Decellularized cancer tissue-derived extracellular matrix scaffold and use thereof

Abstract

The present invention relates to a decellularized cancer tissue-derived extracellular matrix scaffold and a use thereof. The decellularized cancer tissue-derived extracellular matrix according to the present invention contains a large amount of a malignant tumor-related extracellular matrix and proliferation and metastasis components that are more distinct than existing mouse-derived scaffolds or decellularized normal tissue-derived scaffolds, thereby achieving a better cancer tissue-specific microenvironment. A cancer organoid cultured using the decellularized cancer tissue-derived extracellular matrix was found to have improved cancer-specific markers, proliferative capacity, size, and secretion of inflammatory cytokines, and better achieve a configuration and phenotype of cancer tissues at a level similar to the actual configuration and phenotype, and thus can be effectively used in disease modeling, drug screening, new drug toxicity evaluation, personalized drug selection, and the like.

Description

Extracellular matrix scaffold derived from decellularized cancer tissue and its use

[0001] The present invention relates to an extracellular matrix support derived from decellularized cancer tissue and its use.

[0002]

[0003] Cells, the smallest units that make up the human body, can be treated to function as normal cells or, if they are not repaired, die. However, if a cell's genes change for various reasons, the cell changes abnormally, maturing incompletely and continuing to divide due to an uncontrolled cell cycle. This is defined as cancer. Furthermore, cancer has the characteristic of invading and destroying surrounding tissues and organs, as well as spreading to other organs. Cancer is the leading cause of death in Korea, and the number is increasing every year. Cancer cells grow and interact with other factors in the tumor microenvironment (TME), which is composed of various types of normal cells, the extracellular matrix, and blood vessels. In the tumor microenvironment, cancer cells grow and evolve, maintaining heterogeneity (Curr Biol. 2020 Aug 17;30(16):R921-R925). In the tumor microenvironment, cancer cells influence the microenvironment by releasing extracellular signals, promoting cancer cell angiogenesis, and inducing peripheral immune tolerance. Therefore, cancer cells can change the microenvironment, and the microenvironment can affect cancer cell growth or metastasis. In particular, the tumor microenvironment, which is related to immune cells such as lymphocytes, is highly related not only to cancer development and metastasis but also to cancer evasion of the immune system. Therefore, it influences the treatment response to anticancer drugs and is a major target of anticancer immunotherapy (Robert L Ferris, J Clin Oncol. 2015 Oct 10;33(29):3293-304). In addition, with the recent rapid increase in cancer patients due to the aging population, research is being conducted competitively to overcome cancer through patient-tailored anticancer treatments.In particular, targeted therapies can dramatically increase the response rate to anticancer drugs in patients with terminal cancer while minimizing the side effects of anticancer drugs, and can also be used to predict the cancer's response to the treatment using molecular signals, making them a key component of personalized anticancer treatment research.

[0004] In cancer research, cancer models that accurately represent patient characteristics are crucial. Until now, the most commonly used in vitro cancer models have been cancer cell lines. Cancer cell lines are models created by adapting cancer cells derived from patient cancer tissue to a two-dimensional culture. These models offer the advantages of being experimentally easy to handle and suitable for conducting numerous genetic and pharmacological screenings simultaneously. However, the success rate of generating cancer cells from cancer tissue is low. Furthermore, prolonged two-dimensional passage of patient-derived cancer cells often results in the loss of histological characteristics, such as heterogeneity and mutations, that can arise from the patient's tumor. Therefore, even if a cancer model is created by injecting a cell line that has lost its heterogeneity into immunodeficient mice, it cannot accurately represent the patient's characteristics. In contrast, cancer organoids are comprised of various cells that comprise a specific cancer tissue within a three-dimensional structure, and their interactions can be simulated. Therefore, compared to simple cell line models and other existing models for anticancer drug testing, they can serve as a more accurate in vitro model platform.

[0005] Organoids, which have recently been in the spotlight, are tissue-like models with diverse clinical applications, including new drug screening, drug toxicity assessment, disease modeling, cell therapy, and tissue engineering. Organoids are rapidly growing worldwide as a technology. Organoids are comprised of various cells that comprise specific organs and tissues of the human body within a three-dimensional structure, and their ability to simulate complex interactions among these cells allows them to serve as a more accurate in vitro model platform than conventional drug evaluation models, such as simple cell line models or animal models. Numerous research teams worldwide studying organoids have commonly used Matrigel as a culture support for organoid culture. However, Matrigel, derived from mouse sarcoma tissue, presents numerous challenges, including difficulty in maintaining consistent product quality, high cost, and safety concerns related to zoonotic pathogens and viral transmission. These challenges remain as an organoid culture system. In particular, as a material derived from mouse sarcoma tissue, it does not provide the optimal tissue-specific microenvironment required for specific tissue organoid culture. While some research has been conducted to develop polymer-based hydrogels to replace Matrigel, no material capable of replacing Matrigel has yet been reported.

[0006]

[0007] An object of the present invention is to provide a support composition comprising a decellularized extracellular matrix that mimics a cancer-specific microenvironment.

[0008] In addition, the present invention provides a composition for producing cancer organoids.

[0009] In addition, the present invention provides a method for producing cancer organoids.

[0010] In addition, the present invention provides a cancer organoid.

[0011] In addition, the present invention provides a method for screening cancer therapeutic agents.

[0012] In addition, the present invention provides a method for evaluating drug responsiveness using cancer organoids.

[0013] In addition, the present invention provides a personalized anticancer drug screening method.

[0014]

[0015] To achieve the above purpose, the present invention provides a support composition comprising an extracellular matrix derived from decellularized cancer tissue.

[0016] In addition, the present invention provides a composition for producing cancer organoids comprising the support composition.

[0017] In addition, the present invention provides a method for producing cancer organoids.

[0018] In addition, the present invention provides a cancer organoid produced by the above method.

[0019] In addition, the present invention provides a method for screening cancer therapeutic agents using the cancer organoid.

[0020] In addition, the present invention provides a method for evaluating drug responsiveness using the cancer organoid.

[0021] In addition, the present invention provides a personalized anticancer drug screening method.

[0022]

[0023] The extracellular matrix derived from decellularized cancer tissue according to the present invention contains a large amount of distinct malignant tumor-related extracellular matrix, proliferation, and metastasis components compared to conventional mouse-derived scaffolds or decellularized normal tissue-derived scaffolds, thereby better realizing a cancer tissue-specific microenvironment. It was confirmed that cancer organoids cultured using the same had improved cancer-specific markers, proliferation capacity, size, and inflammatory cytokine secretion amount, and better realizing the composition and phenotype of cancer tissue at a level similar to reality, and thus can be usefully utilized for disease modeling, drug screening, new drug toxicity evaluation, personalized drug screening, etc.

[0024]

[0025] Figure 1 is a diagram showing the process of manufacturing decellularized liver cancer tissue-derived extracellular matrix (Liver cancer ECM, LCEM) and hydrogel scaffolds by decellularizing liver cancer tissue derived from a liver cancer patient:

[0026] A: Schematic diagram of the production of extracellular matrix derived from decellularized liver cancer tissue of a liver cancer patient;

[0027] B: Images before and after decellularization of normal liver tissue and liver cancer tissue; and

[0028] C: A hydrogel scaffold derived from decellularized liver cancer tissue, manufactured by gelling decellularized extracellular matrix.

[0029] Figure 2 is a diagram showing the process of manufacturing a decellularized colorectal cancer tissue-derived extracellular matrix (Colorectal cancer ECM, CRCEM) and hydrogel scaffold by decellularizing colorectal cancer tissue derived from a colorectal cancer patient:

[0030] A: Schematic diagram of the production of extracellular matrix derived from decellularized colon cancer tissue of a colon cancer patient; and

[0031] B: Images of colon cancer tissue before and after decellularization.

[0032] Figure 3 is a diagram analyzing the decellularization of extracellular matrix (LCEM) derived from decellularized liver cancer tissue:

[0033] Native: Pre-decellularized tissue;

[0034] LEM: Extracellular matrix derived from decellularized normal liver tissue;

[0035] LCEM: Decellularized hepatocellular carcinoma tissue-derived extracellular matrix;

[0036] A: Preservation rate of dsDNA components and glycosaminoglycan (GAG) before and after decellularization of normal liver tissue;

[0037] B: Preservation rate of dsDNA components and glycosaminoglycans in liver cancer tissue before and after decellularization;

[0038] C: Histological analysis of normal liver tissue before and after decellularization; and

[0039] D: Histological analysis of liver cancer tissue before and after decellularization.

[0040] Figure 4 is a diagram analyzing the decellularization of extracellular matrix (CRCEM) derived from decellularized colon cancer tissue:

[0041] Native: Pre-decellularized tissue; and

[0042] CRCEM: Decellularized colon cancer tissue-derived extracellular matrix.

[0043] Figure 5 is a diagram showing the results of proteomic analysis of MAT, LEM, and LCEM:

[0044] MAT: Matrigel;

[0045] LEM: Extracellular matrix derived from decellularized normal liver tissue;

[0046] LCEM: Decellularized hepatocellular carcinoma tissue-derived extracellular matrix;

[0047] 5a: Liver tissue-specific protein ratio; and

[0048] 5b: Number of liver tissue-specific proteins.

[0049] Figure 6 is a diagram showing the results of extracellular matrix protein analysis of MAT, LEM, and LCEM:

[0050] MAT: Matrigel;

[0051] LEM: Extracellular matrix derived from decellularized normal liver tissue;

[0052] LCEM: Decellularized hepatocellular carcinoma tissue-derived extracellular matrix;

[0053] A: Ratio of matrisome proteins and non-matrisome proteins;

[0054] B: ECM protein composition ratio; and

[0055] C: Composition of the 10 most abundant extracellular matrix proteins in MAT, LEM, and LCEM.

[0056] Figure 7 shows the results of the full proteome analysis of MAT, LEM, and LCEM:

[0057] MAT: Matrigel;

[0058] LEM: Extracellular matrix derived from decellularized normal liver tissue;

[0059] LCEM: Decellularized hepatocellular carcinoma tissue-derived extracellular matrix;

[0060] A: Comparison of protein composition between MAT, LEM, and LCEM;

[0061] B: Principal component analysis results for the entire protein; and

[0062] C: Hierarchical clustering analysis results for total proteins and extracellular matrix (ECM) proteins.

[0063] Figure 8 is a diagram showing the results of total protein analysis of LEM and LCEM:

[0064] LEM: Extracellular matrix derived from decellularized normal liver tissue;

[0065] LCEM: Decellularized hepatocellular carcinoma tissue-derived extracellular matrix;

[0066] 8a: Results of total protein analysis;

[0067] 8b: Results of differentially expressed protein analysis; and

[0068] 8c: Gene ontology analysis results of LCEM-specific expressed proteins.

[0069] Figure 9 shows the results of gene ontology analysis for proteins differentially expressed in LEM and LCEM:

[0070] 9a: Gene ontology analysis results of proteins overexpressed in LCEM; and

[0071] 9b: Gene ontology analysis results of proteins underexpressed in LCEM.

