Pancreatic cancer patient-derived organoid and method for manufacturing same

Abstract

The present invention relates to a method for manufacturing an organoid using a pancreatic cancer patient-derived cell line and, more specifically, to a method for manufacturing a pancreatic cancer organoid, the method comprising the steps of: preparing a mixture for seeding by mixing, with Matrigel, a 2D CRC cell line established by two-dimensionally culturing a pancreatic cancer patient-derived cell line; and three-dimensionally culturing the mixture. The method, by comprising the steps, enables prediction of a patient's drug response and provides improved efficiency in terms of time and cost.

Description

Pancreatic cancer patient-derived organoid and method for manufacturing the same

[0001] The present invention relates to a method for manufacturing organoids using a cell line derived from a pancreatic cancer patient.

[0002] Currently, cell lines or animal models are primarily utilized in the development of new drugs, such as anticancer agents. Cell lines have the advantage of being easy to handle experimentally, but tissues obtained from two-dimensional cultures are not similar to actual human tissues and have the disadvantage of being difficult to reflect the characteristics of the patient. As for animal models, there are models created by transplanting cancer cell lines or cancer cells derived from cancer patients into animals (patient-derived tumor xenograft, PDTX), but these have the disadvantages of requiring a lot of time and cost for production, having low stability, and raising bioethical issues.

[0003] To overcome the aforementioned drawbacks, organoid models can be utilized as an alternative. Also known as organ analogs or mini-organs, organoids refer to three-dimensional cell cultures capable of self-organization that mimic the function and structure of actual in vivo organs. Organoids contain one or more cell types among the various types of cells that constitute organs or tissues, and must be able to reproduce the form and function of the organ or tissue. Organoids can be applied in various fields, including new drug development, artificial organ development, drug toxicity evaluation, and cell therapy development.

[0004] Meanwhile, the pancreas is an organ that performs both exocrine and endocrine functions, such as sending pancreatic fluid to the duodenum through the pancreatic duct and injecting hormones into the bloodstream; cancer cells that develop in the pancreas are called pancreatic cancer. Pancreatic cancer is difficult to diagnose early due to the absence of specific initial symptoms. Furthermore, the pancreas is characterized by being very thin—approximately 2 cm in thickness—and covered only by a capsule, which allows for easy invasion by cancer. Additionally, it is known as a cancer with a poor prognosis, with a 5-year relative survival rate of only 15.9%.

[0005] Pancreatic cancer can be classified into exocrine tumors and neuroendocrine tumors, and in most cases, it is an exocrine tumor, which is called pancreatic ductal adenocarcinoma (PDAC). Pancreatic ductal adenocarcinoma is characterized by a low response and strong resistance to cancer treatments such as chemotherapy or radiation therapy.

[0006] Anticancer drugs used in the treatment of pancreatic cancer include gemcitabine, abraxane, folfirinox, and 5-fluorouracil (5-FU). The decision to change anticancer drugs is made based on drug responsiveness after treatment with these drugs, but in many cases, the cancer progresses and worsens during this process. Furthermore, even among patients with similar clinical characteristics and stages of progression, the degree of response to anticancer drugs varies, making personalized anticancer treatment necessary.

[0007] Accordingly, the inventors have completed the present invention by devising a method for manufacturing organoids derived from pancreatic cancer patients that is efficient in terms of time and cost while effectively predicting clinical results.

[0008]

[0009] The present invention aims to provide a method for manufacturing organoids using cell lines derived from pancreatic cancer patients.

[0010] 1. A step of preparing a mixture for seeding by mixing a 2D CRC cell line established by 2D culturing a cell line derived from a pancreatic cancer patient with Matrigel; and a step of 3D culturing the mixture; comprising a method for preparing a pancreatic cancer organoid.

[0011] 2. A method for producing a pancreatic cancer organoid according to 1 above, wherein the established 2D CRC cell line is cultured with irradiated fibroblasts on a medium supplemented with a ROCK inhibitor.

[0012] 3. A method for preparing a pancreatic cancer organoid according to 2 above, wherein the medium comprises at least one selected from the group consisting of Ham's F-12 nutrient mixture, DMEM, hydrocortisone, insulin, cholera toxin, epidermal growth factor, fetal bovine serum (FBS), adenine, gentamicin, and amphotericin B.

[0013] 4. A method for preparing a pancreatic cancer organoid according to 1 above, wherein the mixture comprises mixing the 2D CRC cell line and the Matrigel in a volume ratio of 1:7 to 1:11.

