KR20220117170A - High purity cancer organoids and a method of producing thereof - Google Patents

Abstract

In order to predict drug reactivity of cancer patients, it is desirable that the in vivo environment for a cancer tissue is established for cancer organoids. However, there is a problem that, during organoid cultivation, normal organoids invade and overgrow in addition to cancer organoids to hinder the growth of cancer organoids. The present invention conceived to solve the above problem relates to pure cancer organoids uncontaminated by normal cells and a preparation method thereof. The cancer organoids prepared according to the preparation method of the present invention are expected to be widely used in the medical field, such as improving the prognosis of cancer patients, because the cancer organoids enable patient-specific precise treatment when the in vivo environment for cancer is established. The preparation method of the present invention comprises: a step (a) of dissociating cells from cancer tissues separated from a subject; a step (b) of mixing cell pellets obtained after centrifugation with a porous medium to sow the cells; and a step (c) of inhibiting normal cells.

Description

High purity cancer organoids, and a method for producing the same

The present invention relates to a high-purity cancer organoid, and a method for preparing the same.

Recently, as the number of cancer patients is rapidly increasing due to an aging population, research to overcome cancer through patient-specific chemotherapy is being conducted competitively. In particular, targeted therapies are the core of patient-specific anticancer treatment research in that they can dramatically increase the response rate to anticancer drugs in terminal cancer patients, minimize the side effects of anticancer drugs, and predict the cancer response to the treatment with molecular signals. can do. However, the development of organoids for testing the response before actually administering a patient-specific targeted therapy to a patient has many limitations.

In order to predict the drug reactivity of a cancer patient, it is preferable that the cancer organoids in the body mimic the environment of the cancer tissue, but normal cells in the periphery are collected together in the process of extracting the cancerous tissue from the patient for the manufacture of cancer organoids. Accordingly, during organoid culture, normal organoids other than cancer organoids invade and overgrowth, thereby inhibiting the growth of cancer organoids. In order to suppress this, the prior art has artificially separated and cultured normal organoids and cancer organoids under a microscope.

Accordingly, the present invention is to solve the above problems, and relates to a method for selectively inhibiting growth of only normal cells in the preparation of cancer organoids. The present invention is expected to be widely used in the medical field, such as improving the prognosis of cancer patients, since it is possible to prepare pure cancer organoids that are not contaminated by normal cells.

The present invention has been devised to solve the problems in the prior art, and relates to a high-purity cancer organoid, and a method for manufacturing the same.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS Various embodiments described herein are described below with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

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

In one embodiment of the present invention, "cancer" is characterized by uncontrolled cell growth, and by such abnormal cell growth, a cell mass called a tumor is formed, penetrates into surrounding tissues, and in severe cases, into other organs of the body. It is said to be transferable. Scientifically, it is also called a neoplasm. Cancer is an intractable chronic disease that in many cases cannot be cured fundamentally even if it is treated with surgery, radiation, and chemotherapy but causes pain and ultimately death. and external factors. It is not known exactly how normal cells are transformed into cancer cells, but it is known that a significant number of cancers are affected by external factors such as environmental factors. Internal factors include genetic factors and immunological factors, and external factors include chemicals, radiation, and viruses. In general, cancer cells exhibit different genetic characteristics from normal cells. Genes related to the development of cancer are called oncogenes, and genes that suppress tumor development are called tumor suppressor genes. Cancer occurs when the balance between oncogenes and tumor suppressor genes is disrupted by the intrinsic or extrinsic factors described above. Specifically, cancer occurs when an oncogene is overexpressed or when a tumor suppressor gene fails to function due to a mutation or the like. Representative oncogenic genes include c-myc (Proto-oncogene bHLH transcription factor), c-src (Proto-oncogene tyrosine-protein kinase), NOTCH (Notch Receptor), FGFR (Fibroblast Growth Factor Receptor), EGFR (Epidermal Growth Factor Receptor) ) and the like, and representative tumor suppressor genes include transforming growth factor-beta (TGF-β) and tumor protein p53 (TP53). Depending on the site of occurrence, cancers include oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, cervical cancer, skin cancer, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, and anal cancer. Peripheral cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, Prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma, The cancer of the present invention is preferably a female cancer (breast cancer, cervical cancer, ovarian cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, thyroid cancer, parathyroid cancer), but is not limited thereto.