[0072] Figure 10 is a diagram showing the results of liver cancer-related protein analysis of MAT and LCEM:

[0073] MAT: Matrigel;

[0074] LCEM: Decellularized hepatocellular carcinoma tissue-derived extracellular matrix;

[0075] A: Number of liver cancer-related proteins; and

[0076] B: Expression rate of liver cancer protein biomarkers.

[0077] Figure 11 shows the results of proteomic analysis for MAT and LCEM:

[0078] MAT: Matrigel;

[0079] LCEM: Decellularized hepatocellular carcinoma tissue-derived extracellular matrix;

[0080] 11a: Results of protein expression analysis of MAT and LCEM; and

[0081] 11b: Reactome pathway analysis results for LCEM-specific proteins.

[0082] Figure 12 is a diagram analyzing the mechanical properties of a hydrogel support according to the concentration of LCEM.

[0083] Figure 13 is a diagram analyzing the formation efficiency (A) and liver cancer organoid gene expression level (B) of liver cancer organoids cultured using a decellularized liver cancer tissue-derived extracellular matrix (LCEM) hydrogel support.

[0084] Figure 14 is a diagram analyzing the optimal LCEM concentration conditions of the LCEM hydrogel support for liver cancer organoid culture:

[0085] MAT: Liver cancer organoids cultured in Matrigel;

[0086] 20% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 20% LCEM and 80% MAT (v / v);

[0087] 30% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 30% LCEM and 70% MAT (v / v);

[0088] 50% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 50% LCEM and 50% MAT (v / v);

[0089] 100% LCEM: Liver cancer organoids cultured on a hydrogel support composed of 100% LCEM;

[0090] Tumor: Primary cancer tissue;

[0091] A: Gene expression levels in liver cancer organoids and actual liver cancer tissue from the corresponding patient;

[0092] B: Size of liver cancer organoids; and

[0093] C: Immunostaining results of liver cancer organoids.

[0094] Figure 15 is a diagram comparing the proliferation and growth of liver cancer organoids produced from liver cancer tissues of four different patients according to the culture support.

[0095] Figure 16 shows the analysis of the formation efficiency (A) and the gene expression level (B) of colon cancer organoids cultured on a decellularized colon cancer tissue-derived extracellular matrix (CRCEM) hydrogel support:

[0096] IEM: Intestinal extracellular matrix (IEM) hydrogel scaffold derived from normal small intestinal tissue; and

[0097] CRCEM: Decellularized colon cancer tissue-derived extracellular matrix (CRCEM) hydrogel scaffold.

[0098] Figure 17 is a diagram analyzing the optimal CRCEM concentration conditions of the CRCEM hydrogel support for colon cancer organoid culture:

[0099] MAT: Colorectal cancer organoids cultured in Matrigel;

[0100] 20% CRCEM: Colon cancer organoids cultured on a hydrogel support composed of 20% CRCEM and 80% MAT (v / v);

[0101] 30% CRCEM: Colon cancer organoids cultured on hydrogel supports composed of 30% CRCEM and 70% MAT (v / v);

[0102] 50% CRCEM: Colon cancer organoids cultured on hydrogel supports composed of 50% CRCEM and 50% MAT (v / v);

[0103] 100% CRCEM: Colon cancer organoids cultured on hydrogel supports composed of 100% CRCEM;

[0104] A: Size of colon cancer organoids; and

[0105] B: Gene expression in colon cancer organoids.

[0106] Figure 18 is a diagram analyzing the expression of liver cancer-specific differentiation markers according to the support on which liver cancer organoids are cultured:

[0107] MAT: Liver cancer organoids cultured on Matrigel; and

[0108] 50% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 50% LCEM and 50% MAT (v / v).

[0109] Figure 19 is a diagram analyzing the gene expression similarity of liver cancer organoids according to the cultured support:

[0110] MAT: Liver cancer organoids cultured in Matrigel;

[0111] 20% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 20% LCEM and 80% MAT (v / v);

[0112] 30% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 30% LCEM and 70% MAT (v / v);

[0113] 50% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 50% LCEM and 50% MAT (v / v);

[0114] A: Hierarchical clustering analysis results; and

[0115] B: Results of gene expression similarity comparison with the primary cancer tissue of the same patient.

[0116] Figure 20 is a diagram showing the results of mRNA transcript hallmark pathway analysis of liver cancer organoids according to the cultured support:

[0117] MAT: Liver cancer organoids cultured in Matrigel;

[0118] 20% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 20% LCEM and 80% MAT (v / v);

[0119] 30% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 30% LCEM and 70% MAT (v / v);

[0120] 50% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 50% LCEM and 50% MAT (v / v).

[0121] Figure 21 is a diagram showing the results of transcriptome analysis of liver cancer development-related genes in liver cancer organoids:

[0122] MAT: Liver cancer organoids cultured in Matrigel;

[0123] 50% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 50% LCEM and 50% MAT (v / v);

[0124] 21a: Volcano plot of genes showing significant expression differences between the two groups through transcriptome analysis;

[0125] 21b: Gene ontology analysis results for genes whose expression levels were significantly increased under 50% LCEM conditions compared to MAT; and

[0126] 21c: Clustering results of genes with strong interactions in the GOBP results.

[0127] Figure 22 is a diagram showing the heatmap analysis results for genes belonging to representative biological processes of gene ontology analysis related to liver cancer development:

[0128] MAT: Liver cancer organoids cultured on Matrigel; and

[0129] 50% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 50% LCEM and 50% MAT (v / v).

[0130] Figure 23 shows images of liver cancer organoids co-cultured on Kupffer cells and LCEM supports (A), the expression levels of inflammatory immune cells and cancer-specific markers (B), and the secretion levels of inflammatory cytokines (C):

[0131] MAT-KC: Liver cancer organoids cultured on Matrigel;

[0132] MAT+KC: Liver cancer organoids co-cultured with Kupffer cells in Matrigel;

[0133] LCEM-KC: Liver cancer organoids cultured on a hydrogel support composed of 50% LCEM and 50% MAT (v / v); and

[0134] LCEM+KC: Liver cancer organoids co-cultured with Kupffer cells in a hydrogel scaffold composed of 50% LCEM and 50% MAT (v / v).

[0135] Figure 24 shows an immunofluorescence image (24a) of a liver cancer organoid co-cultured with hepatic stellate cells on an LCEM support, a quantitative analysis graph of the VIM marker (25b), and the amount of collagen accumulation (25c):

[0136] MAT: Liver cancer organoids cultured on Matrigel; and

[0137] 50% LCEM: Liver cancer organoids cultured on hydrogel supports composed of 50% LCEM and 50% MAT (v / v).

[0138] Figure 25 is a diagram showing the results of drug response evaluation of liver cancer organoids co-cultured with hepatic stellate cells and Kupffer cells, which constitute a liver cancer tissue-specific microenvironment, in a 50% (v / v) LCEM hydrogel support of the present invention:

[0139] 25a: Results of optical microscopy analysis;

[0140] 25b: Results of immunofluorescence staining analysis; and

[0141] 25c: Quantitative evaluation of the results of immunofluorescence staining analysis.

[0142]

[0143] This application was conducted as a result of the research project SeratGen Co., Ltd._Development of decellularized tissue matrix for organoid culture (2022-11-1752 / 2022-11-01 ~ 2024-10-31).

[0144] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are provided as examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0145] Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the practices of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless otherwise specifically stated.

[0146] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited as references herein are incorporated herein by reference.

[0147]

[0148] In one aspect, the present invention provides a support composition comprising an extracellular matrix derived from decellularized cancer tissue.

[0149] In one embodiment, the cancer tissue may be a solid cancer tissue.

[0150] In one embodiment, the solid cancer tissue may be liver cancer tissue, biliary tract cancer tissue, bile duct cancer tissue, gallbladder cancer tissue, thyroid cancer tissue, adrenal cancer tissue, parathyroid cancer tissue, oral cancer tissue, salivary gland cancer tissue, laryngeal cancer tissue, tongue cancer tissue, endometrial cancer tissue, tonsil cancer tissue, peritoneal cancer tissue, spinal tumor tissue, breast cancer tissue, colon cancer tissue, rectal cancer tissue, colon cancer tissue, head and neck cancer tissue, lung cancer tissue, stomach cancer tissue, skin cancer tissue, prostate cancer tissue, bladder cancer tissue, urethra cancer tissue, urothelial cancer tissue, kidney cancer tissue, thyroid cancer tissue, uterine cancer tissue, cervical cancer tissue, brain cancer tissue, anal cancer tissue, ovarian cancer tissue, neuroblastoma tissue, intraperitoneal tumor tissue, osteosarcoma tissue, ocular tumor tissue, esophageal cancer tissue, or pancreatic cancer tissue.

[0151] In one embodiment, the extracellular matrix derived from acellular cancer tissue may include glycosaminoglycan, collagen, glycoprotein, proteoglycan, ECM-affiliated proteins, ECM regulators, and secreted factors.

[0152] In one embodiment, the decellularized cancer tissue-derived extracellular matrix has, compared to the normal tissue-derived decellularized extracellular matrix, an increased concentration of Collagen type 1 alpha 1 (COL1A1), Collagen type 1 alpha 2 (COL1A2), Collagen type 6 alpha 1 (COL6A1), Collagen type 6 alpha 2 (COL6A2), Collagen type 6 alpha 3 (COL6A3), Collagen type 6 alpha 6 (COL6A6), Annexin A2 (ANXA2), Poly(ADP-ribose) polymerase 1 (PARP1), Catenin Beta 1 (CTNNB1), Clusterin (CLU), High mobility group box 1 (HMGB1), Lamin A / C (LMNA), Inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), Complement component 3 (C3), Betaine homocysteine ​​S-methyltransferase (BHMT), The content of Phosphoenolpyruvate carboxykinase 2 (PCK2), Versican (VCAN), Complement C1q binding protein (C1QBP), Heat shock protein family E member 1 (HSPE1), Pyruvate kinase L / R (PKLR), Myeloperoxidase (MPO), Fibronectin (FN1), Vitronectin (VTN), Fibrillin 1 (FBN1), or Galectin-1 (LGALS1) may increase.

[0153] In one embodiment, the decellularized cancer tissue-derived extracellular matrix has higher levels of Annexin A2 (ANXA2), Poly(ADP-ribose) polymerase 1 (PARP1), Catenin Beta 1 (CTNNB1), Clusterin (CLU), High mobility group box 1 (HMGB1), Lamin A / C (LMNA), Inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), Complement component 3 (C3), Betaine homocysteine ​​S-methyltransferase (BHMT), Phosphoenolpyruvate carboxykinase 2 (PCK2), Versican (VCAN), Complement C1q binding protein (C1QBP), Heat shock protein family E member 1 (HSPE1), Pyruvate kinase L / R (PKLR), Myeloperoxidase (MPO), Fibronectin (FN1), Vitronectin (VTN), Fibrillin compared to the decellularized normal tissue-derived extracellular matrix. Expression of FBN1 (FBN1) or Galectin-1 (LGALS1) may be increased.