[0014] 5. In the above 1,

[0015] A method for manufacturing a pancreatic cancer organoid in which the above-mentioned pancreatic cancer patient-derived cell line is a KRAS gene mutation cell line.

[0016] 6. A method for preparing a pancreatic cancer organoid according to 1 above, wherein the 2D CRC cell line is a cell line deposited under accession number KCLRF-BP-00562.

[0017] 7. Pancreatic cancer organoid prepared by any one of the methods of 1 to 6 above.

[0018] 8. In the above 7, the pancreatic cancer organoid is a pancreatic cancer organoid that expresses α-Amylase, Cytokeratin-19, Insulin, and Vimentin.

[0019] 9. A method for evaluating the efficacy of an anticancer drug comprising the step of treating the pancreatic cancer organoid of 7 with an anticancer drug candidate.

[0020] The present invention can provide an organoid using a cell line derived from a pancreatic cancer patient and a method for manufacturing the same.

[0021] The degree of a patient's drug response can be predicted using an organoid derived from a cell line of a pancreatic cancer patient according to the present invention.

[0022] By treating the organoid of the present invention with an anticancer drug candidate and predicting clinical results, the efficacy of the anticancer drug against cell lines derived from pancreatic cancer patients can be evaluated.

[0023] Figure 1 briefly illustrates the process of producing Matrigel-based organoids using pancreatic cancer patient-derived cell lines (CRC) and F-media.

[0024] Figure 2 shows the results of comparing tumor formation in primary cancer of pancreatic cancer patient-derived cell lines (YPAC-2 and YPAC-5), pancreatic cancer patient-derived cell lines (CRC), xenografts, and pancreatic cancer patient-derived organoids (CRC organoid).

[0025] Figures 3a and 3b show the results of comparing the morphology of organoids prepared using Wnt / R-spondin-containing media (Organoid culture media) and F-media (CRC culture media), respectively, when using cell lines derived from pancreatic cancer patients (YPAC-05 and YPAC-35), through bright-field microscopy and H&E staining images. It can be seen that there is no difference in the morphology of the organoids in the two media.

[0026] Figures 4a and 4b show the results of confirming the morphology of organoids of pancreatic cancer patient-derived cell lines YPAC-80 and YPAC-67, respectively, through bright-field microscopy and H&E staining images.

[0027] Figures 5a and 5b show the results of confirming K-RAS mutations in cell lines derived from pancreatic cancer patients (YPAC-23 and YPAC-35) and organoids prepared using them via PCR, respectively.

[0028] Figures 6a and 6b show the results of confirming the morphology, K-RAS mutation, and Tumorigenesis in vitro of pancreatic cancer patient-derived cell lines YPAC-80 and YPAC-67, respectively.

[0029] Figure 7 shows the results of confirming the expression of each marker (α-Amylase, Insulin, CK-19 (Cytokeratin-19), and Vimentin) in pancreatic cancer patient-derived cell lines (YPAC-87 and YPAC-91) and organoids prepared using them through fluorescence staining.

[0030] Figures 8a and 8b show the results of confirming the expression of each marker (CK-19 (Cytokeratin-19), α-Amylase, Insulin, and Vimentin) in cell lines derived from pancreatic cancer patients (YPAC-80 and YPAC-67) and organoids prepared using them, respectively, through fluorescence staining.

[0031] Figure 9 briefly illustrates the anticancer drug sensitivity screening process using a two-dimensional medium and organoids.

[0032] Figures 10a and 10b show the results of confirming drug sensitivity by measuring cell viability after treating two-dimensional culture media and organoids, respectively, of pancreatic cancer patient-derived cell lines (YPAC-12, YPAC-31, YPAC-39, YPAC-43, YPAC-46, YPAC-52, YPAC-59, and YPAC-82) with the anticancer drugs gemcitabine and Abraxane (Gemcitabine plus Abraxane).

[0033] Figure 11 shows the results of confirming drug sensitivity by measuring cell viability after treating two-dimensional culture media and organoids of pancreatic cancer patient-derived cell lines (YPAC-67) with the anticancer drugs gemcitabine and Abraxane (Gemcitabine plus Abraxane), respectively.