In one embodiment of the present invention, "cancer tissue" refers to a set of cancer cells for examination or disposal separated from the human body by incision or fine needle method when cancer incision is required for the purpose of benefiting the human body in a cancer patient do. The cancer tissue may be used to prepare an organoid for a specific cancer tissue and develop a customized therapeutic agent for the purpose of benefiting the human body of a cancer patient.

In one embodiment of the present invention, "metastasis" refers to cancer cells leaving a primary organ and moving to another organ. The spread of cancer to other parts of the body is largely divided into growth of cancer tissue from the primary cancer and directly infiltrating surrounding organs, and distant metastasis along blood vessels or lymphatic vessels to other distant organs. The rate or pattern of metastasis may vary depending on the intrinsic nature of cancer, and may be regulated by suppressing the expression of a gene related to metastasis or suppressing the protein activity of the gene.

In one embodiment of the present invention, "organoid" refers to a structure made by culturing or recombining cells isolated from stem cells or organ cells, and maintaining the shape of the tissue in vitro, artificial organs, bio-organs, It can be interpreted in the same sense as mini-organ, spheroid, spheroid, or embryoid body. When the cell of origin for producing the organoid is collected from cancer tissue, the organoid is a cancer organoid and is characterized by being composed of cancer cells.

In one embodiment of the present invention, "porous medium" refers to a liquid that is initially liquid and can be solidified under various conditions, and the conditions include temperature, light, pH, pressure, vibration, and the like. For example, a porous medium that is liquid at high temperatures may gel at low temperatures. The porous medium may be a material such as a hydrogel or a porous membrane. The hydrogel may be a natural or synthetic polymer material. The hydrogel may include collagen, fibrin, agarose, agar, matrigel, alginate, gelatin, and the like.

In one embodiment of the present invention, "Matrigel" is an artificially synthesized extracellular matrix that is commercially sold, and is composed of laminin, collagen type IV, heparin sulfate proteoglycan and entactin/nidogen. , but is not limited thereto.

In one embodiment of the present invention, "diagnosis" refers to confirming the presence or characteristics of a pathological state, and for the purpose of the present invention, diagnosis is to determine whether or not cancer has occurred, progressed, and metastasized. Cancer can be diagnosed by visual or cytological confirmation of tissue from a patient with cancer onset or suspected of metastasis. (膿), secretion, bile, pharyngeal mucus, urine (尿), 　 bile, 　 feces, etc.) a method using a cancer-associated antibody, a method for directly detecting a cancer-related protein in the sample, or a method for encoding a cancer-related protein Cancer can be diagnosed by a method of directly detecting a nucleic acid. Diagnostic methods using antigen-antibody binding or direct detection of cancer-associated proteins include Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, octero Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Protein Chip ( protein chip), but is not limited thereto, and methods for directly detecting a nucleic acid encoding a cancer-related protein include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time Reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting (Northern blotting) or DNA chip, but is not limited thereto.

In one embodiment of the present invention, "screening" refers to selecting a substance having a specific desired property from a candidate group consisting of several substances by a specific manipulation or evaluation method. For the purpose of the present invention, the screening of the present invention is to treat the cancer organoid according to the present invention with an anticancer candidate material, and when the size of cancer tissue is reduced or metastasis is inhibited by the anticancer candidate material, the candidate material is used as a cancer treatment agent or a cancer metastasis inhibitor.

In one embodiment of the present invention, the term "anticancer drug candidate" refers to an unknown substance used in screening to test whether it has inhibitory activity against the growth or metastasis of tumor tissue. The candidate substances include, but are not limited to, chemicals, peptides, proteins, antibodies, and natural extracts. Candidates analyzed by the screening methods of the present invention are single compounds or mixtures of compounds (eg, natural extracts or cell or tissue cultures). Candidates can be obtained from libraries of synthetic or natural compounds. Methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and natural compounds libraries are available from Pan Laboratories (USA). ) and commercially available from MycoSearch (USA), but is not limited thereto. Candidate substances can be obtained by various combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution It can be obtained by the required synthetic library method, the 1-bead 1-compound library method, and the synthetic library method using affinity chromatography selection. Methods for synthesizing molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci.