[0154] In one embodiment, the support composition may comprise a protease, wherein the protease may be pepsin or trypsin.

[0155] In one embodiment, the extracellular matrix or support composition may be in the form of a surface coating substrate or hydrogel for cell culture and differentiation.

[0156] In one embodiment, the support composition may be a 3D hydrogel support in which the extracellular matrix derived from the decellularized cancer tissue is gelled.

[0157] In one embodiment, the support composition may comprise the extracellular matrix derived from the decellularized cancer tissue at a concentration of 0.005 to 20 mg / mL.

[0158] In one embodiment, when the support composition is a hydrogel support including an extracellular matrix derived from decellularized cancer tissue, the decellularized extracellular matrix may be included in an amount of 0.01 to 10 mg / mL, and in an optimized embodiment, the decellularized extracellular matrix may be included in an amount of 5 mg / mL.

[0159] In one embodiment, the support composition may further include polyethylene glycol (PEG), pectin, collagen, gelatin, Matrigel, methacrylated gelatin (GelMA), hyaluronic acid, alginate, fibrin, chitosan, cellulose, agarose, or a combination thereof.

[0160] In one embodiment, the support composition may comprise Matrigel and decellularized cancer tissue-derived extracellular matrix at a concentration of 9:1 to 0:10 (v / v).

[0161] In one embodiment, the support composition may be for two-dimensional or three-dimensional culture.

[0162] In one embodiment, the support composition may be for culturing cells or organoids, wherein the cells or organoids may be cancer cells, cancer stem cells, or cancer organoids prepared therefrom.

[0163] In one embodiment, the cancer cells or cancer stem cells may be derived from a solid cancer or a blood cancer.

[0164] In one embodiment, the cancer cells or cancer stem cells may be cancer cells or cancer stem cells extracted from cancer tissue, or may be circulating tumor cells isolated from ascites, lymph, or blood.

[0165] In one embodiment, the extracellular matrix derived from the decellularized cancer tissue can be prepared by a method comprising: a) crushing the cancer tissue; b) decellularizing the cancer tissue; and c) lyophilizing the cancer tissue.

[0166] The method for producing an extracellular matrix derived from decellularized cancer tissue of the present invention effectively removes cellular components while minimizing damage to protein components, unlike the existing decellularization process using high-concentration surfactants and ionic surfactants, and this is achieved by using a low-concentration nonionic surfactant and low-temperature and stirring conditions.

[0167] As used herein, the term "extracellular matrix" refers to a natural support for cell growth prepared by decellularization of tissues derived from mammals and multicellular organisms. The extracellular matrix may be further processed through dialysis or cross-linking. The extracellular matrix may be a mixture of structural and non-structural biomolecules, including but not limited to collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors.

[0168] As used herein, the terms "derived" and "derived" refer to components obtained from the source mentioned by a useful method. For example, "decellularized cancer tissue-derived extracellular matrix" may refer to an extracellular matrix component obtained by decellularizing cancer tissue by various methods known in the art.

[0169] The term "hydrogel" in the present invention refers to a hydrophilic network material in which polymer chains form a three-dimensional structure, and through a sol-gel phase transition, a liquid using water as a dispersion medium solidifies, losing fluidity and forming a porous structure. Hydrogels swell and become transparent through the absorption of hydrophilic components, including water, in an aqueous solution, and also have appropriate mechanical properties. This swelling degree and the mechanical properties of the polymer mainly depend on the properties of the polymer network structure. Polymers are mainly used as materials for making such hydrogels, and these polymers are divided into natural polymers and synthetic polymers depending on the origin of the material.

[0170] In the present invention, the term "biodegradable polymer" refers to a polymer that is slowly and spontaneously decomposed in a living body after a certain period of time, and has one or more of the following properties: biocompatibility, blood compatibility, anti-calcification properties, cellular nutritional properties, and intercellular matrix formation ability. The types of these biodegradable polymers include, but are not limited to, fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly-ε-(caprolactone) (PCL), polyanhydride, polyorthoester, polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyurethane, polyacrylic acid (PAA), poly-N-isopropylacrylamide (PNIPAAm), Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymers, copolymers thereof, and mixtures thereof are included.

[0171] In the present invention, the term "organoid" refers to a cultured body created by culturing or recombining cells isolated from stem cells or tissues, and refers to a structure that maintains the shape of a tissue in vitro, and can be interpreted as having the same meaning as artificial organs, bio-organs, mini-organs, spheroids, spheroids, or embryoid bodies. Organoids are "mini-organs" that are designed to perform minimal functions, and are made in a three-dimensional structure, so that an environment similar to an actual body organ can be created in a laboratory, allowing cells to interact with the surrounding environment during their growth process. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro. Accordingly, the 3D organoids of the present invention can almost perfectly mimic organs that actually interact in vivo, and can serve as excellent models for observing the development of treatment agents for diseases, etc. When the source cells for producing the organoids are collected from cancer tissue, the organoids are cancer organoids and are characterized by being composed of cancer cells.

[0172] In one aspect, the present invention relates to a composition for producing cancer organoids, comprising a support composition of the present invention and cells seeded on the support composition.

[0173] In one embodiment, the cell may be a cancer cell or a cancer stem cell, and may be derived from a solid cancer or a blood cancer.

[0174] In one embodiment, the cancer cells or cancer stem cells may be cancer cells or cancer stem cells extracted from cancer tissue, or may be circulating tumor cells isolated from ascites, lymph, or blood.

[0175] In one embodiment, the composition for producing cancer organoids may additionally include tissue-specific cells, mesenchymal cells, fibroblasts, pericytes, vascular cells, or immune cells.

[0176] In one embodiment, the tissue-specific cells, mesenchymal cells, fibroblasts, pericytes, vascular cells, or immune cells may be cells produced by differentiating pluripotent stem cells.

[0177] In the present invention, the term "pluripotent stem cell (PSC)" refers to a stem cell that can be induced to differentiate into any type of cell that constitutes the body, and pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Specifically, embryonic stem cells are induced from the inner cell mass of a blastocyst in the pre-implantation stage. The induced cells are maintained in a specific environment and are capable of unlimited culture and pluripotent differentiation. Furthermore, induced pluripotent stem cells may refer to pluripotent differentiated cells created through induced differentiation from somatic cells, and are formed by making somatic cells into a state very similar to embryonic stem cells through a process called reprogramming, such as cell fusion, nuclear transfer, and overexpression of pluripotency regulatory factors. However, preferably, the pluripotent stem cells may be mammalian cells, more preferably human-derived pluripotent stem cells.

[0178] The term "differentiation" used in the present invention refers to the phenomenon in which the structure or function of a cell becomes specialized while the cell divides, proliferates, and the entire organism grows. In other words, it refers to the process in which the cells, tissues, etc. of a living organism change into a form and function suitable for performing their respective roles. For example, differentiation can include not only the process in which pluripotent stem cells change into ectoderm, mesoderm, and endoderm cells, but also the process in which progenitor cells express specific differentiation traits.

[0179] In one aspect, the present invention relates to a method for producing a cancer organoid, comprising: 1) preparing an extracellular matrix derived from decellularized cancer tissue; 2) preparing a 3D hydrogel scaffold by mixing the decellularized extracellular matrix with polyethylene glycol (PEG), pectin, collagen, gelatin, Matrigel, methacrylated gelatin (GelMA), hyaluronic acid, alginate, fibrin, chitosan, cellulose, agarose, or a combination thereof; and 3) culturing cancer cells or cancer stem cells in the 3D hydrogel scaffold to induce formation and development of the organoid.

[0180] In one embodiment, the extracellular matrix derived from the decellularized cancer tissue can be prepared by a method comprising: a) crushing the cancer tissue; b) decellularizing the cancer tissue; and c) lyophilizing the cancer tissue.

[0181] In one embodiment, a 3D hydrogel scaffold can be manufactured by a method comprising: a) treating a pepsin solution on a decellularized cancer tissue-derived extracellular matrix; b) stirring to form a solution; c) adjusting a pH; and d) gelling.

[0182] In one embodiment, the step of inducing the formation and development of the organoid in step 3) may be co-culturing cancer cells or cancer stem cells with tissue-specific cells, mesenchymal cells, fibroblasts, pericytes, vascular cells, or immune cells.

[0183] In one embodiment, the cancer organoids prepared above may further include a step of co-culturing cancer cells, cancer stem cells, or tissue-specific cells, mesenchymal cells, fibroblasts, pericytes, vascular cells, or immune cells.

[0184] In one embodiment, the immune cell may be a tumor infiltrating lymphocyte (TIL), a T cell, a cytotoxic T lymphocyte (CTL), a B cell, a NK cell, a mononuclear phagocyte, an α / β receptor T-cell, or a γ / δ receptor T cell, may be a tissue-specific immune cell, and in the case of a liver organoid, may be a Kupffer cell (KC).

[0185] In one embodiment, the tissue-specific cells may be pericytes found in a specific tissue, and in the case of liver organoids, hepatic stellate cells (HSCs).

[0186] In one embodiment, the tissue-specific cells, mesenchymal cells, fibroblasts, pericytes, vascular cells, or immune cells may be cells produced by differentiating pluripotent stem cells.

[0187] In one embodiment, co-culturing the human cancer cells or cancer stem cells with one or more of the above cells may increase the expression of cancer-specific markers and the secretion of inflammatory cytokines or collagen accumulation in the produced cancer organoids.

[0188] In one aspect, the present invention relates to cancer organoids produced by the method of the present invention.

[0189] In one embodiment, the cancer organoid may exhibit intra-tissue heterogeneity.

[0190] In one embodiment, the cancer organoid may have increased expression of MUC2, AFP, Ki67, α-SMA, KRT19, PDGFRB, HIF1α, TNF-α, or IL-10.

[0191] In one aspect, the present invention relates to a cancer animal model in which the cancer organoid of the present invention is xenografted.

[0192] In one aspect, the present invention relates to a method for screening a cancer therapeutic agent, comprising the step of treating a cancer organoid or cancer animal model of the present invention with a test substance.

[0193] In one aspect, the present invention relates to a method for evaluating drug responsiveness using cancer organoids, comprising the step of treating a cancer organoid or cancer animal model of the present invention with a therapeutic agent candidate.

[0194] In one aspect, the present invention relates to a personalized anticancer drug screening method, comprising the steps of: 1) isolating cancer tissue from a patient with cancer; 2) preparing a decellularized cancer tissue-derived extracellular matrix from the cancer tissue; 3) preparing a 3D hydrogel scaffold including the decellularized cancer tissue-derived extracellular matrix; 4) culturing cancer cells or cancer stem cells isolated from the patient in the scaffold to prepare cancer organoids; and 5) treating the cancer organoids with a candidate anticancer drug.

[0195]

[0196] The present invention is described in more detail through the following examples. However, the following examples are intended only to concretize the content of the present invention and are not intended to limit the present invention.