[0034] Figures 12a and 12b show cell viability and patient best response to the anticancer drugs gemcitabine and Abraxane in two-dimensional culture media and organoids, respectively, for pancreatic cancer patient-derived cell lines (YPAC-28, YPAC-31, YPAC-43, YPAC-44, YPAC-47, YPAC-61, YPAC-75, YPAC-82, YPAC-83, YPAC-86, YPAC-89, and YPAC-94). (PR: Partial response; SD: Stable disease; PD: Progressive disease)

[0035] The present invention provides a method for manufacturing organoids using a cell line derived from a pancreatic cancer patient.

[0036] The present invention provides a method for manufacturing a pancreatic cancer organoid that is more efficient in terms of time and cost and can predict the degree of drug response of a patient by including the steps of: mixing a 2D CRC cell line established by 2D culturing a cell line derived from a pancreatic cancer patient with Matrigel to create a mixture for seeding; and culturing the mixture in 3D.

[0037] A "pancreatic cancer patient-derived cell line" refers to a clone of cultured cells derived from a patient suffering from pancreatic cancer. It has the advantage of being able to continue generational succession by continuously dividing and proliferating through cell culture without containing other cells.

[0038] "Conditional reprogramming (CR)" refers to a cell culture technique that enables the rapid and efficient establishment of patient-derived cells from normal and tumor cells. This technique offers the advantage of rapidly and efficiently scaling up cell cultures from patient-derived samples.

[0039] "Conditionally reprogrammed cells (CRCs)" refer to cells established from patient-derived cell samples through conditional reprogramming. They offer the advantage of maintaining a viable cell state for as long as necessary and allowing for culture in desired numbers. Conditionally reprogrammed cells can be treated with specific substances to resemble stem cells or to form structures similar to original tissues or tumors. Established conditionally reprogrammed cells can be utilized in applications such as tumor formation and new drug screening.

[0040] The 2D CRC cell line of the present invention can be established by conditionally reprogramming a cell line derived from a pancreatic cancer patient and then culturing it in two dimensions. The 'pancreatic cancer patient-derived cell line (CRC)' or '2D CRC cell line' of the present invention may refer to a 2D CRC cell line derived from a pancreatic cancer patient.

[0041] In a method for preparing a pancreatic cancer organoid according to one embodiment of the present invention, the culture medium for culturing a 2D CRC cell line may include at least one selected from the group consisting of Ham's F-12 nutrient mixture, DMEM, hydrocortisone, insulin, cholera toxin, epidermal growth factor, fetal bovine serum (FBS), adenine, gentamicin, and amphotericin B.

[0042] For example, the organoid preparation method of the present invention may use F-media as a culture medium.

[0043] In this specification, F-media may include Ham's F-12 nutrient mixture, DMEM, hydrocortisone, insulin, cholera toxin, epidermal growth factor, fetal bovine serum, adenine, gentamicin, and amphotericin B.

[0044] DMEM is a medium containing 4.0 mM L-Glutamine, 4500 mg / L Glucose, and Sodium Pyruvate, and Ham's F-12 nutrient mixture is a nutrient mixture containing 1.0 mM L-Glutamine.

[0045]

[0046] According to one embodiment, the 2D CRC cell line can be prepared by a method comprising the step of culturing the pancreatic cancer-derived cell line together with irradiated fibroblasts on a medium supplemented with a ROCK inhibitor.

[0047] A "fibroblast" is a type of cell involved in the synthesis of the extracellular matrix and collagen, and is a cell type commonly found in connective tissue.

[0048] Since fibroblasts have the ability to divide and proliferate on their own while supplying nutrients and growth factors to surrounding cells, there is a problem that they proliferate faster than the cell lines derived from pancreatic cancer patients when cultured together. In one embodiment of the present invention, cell division of fibroblasts is deactivated when irradiated with radiation, so they can supply nutrients and growth factors to the cell lines derived from pancreatic cancer patients but do not proliferate on their own, thereby allowing 2D CRC cell lines to be easily established.

[0049] For example, the fibroblast according to one embodiment of the present invention may be a J2 mouse fibroblast.

[0050] "ROCK (Rho-associated kinase)" is a substance that acts in autoimmune and inflammation-related signaling pathways and is involved in the development of various cardiovascular and cerebrovascular diseases, including hypertension, atherosclerosis, ischemic stroke, heart disease, diabetic kidney disease, ophthalmic diseases, tumors, neurological damage, radiation damage, and autoimmune diseases.

[0051] "ROCK inhibitor" refers to a substance that inhibits the activity of ROCK. In one embodiment, Y-27632 was used as a ROCK inhibitor, but is not limited thereto.