U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, et al.

Current anticancer candidates are classified into the following six categories according to their biochemical mechanism of action.

(1) alkylating agents: highly reactive substances with the ability to introduce an alkyl group R-CH ₂ to a certain compound. When acted on cells, most of them react with N7 of guanine in DNA to modify the DNA structure, It causes chain cutting [鎖切斷] to show anticancer and cytotoxic effects. Drugs belonging to this category include: ① Nitrogen mustard: Nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, etc. ② Ethyleneimine: thiotepa ③ Alkylsulfonate: Busulfan ④ Tri Azine-hydrazine: DTIC (dacarbazine), procarbazine ⑤ Nitrosourea: BCNU, CCNU, methyl-CCNU, etc.

(2) Antimetabolites: Drugs belonging to this group inhibit the metabolic process necessary for the proliferation of cancer cells. ① Folate derivative: Mesotrexate (MTX) ② Purine derivatives: 6-mercaptopurine (6-MP), 6-thioguanine ③ Pyrimidine derivatives: 5-fluorouracil and cytarabine.

(3) Antibiotics (抗生物質: antibiotics): Adriamycin, daunorubicin, bleomycin, mitomycin-C, actinomycin-D, etc. are among the antibiotics produced by bacteria that exhibit anticancer action.

(4) Mitosis inhibitors (有絲分裂抑制劑: vinca alkaloids): These drugs are mitotic-specific drugs that stop cell division in the metaphase of mitosis. vincristine, vinblastine, VP-16-213 and VM-26.

(5) Hormones: Some types of cancer can be treated by administering hormones. When male hormones are used, breast cancer, female hormones are effective against prostate cancer, and progesterone is effective against endometrial cancer. is used for the treatment of acute lymphoblastic leukemia or lymphoma, and for breast cancer, tamoxifen, an anti-female hormone, is used.

(6) Others: cisplatin, L-asparaginase, o,p-DDD, and the like. As described above, there are about 40 types of anticancer drugs currently used for cancer treatment, and each drug has a large difference in its anticancer scope.

In one embodiment of the present invention, "administration" means introducing the composition to the patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration, intraperitoneal administration, intraperitoneal administration, intrathecal administration may be made, but is not limited thereto. does not In the present invention, the effective amount to be administered is the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health status, sex and diet, administration time, It can be adjusted according to various factors including the route of administration and secretion rate of the composition, duration of treatment, and drugs used concurrently. For adults, the therapeutic pharmaceutical composition can be administered into the body in an amount of 50ml to 500ml at a time, 0.1ng/kg-10mg/kg in the case of a compound, and 0.1ng/kg/kg in the case of a monoclonal antibody to the protein It may be administered at a dose of kg-10 mg/kg. The administration interval may be 1 to 12 times a day, and if administered 12 times a day, it may be administered once every 2 hours. In addition, anticancer peptides or nucleic acids, including antibodies or siRNAs, may be administered alone or with other therapies known in the art, for example, chemotherapeutic agents, radiation and surgery for the treatment of desired cancer. In addition, the peptides and nucleic acids of the present invention can be administered in admixture with other treatments designed to enhance the immune response, for example adjuvants or cytokines (or nucleic acids encoding cytokines) as well known in the art. . Other standard delivery methods may be used, such as biolistic delivery or ex vivo treatment. In ex vivo treatment, for example, antigen-presenting cells (APCs), dendritic cells, peripheral blood mononuclear cells, or bone marrow cells can be obtained from a patient or an appropriate donor and activated ex vivo with the present immune composition and then administered to the patient. have.

