the system comprises:
a. the biomimetic 3D models to culture cells, the biomimetic 3D models comprise (i) an organoid culture, the cells are grown in a biomimetic basal membrane extract, (ii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 0.3kPa to 0.6kPa forming soft hydrogels, (iii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 1.0kPa to 5.0kPa forming stiff hydrogels, (iv) a spheroid cultures, the cells are cultured with a serum-free medium under ultra-low attachment conditions, the cultured cells comprises in-vitro formed micro-tumors, the biomimetic 3D models comprising the in-vitro formed micro-tumors are subjected to a radiation ranging from 0 to 8gy in a single-dose and a chemotherapeutic agent for about 72 hours,
b. a server, wherein the server stores instructions in a database to perform the following:
c. obtaining from an external device, data related to an ability of the cells to form the spheroids, an extent of the DNA damage, a resistance index, a proliferative index, and an apoptosis rate of the cells on the biomimetic 3D models after the treatment with the radiation and the chemotherapeutic agent,
d. the ability of the cells to form spheroids in the hydrogels of the biomimetic 3D models is determined by a morphological analysis, the ability of the cells to form the spheroids is determined by measuring a time taken to form the spheroids, the time taken to form spheroids is inversely proportional to an aggressiveness of the cells, the aggressive cells form spheroids within 5-days from the day of seeding, the less aggressive cells take about 7 days to form the spheroids,
e. the extent of DNA damage of the cells is determined by performing a TUNEL assay,
f. the resistance index of the cells is determined by performing a cytotoxicity assay, the cytotoxicity assay comprises MTT or CCK-8 assay, an inhibitory concentration 50 for the chemotherapeutic agent is calculated against a 2D model, the resistance index of greater than or equal to 3 is considered a non-responder;
g. the proliferative index of the cells is determined using Ki-67 stain, the proliferative index is measured by stained the cells with Ki-67, a value of 30% or above is considered as no response to the chemotherapeutic agent, the value of lesser than 30% score is considered a response to the chemotherapeutic agent,
h. the cellular apoptosis rate is determined by performing anti-cleaved caspace-3 assay, wherein the cellular apoptosis is generated by measuring the cleaved caspase-3 before and after the treatment with the chemotherapeutic agent, cellular apoptosis rate is determined based on percentage of cells having the expression for cleaved caspase-3,
i. generating a response score of the cells based on the data related to the ability to form the spheroids, the extent of the DNA damage, the resistance index, the proliferative index, and the apoptosis rate; and
j. generating a nomogram based on the generated response score of the cells and predicting the subject-specific response of progression, recurrence or complete remission of the condition, the subject-specific response is predicted based on the response score of the cells along with habit history, clinic-pathological staging of the subject.
[095] According to the one aspect of the disclosure, the condition is head and neck squamous cell carcinoma.
[096] According to an aspect of the disclosure, the cells comprise neoplastic cells, fibroblasts, immune cells and endothelial cells, the cells are obtained from the subject with head and neck squamous cell carcinoma, the cells comprise Cal27, HSC3 or other HNSCC cell lines.
[097] According to another aspect of the disclosure, the chemotherapeutic agent comprises a concentration ranging from nano molar concentrations to micro molar, the chemotherapeutic agent is selected from Cisplatin (Platinum based drugs), 5-fluro uracil, and Taxol.
[098] According to the further aspect the present disclosure, the biomimetic 3D models are subjected to the chemotherapeutic agent along with one or more therapeutic drugs selected from ALDH1 inhibitor/NCT-501, Notch1 inhibitor/begacestat, PLD2 inhibitor/ML298, mTOR inhibitor/rapamycin, methotrexate, celecoxib, immunotherapy drugs (anti-PD1, anti-PDL1, anti-CTLA4), Celecoxib, targeted therapies (anti-EGFR, anti-mTOR, anti-VEGF, anti-PI3K, anti-CDKs inhibitors of the PI3-AKT, JAK-STAT, RAS-RAF, anti-NOTCH, anti-FGFR, anti-MET, anti-RET pathways) or combinations thereof.
[099] According to the aspect of the present disclosure, the biomimetic 3D model is prepared by,
a. dissolving gelatin in PBS comprising a pH of 7.4 and stirring at 50°C for 1 hour to obtain a first mixture;
b. adding 0.06 to 0.6g per 1g of (w/v) of methacrylic anhydride dropwise to the first mixture;
c. adding the PBS to the first mixture comprising the methacrylic anhydride to stop the reaction and dialyzing for 3 days with distilled water at 37 °C to obtain a second mixture;
d. freeze-drying the second mixture in a lyophilizer to obtain a methacrylamide-modified gelatin as a dry white powder and storing at -20°C;
e. dissolving the methacrylamide-modified gelatin in 1X PBS at a concentration of 5% - 10% along with a photo-initiator comprising 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone of 0.5 mg/mL to obtain a third mixture; and
f. placing the third mixture in culture vessels followed by 10 minutes of UV-365 exposure to obtain a microporous 3D hydrogel of the methacrylamide-modified gelatin forming the biomimetic 3D model.
[100] According to the aspect of the present disclosure, the microporous 3D hydrogel of the methacrylamide-modified gelatin is equilibrated with 500µL of a culture medium for 24 hours before performing seeding of the cells.
[101] A. THE 3D MODEL SYSTEM
[102] The system comprises of,
[103] a. Sample collection unit, b. Sample preparation, d. Culture platforms, e. Treatment with chemotherapeutic drugs or ionizing radiation, f. Microplate reader, g. Histological analysis unit, h. Prediction and corelation outcome or efficacy of novel drugs across the patient-derived 3D models.
[104] Steps involves
[105] The assay can be used to test the standard of care chemotherapeutic drugs and/or novel drugs, radiation therapy, or in combination to predict the clinical response of the patient.
[106] Step 1 – Sample collection unit the patient’s tumor sample is collected (202) on the day of surgery cleaned and decontaminated (204, 206). Step 2 – Sample preparation the tissue is minced and subjected to enzymatic degradation (208) to form single cell suspension (210). The cells are passes through 70micron filter to remove the debris. The single cells thus obtained are counted and seeded on to different 3D models 2D monolayer (214), organoids (216) 3D Soft GelMA (218), 3D Stiff GelMA (220) including GelMA hydrogels of different stiffness and Step 3 - Cultured platforms (212) for 7-10 days, varying from patient-to-patient. This allows the cells to form spheroids/micro-tumors in-vitro, that are subjected to Step 4 - Treatment with chemotherapeutic drugs or ionizing radiation - chemotherapy (upto 72hrs) or radiotherapy (222) e.g. (0-8gy) (224), cisplatin (226). Step 5 - Microplate reader, Step – 6 Histological analysis unit Further, the cells will be assessed for various parameters as listed in (FIG. 2), and a response score is calculated. The response score is compared with the patients’ clinical response. The patients’ test report includes the response of tumor sample to tested chemotherapeutic drugs, radiotherapy, and a calculated chances of survival beyond 5-years (FIG. 2).