[0052] In the method for preparing pancreatic cancer organoids according to one embodiment of the present invention, the 2D CRC cell line may be the cell line deposited under accession number KCLRF-BP-00562.

[0053] The cell line YPAC-67, deposited under accession number KCLRF-BP-00562, was deposited on August 27, 2024 (Accession number: KCLRF-BP-00562).

[0054] In the present invention, "organoid" refers to an organ analog as a three-dimensional cell structure produced by culturing or recombining cells in three dimensions. The organoid includes specific cells of a tissue or organ used as a model and is produced to reproduce its function and structure.

[0055] Organoids can be utilized in new drug development and disease treatment. While the use of conventional animal models has the disadvantage that results from animal experiments may differ from those of clinical trials, organoids can overcome this drawback. Furthermore, organoids produced using patient-derived cell lines can be used in personalized clinical trials.

[0056] "Matrigel" is a protein complex extracted from sarcoma cells of EHS (Engelbreth-Holm-Swarm) mice (a product name of BD Bioscience) and contains an extracellular matrix (ECM) such as laminin, collagen, and heparin sulfate proteoglycan, as well as various growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), TGF-β, or platelet-derived growth factor (PDGF). The complexes constituting Matrigel provide a complex extracellular environment found in many tissues, and are therefore used as substrates for cell culture.

[0057] The term "patient-derived" means obtaining biological samples from a pancreatic cancer patient, and these biological samples include, for example, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, blood (including plasma and serum), sputum, tears, mucus, nasal washes, nasal aspirates, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirates, and bronchi. It may include, but is not limited to, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract, or cerebrospinal fluid.

[0058] For the purposes of the present invention, biological samples may be tissues or cancer cell lines isolated from cancer patients, but are not limited thereto.

[0059] In one embodiment, the biological sample may be tumor tissue isolated from a pancreatic cancer patient.

[0060] The method for manufacturing organoids according to the present invention may add antibiotics, antifungal agents, and agents that prevent the growth of Mycoplasma to the organoid culture medium.

[0061] The method for preparing organoids according to the present invention may involve mixing a cell line derived from a pancreatic cancer patient and Matrigel in a volume ratio of 1:7 to 1:11, 1:8 to 1:10, 1:9 to 1:10, or 1:9, but is not limited thereto.

[0062] The present invention provides a method for preparing a pancreatic cancer organoid by mixing a 2D CRC cell line and Matrigel in a volume ratio of 1:7 to 1:11.

[0063] Matrigel is a gel-type substrate that serves as a support for cells to grow in three dimensions. The 2D CRC cell line of the present invention can be cultured in three dimensions after being mixed with Matrigel.

[0064] For example, the concentration of Matrigel and the volume ratio of 2D CRC cell lines to Matrigel can be adjusted according to the cell growth rate.

[0065] In a method for manufacturing pancreatic cancer organoids according to one embodiment of the present invention, the cell line derived from a pancreatic cancer patient may be a cell line with a KRAS gene mutation. Among the cell lines derived from pancreatic cancer patients, the cell line with a KRAS gene mutation, the 2D CRC cell line derived therefrom, and the 3D organoid are all KRAS gene mutants, and such mutants are very effective in manufacturing 3D organoids.

[0066]

[0067] The present invention provides a pancreatic cancer organoid produced by an organoid manufacturing method according to one embodiment of the present invention.

[0068] A pancreatic cancer organoid according to one embodiment of the present invention can express α-Amylase, Cytokeratin-19, Insulin, and Vimentin.

[0069] "α-amylase" is a type of amylase and is a pancreatic enzyme capable of breaking down polysaccharides such as starch and glycogen. α-amylase can be used as a marker in the diagnosis of pancreatitis.

[0070] Cytokeratin-19 (CK-19) is a keratin-19 protein expressed by the KRT19 gene and is involved in the structural rigidity and skeleton of cells. Cytokeratin-19 is highly expressed in gastrointestinal epithelium and can be used as a marker for pancreatic ductal adenocarcinoma (PDAC).

[0071] Insulin is a hormone secreted by beta cells in the islets of Langerhans of the pancreas, which plays a role in regulating blood sugar by converting glucose into glycogen.

[0072] "Vimentin" is an intermediate filament protein expressed by the VIM gene, is a marker of fibroblasts, and is involved in maintaining cell shape and cell flexibility.