In one embodiment of the present invention, (a) dissociating cells from the cancer tissue isolated from the subject; (b) mixing and seeding the cell aggregate (pettet) obtained after centrifugation with a porous medium; And, (c) inhibiting the growth of normal cells; culturing in a culture medium comprising; provides a method for producing cancer organoids comprising; dissociation in step (a) is performed with collagenase It provides a method for producing a cancer organoid, wherein the porous medium in step (b) provides a method for producing a cancer organoid that is a hydrogel or a membrane, wherein the hydrogel is collagen, Fibrin (fibrin), agarose (agarose), agar (agar), matrigel (matrigel), alginate (alginate), and production of cancer organoids that are at least one selected from the group consisting of gelatin (gelatin) It provides a method for producing a cancer organoid, wherein the inhibition of normal cell growth in step (c) is performed with an anticancer agent, wherein the anticancer agent is an epidermal growth factor receptor-tyrosine kinase inhibitor (Epidermal Growth Factor Receptor-) It provides a method for producing a cancer organoid that is a Tyrosine kinase Inhibitor; EGFR-TKI), and the anticancer agent provides a method for producing a cancer organoid that is gefitinib or erlotinib .

In another embodiment of the present invention, there is provided a culture medium for cancer organoid culture comprising an anticancer agent, wherein the anticancer agent is an Epidermal Growth Factor Receptor-Tyrosine kinase inhibitor (EGFR-TKI). It provides a culture medium for culturing cancer organoids, and the anticancer agent provides a culture medium for culturing cancer organoids that is gefitinib or erlotinib.

In another embodiment of the present invention, there is provided a cancer organoid treated with an anticancer agent, wherein the anticancer agent is an Epidermal Growth Factor Receptor-Tyrosine kinase inhibitor (EGFR-TKI). It provides, and the anticancer agent provides a cancer organoid that is gefitinib (gefitinib), or erlotinib (Erlotinib).

In another embodiment of the present invention, the step of treating an anticancer candidate substance to the cancer organoid treated with an anticancer agent; And (b) when the size of the tissue is reduced or maintained by the anticancer candidate substance, it provides a screening method of an anticancer candidate material comprising the step of determining the candidate material as a cancer therapeutic agent, wherein the anticancer agent is epidermal growth factor receptor- It provides a screening method of an anticancer candidate that is a tyrosine kinase inhibitor (Epidermal Growth Factor Receptor-Tyrosine kinase Inhibitor; EGFR-TKI), wherein the anticancer agent is gefitinib, or erlotinib A method for screening an anticancer candidate is provided.

In another embodiment of the present invention, the step of treating an anticancer candidate substance to the cancer organoid treated with an anticancer agent; And (b) when the size of the cancer tissue is reduced or maintained by the anticancer candidate substance, the information providing method for selecting a patient-specific cancer treatment method comprising the step of selecting the candidate substance as a target patient's cancer therapeutic agent It provides, and the selection of the cancer treatment method is chemotherapy, radiation therapy, surgical therapy, immune cell therapy, or a combination thereof provides an information providing method for the selection of a patient-specific cancer treatment method, the anticancer agent Epidermal Growth Factor Receptor-Tyrosine kinase inhibitor (Epidermal Growth Factor Receptor-Tyrosine kinase Inhibitor; EGFR-TKI) provides an information providing method for selecting a patient-specific cancer treatment method, the anticancer agent gefitinib (gefitinib) , or erlotinib (Erlotinib) provides an information providing method for the selection of a patient-specific cancer treatment method.

Hereinafter, the present invention will be described in detail step by step.

The present invention is designed to solve the conventional problem of inhibiting the growth of cancer organoids by intrusion and overproliferation of normal cells other than cancer cells when preparing cancer organoids. it's about

Since the high-purity cancer organoid prepared by the method of the present invention can accurately predict drug reactivity in cancer patients, it is expected to be widely used in the medical field, such as improving the prognosis of cancer patients.

1 shows the organoid production results when culturing cancer organoids continuously in EM culture medium, or in gefitinib culture medium for 2 weeks, 4 weeks, or 8 weeks according to an embodiment of the present invention. .
Figure 2 is, according to an embodiment of the present invention, when the cancer organoids are continuously cultured in the EM culture medium, or the organoid production results when cultured for 2 weeks, 4 weeks, or 8 weeks with the gefitinib culture medium, and the The results of H&E staining of organoids are shown.
3 to 5 are, according to an embodiment of the present invention, A83-01 is removed from the EM culture medium and TGF ^- β is added (-A83-01 ⁺ TGFb), Nutlin is added to the culture medium ( ⁺ Nutlin), or shows the results of culturing cancer organoids in gefitinib culture. 3 to 5 , “T” denotes a cancer organoid, and “N” denotes a normal organoid.
6 shows a total of 195 genes to be analyzed for genetic variation according to an embodiment of the present invention.
7 is a diagram showing the results of analysis of genetic mutations in a patient-derived cancer tissue sample according to an embodiment of the present invention.
8 is a diagram showing the results of gene mutation analysis in cancer organoids cultured for 8 weeks in EM culture medium according to an embodiment of the present invention.
9 is a diagram showing the results of gene mutation analysis in cancer organoids cultured for 8 weeks in gefitinib culture medium according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Manufacture and evaluation of high-purity cancer organoids with growth inhibition of normal cells