[0197]

[0198] Example 1. Preparation of extracellular matrix derived from decellularized cancer tissue

[0199] 1-1. Preparation of extracellular matrix and hydrogel scaffolds derived from decellularized cancer tissue

[0200] 1-1-1. Preparation of extracellular matrix and hydrogel scaffolds derived from decellularized liver cancer tissue

[0201] To create a liver cancer organoid culture scaffold that effectively mimics the microenvironment of liver cancer, decellularized extracellular matrix (Liver cancer ECM, LCEM) was prepared from human liver cancer tissues (Fig. 1A). Specifically, liver cancer tissues were obtained after surgery from patients with various liver cancer lesions, cut into 0.5 cm x 0.5 cm x 0.5 cm pieces at 4°C, and then stirred in sterile water for more than 12 hours at 180 rpm in a shaker to remove tissue impurities. A mixed solution of 1% (v / v) Triton X-100 and 0.1% (v / v) ammonium hydroxide, a nonionic surfactant, was treated at 4°C for 48 hours under strong stirring at 180 rpm to remove only cellular components while preserving protein components in liver cancer tissues. After removing all surfactant foam with 3 times the water, sterilization was performed with 1% Penicillin-Streptomycin for 2 hours, and then freeze-drying to prepare decellularized liver cancer tissue-derived extracellular matrix (LCEM) and decellularized normal liver tissue-derived extracellular matrix (Liver ECM, LEM) as a control (Fig. 1B). 10 mg of the decellularized extracellular matrix LCEM prepared above was finely chopped with scissors to make a powder, and then treated with a 4 mg / ml pepsin solution (a solution of pepsin powder (4 mg) dissolved in 0.02 M HCL (1 ml)) and stirred in a shaker at 300 rpm for 24 hours to make a solution. After that, LCEM (5 mg / ml) was adjusted to neutral pH using 10X PBS (Phosphate buffered saline) and 1 M NaOH (Sodium hydroxide), and then gelled at 37°C for 30 minutes to produce a hydrogel scaffold derived from extracellular matrix of decellularized liver cancer tissue (Fig. 1C).

[0202]

[0203] 1-1-2. Preparation of extracellular matrix and hydrogel scaffolds derived from decellularized colon cancer tissue

[0204] To create a colorectal cancer organoid culture support that effectively mimics the colorectal cancer microenvironment, decellularized extracellular matrix (CRCEM) was prepared from human colorectal cancer tissues (Fig. 2A). Specifically, colorectal cancer tissues were obtained from patients with various colorectal cancer lesions after surgery, cut into 0.5 cm x 0.5 cm x 0.5 cm pieces at 4°C, and then stirred in sterile water for more than 12 hours at 180 rpm in a shaker to remove tissue impurities. A mixed solution of 1% (v / v) Triton X-100 and 0.1% (v / v) ammonium hydroxide, a nonionic surfactant, was treated at 4°C for 48 hours under strong stirring at 180 rpm to remove only the cellular components while preserving the protein components in the colorectal cancer tissues. After removing all surfactant foam with 3 times the water, sterilization with 1% Penicillin-Streptomycin for 2 hours and freeze-drying were performed to prepare decellularized colorectal cancer tissue-derived extracellular matrix (CRCEM) (Fig. 2B). 10 mg of the decellularized extracellular matrix CRCEM prepared above was finely chopped with scissors into powder form, and then treated with 4 mg / ml pepsin solution (pepsin powder (4 mg) dissolved in 0.02 M HCl (1 ml)) and stirred in a shaker at 300 rpm for 24 hours to dissolve. After that, CRCEM (5 mg / ml) was adjusted to neutral pH using 10X PBS and 1 M NaOH and gelled at 37 ℃ for 30 minutes to prepare a decellularized colorectal cancer tissue-derived extracellular matrix (CRCEM) hydrogel scaffold.

[0205]

[0206] 1-2. Confirmation of decellularization of extracellular matrix derived from decellularized cancer tissue

[0207] 1-2-1. Confirmation of decellularization of extracellular matrix derived from decellularized liver cancer tissue

[0208] In order to confirm whether the human decellularized normal liver tissue-derived extracellular matrix (LEM) and the human decellularized liver cancer tissue-derived extracellular matrix (LCEM) prepared in Example 1-1-1 were decellularized, the preservation rate of residual cellular components (dsDNA) and extracellular matrix components, glycosaminoglycan (GAG), was evaluated. In addition, histological staining analysis using H&E (Hematoxylin and Eosin), TB (Toluidine Blue), and MT (Masson's Trichrome) was performed.

[0209] As a result of quantitative evaluation of cellular components (dsDNA), 219.90 ng DNA / mg and 9.54 ng DNA / mg were detected in normal liver tissues before and after decellularization, respectively (Fig. 3A), and 336.63 ng DNA / mg and 49.05 ng DNA / mg were detected in human liver cancer tissues before and after decellularization, respectively (Fig. 3B), confirming that the dsDNA component was reduced to below 50 ng DNA / mg, which is the acceptable level for decellularization for clinical application. In addition, as a result of evaluating the preservation rate of glycosaminoglycans, 2.13 μg / mg and 2.15 μg / mg were detected in normal liver tissues before and after decellularization, respectively (Fig. 3A), and 3.97 μg / mg and 4.46 μg / mg were detected in liver cancer tissues before and after decellularization, respectively (Fig. 3B), confirming that the extracellular matrix components were well preserved. In addition, the H&E staining results confirmed that the cell nuclei were completely removed in both LEM and LCEM after decellularization, and that the extracellular matrix components, glycosaminoglycan and collagen, were well preserved even after decellularization (Fig. 3C and D).

[0210] Through this, it was confirmed that both human normal liver tissue and human liver cancer tissue were effectively decellularized by the decellularization process, and that the extracellular matrix components were well preserved.

[0211]

[0212] 1-2-2. Confirmation of decellularization of extracellular matrix derived from decellularized colon cancer tissue

[0213] In order to confirm whether the human decellularized colon cancer tissue-derived extracellular matrix (Colorectal cancer ECM, CRCEM) prepared in Example 1-1-2 above was decellularized, histological staining analysis using H&E (Hematoxylin and Eosin), TB (Toluidine Blue), and MT (Masson's Trichrome) was performed on the decellularized tissue (Native) and CRCEM.

[0214] As a result, it was confirmed that the nuclear component was successfully removed from the CRCEM, and it was confirmed that the glycosaminoglycan and collagen components, which are extracellular matrix components important for organoid culture, were well preserved in the CRCEM even after the decellularization process (Fig. 4).

[0215] Through this, it was confirmed that all cellular components within the colon cancer tissue were removed through the decellularization process and that the major extracellular matrix components were well preserved.

[0216]

[0217] Example 2. Characterization of extracellular matrix derived from decellularized liver and liver cancer tissues.

[0218] 2-1. Liver tissue-specific proteomic analysis

[0219] In order to analyze the protein composition and role of Matrigel (MAT), a commercially available support, human decellularized normal liver tissue-derived extracellular matrix (LEM) and human decellularized liver cancer tissue-derived extracellular matrix (LCEM) prepared in Example 1-1-1, liquid chromatography tandem mass spectrometry (LC-MS / MS) was used to analyze liver tissue-specific proteins (liver-enriched proteins: proteins expressed more than four times more in the liver than in other tissues).

[0220] As a result, MAT, LEM, and LCEM were found to contain 1.83%, 41.42%, and 9.68%, respectively, of liver tissue-specific proteins (Fig. 5a). In particular, MAT, derived from mouse sarcoma, was found to contain a significantly lower proportion of liver-specific proteins than LEM and LCEM, and LCEM was found to contain a lower proportion of liver-specific proteins than LEM (Fig. 5a). An average of 31 liver tissue-specific proteins were detected in MAT, an average of 107 in LEM, and an average of 91 in LCEM (Fig. 5b), suggesting that LCEM may better mimic the microenvironment of human liver cancer in vivo than MAT.

[0221] Through this, it was confirmed that LCEM, which reflects the cancer cell proliferation and survival environment, has a different protein composition from MAT, a conventional culture support, and LEM, a normal tissue, and it was predicted that LCEM would implement a microenvironment similar to actual liver cancer tissue due to these compositional differences.

[0222]

[0223] 2-2. Extracellular matrix protein analysis

[0224] A comparative analysis was performed on the matrisome proteins of Matrigel (MAT), a commercially available support, human decellularized normal liver tissue-derived extracellular matrix (LEM), and human decellularized liver cancer tissue-derived extracellular matrix (LCEM).

[0225] As a result, MAT was composed of 80.76% matrisome proteins and 19.23% non-matrisome proteins, while LEM and LCEM were composed of 27.01% and 9.90% matrisome proteins and 73.02% and 90.10% non-matrisome proteins, respectively (Fig. 6A), confirming that LCEM contains a smaller amount of matrisome components than MAT and LEM. In addition, the compositional ratios of ECM proteins constituting LEM and LCEM were significantly different from those of MAT, and the compositional ratios of ECM proteins were also significantly different between LEM and LCEM (Fig. 6B). Specifically, MAT was found to be composed mostly of glycoproteins, with very small amounts of ECM regulators and proteoglycans in that order (Fig. 6C). On the other hand, LEM and LCEM were composed of six types of extracellular matrix proteins: collagen, glycoproteins, proteoglycans, ECM-affiliated proteins, ECM regulators, and secreted factors, with glycoproteins, collagen, and proteoglycans accounting for a large proportion in that order (Fig. 6C). In particular, LCEM was found to have extracellular matrix components that are different from LEM, as more than 50% of the extracellular matrix components were detected as glycoprotein components.When examining the extracellular matrix proteins of MAT, LEM, and LCEM, MAT was mostly composed of glycoproteins such as Laminins (LAM) and Nidogen 1 (NID1), whereas LEM and LCEM were found to contain a large number of proteins related to the actual extracellular matrix components of the liver and the composition of liver cells (Collagen type 1 (COL1A1, COL1A2), Collagen type 6 (COL6A1, COL6A2, COL6A3, COL6A6), Fibronectin (FN1), and Vitronectin (VTN)). In particular, among the top 10 ECM components of LEM and LCEM, FN1 and FBN1, proteins related to migration and metastasis, which are major characteristics of cancer, and LGALS1, a protein related to the progression and aggressiveness of liver cancer, were found to exist only in LCEM (Fig. 6C).

[0226] Through this, it can be seen that a scaffold containing extracellular matrix derived from decellularized liver cancer tissue containing a number of proteins related to the characteristics of cancer tissue is suitable for liver cancer organoid culture.

[0227]

[0228] 2-3. Analysis of differences in protein content and expression patterns

[0229] The total proteins of existing commercialized supports, Matrigel (MAT), human decellularized normal liver tissue-derived extracellular matrix (LEM), and human decellularized liver cancer tissue-derived extracellular matrix (LCEM), were analyzed, and principal component analysis (PCA) and hierarchical clustering analysis were performed on them.