[107] The assay can also be used as a drug testing platform in pharmaceutical industries, where, it can bridge the gap between currently used 2D platform and animal models. Resulting in conservation of time, efforts, and money.
[108] The present disclosure has used various 3D models to culture cancer cells derived from HNSCC (Head and neck squamous cell carcinoma) patients. Tumor microenvironment includes various cell types such as neoplastic cells, fibroblasts, immune cells, endothelial cells, and others. Neoplastic cells drive the disease progression whereas, fibroblasts support the neoplastic cells. Therefore, they have proposed using both the cell types in the 3D model. Four different 3D models are used, including:
[109] a. Organoid culture (in which cells are grown in a biomimetic basal membrane extract).
[110] b. Gelatin-based biomimetic hydrogels with mechanical stiffnesses <1kPa, soft.
[111] c. Gelatin-based biomimetic hydrogels with mechanical stiffnesses >5kPa, stiff.
[112] d. Spheroids cultures (in which cells are cultured with a specific medium under ultra-low attachment conditions to enrich cancer stem cells.
[113] All these 3D models have specific roles- Matrigel is a solubilized extract derived from mouse tail that contains ECM components and growth factors. Although cancer cells form spheroids/organoids in this 3D model, the mechanical properties cannot be altered to mimic the native tissue. Thus, present disclosure GelMA hydrogels of varying mechanical stiffness to represent the heterogeneous microenvironment, with the infiltrative edge of the tumor being the stiffer and the hypoxic/necrotic regions, and the normal tissue regions being mechanically softer. Therefore, the present disclosure chosen to work with hydrogels for two different stiffness: hydrogels stiffer than 5kPa represents the infiltrative edge, while hydrogels of stiffness less than 1kPa represent the normal tissue. In spheroid cultures, cancer cells are cultured in serum-free media enriched with growth factors in ultra-low attachment conditions, which have been shown to enrich for cancer-stem cells. These hydrogels will enrich for all neoplastic cell types present in a heterogeneous TME representing all the neoplastic cell types in order to assess response to chemotherapeutic drugs and radiotherapy.
[114] Following treatment with respective therapies (either radiotherapy or chemotherapy), the cells will be comprehensively assessed for the following parameters: (232).
[115] 1. The ability to form spheroids in 3D hydrogels (Morphological analysis).
[116] 2. TUNEL assay to determine the extent of DNA damage upon targeting with CT and RT.
[117] 3. Cytotoxicity assay (MTT/CCK-8 assay) for resistance index upon targeting with CT and RT.
[118] 4. Ki-67 stain is used to assess the proliferative index upon targeting with CT and RT.
[119] 5. Anti-cleaved caspase -3 assay for apoptosis upon targeting with CT and RT.
[120] A comprehensive analysis will be performed considering the response of the cancer cells on 3D models to CT and RT using different assays. Step 7 - A response score will be calculated that will be correlated with the patients’ clinical correlation matrix. (234). For novel drugs, this score will be generated across multiple 3D models to assess efficacy in HNSCC.
[121] 3. COMPOSITION OF DISCLOSED EMBODIMENT
[122] According to the aspect of the present disclosure, a composition for preparing a biomimetic 3D model for predicting a subject-specific response of progression, recurrence or complete remission of a condition, the composition comprises, (i) gelatin in an amount of xxx, (ii) 0.06 to 0.6g per 1g of (w/v) of methacrylic anhydride, (iii) PBS comprising a pH of 7.4, and (iv) 0.5 mg/mL of a photo-initiator comprising 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.
[123] A. Development and characterization of biomimetic 3D models
[124] Protocol optimization to develop soft (<1kPa) and stiff (>5kPa) 3D GelMA hydrogels.
[125] Characterization of the 3D models for mechanical stiffness, porosity, and diffusive properties.
[126] B. Assessment of cellular behaviour/molecular signature of cancer cells cultured on in-vitro 3D models
[127] Assessment of the cellular behaviour of cancer cells cultured on 3D models.
[128] Assessment of molecular signature of cancer cells cultured on 3D models and representation of intra-tumoral heterogeneity.
[129] C. Evaluation of cancer cells cultured on in-vitro 3D models to chemotherapeutic drugs
[130] RESULTS
[131] I. DEVELOPMENT AND CHARACTERIZATION OF BIOMIMETIC 3D MODELS
[132] The 3D hydrogels were prepared as per the protocol (FIG. 3), and optimized by tweaking the concentration of GelMA and the cross-linker13. Briefly, methacrylic anhydride (304) (SigmaAldrich, USA), pendants are added on to Gelatin (302) (type A, isolated from porcine skin; MP Biomedicals, USA), hydrolyzed Type1 collagen, dissolved in PBS (pH 7.4) stirred at 50°C for 1 hour, 0.06 to 0.6g per 1g of (w/v) of methacrylic anhydride will be added dropwise. The reaction will be aborted by the addition of double the quantity of PBS and dialyzed for 3 days against distilled water at 37 °C, followed by freeze-drying in a lyophilizer (Alpha 1-2/LD plus, Martin Christ, Germany) to obtain methacrylamide-modified gelatin as a dry white powder and stored at -20°C.
[133] Freeze-dried GelMA (306) will be dissolved in 1X PBS (308) at required concentration along with a photo- initiator (310) of 0.5 mg/mL of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone precursor solution (312) (Irgacure2959, Sigma-Aldrich, USA), and placed in culture vessels 24-well plate (314) (Eppendorf, USA), followed by 10 min of UV-365 (316) exposure to obtain a microporous 3D hydrogel (318) of methacrylamide-modified gelatin, as and when required. Hydrogels thus obtained will be equilibrated with 500µL of a culture medium (320) for 24 hours prior to seeding of the cells (322) for experiments.