[0073]

[0074] The present invention provides a method for evaluating the efficacy of an anticancer drug, comprising the step of treating a pancreatic cancer organoid of the present invention with an anticancer drug candidate.

[0075] Through the method for evaluating the efficacy of the anticancer drug of the present invention, the sensitivity and resistance of the drug in cell lines derived from pancreatic cancer patients can be confirmed and clinical trial results can be predicted.

[0076] In the present invention, the method for evaluating the efficacy of an anticancer drug may be a screening method for an anticancer drug.

[0077] In this specification, the term "candidate substance" refers to a substance expected to be used as an anticancer agent, which is expected to perform a role such as inhibiting the growth of cancer tissue, inhibiting cancer metastasis, or inducing apoptosis of cancer cells. Examples may include, but are not limited to, compounds, DNA, RNA, enzymes, ligands, peptides, proteins, antibodies, and natural extracts.

[0078] Treatment of anticancer drug candidates may be performed using methods known in the art. For example, methods such as treating and co-culturing the anticancer drug candidates may be used, but are not limited thereto.

[0079] In one embodiment, the efficacy of an anticancer drug can be evaluated by measuring cell viability when an organoid derived from a pancreatic cancer patient is treated with an anticancer drug candidate and cultured for 3 days.

[0080]

[0081] Hereinafter, in order to specifically explain the present invention, it will be described in detail with reference to examples.

[0082]

[0083] Examples

[0084] I. Establishment of 2D CRC Cell Line and 3D Organoid Culture

[0085] 1. Establishment and Characterization of Pancreatic Cancer CRC Cell Lines

[0086] 1-1. Culture of cell lines derived from pancreatic cancer patients

[0087] Tumor tissue obtained from a pancreatic cancer patient was treated with collagenase (1 mg / mL, Sigma, St. Louis, MO, USA) in a culture medium, stirred at 37°C for 30 minutes, and stored in a suspended state for separation of each cell. Next, the cell suspension on F-media was inoculated into a feeder layer of J2 mouse fibroblasts irradiated with a lethal dose (30 Gy).

[0088] F-media was prepared by adding 70% Ham's F-12 nutrient mixture (Hyclone, Logan, UT, USA) and 25% complete Dulbecco's modified Eagle's medium (DMEM), 0.4 mg / mL hydrocortisone (Sigma), 5 mg / mL insulin, 8.4 ng / mL cholera toxin (Sigma-Aldrich, St. Louis, MO, USA), 10 ng / mL epidermal growth factor, 5% fetal bovine serum (FBS) (Hyclone), 24 mg / mL adenine (Sigma), 10 mg / mL gentamicin (Life Technologies), and 250 ng / mL amphotericin B (Thermo Fisher Scientific, Waltham, MA, USA). DMEM consists of 4.0 mM L-Glutamine, 4500 mg / L Glucose, and Sodium Pyruvate, and Ham's F-12 nutrient mixture contains 1.0 mM L-Glutamine.

[0089] Cells were cultured at 37°C in a humid atmosphere with 5% carbon dioxide in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632 at a final concentration of 5 μM. To separate the tumor tissue and the collagen matrix, 5 times the concentration of F-meida was added and centrifuged at 1500 rpm for 3 minutes, after which the supernatant was filtered through a cell filter (70 μm, Falcon).

[0090] Tumor cells on the dish were morphologically distinct compared to fibroblasts. If necessary, contaminating fibroblasts were removed by trypsinization or selective dish scraping. Established conditionally reprogrammed (CRC) cells were pretreated with 500 ng / mL of a mycoplasma scavenger (MP Biomedicals, Santa Ana, CA, USA) to inhibit mycoplasma infection, and mycoplasma infection of the generated CRC cell lines was checked regularly.

[0091]

[0092] 1-2. Characterization of Cell Lines Derived from Pancreatic Cancer Patients

[0093] To identify the KRAS mutation, one of the representative somatic mutations in pancreatic cancer, DNA was extracted from cell lines derived from pancreatic cancer patients using the QIAGEN QIAamp® DNA Mini Kit (Hilden, Germany). PCR amplification was performed on the extracted DNA targeting exon 1 (codons 12, 13, and 61) of the KRAS gene to achieve a total length of 164 bp, and the sequences of the primers used for PCR amplification are shown in Table 1 below.