[0230] When we checked the total protein counts for MAT, LEM, and LCEM, there were more overlapping proteins in LEM and LCEM than in MAT, but many different protein components were also identified between LEM and LCEM (Fig. 7A). In addition, the principal component analysis results showed that although LEM and LCEM showed higher similarity than MAT, there were differences in overall protein components and expression in the distribution maps between LEM and LCEM (Fig. 7B). In particular, LCEM showed large variations between samples, confirming intra-tissue heterogeneity, a major characteristic of cancer. In addition, the hierarchical clustering analysis of total proteins and ECM proteins showed that, similar to the principal component analysis, MAT showed a different protein expression pattern from LEM and LCEM, and although there were some overlapping protein components between LEM and LCEM, different protein expression patterns were also identified (Fig. 7C). In particular, LCEM showed greater differences in protein expression patterns between samples compared to MAT and LEM, indicating intra-tissue heterogeneity, a key characteristic of cancer.

[0231] Through this, it was confirmed that LCEM has different overall protein content and expression pattern from MAT, a conventional culture support, and LEM derived from normal liver tissue, and that it exhibits heterogeneity, which is a characteristic of cancer tissue.

[0232]

[0233] 2-4. Comparative analysis of cancer tissue-specific proteomes

[0234] We conducted a comparative proteome analysis of human decellularized normal liver tissue-derived extracellular matrix (LEM) and human decellularized hepatocellular carcinoma tissue-derived extracellular matrix (LCEM). Afterwards, we identified differentially expressed proteins using volcano plots, targeting proteins commonly expressed in LEM and LCEM. Furthermore, we performed a gene ontology (GEO) analysis on proteins specifically expressed in LCEM.

[0235] Whole proteome analysis revealed that 526 proteins were commonly expressed in LEM and LCEM, while 26 proteins were specifically detected in LEM and 81 proteins were specifically detected in LCEM, respectively (Fig. 8a). In addition, volcano plot analysis revealed that among the 526 proteins commonly expressed in LEM and LCEM, 41 proteins were significantly overexpressed by more than 2-fold in LCEM compared to LEM, and 184 proteins were significantly underexpressed by less than 0.5-fold in LCEM compared to LEM (Fig. 8b). In addition, gene ontology analysis was performed on 81 proteins specifically expressed in LCEM, and it was confirmed that the proteins perform roles that appear in actual liver cancer tissues, such as cell death, programmed cell death, and regulation of cell death. It was also confirmed that the proteins are related to protein synthesis (translation) essential for cell proliferation in cancer tissues (Fig. 8c), confirming that the proteins specifically expressed in LCEM perform functions related to biological processes that are actively occurring in actual liver cancer tissues.

[0236] Through this, it was confirmed that LCEM, an extracellular matrix derived from decellularized liver cancer tissue, can be used as an optimal platform for culturing liver cancer organoids because it contains cancer tissue-specific proteins and well embodies the characteristics of the cancer microenvironment due to differences in protein components from LEM, an extracellular matrix derived from decellularized normal liver tissue.

[0237]

[0238] 2-5. Gene Ontology Analysis

[0239] 2-5-1. Gene Ontology Analysis of Differentially Expressed Proteins in Cancer Tissues

[0240] Gene ontology analysis was performed on differentially expressed proteins in human decellularized normal liver tissue-derived extracellular matrix (LEM) and human decellularized hepatocellular carcinoma tissue-derived extracellular matrix (LCEM).

[0241] Gene ontology analysis of 41 proteins significantly overexpressed in LCEM compared to LEM revealed that they are involved in biological processes such as chromatin remodeling, protein synthesis (Translation), and macromolecular biosynthetic process, which are closely related to cancer cell survival and proliferation, and are involved in the regulation of DNA repair patterns associated with aggressiveness, intratumor heterogeneity, and mutation of liver cancer (Fig. 9a). In addition, gene ontology analysis was performed on 184 proteins that were significantly underexpressed in LCEM compared to LEM, and major liver-related metabolic functions such as lipid metabolism, lipid oxidation, carbohydrate metabolic process, alcohol metabolic process, and ethanol metabolic process were identified. In particular, as it is known that major metabolisms such as lipid oxidation are reduced due to cancer proliferation in actual liver cancer, major metabolic functions were found to be reduced in LCEM compared to LEM (Fig. 9b).

[0242] It was confirmed that LCEM has enhanced functions related to cancer proliferation and decreased major liver-related metabolic functions similar to actual liver cancer tissue compared to LEM, thereby confirming that LCEM well embodies the microenvironment of actual cancer tissue.

[0243]

[0244] 2-5-2. Gene Ontology Analysis of Differentially Expressed Proteins in Liver Cancer Tissues

[0245] We compared and analyzed the proteome related to hepatocellular carcinoma (HCC) between Matrigel (MAT), a commercially available support, and human decellularized liver cancer tissue-derived extracellular matrix (LCEM). In addition, the expression rates of liver cancer protein biomarkers known to be frequently identified in liver cancer, such as Annexin A2 (ANXA2), Poly(ADP-ribose) polymerase 1 (PARP1), Catenin Beta 1 (CTNNB1), Clusterin (CLU), High mobility group box 1 (HMGB1), Lamin A / C (LMNA), Inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), Complement component 3 (C3), Betaine homocysteine ​​S-methyltransferase (BHMT), Phosphoenolpyruvate carboxykinase 2 (PCK2), Versican (VCAN), Complement C1q binding protein (C1QBP), Heat shock protein family E member 1 (HSPE1), Pyruvate kinase L / R (PKLR), Myeloperoxidase (MPO), Fibronectin (FN1), and Vitronectin (VTN), were compared.

[0246] As a result of proteomic analysis related to liver cancer, an average of 336 and 478 proteins were detected in MAT and LCEM, respectively, confirming that LCEM contains approximately 1.42 times more HCC-related proteins than MAT (Fig. 10A). In addition, as a result of expression analysis of liver cancer protein biomarkers, it was confirmed that all 17 major liver cancer protein biomarkers were expressed at significantly higher levels in LCEM than in MAT (Fig. 10B).

[0247] Thus, the extracellular matrix derived from human decellularized liver cancer tissue contains various liver cancer-related proteins, suggesting that LCEM can effectively reflect key liver cancer characteristics. Therefore, the LCEM scaffold is more suitable for liver cancer organoid culture than the existing MAT scaffold.

[0248]

[0249] 2-6. Gene Ontology Molecular Function Analysis

[0250] We performed proteomic analysis on Matrigel (MAT), a commercially available support, and human decellularized liver cancer tissue-derived extracellular matrix (LCEM), and analyzed the reactome pathway through protein-protein interactions specifically detected in LCEM.

[0251] Proteomic analysis revealed that 168 proteins were commonly expressed in MAT and LCEM, while 229 proteins were specifically detected in MAT and 439 proteins were specifically detected in LCEM, respectively (Fig. 11a). Reactome pathway analysis of the 439 proteins specifically detected in LCEM revealed that the proteins specifically detected only in LCEM were significantly involved in signaling pathways such as Apoptosis, Signaling by WNT, VEGFA-VEGFR2 pathway, and Signaling by NOTCH (Fig. 11b). The four detected signaling pathways are known to be closely involved in important signaling pathways related to cancer cell proliferation and metastasis in actual liver cancer tissues.

[0252] Through this, it can be inferred that the interaction of proteins specifically expressed in LCEM will contribute to the liver cancer organoids cultured in LCEM having the characteristics of actual liver cancer tissue by precisely simulating the actual liver cancer tissue microenvironment.

[0253]

[0254] Example 3. Analysis of mechanical properties according to concentration

[0255] To investigate the difference in mechanical properties between hydrogel scaffolds manufactured from decellularized liver cancer tissue-derived extracellular matrix (LCEM) and the conventional scaffold MAT according to concentration, 5 mg / ml LCEM (100% LCEM) or 5 mg / ml LCEM was mixed with MAT at concentrations of 20%, 30%, and 50% (v / v), respectively, to induce hydrogel formation, and then rheological analysis was performed to measure the mechanical properties. MAT was used as a control.

[0256] As a result, both the support and MAT containing LCEM at concentrations of 20%, 30%, 50%, and 100% showed consistently higher G' (Storage modulus) values ​​than G" (Loss modulus) values, confirming that LCEM formed a stable polymer network through cross-linking within the hydrogel under all concentration conditions. In addition, as the concentration of added LCEM increased, the mechanical properties (Elastic modulus) decreased compared to MAT (Fig. 12).

[0257]

[0258] Example 4. Production of cancer organoids using extracellular matrix derived from decellularized cancer tissue.

[0259] 4-1. Production of liver cancer organoids

[0260] 4-1-1. Comparison of liver cancer organoid formation efficiency

[0261] To evaluate the suitability of hydrogel scaffolds manufactured from decellularized liver cancer tissue-derived extracellular matrix (LCEM) for culturing liver cancer organoids, 30 μL of Matrigel (MAT), a commercially available scaffold, 30 μL of decellularized normal liver tissue-derived extracellular matrix (LEM) hydrogel scaffolds at a concentration of 5 mg / mL, and 30 μL of hydrogel scaffolds manufactured from decellularized liver cancer tissue-derived extracellular matrix (LCEM) at a concentration of 5 mg / mL were mixed with 100,000 tumor cells extracted from human liver cancer tissues and cultured three-dimensionally for 17 days to culture liver cancer organoids. The formation efficiency and the expression of liver cancer-related markers (AFP, a major biomarker of liver cancer, Ki67, a proliferation marker, and α-SMA, a fibrosis marker) were compared and analyzed.

[0262] As a result of measuring the formation efficiency of liver cancer organoids formed for 17 days on each support, the formation efficiency of liver cancer organoids was found to be lower on the decellularized normal liver tissue-derived extracellular matrix (LEM) hydrogel support than on the control, Matrigel (MAT), whereas the formation efficiency of liver cancer organoids was found to be significantly increased on the hydrogel support manufactured with decellularized liver cancer tissue-derived extracellular matrix (LCEM) (Fig. 13A). In addition, when the expression of liver cancer-related markers was analyzed in the formed liver cancer organoids, the expression of AFP, Ki67, and α-SMA was significantly increased in liver cancer organoids cultured on hydrogel scaffolds prepared from MAT and decellularized liver cancer tissue-derived extracellular matrix (LCEM) compared to liver cancer organoids cultured on decellularized normal liver tissue-derived extracellular matrix (LEM) hydrogel scaffolds, confirming that a cancer-specific microenvironment was not implemented in the decellularized normal liver tissue-derived extracellular matrix (LEM) hydrogel scaffolds. In particular, the expression in liver cancer organoids cultured on hydrogel scaffolds prepared from decellularized liver cancer tissue-derived extracellular matrix (LCEM) was significantly increased compared to MAT (Fig. 13B), confirming that the hydrogel scaffold prepared from decellularized liver cancer tissue-derived extracellular matrix (LCEM) better embodies the liver cancer microenvironment than existing commercial culture scaffolds, making it the most suitable for liver cancer organoid culture.