[134] 4. METHOD FOR PREPARING A BIOMIMETIC 3D MODEL
[135] According to another aspect of the present disclosure, a method for predicting a subject-specific response of progression, recurrence or complete remission of a condition using biomimetic 3D models, the method comprises:
a. generating the biomimetic 3D models to culture cells, the biomimetic 3D models comprise (i) an organoid culture, the cells are grown in a biomimetic basal membrane extract, (ii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 0.3kPa to 0.6kPa forming soft hydrogels, (iii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 1.0kPa to 5.0kPa forming stiff hydrogels, (iv) a spheroid cultures, the cells are cultured with a serum-free medium under ultra-low attachment conditions, the cultured cells comprises in-vitro formed micro-tumors;
b. subjecting the biomimetic 3D models with the in-vitro formed micro-tumors to radiation ranging from 0 to 8gy in a single dose and a chemotherapeutic agent for about 72 hours,
c. determining (i) an ability of the cells to form spheroids in the hydrogels of the biomimetic 3D models by performing a morphological analysis, (ii) an extent of DNA damage of the cells by performing a TUNEL assay; (iii) a resistance index of the cells by performing a cytotoxicity assay, the cytotoxicity assay comprises MTT or CCK-8 assay, (iv) a proliferative index of the cells, the proliferative index of the cells are determined using Ki-67 stain, (v) an apoptosis rate by performing anti-cleaved caspace-3 assay;
d. the ability of the cells to form spheroids in the hydrogels of the biomimetic 3D models are determined by a morphological analysis, wherein the ability of the cells to form the spheroids is determined by measuring a time taken to form the spheroids, wherein the time taken to form spheroids is inversely proportional to an aggressiveness of the cells, wherein the aggressive cells form spheroids within 5-days from the day of seeding, wherein the less aggressive cells take about 7 days to form the spheroids,
e. the extent of DNA damage of the cells is determined by performing a TUNEL assay,
f. the resistance index of the cells is determined by performing a cytotoxicity assay, wherein the cytotoxicity assay comprises MTT or CCK-8 assay, wherein an inhibitory concentration 50 for the chemotherapeutic agent is calculated against a 2D model stored in the database, wherein the resistance index of greater than or equal to 3 is considered a non-responder;
g. the proliferative index of the cells is determined using Ki-67 stain, wherein the proliferative index is measured by stained the cells with Ki-67, wherein a value of 30% or above is considered as no response to the chemotherapeutic agent, wherein the value of greater than 30% score is considered a response to the chemotherapeutic agent,
h. the cellular apoptosis rate is determined by performing anti-cleaved caspace-3 assay, wherein the cellular apoptosis is generated by measuring the cleaved caspase-3 before and after the treatment with the chemotherapeutic agent, wherein cellular apoptosis rate is determined based on percentage of cells having the expression for cleaved caspase-3,
i. generating a response score of the cells based on the data related to the ability to form the spheroids, the extent of the DNA damage, the resistance index, the proliferative index, and the apoptosis rate; and
j. generating a nomogram based on the generated response score of the cells and predicting the subject-specific response of progression, recurrence or complete remission of the condition, wherein the subject-specific response is predicted based on the response score of the cells along with habit history, clinic-pathological staging of the subject.
[136] According to one another aspect of the present disclosure, the condition is head and neck squamous cell carcinoma.
[137] According to the yet another aspect of the present disclosure, the cells comprise neoplastic cells, fibroblasts, immune cells and endothelial cells, the cells are obtained from the subject with head and neck squamous cell carcinoma, the cells comprise Cal27, HSC3 or other HNSCC cell lines.
[138] According to the one aspect of the present disclosure, the chemotherapeutic agent comprises a concentration ranging from nano molar concentrations to micro molar, wherein the chemotherapeutic agent is selected from Cisplatin (platinum-based drugs), 5-fluro uracil, and Taxol.
[139] According to the further aspect the present disclosure, the biomimetic 3D models are subjected to the chemotherapeutic agent along with one or more therapeutic drugs selected from ALDH1 inhibitor/NCT-501, Notch1 inhibitor/begacestat, PLD2 inhibitor/ML298, mTOR inhibitor/rapamycin, methotrexate, celecoxib, immunotherapy drugs (anti-PD1, anti-PDL1, anti-CTLA4), Celecoxib, targeted therapies (anti-EGFR, anti-mTOR, anti-VEGF, anti-PI3K, anti-CDKs inhibitors of the PI3-AKT, JAK-STAT, RAS-RAF, anti-NOTCH, anti-FGFR, anti-MET, anti-RET pathways) or combinations thereof.
[140] 5. METHOD FOR PREDICTING A SUBJECT-SPECIFIC RESPONSE
[141] According to the one aspect of the present disclosure, the biomimetic 3D model is prepared by,
a. dissolving gelatin in PBS comprising a pH of 7.4 and stirring at 50°C for 1 hour to obtain a first mixture;
b. adding 0.06 to 0.6g per 1g of (w/v) of methacrylic anhydride dropwise to the first mixture;
c. adding the PBS to the first mixture comprising the methacrylic anhydride to stop the reaction and dialyzing for 3 days with distilled water at 37 °C to obtain a second mixture;
d. freeze-drying the second mixture in a lyophilizer to obtain a methacrylamide-modified gelatin as a dry white powder and storing at -20°C;
e. dissolving the methacrylamide-modified gelatin in 1X PBS at a concentration of 5% - 10% along with a photo-initiator comprising 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone of 0.5 mg/mL to obtain a third mixture; and
f. placing the third mixture in culture vessels followed by 10 minutes of UV-365 exposure to obtain a microporous 3D hydrogel of the methacrylamide-modified gelatin forming the biomimetic 3D model.
[142] According to the one aspect of the present disclosure, the microporous 3D hydrogel of the methacrylamide-modified gelatin is equilibrated with 500µL of a culture medium for 24 hours before performing seeding of the cells.
[143] e. generating a nomogram based on the generated response score of the cells and predicting the subject-specific response of progression, recurrence or complete remission of the condition.
[144] Present disclosure has worked with three patient samples B12, M08, and P7 as listed below. All the three patient samples are from oral cavity (Tongue and alveolus) from advanced pathological conditions.
[152] To assess the response of chemotherapy in patient samples, patient-derived cells B12, M08, and P7 were isolated and were treated with various doses of standard chemotherapeutic agents (Cisplatin, 5-fluro uracil) and novel therapeutic agents (ALDH1 inhibitor/NCT-501, Notch1 inhibitor/begacestat, PLD2 inhibitor/ML298, mTOR inhibitor/rapamycin). The treated cells were then incubated for 72-h at normal culture conditions. Then to assess the fraction of cell survival, MTT assay was performed, and the IC50 was derived and calculated as given in table-5. All the three patient-derived cells showed an aggressive behaviour to standard chemotherapeutic agent cisplatin, clinically.
[145] Example of P07-3D model
a. Even though we see P07 cells are sensitive to cisplatin on 2D platform, which is not a representative of clinical response (FIG. 9). We see an increase in the resistance index (>23X) on 3D platform, supporting the fact that our 3D hydrogels models are better representative of the in-vivo physiology and are able to mimic the patients’ clinical response (FIG.9).
b. The morphological analysis of patient-derived OSCC cells cultured on 3D GelMA hydrogels differ from the cells cultured on 2D platform. Further, it can be seen that the cells cultured on 3D-soft hydrogels demonstrate a rounded spheroid morphology with no elongation (Figure-14). Whereas, cells cultured on 3D-stiff hydrogels show a change in morphological changes as they try to spread and migrate out of the spheroids, demonstrating the aggressive phenotype of cancer (FIG. 9).