Patient-derived cancer tissues were prepared by receiving endometrial cancer patient lesion tissues from a domestic university hospital (Yonsei Severance Hospital) (n=61). A control group for cancer organoids was prepared by preparing endometrial normal organoids.

For organoid preparation, the obtained tissue was washed with RPMI 1640 culture medium, pulverized into small units, and 2 mg/ml collagenase type I 5 ml was added and stirred at 37° C. for 30 minutes. . Then, the collagenase was neutralized with 20 ml of RPMI1640, and centrifuged at 1000 rpm for 5 minutes to obtain a cell aggregate (pellet). When blood was mixed, the cell aggregates were diluted with 1 ml of distilled water and reacted on ice for 3 minutes, after which 9 ml of RPMI1640 culture solution was added and centrifuged to re-obtain cell aggregates. The obtained petlets were diluted in 1 ml of RPMI1640 culture solution, counted, and centrifuged again to mix the cell aggregate with Matrigel (15 μl/well), and then incubated at 37° C. for 1 hour after dispensing in a 24-well plate. Then, a custom culture medium (complete medium) was added and cultured in a 37° C. CO ₂ incubator. The culture medium was freshly replaced every 2-3 days.

The customized culture medium is applied as a basic EM culture medium, and when normal cell contamination is confirmed during culture (usually on day 3-4), it was replaced with a gefitinib culture medium. The composition of the EM culture solution and the gefitinib culture solution is shown in Table 1 and Table 2, respectively.

agent name Final
concentration ADF (Advanced DMEM/F12, -4℃) up to 50 mL Antibiotic-Antimycotic (100X, 100ml) One% R-spondin1 conditioned medium (R-spondin CM, stock : 2 μg/mL) 10% B-27 supplement (50X -20℃) - B One% N-2 supplement (100x, 50ml) One% L-glutamine (200Mm, 100ml) 2 mM N-acetyl-l-CYSTEIN (5 g, in water) 1.25mM Human EGF (Hegf STOCK: 10ug/ml) 50ng/ml Nicotinamide-Ni (stock 1M) 10 mM human Noggin - N (stock con. : 100 μg/mL) 100 ng/mL Human FGF10 -F (stock 100ug/ml) 100ug/ml Human HGF (stock 500ug/ml) 50ng/ml Primocin (antimicrobial agent for primary cells), stock 50mg/ml 100ug/ml A83-01 stock 0.5mM 0.5 μM ROCK inhibitor (Y-27632, -20℃) (stock con. : 10mM) 10 μM GlutaMAX supplement (100X, RT) 1X HEPES buffer solution (1M in H2O, 100 mL, -4℃) 2 mM

agent name Final
concentration ADF (Advanced DMEM/F12, -4℃) up to 50 mL Antibiotic-Antimycotic (100X, 100ml) One% R-spondin1 conditioned medium (R-spondin CM, stock : 2 μg/mL) 10% B-27 supplement (50X -20℃) - B One% N-2 supplement (100x, 50ml) One% L-glutamine (200Mm, 100ml) 2 mM N-acetyl-l-CYSTEIN (5 g, in water) 1.25mM Nicotinamide-Ni (stock 1M) 10 mM human Noggin - N (stock con. : 100 μg/mL) 100 ng/mL Gefitinib 10 mM 10 mM Human HGF (stock 500ug/ml) 50ng/ml Primocin (antimicrobial agent for primary cells), stock 50mg/ml 100ug/ml A83-01 stock 0.5mM 0.5 μM ROCK inhibitor (Y-27632, -20℃) (stock con. : 10mM) 10 μM GlutaMAX supplement (100X, RT) 1X HEPES buffer solution (1M in H2O, 100 mL, -4℃) 2 mM

1 and 2 show the organoid production results when the cancer organoids were continuously cultured in the EM culture medium or cultured for 2 weeks, 4 weeks, or 8 weeks with the gefitinib culture medium. As a result of the experiment, when the gefitinib culture medium was treated for 2 weeks, 4 weeks, or 8 weeks or more, it was confirmed that the distribution of cancer organoids proportionally increased according to the treatment period.