[0263]

[0264] 4-1-2. Deriving the optimal concentration conditions for the culture support

[0265] To determine the optimal concentration conditions of the decellularized liver cancer tissue-derived extracellular matrix (LCEM) hydrogel scaffold for liver cancer organoid culture, 30 μL of hydrogel scaffolds containing 20%, 30%, and 50% (v / v) LCEM (5 mg / ml) mixed with MAT, and 30 μL of 100% LCEM hydrogel scaffolds containing 5 mg / ml were mixed with 100,000 tumor cells extracted from human liver cancer tissue. The scaffolds were cultured for 17 days to form liver cancer organoids. The expression levels of liver cancer-specific markers and proliferation-related genes were compared and analyzed with those of the primary cancer tissue from which the tumor cells were extracted. In addition, the size of the formed liver cancer organoids was confirmed, and the expression of liver cancer-specific markers was verified through immunostaining.

[0266] As a result, the expression of AFP, a major biomarker of liver cancer, and Ki67, a proliferation marker, increased in liver cancer organoids cultured in hydrogels containing 20% ​​and 30% LCEM mixed with MAT compared to liver cancer organoids cultured in MAT. In particular, in the case of liver cancer organoids cultured in hydrogels containing 50% LCEM, the expression levels of not only AFP and Ki67 but also the liver fibrosis marker α-SMA and the activated hepatic stellate cell marker PDGFRB were significantly increased (Fig. 14A). Compared to other concentration conditions, liver cancer organoids cultured in hydrogels containing 50% LCEM showed the most similar level of expression of each marker gene to that of the primary cancer tissue. Through this, the 50% LCEM concentration condition was selected as the optimal support condition for liver cancer organoid culture. In addition, when the size of liver cancer organoids was measured according to the culture period, the size of liver cancer organoids increased significantly as the concentration of LCEM increased in the hydrogel support containing LCEM compared to MAT, and the highest cancer organoid size growth was confirmed under the condition of 50% LECM concentration (Fig. 14B). In addition, the immunostaining results showed that as the ratio of LCEM increased in the hydrogel support containing LCEM compared to liver cancer organoids formed in MAT, the expression of AFP and Ki67 in liver cancer organoids increased, and liver cancer organoids cultured in the hydrogel support with a 50% LCEM concentration showed the highest expression of AFP and Ki67 (Fig. 14C).

[0267] Through this, a 50% (v / v) LCEM hydrogel scaffold prepared by mixing 5 mg / ml decellularized liver cancer tissue-derived extracellular matrix (LCEM) and Matrigel (MAT) in a 1:1 ratio was selected as the optimal concentration condition for liver cancer organoid culture and used in subsequent experiments.

[0268]

[0269] 4-1-3. Liver cancer organoid proliferation and growth analysis

[0270] To compare the proliferation capacity of cancer organoids according to the support on which the liver cancer organoids were cultured, liver cancer organoids were cultured using tumor cells derived from four different patient tissues in MAT and a 50% (v / v) LCEM hydrogel support prepared by mixing 5 mg / ml LCEM and MAT in a 1:1 ratio, and their areas were compared.

[0271] Compared to the four types of liver cancer organoids cultured on MAT (liver cancer organoids derived from four patients), the size of liver cancer organoids cultured on LCEM hydrogel scaffolds containing 50% LCEM was significantly larger (Fig. 15).

[0272] Through this, it was confirmed that the proliferation and growth of cancer organoids can be improved by using a support containing LCEM components compared to a MAT-only support.

[0273]

[0274] 4-2. Manufacturing of colon cancer organoids

[0275] 4-2-1. Comparison of colon cancer organoid formation efficiency

[0276] To evaluate the suitability of decellularized colon cancer tissue-derived extracellular matrix (CRCEM) hydrogel scaffolds for colon cancer organoid culture, 500 crypts / fragments (1000 cells / 30μL gel) of tumor cells extracted from human colon cancer tissues were cultured in 3D for 7 days in MAT, 30 μL of decellularized intestinal extracellular matrix (IEM) hydrogel scaffolds derived from normal small intestine at a concentration of 5 mg / mL, and 30 μL of decellularized colon cancer tissue-derived extracellular matrix (CRCEM) hydrogel scaffolds at a concentration of 5 mg / mL, respectively, to form colon cancer organoids. The organoid formation efficiency and the expression levels of colon cancer-related markers were compared and analyzed.

[0277] While the formation efficiency of colorectal cancer organoids was similar in MAT and decellularized extracellular matrix (IEM) hydrogel scaffolds derived from normal intestinal tissue, the formation efficiency of colorectal cancer organoids cultured in decellularized colorectal cancer tissue-derived extracellular matrix (CRCEM) hydrogel scaffolds was significantly higher than those in MAT (Fig. 16A). In addition, analysis of the expression of colorectal cancer-related markers showed that the expression of MUC2, a major colorectal cancer biomarker, and Ki67, a proliferation marker, were increased in colorectal cancer organoids cultured in decellularized extracellular matrix (IEM) hydrogel scaffolds derived from normal intestinal tissue and decellularized colorectal cancer tissue-derived extracellular matrix (CRCEM) hydrogel scaffolds compared to colorectal cancer organoids cultured in MAT. In particular, colorectal cancer organoids cultured in decellularized colorectal cancer tissue-derived extracellular matrix (CRCEM) hydrogel scaffolds exhibited the highest cancer differentiation (Fig. 16B). For the fibrosis marker α-SMA, lower expression was observed in colon cancer organoids cultured on decellularized extracellular matrix (IEM) hydrogel scaffolds derived from normal intestinal tissues compared to colon cancer organoids cultured on MAT. Through this, it was confirmed that colon cancer organoids cultured on decellularized extracellular matrix (IEM) hydrogel scaffolds derived from normal intestinal tissues showed higher cancer differentiation than organoids cultured on MAT, but their formation efficiency and fibrosis marker expression were lower than those on MAT. It was confirmed that colon cancer organoids cultured on decellularized colon cancer tissue-derived extracellular matrix (CRCEM) hydrogel scaffolds that implemented colon cancer microenvironment showed the highest level of cancer tissue-specific marker expression and formation efficiency.

[0278] Therefore, it can be seen that the decellularized colon cancer tissue-derived extracellular matrix (CRCEM) hydrogel scaffold is most suitable for colon cancer organoid culture by best reproducing the colon cancer microenvironment compared to existing commercial scaffolds or the decellularized colon cancer tissue-derived extracellular matrix (IEM) hydrogel scaffold.

[0279]

[0280] 4-2-2. Deriving the optimal concentration conditions for the culture support

[0281] To determine the optimal concentration conditions of decellularized colorectal cancer tissue-derived extracellular matrix (CRCEM) hydrogel scaffolds for colorectal cancer organoid culture, 30 μL of hydrogel scaffolds containing 20%, 30%, and 50% (v / v) of 5 mg / mL CRCEM mixed with MAT, and 30 μL of 100% CRCEM hydrogel scaffolds containing 5 mg / mL CRCEM were mixed with 500 crypts / fragments (1000 cells / 30 μL gel) of tumor cells extracted from human colorectal cancer tissues. Colorectal cancer organoids were formed by 3D culture for 7 days, and the expression levels of colorectal cancer-specific markers and proliferation-related genes were analyzed. In addition, the size of the formed colorectal cancer organoids was confirmed.

[0282] Analysis of the formation efficiency of colon cancer organoids formed for 7 days under each culture support condition showed that as the concentration of CRCEM increased, the formation efficiency and size of colon cancer organoids significantly increased compared to MAT, and the highest formation efficiency and size of colon cancer organoids were confirmed at 50% and 100% CRCEM concentration conditions (Fig. 17A). In addition, gene expression analysis results confirmed that the expression levels of the proliferation marker Ki67 and the fibrosis marker α-SMA increased in colon cancer organoids cultured on hydrogel supports in which CRCEM was additionally mixed with MAT compared to colon cancer organoids cultured on MAT (Fig. 17B). In addition, the expression of MUC2 and VILL markers, which are major biomarkers of colon cancer, increased in colon cancer organoids cultured on hydrogels containing 20% ​​and 30% CRCEM scaffolds mixed with MAT. In particular, the expression levels increased more significantly as the concentration increased to 50% and 100% CRCEM hydrogel groups, and the highest marker gene expression level was shown in the 100% CRCEM hydrogel composed of only 5 mg / mL CRCEM scaffolds (Fig. 17B). Through this, the hydrogel scaffold containing 100% 5 mg / mL CRCEM was selected as the optimal hydrogel for colon cancer organoid culture.

[0283]

[0284] Example 5. Characterization of cancer organoids

[0285] 5-1. Expression analysis of liver cancer-specific differentiation markers in liver cancer organoids

[0286] To determine the difference in the expression of liver cancer-specific differentiation markers depending on the support on which liver cancer organoids were cultured, liver cancer organoids were cultured for 17 days on MAT and 50% (v / v) LCEM hydrogel supports (prepared by mixing 5 mg / ml LCEM with MAT in a 1:1 ratio), and immunostaining analysis was performed on the 17th day of culture for AFP, the most representative biomarker of liver cancer, Ki67, a marker related to cancer proliferation, α-SMA, a fibrosis marker, and KRT19, a liver cancer marker.

[0287] As a result, it was confirmed that the expression levels of liver cancer-related markers were increased in liver cancer organoids cultured on a 50% (v / v) LCEM hydrogel support compared to liver cancer organoids cultured on MAT (Fig. 18).

[0288] Through this, it was confirmed that the scaffold including the extracellular matrix derived from decellularized liver cancer tissue of the present invention better implements a liver cancer-specific microenvironment compared to conventional scaffolds, thereby increasing the size of liver cancer organoids and the expression level of liver cancer-related markers.

[0289]

[0290] 5-2. Gene Expression Similarity Analysis of Liver Cancer Organoids

[0291] mRNA transcriptome analysis was performed on liver cancer organoids cultured for 21 days under conditions of hydrogel supports containing MAT and various concentrations of LCEM (hydrogels containing 5 mg / ml LCEM mixed with MATRIGEL at a ratio of 20%, 30%, or 50% (v / v)). The expression distribution of genes expressed in each group was analyzed by hierarchical clustering, and the gene expression similarity with the primary cancer tissue of the same patient was compared and analyzed through Pearson coefficient, Spearman coefficient, and Cosine similarity score.

[0292] Hierarchical clustering analysis results showed that groups with similar gene expression patterns were located in close clusters depending on the support on which the liver cancer organoids were cultured (Fig. 19A). Specifically, liver cancer organoids cultured on MAT and liver cancer organoids cultured on low-concentration LCEM supports showed relatively similar gene expression patterns, and it was confirmed that the genetic similarity distance from liver cancer organoids cultured on MAT increased as the LCEM concentration increased (Fig. 19A). In addition, the similarity analysis results showed that the similarity was measured differently depending on the support conditions on which the organoids were cultured, even though they were derived from the primary cancer tissue of the same patient. In particular, the similarity difference was significantly different between the MAT group and the 50% LCEM group (0.10, 0.12, and 0.017, respectively, according to the similarity analysis coefficient) (Fig. 19B). The higher the Cosine similarity score value of the analysis results, the more similar it is to the primary cancer tissue, and the liver cancer organoid group cultured on a support mixed with 5 mg / ml LCEM at a 50% ratio was found to have a higher similarity to the patient's primary cancer tissue than the MAT group (Fig. 19B).