c. Further, to assess the role of 3D GelMA hydrogels as a drug testing platform, the patient-derived OSCC cells (P7) were cultured on 3D-stiff, and 3D-soft GelMA hydrogels. These cells were treated with standard chemotherapeutic drugs such as cisplatin (platinum-based drugs), and 5-FU, and with novel therapeutic agents such as NCT-501 (ALDH1 inhibitor), Begacestat (Notch1 inhibitor), ML298 (PLD2 inhibitor), and rapamycin (mTOR inhibitor). The drug panel can also include include taxol, immunotherapy drugs (anti-PD1, anti-PDL1, anti-CTLA4), Celecoxib, methotrexate, targeted therapies (anti-EGFR, anti-mTOR, anti-VEGF, anti-PI3K, anti-CDKs inhibitors of the PI3-AKT, JAK-STAT, RAS-RAF, anti-NOTCH, anti-FGFR, anti-MET, anti-RET pathways) or combinations thereof. The patient-derived cells cultured on 3D GelMA hydrogels of varying mechanical stiffness demonstrated differing response in contrast to cells cultured on 2D platform.
d. Both cisplatin and 5-FU were inefficient in arresting in the proliferation of patient-derived OSCC cells, as the resistance index of cells cultured on 3D hydrogels increased significantly (23X on 3D-stiff, and 9X on 3D-soft for cisplatin). Whereas, the cells treated with PLD2 inhibitor and Notch1 inhibitors were efficient in arresting the proliferation of patient-derived OSCC cells on both 3D-soft, and 3D-stiff hydrogels, with the resistance index of cells cultured on 3D hydrogels =1 (0.92X on 3D-stiff, and 0.38X on 3D-soft for ML298, a PLD2 inhibitor).
[158] Further we explored the scope of combinatorial therapy. We observed that the treatment of cisplatin along with novel therapeutic agents (with IC50 of 2D platform) NCT-501 (ALDH1 inhibitor), Begacestat (Notch1 inhibitor), and ML298 (PLD2 inhibitor) decreases the IC50 of cisplatin displaying the significance of combinatorial therapy. Likewise, the IC50 of mTOR inhibitor is significantly reduced in response to treatment along with ML298 (PLD2 inhibitor). The resistance index of cisplatin on 3D hydrogels reduced to 10X from 23X on 3D-stiff hydrogels. In case of rapamycin, a mTOR inhibitor, PLD2 inhibitor was able to successfully sensitize the OSCC cells for rapamycin treatment as the resistance index reduced from 72X to 2X on 3D-stiff hydrogels.
a. P07-3D model predicted a no-response to platinum-based drugs. However, the patient model indicated sensitivity to PLD2i and Notch inhibitor and to combinatorial therapy.
b. In correlation with patient clinical response, a clinical progression of the disease to nodal metastasis indicated a lack of response to platinum-based therapy.
f. assessing the response of novel drugs across multiple 3D models to assess the efficacy in HNSCC. In this case the score as mentioned before will be assessed across multiple patient-derived models and efficacy of the drugs in the models will be assessed based on the score of >80% of the models; drugs with efficacy in >80% of 3D models will be considered validated in the platform.
[146] 6. EXAMPLE EMBODIMENT
[147] The disclosed embodiment will be further described in the following examples. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this disclosed embodiment in any manner.
[148] EXAMPLE 1: CHARACTERIZATION OF THE PHYSICAL PROPERTIES OF GELMA
[149] The GelMA hydrogels prepared were characterized to be porous in nature of dry GelMA scaffolds using scanning electron microscopy (SEM) (FIG- 4a, 4c) and of live cells cultured in GelMA hydrogels using confocal microscopy (FIG- 4d). The GelMA hydrogels are mechanically tunable, 10% and 15% GelMA hydrogels demonstrate a mechanical stiffness of 5kPa and 20kPa respectively (FIG-4 b).
[150] EXAMPLE: 2 PHENOTYPIC PROPERTIES OF OSCC CELL LINES CULTURED ON 2D TCPS AND GELMA HYDROGELS OF VARYING MECHANICAL STIFFNESS
[151] Results
[152] Next, present disclosure cultured OSCC cell lines Cal27, and HSC3 on both soft and stiff 3D hydrogels. Cal 27 cells form proper spheroid on stiff hydrogels whereas they form smaller spheroids on soft hydrogels with some cells elongated and trying to migrate out of the hydrogels. In contrast, HSC3 cells form proper spheroids on soft hydrogels whereas they form smaller spheroids on stiff hydrogels with some cells elongated with fibroblastic-like structures and trying to migrate out of the hydrogels (FIG. 5A). This change in cellular behavior explains not just the cell-extrinsic property (mechanical property of the ECM), but also the cell-intrinsic properties (mutational status of the cell) influence the behavior of the cancer cells which are being captured in the 3D hydrogels). Both the cells cultured in 3D hydrogels (both soft and stiff) display a higher proliferate index when compared to cells cultured on 2D platform. The cells cultured on 2D attain confluency by 4 days whereas, in 3D cells keep proliferating even upto day-18 (beyond day-18 is not assessed) (FIG. 5B, 5C).
[153] EXAMPLE 3: OSCC CELL LINES CAL27 AND HSC3 BEHAVE DIFFERENTLY UPON VARYING THE MECHANICAL PROPERTY OF 3D GELMA
[154] Next, the OSCC cell line Cal27, and HSC3 cultured on 3D hydrogels soft and stiff were treated with chemotherapeutic drug Docetaxel, Cisplatin, and 5-FU for 72hrs and MTT was performed to calculate the percentage cell viability. It was found that the cells cultured in 3D hydrogels (soft, and stiffness) are less sensitive to the chemotherapeutic drug in contrast to cells cultured in 2D platform (FIG. 6A). The Cal27 cells cultured on stiff hydrogels were less sensitive to the chemotherapeutic drugs than the cells cultured in soft hydrogels. In contrast, the HSC3 cells cultured on soft hydrogels were less sensitive to the chemotherapeutic drugs than the cells cultured in stiff hydrogels (FIG-6B). The change in the mechanical stiffness of the 3D hydrogels influences the cells response to different chemotherapeutic drugs.
[155] EXAMPLE 4: RADIO-RESPONSE OF PATIENT-DERIVED ORAL CANCER CELLS CULTURED ON 2D PLATFORM
[156] Further, to assess the response of radiotherapy in patient samples, patient-derived cells were isolated and were treated with various doses of ionization radiation (0.5Gy, 1Gy, 2Gy, 4Gy, 6Gy, and 8Gy). The treated cells were then incubated for 72-h at normal culture conditions. This allowed the cell death of affected cells and growth of the radioresistant cells. Then to assess the fraction of cell survival, MTT assay was performed, and the IC50 was derived. For the above patient-derived cells, the IC50 was calculated to be 15.35Gy (N=1). (FIG. 7).
