Method for manufacturing pancreatic cancer organoids

Abstract

The method for producing pancreatic cancer organoids of the present invention fully reflects the interaction between cancer cells and endothelial cells, i.e., the cross-talk of the vascular niche. Therefore, compared to existing cancer organoids, it is possible to exhibit the characteristics of cancer initiating cells (CICs) present in the organic environment, thereby significantly improving the clinical applicability and reliability of the screened drugs.

Description

Technical Field

[0001] The present invention relates to a method for producing pancreatic cancer organoids. More specifically, the present invention relates to pancreatic cancer organoids produced by co-culturing with endothelial cells or culturing endothelial cells so as to be able to contain cancer initiating cells. Supernatant

Background Art

[0002] Out of a total of 65,479 people who died of diseases in 2005, 26.7% of all deaths were due to cancer. Among such cancers, in particular, pancreatic cancer has a very poor prognosis. According to the National Cancer Center of Korea, the 5-year survival rate is less than 10%. Although surgical resection can be performed for the treatment of such pancreatic cancer, there is a limitation that only 15% of newly diagnosed patients can undergo such surgery.

[0003] On the other hand, due to the aging population, the number of cancer patients has been rapidly increasing both in Korea and abroad, and research to overcome cancer through anti-cancer treatment tailored to each patient is actively underway. In particular, in the case of targeted therapeutic agents, not only can the side effects of anti-cancer agents be minimized, but also the reactivity of cancer cells to therapeutic agents can be predicted by cell signal transduction, so their clinical utility is very high.

[0004] However, in the case of cancer cell lines prepared from primary patient specimens, it is very inefficient to establish cell lines from patient samples, and there is a limitation that genetic mutations occur during the adaptation and selection process for two-dimensional culture, and genetic heterogeneity can disappear. Furthermore, in the case of the cancer cell line, there is also a limitation that the components of other substrates in the tissue that can be used as a standard are lacking.

[0005] ​In the case of HRDA (Histoculture drug response assay), which can test a patient's sensitivity to cancer treatment drugs in the preclinical stage, there are limitations: it uses only a small amount of tissue, making it difficult to confirm sensitivity to a variety of anticancer drugs; and because the test can only be performed once, there is a significant loss of the main cancer tissue.

[0006] Furthermore, while patient-derived tumor xenografts (PDTX), which involve transplanting cancer cell lines or cancer cells from cancer patients into animal models, offer the advantage of better mimicking the biological characteristics of tumor tissue compared to cell-based models, they are limited by high costs, time, and resource consumption, as well as bioethical concerns. In addition, some cancer organoids lack sufficient cancer-supporting cells, including immune components, vascular components, or normal epithelial cells, which can prevent adequate support for clinical outcomes.

[0007] To overcome these limitations, organoids (ex vivo tissue structures) are being developed by culturing or recombining cells isolated from stem cells or organ cells. While such organoids offer the advantage of allowing experimental treatment to be administered to the organoid first, and then the selection of effective anticancer drugs based on the results, existing methods suffer from a very low success rate in manufacturing these structures. [Overview of the project] [Problems that the invention aims to solve]

[0008] One object of the present invention is to provide a method for producing pancreatic cancer organoids; and pancreatic cancer organoids produced by the said method. Another object of the present invention is to provide a method for screening pancreatic cancer therapeutic agents using the pancreatic cancer organoids according to the present invention. However, the technical problems that this invention aims to solve are not limited to those mentioned above, and any other problems not mentioned will be clearly understood by those with ordinary skill in the art from the following description. [Means for solving the problem]

[0009] One embodiment of the present invention provides a method for producing pancreatic cancer organoids. The present invention provides the following manufacturing method: a) dissociating a biological sample isolated from a pancreatic cancer patient and immobilizing it on growth factor reduced Matrigel®; b) culturing the sample immobilized on growth factor reduced Matrigel® in step a) to form pancreatic cancer organoids; c) dissociating the pancreatic cancer organoids formed in step b); and d) co-culturing the dissociated pancreatic cancer organoids with endothelial cells; or culturing endothelial cells on the dissociated pancreatic cancer organoids. Supernatant This includes the step of adding and culturing the substance.

[0010] The "pancreatic cancer" of this invention is divided into exocrine tumors and neuroendocrine tumors, with the majority being exocrine tumors, corresponding to pancreatic ductal adenocarcinomas (PDAC). Cancer cells or tissues from such PDAC patients have subpopulations of cancer-initiating cells with the phenotypes CD44(+)CD24(+) and CD44(+)CD24(+)EpCAM(+), possessing drug resistance, regenerative capacity, and differentiated capabilities. As a result, the cancer cells of such PDAC patients are largely metastatic and highly resistant to classical treatments such as chemotherapy, radiotherapy, and immunotherapy. In particular, patients with cells exhibiting the CD44(+)CD24(+) phenotype have a significantly worse prognosis compared to patients without it. For the purposes of this invention, the pancreatic cancer may, but is not limited to, pancreatic ductal adenocarcinoma.