[0094] Forward primer 5'-aggcctgctgaaaatgactga-3'(SEQ No. 1) Reverse primer 5'-ggtcctgcaccagtaatatgca-3'(SEQ No. 2)

[0095] KRAS mutations were evaluated by direct sequencing analysis after PCR amplification, and mutations in the KRAS gene were identified by confirming the presence and type of mutations. Specifically, mutations in the KRAS gene were identified in pancreatic cancer patient-derived cell lines YPAC-23, YPAC-35, YPAC-80, and YPAC-67 (Figs. 5a, 5b, 6a, and 6b).

[0096]

[0097] 1-3. Confirmation of marker gene expression in pancreatic cancer patient-derived cell lines via fluorescent staining

[0098] The expression of pancreas-related gene markers in cell lines derived from pancreatic cancer patients was confirmed.

[0099] After washing the established cells three times with PBS, fluorescence staining was performed. The primary antibodies used were α-Amylase (A8273, 1:100; Sigma), Cytokeratin-19 (A53-B / A2: sc-6278, 1:100; Santa Cruz Biotechnology), Insulin (1:100; Zymed), and Vimentin (V9: sc-6260, 1:100; Santa Cruz), and the auxiliary antibody conjugated with Invitrogen's Alexa Fluor was used. Specifically, the expression of pancreas-related gene markers was confirmed in pancreatic cancer patient-derived cell lines YPAC-87, YPAC-91, YPAC-80, and YPAC-67 (Figs. 7, 8a, and 8b).

[0100]

[0101] 2. Fabrication and Evaluation of Organoid Models via 3D Culture

[0102] 2-1. Fabrication of Organoid Models via 3D Culture

[0103] After counting the cells, a mixture of 30 μl of cells and 270 μl of Matrigel was prepared, calculated to seed a minimum of 5,000 to a maximum of 10,000 cells per well of a 24-well cell culture plate. Carefully dispensed 30 μl of the mixture into each well of the 24-well cell culture plate, taking care to avoid creating bubbles.

[0104] After dispensing the mixture, the plate cover was closed inside a clean bench and waited for 5 minutes to allow the Matrigel to harden sufficiently so that it would not flow or drip even if the plate was inverted. Afterward, the plate was inverted inside the clean bench and the Matrigel was allowed to harden for another 5 minutes to form a dome shape. After 5 minutes, the plate was placed in an incubator with the plate inverted and the Matrigel was allowed to harden for 15 minutes to further solidify the dome shape.

[0105] After 15 minutes, 600 μl of culture medium was added to each well of the plate inside a clean bench. Subsequently, the plates were transferred to an incubator and checked and observed at intervals of 3 to 4 days. As a result, it was confirmed that the appearance of the organoids was observed as early as 3 to 5 days after seeding, or at the latest, within about 7 days. If no appearance of the organoids or no change in size was observed for up to 2 weeks, they were discarded.

[0106] Subculture was performed when tumor organoids of at least 200 μm were observed approximately 3 to 4 weeks after seeding. During the experiment, cell culture plates may be stored in an incubator as needed by the researcher, and ep tubes and tips may be stored in a refrigerator or freezer. For a 24-well plate, a minimum of 20 μl and a maximum of 30 μl of Matrigel per well, with at least 5,000 and no more than 10,000 cells, is appropriate; for a 12-well plate, up to 3 wells of Matrigel with a minimum of 20 μl and a maximum of 30 μl can be dispensed per well, and for a 6-well plate, up to 7 wells of Matrigel with a minimum of 20 μl and a maximum of 30 μl can be dispensed per well.

[0107]

[0108] 2-2. Characterization of organoids derived from pancreatic cancer patients and confirmation of marker gene expression via fluorescence staining

[0109] The morphology of organoids fabricated using YPAC-80 and YPAC-67 was confirmed through bright-field microscopy and H&E staining images (Figs. 4a and 4b).

[0110] In addition, KRAS mutations in YPAC-23 and YPAC-35 pancreatic cancer patient-derived organoids were confirmed in the same manner as in Example 1-2 (Figs. 5a and 5b), and the expression of marker genes α-Amylase, Cytokeratin-19, Insulin, and Vimentin in YPAC-87, YPAC-91, YPAC-80, and YPAC-67 organoids was confirmed in the same manner as in Example 1-3 (Figs. 7, 8a, and 8b).

[0111]

[0112] II. Drug Sensitivity Analysis

[0113] To determine drug sensitivity, cell viability was measured in cell lines and organoids after treatment with an anticancer drug (Fig. 9).