[0293] Through this, it was confirmed that even liver cancer organoids derived from primary cancer tissue of the same patient showed significant differences in the gene expression profile of liver cancer organoids depending on the support, and in all similarity analyses, it was confirmed that liver cancer organoids cultured under MAT conditions and 50% LCEM conditions had the lowest similarity, while liver cancer organoids cultured under 50% LCEM conditions had the highest similarity to primary cancer tissue.

[0294]

[0295] 5-3. mRNA transcript hallmark pathway analysis

[0296] mRNA transcriptome analysis was performed on liver cancer organoids cultured for 21 days under conditions of hydrogel supports containing MAT and various concentrations of LCEM (hydrogels containing 5 mg / ml LCEM mixed with MATRIGEL at a ratio of 20%, 30%, or 50% (v / v)). Differences in gene expression patterns of each liver cancer organoid were analyzed for pathways highly related to HCC (Hepatocellular carcinoma).

[0297] As a result, compared to liver cancer organoids cultured on MAT, liver cancer organoids cultured on hydrogel supports containing LCEM showed an overall significant increase in the expression of genes related to pathways with high expression levels in actual HCC as the concentration of LCEM contained in the support increased (Fig. 20). In particular, a significant increase in pathways related to cancer growth and metastasis, such as inflammatory responses, epithelial to mesenchymal transition (EMT) signaling, hypoxia, KRAS signaling, p53 signaling, and protein phosphorylation, was confirmed.

[0298] Through this, similar to the genetic similarity analysis results of Example 5-1 above, it was verified that as the ratio of LCEM increases, the difference in gene expression pattern between liver cancer organoids cultured in MAT and MAT increases, and it was proven that the hydrogel support including LCEM of the present invention provides a liver cancer-specific microenvironment, thereby implementing an actual liver cancer-related pathway and producing liver cancer organoids with a similar liver cancer-specific gene expression pattern.

[0299]

[0300] 5-4. Transcriptome analysis of genes related to liver cancer development

[0301] For an in-depth gene ontology comparison of the two groups (MAT and 50% LCEM) showing the most significant expression differences in the above mRNA transcriptome analysis, gene mRNA transcriptome analysis was performed to identify gene ontologies and liver cancer-related gene expression levels that are majorly involved in cancer growth, metastasis, immune response, and inflammatory response in liver cancer organoids cultured in MAT for 21 days and liver cancer organoids cultured in 50% (v / v) LCEM hydrogel supports, and gene ontology biological process (GOBP) analysis was performed on genes whose expression levels were significantly increased under the 50% LCEM condition compared to MAT.

[0302] The genes showing significant differences in expression levels (fold change > 2 or < 0.5, p-value < 0.05) between liver cancer organoids cultured in MAT and liver cancer organoids cultured in 50% (v / v) LCEM hydrogel support were expressed in a volcano plot, and it was confirmed that the number of genes with significantly increased expression levels (differentially expressed genes, DEGs) was higher in liver cancer organoids cultured in 50% LCEM than in liver cancer organoids cultured in MAT (Fig. 21a). The gene ontology analysis of genes whose expression levels were significantly increased under the 50% LCEM condition compared to MAT was displayed in a semantic plot, and it was confirmed that GOBPs, which are highly found in actual liver cancer tissues, were observed, such as Epithelial to mesenchymal transition (EMT), Cell-cell adhesion, Phosphorylation, Immune response, Vasculature development, Extracellular matrix organization, and p38MAPK cascade regulation (Fig. 21b). As a result of clustering the genes that had strong interactions with the GOBPs related to the genes whose expression levels were significantly increased under the 50% LCEM condition compared to MAT, three major strong interaction clusters were additionally identified: (1) immune response-related cluster, (2) protein phosphorylation-related cluster, and (3) cell-cell adhesion-related cluster. In particular, strong interactions were confirmed in cancer development biological processes such as immune evasion regulation (antigen receptor-mediated signaling pathway and T cell receptor signaling pathway), EMT, and cell-cell adhesion (Fig. 21c).

[0303] Through this, it was confirmed that organoids closer to actual liver cancer can be produced than existing supports by controlling the liver cancer-specific microenvironment using a hydrogel support containing LCEM.

[0304]

[0305] 5-5. Gene ontology analysis related to liver cancer development

[0306] mRNA transcriptome analysis was performed on liver cancer organoids cultured on MAT for 21 days and liver cancer organoids cultured on 50% (v / v) LCEM hydrogel supports. Gene ontology analysis related to liver cancer development was performed on genes that showed a significant increase in expression (fold change > 2, p-value < 0.05) in liver cancer organoids cultured on 50% (v / v) LCEM hydrogel supports, and heatmap analysis was performed to compare the differences in expression levels of genes belonging to representative biological processes.

[0307] As a result of gene ontology (GOBP) analysis of liver cancer organoids cultured on MAT and liver cancer organoids cultured on 50% (v / v) LCEM hydrogel scaffolds, six representative biological processes related to the actual liver cancer tissue microenvironment and liver cancer development were discovered. As a result of comparative analysis of the expression levels of each gene corresponding to the gene ontology, the expression of genes belonging to Positive regulation of cell-cell adhesion (GO:0022409), Vasculature development (GO:0001944), Positive regulation of apoptotic signaling pathway (GO:2001235), Cellular response to glucose starvation (GO:0042149), Extracellular matrix organization (GO:1903053), and Regulation of epithelial to mesenchymal transition (GO:0010717) was found to be higher in liver cancer organoids cultured on 50% (v / v) LCEM hydrogel scaffolds compared to liver cancer organoids cultured on MAT. A significant increase was confirmed in liver cancer organoids (Figs. 22a to 22e). In addition, in liver cancer organoids cultured on 50% (v / v) LCEM hydrogel supports, we confirmed that not only genes related to the liver cancer microenvironment and cancer development, but also genes related to actual cancer metastasis, such as vasculature development and epithelial to mesenchymal transition (EMT), were significantly increased (Figs. 22a to 22e).

[0308] Through this, it was confirmed that it is possible to produce cancer organoids with enhanced liver cancer phenotypes using a hydrogel support containing LCEM.

[0309]

[0310] Example 6. Establishment of coculture conditions for creating a microenvironment for cancer organoids.

[0311] 6-1. Establishment of immune cell co-culture conditions for liver cancer organoids

[0312] To establish a microenvironment for liver cancer organoids through co-culture with liver-specific immune cells, 40,000 Kupffer cells (KC) differentiated from human induced pluripotent stem cells and 100,000 tumor cells extracted from human liver cancer tissues (tumor cells extracted from liver cancer tissues:Kupffer cells = 10:4) were co-cultured in 30 μL of MAT or 50% (v / v) LCEM hydrogel supports to form liver cancer organoids. The effects of co-culture with immune cells and the type of culture support were analyzed by examining the amount of inflammatory cytokines secreted by liver cancer organoids and the expression levels of cancer-specific differentiation markers.

[0313] As a result, in the group co-cultured with liver-specific Kupffer cells, it was confirmed that cancer organoids and surrounding Kupffer cells were cultured together, and larger liver cancer organoids were formed in the group using LCEM as a support compared to the group using MAT as a support (Fig. 23A). In addition, as a result of confirming the cancer-specific gene expression level by quantitative PCR analysis, the expression level of inflammatory immune cell-related markers (TNF-α and IL-10) was found to be increased in the LCEM group compared to the MAT group, and was found to increase more significantly in the MAT+KC and LCEM+KC groups co-cultured with immune cells (Fig. 23B). In particular, the highest expression of inflammatory markers was confirmed in liver cancer organoids co-cultured with immune cells in a 50% (v / v) LCEM hydrogel support (LCEM+KC group) (Fig. 23B). In addition, the LCEM group showed higher expression levels of the proliferation marker (Ki67), which is a major indicator of cancer functionality, and the hypoxia and metastasis-related marker (HIF1α) compared to the MAT group, and when immune cells were co-cultured, the marker expression was confirmed to increase the most in the LCEM group (Figure 23B). In addition, as a result of comparing the secretion amount of TNF-α, an inflammatory cytokine, of each liver cancer organoid, when liver cancer organoids were co-cultured with immune cells, the immune cells added to the liver cancer organoid culture environment differentiated into M1 type inflammatory immune cells that secrete inflammatory cytokines, and the secretion of TNF-α was found to significantly increase compared to when liver cancer organoids were cultured alone. In particular, the group using LCEM as a support showed the highest TNF-α secretion level compared to the group using MAT as a support (Figure 23C).

[0314] Through this, it was confirmed that the conditions for co-culturing immune cells using a hydrogel support containing LCEM are the optimal liver cancer organoid culture platform that establishes a liver cancer tissue-specific microenvironment and inflammatory environment.

[0315]

[0316] 6-2. Establishment of coculture conditions for liver cancer organoids with hepatic stellate cells

[0317] To establish a microenvironment for liver cancer organoids through co-culture with liver-specific hepatic stellate cells (HSCs), 30,000 hepatic stellate cells differentiated from human induced pluripotent stem cells and 100,000 tumor cells extracted from human liver cancer tissue (tumor cells extracted from liver cancer tissue: hepatic stellate cells = 10:3) were co-cultured in hydrogel supports containing 30 μL of MAT or various concentrations of LCEM (20%, 30%, and 50% of LCEM at a concentration of 5 mg / ml) to form liver cancer organoids. The effects of co-culture of formed liver cancer organoids with hepatic stellate cells and the type of culture support were confirmed by immunostaining analysis of the expression of ALB, a liver cancer differentiation marker, F-actin, a cell-to-cell junction marker, and VIM (Vimentin), a hepatic stellate cell activation marker. In addition, the amount of collagen, an indicator of fibrosis, one of the characteristics of liver cancer, and accumulated by activated hepatic stellate cells was analyzed.

[0318] Marker expression analysis confirmed that ALB was well expressed in liver cancer organoids of all groups, and F-actin staining confirmed cell-to-cell junctions within the organoids in all groups (Fig. 24A). In addition, VIM immunostaining showed that activated hepatic stellate cells were not present in the group cultured with liver cancer organoids alone (- HSC), whereas co-cultured hepatic stellate cells were confirmed inside and around the liver cancer organoids in the group co-cultured with hepatic stellate cells (+ HSC), and it was confirmed that the proportion of activated hepatic stellate cells increased as the mixing ratio of LCEM increased (Fig. 24A). In addition, quantitative analysis of the immunostaining results of the VIM marker confirmed that the VIM expression level was significantly increased in the groups co-cultured with hepatic stellate cells (+ HSC) compared to the groups not co-cultured with hepatic stellate cells (- HSC). In addition, within the group co-cultured with hepatic stellate cells, it was quantitatively confirmed that the VIM expression level was significantly increased in the hepatic cancer organoids cultured in the high-concentration LCEM group compared to the hepatic cancer organoids cultured in MAT (Fig. 24b). In addition, a comparison of the collagen accumulation amount confirmed that collagen accumulation increased approximately 2-fold in the hepatic cancer organoids co-cultured with hepatic stellate cells in the hydrogel support of 50% LCEM compared to the hepatic cancer organoids cultured alone in MAT (Fig. 24c).