[157] TABLE 1 PATIENT DETAILS AND CLINICAL RESPONSE
SI. NO. Pathological stage Clinical response
1 B12 Pt3n3bMx Patient expired
2 M08 pT4N1Mx Developed lung disease
3 P7 pT3N3b Patient expired
[158] EXAMPLE 5: CHEMO-RESPONSE OF PATIENT-DERIVED OSCC CELLS CULTURED UPON 2D PLATFORM
[159] Further, to assess the response of chemotherapy in patient samples, patient-derived cells B12 (FIG.8A), M08 (FIG. 8B), and P7 (FIG.8C) were isolated and were treated with various doses of chemotherapeutic agents (Cisplatin, 5-fluro uracil, ALDH1 inhibitor/NCT-501, Notch1 inhibitor/begacestat, PLD2 inhibitor/ML298, mTOR inhibitor/rapamycin). The treated cells were then incubated for 72-h at normal culture conditions. Then to assess the fraction of cell survival, MTT assay was performed, and the IC50 was derived and calculated as given in (FIG.8). All the three patient-derived cells showed an aggressive behavior to standard chemotherapeutic agent cisplatin.
[160] EXAMPLE 6: PHENOTYPIC PROPERTIES OF PATIENT-DERIVED OSCC CELLS CULTURED ON 2D TCPS AND GELMA HYDROGELS OF VARYING MECHANICAL STIFFNESS
[161] The morphological analysis of patient-derived OSCC cells cultured on 3D GelMA hydrogels differ from the cells cultured on 2D platform. Further, it can be seen that the cells cultured on 3D-soft hydrogels demonstrate a rounded spheroid morphology with no elongation (Figure-14). Whereas, cells cultured on 3D-stiff hydrogels show a change in morphological changes as they try to spread and migrate out of the spheroids, demonstrating the aggressive phenotype of cancer (FIG. 9).
[162] EXAMPLE 7: CHEMO-RESPONSE OF PATIENT-DERIVED OSCC CELLS CULTURED UPON 2D, 3D GELMA HYDROGELS OF VARYING THE MECHANICAL PROPERTY
[163] The chemo-response of patient-derived OSCC cells cultured upon 2D, 3D GelMA hydrogels of varying the mechanical property. Effect of chemotherapeutic drugs Cisplatin, 5-FU, NCT-501, Notch1 inhibitor, PLD2 inhibitor, and mTOR inhibitor on the viability of patient-derived OSCC cells P7 cultured under 2D, 3D-soft, and 3D-stiff conditions was monitored using MTT assay, cells incubated with various concentrations of chemotherapeutic drugs for 72-h were analyzed. (FIG. 10).
[164] EXAMPLE 8: CHEMO-RESPONSE OF PATIENT-DERIVED OSCC CELLS CULTURED UPON 2D, 3D GELMA HYDROGELS OF VARYING THE MECHANICAL PROPERTY.
[165] Further, to assess the role of 3D GelMA hydrogels as a drug testing platform, the patient-derived OSCC cells (P7) were cultured on 3D GelMA stiff, and 3D GelMA soft hydrogels. These cells were treated with standard chemotherapeutic drugs such as cisplatin, and 5-FU, and with novel chemotherapeutic agents such as NCT-501 (ALDH1 inhibitor), Begacestat (Notch1 inhibitor), ML298 (PLD2 inhibitor), and rapamycin (mTOR inhibitor). The patient-derived cells cultured on 3D GelMA hydrogels of varying mechanical stiffness demonstrated differing response in contrast to cells cultured on 2D platform (FIG. 11A).
[166] Both cisplatin and 5-FU were inefficient in arresting in the proliferation of patient-derived OSCC cells. Whereas, the cells treated with PLD2 inhibitor and Notch1 inhibitors were efficient in arresting the proliferation of patient-derived OSCC cells on both 3D-soft, and 3D-stiff hydrogels (FIG. 11B).
[167] EXAMPLE 9: CHEMO-RESPONSE OF PATIENT-DERIVED OSCC CELLS CULTURED UPON 2D, 3D GELMA HYDROGELS OF VARYING THE MECHANICAL PROPERTY TO COMBINATION THERAPY
[168] Further exploring the scope of combinatorial therapy, we treated the patient-derived OSCC cells with chemotherapeutic drugs in combination with novel therapeutic agents mentioned above. We observed that the treatment of cisplatin along with novel therapeutic agents (with IC50 of 2D platform) NCT-501 (ALDH1 inhibitor), Begacestat (Notch1 inhibitor), and ML298 (PLD2 inhibitor) decreases the IC50 of cisplatin displaying the significance of combinatorial therapy. Likewise, the IC50 of mTOR inhibitor is significantly reduced in response to treatment along with ML298 (PLD2 inhibitor) (FIG. 12A and 12B).
[169] The established primary cells (homologous epithelial and fibroblast) will be cultured on to different 3D models including GelMA hydrogels of different stiffness and cultured for 10 days.
[170] EXAMPLE 10: REPRESENTATION OF THE WORK FLOW FOR TESTING NOVEL CHEMOTHERAPEUTIC DRUGS
[171] The established primary cells (homologous epithelial and fibroblast) will be cultured (1302) on to different 3D models (1306, 1308, 1310, 1312) including GelMA hydrogels of different stiffness and culture platforms (1304) for 10 days.
[172] This allows the primary cells to form spheroids/micro-tumors in-vitro that are subjected to chemotherapy (upto 72hrs) using novel chemotherapeutic drugs. The chemotherapeutic treatment can also be given in combination to radiotherapy (0-8gy) to test the efficacy of the chemotherapeutic drugs. (Novel chemotherapeutic agents, repurposed drugs, compounds of interest alone or in combination with RT) (1314). The chemotherapeutic agent will be one or more therapeutic drugs selected from ALDH1 inhibitor/NCT-501, Notch1 inhibitor/begacestat, PLD2 inhibitor/ML298, mTOR inhibitor/rapamycin, celecoxib, immunotherapy drugs (anti-PD1, anti-PDL1, anti-CTLA4), Celecoxib, targeted therapies (anti-EGFR, anti-mTOR, anti-VEGF, anti-PI3K, anti-CDKs inhibitors of the PI3-AKT, JAK-STAT, RAS-RAF, anti-NOTCH, anti-FGFR, anti-MET, anti-RET pathways) or combinations thereof.
[173] Further, the cells will be assessed for various parameters as listed in (FIG. 2), (1316, 1318, 1322) and a drug-sensitivity score will be calculated. The drug-sensitivity score corresponds to the ability of the chemotherapeutic drug to shrink the micro-tumors. (FIG.13).
[174] The 3D model can be a pre-clinical drug testing platform, which can indicate the actual response of the drugs in a biomimicking model across multiple patient-derived 3D models
[175] 7. HARDWARE
[176] Digital Processing System
[177] (FIG. 14) is a block diagram illustrating the details of a digital processing system (1400) in which various aspects of the present disclosure are operative by execution of appropriate execution modules, firmware or hardware components.
[178] Digital processing system (1400) 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) (1402)), random access memory (RAM) (1404), secondary memory (1406), graphics controller (GPU) (1412), display unit (1414), network interfaces like (WLAN) (1416), and input interfaces (1418).
[179] CPU (1402) executes instructions stored in RAM (1404) to provide several features of the present disclosure. CPU (1402) may contain multiple processing units, with each processing unit potentially being designed for a specific task.