[0114] After calculating the number of counted cells to seed 5,000 in each well of a 48-well cell culture plate, a 300 μl mixture was prepared using 30 μl of cells and 270 μl of Matrigel. Care was taken to avoid the formation of bubbles, and 10 μl of the mixture was dispensed into each well of the 48-well cell culture plate. After dispensing the mixture, the plate was allowed to solidify in an incubator for 20 minutes, after which 300 μl of culture medium was added. After adding the medium, the plate was transferred to an incubator and cultured for 4 days. After 4 days, the formation of organoids was confirmed, and gemcitabine and abraxane (Gemcitabine plus Abraxane) were treated at various concentrations. Cell viability was measured using a Cell Titer 3 days (72 hours) after treatment with the compounds.

[0115] The IC50 values ​​for the anticancer drugs gemcitabine plus Abraxane (G / A) in two-dimensional culture media and organoids of pancreatic cancer patient-derived cell lines (YPAC-28, YPAC-31, YPAC-43, YPAC-44, YPAC-47, YPAC-61, YPAC-75, YPAC-82, YPAC-83, YPAC-86, YPAC-89, and YPAC-94) are shown in Table 2 below, and cell viability is graphed in Figures 10a and 10b. In addition, cell viability for the anticancer drugs gemcitabine plus Abraxane in two-dimensional culture media and organoids of the pancreatic cancer patient-derived cell line YPAC-67 is shown in Figure 11.

[0116]

[0117] As a result, when comparing the sensitivity of drug treatment in 2D cell lines and pancreatic cancer organoids with the actual clinical results (Best response), it was confirmed that the IC50 values ​​measured in pancreatic cancer organoids were more similar to the actual clinical results (Figs. 12a and 12b).

[0118]

[0119] Comprehensive analysis of the matched tumor tissue, CRC, and corresponding organoids confirmed that the constructed organoids maintained a high level of genetic and transcriptomic concordance with the parent tumor tissue. Furthermore, the CRC organoids exhibited distinct morphological features corresponding to the cancer stage and degree of cell differentiation observed in the patient. In particular, the drug response profile of the CRC organoids to G / A accurately reflected the patient's clinical response, demonstrating higher predictive accuracy than that of 2D CRC cells.

[0120] These results demonstrate the utility of the CRC organoid model as a powerful preclinical tool for exploring the molecular biology of pancreatic cancer and evaluating therapeutic agents.

[0121] In other words, through the above experimental results, it can be seen that the organoid of the present invention can be usefully utilized for evaluating drug resistance and therapeutic efficacy.

[0122]

Claims

1. A step of preparing a mixture for seeding by mixing a 2D CRC cell line established by 2D culturing a cell line derived from a pancreatic cancer patient with Matrigel; and A method for preparing a pancreatic cancer organoid comprising the step of three-dimensionally culturing the above mixture.

2. In Claim 1, The above-established 2D CRC cell line is, A method for producing a pancreatic cancer organoid comprising the step of culturing the above-mentioned pancreatic cancer-derived cell line together with irradiated fibroblasts on a medium supplemented with a ROCK inhibitor.

3. In Claim 2, A method for preparing a pancreatic cancer organoid comprising at least one selected from the group consisting of Ham's F-12 nutrient mixture, DMEM, hydrocortisone, insulin, cholera toxin, epidermal growth factor, fetal bovine serum (FBS), adenine, gentamicin, and amphotericin B.

4. In Claim 1, A method for preparing a pancreatic cancer organoid, wherein the above mixing is the 2D CRC cell line and the Matrigel in a volume ratio of 1:7 to 1:

11.

5. In Claim 1, A method for manufacturing a pancreatic cancer organoid in which the above-mentioned pancreatic cancer patient-derived cell line is a KRAS gene mutation cell line.

6. In Claim 1, Method for preparing a pancreatic cancer organoid using the above 2D CRC cell line, which is a cell line deposited under accession number KCLRF-BP-00562.

7. A pancreatic cancer organoid prepared by the method of any one of claims 1 to 6.

8. In Claim 7, The above pancreatic cancer organoid is a pancreatic cancer organoid that expresses α-Amylase, Cytokeratin-19, Insulin, and Vimentin.

9. A method for evaluating the efficacy of an anticancer drug comprising the step of treating a pancreatic cancer organoid of claim 7 with an anticancer drug candidate.

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