[0319] Through this, it was confirmed that the conditions for co-culturing liver-specific hepatic stellate cells using a hydrogel support containing LCEM are the optimal liver cancer organoid culture platform in which the liver cancer tissue-specific microenvironment and fibrosis environment are more similar to actual cancer tissue.

[0320]

[0321] Example 7. Confirmation of the feasibility of drug evaluation using co-cultured liver cancer organoids.

[0322] Liver cancer organoids were cultured for 21 days by mixing 100,000 tumor cells extracted from human liver cancer tissues and co-cultured with hepatic stellate cells (30,000) and Kupffer cells (40,000), which constitute a liver cancer tissue-specific microenvironment, in 30 μL of a 50% (v / v) LCEM hydrogel scaffold (a scaffold in which 5 mg / ml LCEM is mixed with Matrigel at a 50% ratio). The organoids were then treated with clinical candidate drugs (sorafenib or lenvatinib) currently widely used for liver cancer treatment at concentrations of 5, 10, or 20 μM for 3 days, and the apoptotic effect on the liver cancer organoids was confirmed by optical microscopy and immunostaining.

[0323] As a result, the drug sorafenib significantly reduced the size of liver cancer organoids and started to die from a concentration of 10 μM or higher, and almost all organoids were killed at 20 μM. In the case of the drug lenvatinib, the drug significantly reduced the size of liver cancer organoids and started to die from a concentration of 5 μM or higher, and almost all organoids were killed at 10 μM and 20 μM conditions (Fig. 25a). In addition, the results of immunofluorescence staining analysis showed that in the group treated with sorafenib, the expression level of the apoptosis marker (cleaved-caspase 3; C-CASP3) increased in liver cancer organoids and surrounding microenvironment cells from a concentration of 10 μM or higher and started to die, and the expression level was further increased at 20 μM, and almost all organoids were killed (Fig. 25b and C). In addition, the expression of F-actin, a cell junction marker, began to decrease from concentrations above 10 μM, and it was confirmed that tight junctions were almost collapsed at a concentration of 20 μM (Fig. 25b). In the case of VIM, a hepatic stellate cell marker, as the concentration of sorafenib increased, the expression level decreased and the cells began to die, and it was confirmed that the cells were almost dead at the 20 μM condition (Fig. 25b and c). In addition, in the case of the drug lenvatinib, the size of liver cancer organoids was significantly reduced from concentrations above 5 μM, the expression level of C-CASP3 in liver cancer organoids and surrounding microenvironment cells increased, and the cells began to die, and it was confirmed that the tight junctions of F-actin were almost collapsed from the 10 μM concentration condition (Fig. 25b and c). In addition, in the case of VIM, a hepatic stellate cell marker, as the concentration of lenvatinib increased, the expression level decreased and the cells began to die, and it was confirmed that they were almost dead under the 10 μM and 20 μM conditions (Figures 25b and c).In this way, when comparing drug sensitivity to the two drugs in liver cancer organoids with a liver cancer-specific microenvironment, it was confirmed that drug sensitivity to the drug lenvatinib was higher than that to the drug sorafenib, similar to actual clinical results.

[0324]

[0325] Through the above results, it can be seen that the conditions for co-culturing liver cancer organoids and liver-specific microenvironment cells in the decellularized liver cancer tissue-derived support of the present invention enable more precise drug testing by identifying not only the drug-induced liver cancer organoid killing effect but also the influence of microenvironment cells.

[0326]

[0327] The present invention relates to a decellularized cancer tissue-derived extracellular matrix scaffold and its use. Compared to conventional mouse-derived scaffolds or decellularized normal tissue-derived scaffolds, the decellularized cancer tissue-derived extracellular matrix according to the present invention contains a large amount of distinct malignant tumor-related extracellular matrix, proliferation, and metastasis components, thereby better realizing a cancer tissue-specific microenvironment. It was confirmed that cancer organoids cultured using the same had improved cancer-specific markers, proliferation capacity, size, and inflammatory cytokine secretion amount, and better realizing the composition and phenotype of cancer tissue at a level similar to that of actual cancer tissue. According to the present invention, the decellularized cancer tissue-derived extracellular matrix scaffold can be utilized for disease modeling, drug screening, new drug toxicity evaluation, personalized drug screening, etc., and through this, low-cost, high-functionality pharmaceuticals can be manufactured, which can be effectively used for the prevention or treatment of diseases.

[0328] Therefore, the extracellular matrix support derived from decellularized cancer tissue according to one embodiment of the present invention can be considered to have industrial applicability.

Claims

1. A support composition comprising an extracellular matrix derived from decellularized cancer tissue.

2. A support composition comprising an extracellular matrix derived from decellularized solid tumor tissue according to claim 1.

3. In the second paragraph, the solid cancer tissue is a support composition which is bile duct cancer tissue, bile duct cancer tissue, gallbladder cancer tissue, thyroid cancer tissue, adrenal cancer tissue, parathyroid cancer tissue, oral cancer tissue, salivary gland cancer tissue, laryngeal cancer tissue, tongue cancer tissue, endometrial cancer tissue, tonsil cancer tissue, peritoneal cancer tissue, spinal tumor tissue, breast cancer tissue, colon cancer tissue, rectal cancer tissue, colon cancer tissue, head and neck cancer tissue, lung cancer tissue, stomach cancer tissue, skin cancer tissue, prostate cancer tissue, bladder cancer tissue, urethra cancer tissue, urothelial cancer tissue, kidney cancer tissue, thyroid cancer tissue, uterine cancer tissue, cervical cancer tissue, brain cancer tissue, anal cancer tissue, ovarian cancer tissue, neuroblastoma tissue, intraperitoneal tumor tissue, osteosarcoma tissue, ocular tumor tissue, esophageal cancer tissue, or pancreatic cancer tissue.

4. A support composition comprising 0.005 to 20 mg / mL of an extracellular matrix derived from adenomatous tissue according to claim 1.

5. In the first paragraph, the extracellular matrix derived from acellular cancer tissue comprises glycosaminoglycan, collagen, glycoprotein, proteoglycan, ECM-affiliated proteins, ECM regulators, and secreted factors. A support composition.

6. In the first paragraph, the extracellular matrix derived from decellularized cancer tissue has, compared to the extracellular matrix derived from normal tissue, Collagen type 1 alpha 1 (COL1A1), Collagen type 1 alpha 2 (COL1A2), Collagen type 6 alpha 1 (COL6A1), Collagen type 6 alpha 2 (COL6A2), Collagen type 6 alpha 3 (COL6A3), Collagen type 6 alpha 6 (COL6A6), Annexin A2 (ANXA2), Poly(ADP-ribose) polymerase 1 (PARP1), Catenin Beta 1 (CTNNB1), Clusterin (CLU), High mobility group box 1 (HMGB1), Lamin A / C (LMNA), Inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), Complement component 3 (C3), Betaine homocysteine ​​S-methyltransferase (BHMT), A support composition having an increased content of Phosphoenolpyruvate carboxykinase 2 (PCK2), Versican (VCAN), Complement C1q binding protein (C1QBP), Heat shock protein family E member 1 (HSPE1), Pyruvate kinase L / R (PKLR), Myeloperoxidase (MPO), Fibronectin (FN1), Vitronectin (VTN), Fibrillin 1 (FBN1), or Galectin-1 (LGALS1).

7. A support composition according to claim 1, further comprising polyethylene glycol (PEG), pectin, collagen, gelatin, Matrigel, methacrylated gelatin (GelMA), hyaluronic acid, alginate, fibrin, chitosan, cellulose, agarose, or a combination thereof.

8. A support composition for two-dimensional or three-dimensional culture according to claim 1.

9. A support composition for cell or organoid culture according to claim 1.

10. A support composition according to claim 9, wherein the cell or organoid is a cancer cell, a cancer stem cell, or a cancer organoid prepared therefrom.

11. In paragraph 1, the extracellular matrix derived from decellularized cancer tissue is: a) A step of crushing cancer tissue; b) a step of decellularization; and c) A support composition manufactured by a method including a freeze-drying step.

12. A composition for producing cancer organoids, comprising the support composition of claim 1 and cells dispensed onto the support composition.

13. A composition for producing a cancer organoid, wherein the cell is a cancer cell or a cancer stem cell in claim 12.

14. A composition for producing cancer organoids, further comprising tissue-specific cells, mesenchymal cells, fibroblasts, pericytes, vascular cells, or immune cells, in accordance with claim 12. 15.1) Step of producing extracellular matrix derived from decellularized cancer tissue; 2) A step of manufacturing a 3D hydrogel scaffold by mixing the decellularized extracellular matrix with polyethylene glycol (PEG), pectin, collagen, gelatin, Matrigel, methacrylated gelatin (GelMA), hyaluronic acid, alginate, fibrin, chitosan, cellulose, agarose, or a combination thereof; and 3) A method for producing a cancer organoid, comprising a step of culturing cancer cells or cancer stem cells in a 3D hydrogel support to induce formation and development of an organoid.

16. In paragraph 15, the extracellular matrix derived from decellularized cancer tissue is: a) A step of crushing cancer tissue; b) a step of decellularization; and c) A method for producing a cancer organoid, comprising a freeze-drying step.

17. A method for producing a cancer organoid, according to claim 15, further comprising a step of co-culturing the cancer organoid by adding tissue-specific cells, mesenchymal cells, fibroblasts, pericytes, vascular cells, or immune cells.

18. Cancer organoids manufactured by the method of Article 15.

19. A cancer organoid exhibiting heterogeneity within the tissue in accordance with claim 18.

20. A cancer organoid according to claim 18, wherein the expression of MUC2, AFP, Ki67, α-SMA, KRT19, PDGFRB, HIF1α, TNF-α or IL-10 is increased.

21. A method for screening a cancer therapeutic agent, comprising the step of treating a cancer organoid of claim 18 with a test substance.

22. A method for evaluating drug responsiveness using a cancer organoid, comprising the step of treating a therapeutic agent candidate substance to the cancer organoid of claim 18. 23.1) A step of isolating cancer tissue from a patient with cancer; 2) A step of producing a decellularized cancer tissue-derived extracellular matrix from cancer tissue; 3) A step of manufacturing a 3D hydrogel scaffold including an extracellular matrix derived from decellularized cancer tissue; 4) A step of producing cancer organoids by culturing cancer cells or cancer stem cells isolated from a patient on a support; and 5) A personalized anticancer drug screening method comprising a step of treating a candidate anticancer drug to a cancer organoid.

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