[180] Alternatively, CPU (1402) may contain only a single general purpose processing unit. RAM (1404) may receive instructions from secondary/system memory (1410).
[181] Graphics controller (GPU) (1412) generates display signals (e.g., in RGB format) to primary display unit (1414) based on data/instructions received from CPU (1402). Primary display unit contains a display screen (1414) (e.g., monitor, touchscreen) to display the images defined by the display signals. Input interfaces (1418) 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 (1416) provides connectivity to a network (e.g., using Internet Protocol), and may be used to communicate with other connected systems. Network interface (1416) 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).
[182] Secondary memory (1406) may contain hard drive (mass storage) (1406a), flash memory (1406b), and removable storage drive (1406c). Secondary memory (1406) may store the data (e.g., the specific requests sent, the responses received, etc.) and executable modules, which enable the digital processing system (1400) to provide several features in accordance with the present disclosure.
[183] Some or all of the data and instructions may be provided on a removable storage unit (SD card) (1408), and the data and instructions may be read and provided by removable storage drive (1406c) to CPU (1402). 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. (1406c).
[184] Removable storage unit (1408) may be implemented using storage format compatible with removable storage drive (1406c) such that removable storage drive (1406c) can read the data and instructions. Thus, removable storage (1406c) 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 (1402) may retrieve the executable modules, and execute them to provide various features of the present disclosure described above.
[185] 8. IMPORTANT ATTRIBUTES OF THE PRESENT DISCLOSURE
[186] 3D model as a tumor-mimicking model in HNSCC.
[187] Prediction model (The 3D model has a higher resistance, IC50 as compared to the 2D).
[188] Currently available prediction models
[189] At present, clinical response of patient to chemotherapeutic drugs and radiation therapy can be divided majorly into two types: a.) Based on transcriptomic gene signature b.) Protein/peptide detection in saliva c.) Based on ex-vivo culture of tumor tissue. None of these studies have been extensively adapted into clinical practice due to the cost and loss of tumor tissue due to viability issues. These ex-vivo based models majorly assess the live/dead population upon targeting which may not give the exact result.
[190] 9. ADVANTAGES OF THE DISCLOSED EMBODIEMNT
[191] In the present disclosure, the assay/product can be used to culture cancer cells derived from tumor samples and predict the patients’ clinical outcome to both radiotherapy and chemotherapy. This information can be used to guide clinicians in making the decision on therapeutic strategy. The patient can opt for a precise therapeutic regimen resulting in befitting therapy in a personalized manner.
[192] The major advantages of this model/assay are:
[193] A comprehensive analysis of the tumor tissue was performed in order to predict the patients’ clinical response of the therapy.
[194] Assessment was performed on various parameters such as cell viability, proliferative index, DNA damage, apoptosis, and stemness of tumor cells.
[195] Disclosure model uses a method to include both epithelial and fibroblastic cell types along with mechanical stiffness parameter, which better mimics the in vivo tumor.
[196] Unlike other models, multiple platforms were compared to culture tumor cells, which enrich for all the neoplastic cell types present in the in-vivo tumor.
[197] The model enables to enrich, and isolate different neoplastic cells such as to cell-cycling (G1/S, and G2/M), partial EMT cells, terminally differentiated epithelial cells, cells under stress, cells under hypoxia, and cells with phenotypes such as chemoresistance, chemosensitive, invasive, etc.
[198] The assay can be used to test the chemotherapeutic drugs, radiation therapy, or in combination to predict the clinical response of the patient.
[199] The assay can be used to test the chemotherapeutic drugs, radiation therapy, or both to predict the patients’ clinical response.
[200] 10. USES, APPLICATIONS AND BENEFITS OF THE DISCLOSED EMBODIEMNT
[201] The assay involves culturing cancer cells derived from tumor samples and predict the patients’ clinical outcome to both radiotherapy and chemotherapy. This information can be used to guide clinicians in making the decision on therapeutic strategy. The patient can opt for a precise therapeutic regimen resulting in befitting therapy in a personalized manner.
[202] The assay/product can be used to predict the overall survival of the patient post radio or chemotherapy.
[203] The assay/product can predict the disease-free survival and recurrence of the tumor in the patients.
[204] The model enables to enrich, and isolate different neoplastic cells such as to cell-cycling (G1/S, and G2/M), partial EMT cells, terminally differentiated epithelial cells, cells under stress, cells under hypoxia, and cells with phenotypes such as chemoresistance, chemo sensitive, invasive, and incorporates the mechanical stiffness modulation.
[205] The gene signature can be used to identify different neoplastic cells present in the respective tumor microenvironment.
[206] The assay can be used to test the chemotherapeutic drugs, radiation therapy, or both to predict the patients’ clinical response.
[207] The assay/product described above will reduce the morbidity associated with therapy resistance (both radio and chemo) in addition to decreasing the economic burden associated with both radiotherapy and chemotherapy.
[208] 11. BEST MODE TO PRACTICE THE DISCLOSED EMBODIEMNT
[209] A. The assay can be used to test the chemotherapeutic drugs, radiation therapy, or in combination to predict the clinical response of the patient.
[210] The patient’s tumor sample is collected on the day of surgery cleaned and decontaminated. The tissue is minced and subjected to enzymatic degradation to form single cell suspension. The cells are passes through 70-micron filter to remove the debris. The single cells thus obtained are counted and seeded on to different 3D models including GelMA hydrogels of different stiffness and cultured for 7-10 days, varying from patient-to-patient. This allows the cells to form spheroids/micro-tumors in-vitro, that are subjected to chemotherapy (upto 72hrs) or radiotherapy (0-8gy). Further, the cells will be assessed for various parameters as listed in (FIG. 2), and a response score is calculated. The response score is compared with the patients’ clinical response.
[211] The patients’ test report includes the response of tumor sample to tested chemotherapeutic drugs, radiotherapy, and a calculated chances of survival beyond 5-years (FIG. 12). The established primary cells (homologous epithelial and fibroblast) will be cultured on to different 3D models including GelMA hydrogels of different stiffness and cultured for 10 days. This allows the primary cells to form spheroids/micro-tumors in-vitro that are subjected to chemotherapy (upto 72 hrs) using novel chemotherapeutic drugs. The chemotherapeutic treatment can also be given in combination to radiotherapy (0-8gy) to test the efficacy of the chemotherapeutic drugs. Further, the cells will be assessed for various parameters as listed in (FIG. 2), and a drug-sensitivity score will be calculated (FIG. 13). The drug-sensitivity score corresponds to the ability of the chemotherapeutic drug to shrink the micro-tumors. As a pre-clinical platform, the report will include the drug-sensitivity score across multiple patient-derived 3D models.
[212] 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.
[213] While specific embodiments of the disclosure have been shown and described in detail to illustrate the inventive principles, it will be understood that the disclosure may be embodied otherwise without departing from such principles.
[214] 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.
[215] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present disclosure are presented for example purposes only. The present disclosure is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.