In addition, the case of culturing cancer organoids in the culture medium in which A83-01 is removed from the EM culture medium and TGF ^- β is added (-A83-01 ⁺ TGFb) or in the EM culture medium in which Nutlin is added ( ⁺ Nutlin) is excluded. Compared to the case of culturing in a fitinib culture medium, the results are shown in Figures 3 to 5. As a result of the experiment, it was confirmed that pure cancer organoids that were not contaminated by normal cells were cultured even when passages were repeated only when cancer organoids were cultured in gefitinib culture medium in common in various cases.

In addition, a total of 195 gene mutations were identified in patient-derived cancer tissue samples, cancer organoids cultured for 8 weeks in EM culture medium, or cancer organoids cultured for 8 weeks in gefitinib culture medium. Cancer tissue samples derived from the patient and cancer organoids cultured for 8 weeks in EM culture medium have a mixed state of cancer cells and normal cells, and cancer organoids cultured for 8 weeks in gefitinib culture medium contain only cancer cells. After confirming that, the test was carried out. A total of 195 genes to be analyzed are shown in FIG. 6 , and the analysis results of each group are shown in FIGS. 7 to 9 . As a result of the analysis, among the genes to be analyzed, IDH2 (Isocitrate dehydrogenase [NADP]2), FGFR1 (Fibroblast Growth Factor Receptor 1), NOTCH3 (Notch Receptor 3), and ATM (serine/threonine kinase) mutations were confirmed in all groups, ERBB2 (Erb-B2 Receptor Tyrosine Kinase 2) and RUNX1 (runt-related transcription factor 1) were specifically mutated only in cancer organoids cultured in gefitinib culture medium.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

(a) dissociating the cells from the cancer tissue isolated from the subject;
(b) mixing and seeding the cell aggregate (pettet) obtained after centrifugation with a porous medium; and,
(c) inhibiting the growth of normal cells; culturing in a culture medium containing; including, a method for producing cancer organoids.
The method of claim 1,
The dissociation in step (a) is to be performed with collagenase, the method.
The method of claim 1,
The porous medium in step (b) is a hydrogel, or a membrane, the method.
4. The method of claim 3,
The hydrogel is any one selected from the group consisting of collagen, fibrin, agarose, agar, matrigel, alginate, and gelatin. more than one method.
The method of claim 1,
Inhibition of normal cell growth in step (c) is to be performed with an anticancer agent, the method.
6. The method of claim 5,
The anticancer agent is epidermal growth factor receptor-tyrosine kinase inhibitor (Epidermal Growth Factor Receptor-Tyrosine kinase Inhibitor; EGFR-TKI), the method.
6. The method of claim 5,
Wherein the anticancer agent is gefitinib (gefitinib), or erlotinib (Erlotinib), the method.
A culture medium for culturing cancer organoids, comprising an anticancer agent.
Cancer organoids treated with chemotherapy.
(a) treating the cancer organoid of claim 9 with an anticancer candidate; and
(b) when the size of the tissue is reduced or maintained by the anticancer candidate material comprising the step of determining the candidate material as a cancer therapeutic agent, the screening method of an anticancer candidate material.
(a) treating the cancer organoid of claim 9 with an anticancer candidate; and
(b) when the size of the cancer tissue is reduced or maintained by the anticancer candidate material, comprising the step of selecting the candidate material as a target patient's cancer treatment method, information providing method for selecting a patient-specific cancer treatment method.
12. The method of claim 11,
The selection of the cancer treatment method is chemotherapy, radiation therapy, surgical therapy, immune cell therapy, or a combination thereof, the information providing method for the selection of a patient-specific cancer treatment method.

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