KR-20260065534-A - PATIENT-DERIVED PANCREATIC CANCER ORGANOID AND METHOD OF PREPARING THE SAME

Abstract

The present invention relates to a method for manufacturing an organoid using a cell line derived from a pancreatic cancer patient, and more specifically, to a method for manufacturing a pancreatic cancer organoid that is more efficient in terms of time and cost and can predict the degree of the patient's drug response by including the step of making 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 the step of 3D culturing the mixture.

Inventors

• 방승민
• 박찬희
• 임가람
• 이진영
• 김진수

Assignees

• 연세대학교 산학협력단

Dates

Publication Date

20260508

Application Date

20251027

Priority Date

20241030

Claims (9)

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. 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.

Description

Patient-Derived Pancreatic Cancer Organoid and Method of Preparing the Same The present invention relates to a method for manufacturing organoids using a cell line derived from a pancreatic cancer patient. 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. 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. 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%. 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. 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. 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. Figure 1 briefly illustrates the process of producing Matrigel-based organoids using pancreatic cancer patient-derived cell lines (CRC) and F-media. 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). Figures 3a and 3b show the results of comparing the morphology of organoids prepared using Wnt/R-spondin-containing media and F-media (CRC culture media) when using Wnt/R-spondin-containing media and F-media (CRC culture media), respectively, using bright-field microscopy and H&E staining images, when using cell lines (YPAC-05 and YPAC-35) derived from pancreatic cancer patients, respectively. It can be seen that there is no difference in the morphology of the organoids in the two media. 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. 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. 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. 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 YP