[216] 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. B, E., J, B., Y, J., B, J. & T, S. Biophysics of Tumor Microenvironment and Cancer Metastasis - A Mini Review. Comput. Struct. Biotechnol. J. 16, 279–287 (2018).
2. Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 57–70 (2000).
3. Malik, R., Lelkes, P. I. & Cukierman, E. Biomechanical and biochemical remodeling of stromal extracellular matrix in cancer. Trends in Biotechnology 33, 230–236 (2015).
4. Miroshnikova, Y. A. et al. Tissue mechanics promote IDH1-dependent HIF1a-tenascin C feedback to regulate glioblastoma aggression. Nat. Cell Biol. 18, 1336–1345 (2016).
5. Acerbi, I. et al. Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration. Integr. Biol. (United Kingdom) 7, 1120–1134 (2015).
6. Handorf, A. M., Zhou, Y., Halanski, M. A. & Li, W. J. Tissue stiffness dictates development, homeostasis, and disease progression. Organogenesis 11, 1–15 (2015).
7. Mierke, C. T. et al. The two faces of enhanced stroma: Stroma acts as a tumor promoter and a steric obstacle. NMR Biomed. 31, (2018).
8. Zhang, B. et al. Tissue mechanics and expression of TROP2 in oral squamous cell carcinoma with varying differentiation. doi:10.1186/s12885-020-07257-7
9. Lyssiotis, C. A. & Kimmelman, A. C. Metabolic Interactions in the Tumor Microenvironment. Trends Cell Biol. 27, 863–875 (2017).
10. Hachey, S. J. & Hughes, C. C. W. Applications of tumor chip technology. Lab on a Chip 18, 2893–2912 (2018).
11. Wolf, K. J., Chen, J., Coombes, J. D., Aghi, M. K. & Kumar, S. Dissecting and rebuilding the glioblastoma microenvironment with engineered materials. Nature Reviews Materials 4, 651–668 (2019).
12. Rape, A., Ananthanarayanan, B. & Kumar, S. Engineering strategies to mimic the glioblastoma microenvironment. Advanced Drug Delivery Reviews 79, 172–183 (2014).
13. Arya, A. D. et al. Gelatin Methacrylate Hydrogels as Biomimetic Three-Dimensional Matrixes for Modeling Breast Cancer Invasion and Chemoresponse in Vitro. ACS Appl. Mater. Interfaces 8, (2016).
14. Shah, N. et al. Gelatin methacrylate hydrogels culture model for glioblastoma cells enriches for mesenchymal-like state and models interactions with immune cells. Sci. Reports 2021 111 11, 1–18 (2021).

,CLAIMS:I/WE CLAIM:
1. A system for predicting a subject-specific response of progression, recurrence or complete remission of a condition using biomimetic 3D models, wherein the system comprises:
the biomimetic 3D models to culture cells, wherein the biomimetic 3D models comprise (i) an organoid culture, wherein the cells are grown in a biomimetic basal membrane extract, (ii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 0.3kPa to 0.6kPa forming soft hydrogels, (iii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 1.0kPa to 5.0kPa forming stiff hydrogels, (iv) a spheroid cultures, wherein the cells are cultured with a serum-free medium under ultra-low attachment conditions, wherein the cultured cells comprises in-vitro formed micro-tumors, wherein the biomimetic 3D models comprising the in-vitro formed micro-tumors are subjected to a radiation ranging from 0 to 8gy in a single-dose and a chemotherapeutic agent for about 72 hours,
a server, wherein the server stores instructions in a database to perform the following:
obtaining from an external device, data related to an ability of the cells to form the spheroids, an extent of the DNA damage, a resistance index, a proliferative index, and an apoptosis rate of the cells on the biomimetic 3D models after the treatment with the radiation and the chemotherapeutic agent,
wherein the ability of the cells to form spheroids in the hydrogels of the biomimetic 3D models is determined by a morphological analysis, wherein the ability of the cells to form the spheroids is determined by measuring a time taken to form the spheroids, wherein the time taken to form spheroids is inversely proportional to an aggressiveness of the cells, wherein the aggressive cells form spheroids within 5-days from the day of seeding, wherein the less aggressive cells take about 7 days to form the spheroids,
wherein the extent of DNA damage of the cells is determined by performing a TUNEL assay,
wherein the resistance index of the cells is determined by performing a cytotoxicity assay, wherein the cytotoxicity assay comprises MTT or CCK-8 assay, wherein an inhibitory concentration 50 for the chemotherapeutic agent is calculated against a 2D model, wherein the resistance index of greater than or equal to 3 is considered a non-responder;
wherein the proliferative index of the cells is determined using Ki-67 stain, wherein the proliferative index is measured by stained the cells with Ki-67, wherein a value of 30% or above is considered as no response to the chemotherapeutic agent, wherein the value of lesser than 30% score is considered a response to the chemotherapeutic agent,
wherein the cellular apoptosis rate is determined by performing anti-cleaved caspace-3 assay, wherein the cellular apoptosis is generated by measuring the cleaved caspase-3 before and after the treatment with the chemotherapeutic agent, wherein cellular apoptosis rate is determined based on percentage of cells having the expression for cleaved caspase-3,
generating a response score of the cells based on the data related to the ability to form the spheroids, the extent of the DNA damage, the resistance index, the proliferative index, and the apoptosis rate; and
generating a nomogram based on the generated response score of the cells and predicting the subject-specific response of progression, recurrence or complete remission of the condition, wherein the subject-specific response is predicted based on the response score of the cells along with habit history, clinic-pathological staging of the subject.
2. The system as claimed in claim 1, wherein the condition is head and neck squamous cell carcinoma.
3. The system as claimed in claim 1, wherein the cells comprise neoplastic cells, fibroblasts, immune cells and endothelial cells, wherein the cells are obtained from the subject with head and neck squamous cell carcinoma, wherein the cells comprise Cal27, HSC3 or other HNSCC cell lines.
4. The system as claimed in claim 3, wherein the chemotherapeutic agent comprises a concentration ranging from 1nM to 1mM, wherein the chemotherapeutic agent is selected from Cisplatin (platinum-based drugs), 5-fluro uracil, and Taxol.
5. The system as claimed in claim 1, wherein the cells cultured in the biomimetic 3D models are subjected to the chemotherapeutic agent along with one or more therapeutic drugs selected from ALDH1 inhibitor/NCT-501, Notch1 inhibitor/begacestat, PLD2 inhibitor/ML298, mTOR inhibitor/rapamycin, methotrexate, celecoxib, immunotherapy drugs (anti-PD1, anti-PDL1, anti-CTLA4), Celecoxib, targeted therapies (anti-EGFR, anti-mTOR, anti-VEGF, anti-PI3K, anti-CDKs inhibitors of the PI3-AKT, JAK-STAT, RAS-RAF, anti-NOTCH, anti-FGFR, anti-MET, anti-RET pathways) or combinations thereof.
6. The system as claimed in claim 1, wherein the biomimetic 3D model is prepared by,
dissolving gelatin in PBS comprising a pH of 7.4 and stirring at 50°C for 1 hour to obtain a first mixture;
adding 0.06 to 0.6g per 1g of (w/v) of methacrylic anhydride dropwise to the first mixture;
adding the PBS to the first mixture comprising the methacrylic anhydride to stop the reaction and dialyzing for 3 days with distilled water at 37 °C to obtain a second mixture;
freeze-drying the second mixture in a lyophilizer to obtain a methacrylamide-modified gelatin as a dry white powder and storing at -20°C;
dissolving the methacrylamide-modified gelatin in 1X PBS at a concentration of 5% - 10% along (to modulate the stiffness) with a photo-initiator comprising 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone of 0.5 mg/mL to obtain a third mixture; and
placing the third mixture in culture vessels followed by 10 minutes of UV-365 exposure to obtain a microporous 3D hydrogel of the methacrylamide-modified gelatin forming the biomimetic 3D model.
7. The system as claimed in claim 1, wherein the microporous 3D hydrogel of the methacrylamide-modified gelatin is equilibrated with 500µL of a culture medium for 24 hours before performing seeding of the cells.
8. A method for predicting a subject-specific response of progression, recurrence or complete remission of a condition using biomimetic 3D models, wherein the method comprises:
generating the biomimetic 3D models to culture cells, wherein the biomimetic 3D models comprise (i) an organoid culture, wherein the cells are grown in a biomimetic basal membrane extract, (ii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 0.3kPa to 0.6kPa forming soft hydrogels, (iii) a gelatin-based biomimetic hydrogels with mechanical stiffness ranging from 1.0kPa to 5.0kPa forming stiff hydrogels, (iv) a spheroid cultures, wherein the cells are cultured with a serum-free medium under ultra-low attachment conditions, wherein the cultured cells comprises in-vitro formed micro-tumors;
subjecting the biomimetic 3D models with the in-vitro formed micro-tumors to radiation ranging from 0 to 8gy in a single dose and a chemotherapeutic agent for about 72 hours,
determining (i) an ability of the cells to form spheroids in the hydrogels of the biomimetic 3D models by performing a morphological analysis, (ii) an extent of DNA damage of the cells by performing a TUNEL assay; (iii) a resistance index of the cells by performing a cytotoxicity assay, wherein the cytotoxicity assay comprises MTT or CCK-8 assay, (iv) a proliferative index of the cells, wherein the proliferative index of the cells are determined using Ki-67 stain, (v) an apoptosis rate by performing anti-cleaved caspace-3 assay;
wherein the ability of the cells to form spheroids in the hydrogels of the biomimetic 3D models are determined by a morphological analysis, wherein the ability of the cells to form the spheroids is determined by measuring a time taken to form the spheroids, wherein the time taken to form spheroids is inversely proportional to an aggressiveness of the cells, wherein the aggressive cells form spheroids within 5-days from the day of seeding, wherein the less aggressive cells take about 7 days to form the spheroids,
wherein the extent of DNA damage of the cells is determined by performing a TUNEL assay,
wherein the resistance index of the cells is determined by performing a cytotoxicity assay, wherein the cytotoxicity assay comprises MTT or CCK-8 assay, wherein an inhibitory concentration 50 for the chemotherapeutic agent is calculated against a 2D model stored in the database, wherein the resistance index of greater than or equal to 3 is considered a non-responder;
wherein the proliferative index of the cells is determined using Ki-67 stain, wherein the proliferative index is measured by stained the cells with Ki-67, wherein a value of 30% or above is considered as no response to the chemotherapeutic agent, wherein the value of greater than 30% score is considered a response to the chemotherapeutic agent,
wherein the cellular apoptosis rate is determined by performing anti-cleaved caspace-3 assay, wherein the cellular apoptosis is generated by measuring the cleaved caspase-3 before and after the treatment with the chemotherapeutic agent, wherein cellular apoptosis rate is determined based on percentage of cells having the expression for cleaved caspase-3,
generating a response score of the cells based on the data related to the ability to form the spheroids, the extent of the DNA damage, the resistance index, the proliferative index, and the apoptosis rate; and
generating a nomogram based on the generated response score of the cells and predicting the subject-specific response of progression, recurrence or complete remission of the condition, wherein the subject-specific response is predicted based on the response score of the cells along with habit history, clinic-pathological staging of the subject.
9. The method as claimed in claim 8, wherein the condition is head and neck squamous cell carcinoma.
10. The method as claimed in claim 8, wherein the cells comprise neoplastic cells, fibroblasts, immune cells and endothelial cells, wherein the cells are obtained from the subject with head and neck squamous cell carcinoma, wherein the cells comprise Cal27, HSC3 or other HNSCC cell lines.
11. The method as claimed in claim 8, wherein the chemotherapeutic agent comprises a concentration ranging from nano molar concentrations to micro molar, wherein the chemotherapeutic agent is selected from Cisplatin (platinum-based drugs), 5-fluro uracil, and Taxol.
12. The method as claimed in claim 8, wherein the biomimetic 3D models are subjected to the chemotherapeutic agent along with one or more therapeutic drugs selected from ALDH1 inhibitor/NCT-501, Notch1 inhibitor/begacestat, PLD2 inhibitor/ML298, mTOR inhibitor/rapamycin, methotrexate, celecoxib, immunotherapy drugs (anti-PD1, anti-PDL1, anti-CTLA4), Celecoxib, targeted therapies (anti-EGFR, anti-mTOR, anti-VEGF, anti-PI3K, anti-CDKs inhibitors of the PI3-AKT, JAK-STAT, RAS-RAF, anti-NOTCH, anti-FGFR, anti-MET, anti-RET pathways) or combinations thereof.
13. The method as claimed in claim 8, wherein the biomimetic 3D model is prepared by,
dissolving gelatin in PBS comprising a pH of 7.4 and stirring at 50°C for 1 hour to obtain a first mixture;
adding 0.06 to 0.6g per 1g of (w/v) of methacrylic anhydride dropwise to the first mixture;
adding the PBS to the first mixture comprising the methacrylic anhydride to stop the reaction and dialyzing for 3 days with distilled water at 37 °C to obtain a second mixture;
freeze-drying the second mixture in a lyophilizer to obtain a methacrylamide-modified gelatin as a dry white powder and storing at -20°C;
dissolving the methacrylamide-modified gelatin in 1X PBS at a concentration of 5% - 10%, for modulating stiffness, along with a photo-initiator comprising 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone of 0.5 mg/mL to obtain a third mixture; and
placing the third mixture in culture vessels followed by 10 minutes of UV-365 exposure to obtain a microporous 3D hydrogel of the methacrylamide-modified gelatin forming the biomimetic 3D model.
14. The method as claimed in claim 13, wherein the microporous 3D hydrogel of the methacrylamide-modified gelatin is equilibrated with 500µL of a culture medium for 24 hours before performing seeding of the cells.

Dated this 25th day of March, 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)