[0011] In the case of pancreatic cancer patients according to the present invention, endothelial cells constitute a significant proportion of the cancer tissue, and in the case of said endothelial cells, self-regeneration and maintenance of cancer initiation cells can be supported in the tumor microenvironment by Notch, TGF-β, nitric oxide (NO), sonic hedgehog (SHH), integrin, and Wnt cell signaling pathways.

[0012] The pancreatic cancer organoid of the present invention fully reflects the interaction between cancer cells and endothelial cells, i.e., crosstalk of the vascular niche. Therefore, compared to existing pancreatic cancer organoids, it can exhibit the characteristics of cancer-initiating cells (CICs) present in the organic environment, and can be used very effectively to study pancreatic cancer development, drug resistance, etc. Furthermore, using such organoids can significantly improve the clinical applicability and reliability of screened drugs.

[0013] The biological sample of the present invention means any substance, biological fluid, tissue, or cell obtained from or derived from an individual who is a pancreatic cancer patient, such as whole blood, leukocytes, peripheral blood mononuclear cells, or leukocytes. Blood including buffy coat, plasma and serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions. The biological sample may include, but is not limited to, secretions, cells, cell extracts, or cerebrospinal fluid. For the purposes of the present invention, the biological sample may be tissue or pancreatic cancer cell line isolated from a pancreatic cancer patient, for example, tissue isolated from a pancreatic cancer patient, but is not limited to that.

[0014] The pancreatic cancer organoid of the present invention does not undergo primary culture using cancer cells dissociated from the cancerous tissue after obtaining tissue from a patient. Therefore, it is possible to very effectively prevent genetic deformation of cancer cells and the disappearance of phenotypic characteristics in vitro, which can occur in such processes.

[0015] For the purposes of the present invention, the pancreatic cancer may be, but is not limited to, pancreatic ductal adenocarcinoma. In the case of tissue obtained from a patient with pancreatic ductal adenocarcinoma, compared to other types of cancer, histological features similar to the original cancer tissue can be maintained very effectively in vitro, even without mutation analysis to confirm the origin of the cancer.

[0016] The endothelial cells in step d) of the present invention may be vascular endothelial cells, for example, human umbilical vein endothelial cells (HUVECs), but are not limited thereto.

[0017] In step d) of the present invention, the pancreatic cancer organoid and endothelial cells may be mixed in a ratio of 1:1 to 1:6, for example, in a ratio of 1:4, but are not limited thereto. When pancreatic cancer organoids containing endothelial cells are produced by mixing them in a ratio of 1:1 to 1:6 in this way, the interaction between endothelial cells and pancreatic cancer cells in the tumor microenvironment can be fully reflected, and the characteristics of cancer-initiating cells can be more effectively and clearly demonstrated.

[0018] Culturing in step d) of the present invention Supernatant This refers to a product obtained by culturing cells in a known liquid or solid culture medium, and is a concept that does not include cells. In step a) of the present invention, the dissociation step may be performed using collagenase, for example, collagenase D (collagenase type IV), but is not limited thereto. Collagenase D has low trypsin activity and can minimize cell damage during the process of dissociating tissue into cellular units.

[0019] The pancreatic cancer organoid containing the endothelial cells of the present invention may contain cancer-initiating cells. For the purpose of the present invention, even when no other growth factor capable of maintaining cancer-initiating cells is added, the pancreatic cancer organoid can sustain the self-renewal and maintenance of cancer-initiating cells by the interaction with endothelial cells or substances contained in the supernatant of endothelial cells. It can be carried out by culturing the dissociated sample in the step a) in a DMEM / F12 medium further containing one or more selected from the group consisting of an antibiotic, glutamine, B27, N-acetyl-L-cysteine, a growth factor, gastrin, a cell signaling inhibitor, PGE2 (Prostaglandin E2), and an additive. For the purpose of the present invention, when using such a medium, cells can be very effectively cancer-organoidized without the process of primary culturing the cells dissociated from the tissue.

[0020] In the step b) of the present invention, the step of forming an organoid may be carried out by culturing the dissociated sample in the step a) in a DMEM / F12 medium further containing one or more selected from the group consisting of an antibiotic, glutamine, B27, N-acetyl-L-cysteine, a growth factor, gastrin, a cell signaling inhibitor, PGE2 (Prostaglandin E2), and an additive. For the purpose of the present invention, when using such a medium, cells can be very effectively cancer-organoidized without the process of primary culturing the cells dissociated from the tissue.

[0021] The "antibiotic" of the present invention is for preventing contamination by bacteria, molds, mycoplasmas, enzymes, etc. during the culturing of pancreatic cancer organoids, and may be, for example, ampicillin, penicillin, streptomycin, spectinomycin, tetracycline, neomycin, etc., or may be penicillin and streptomycin, but is not limited thereto.

[0022] The "glutamine" of the present invention is an essential component that provides energy during cell culture, and may be, for example, alanyl glutamine or glutamine dipeptide, and may be, for example, L-alanyl-L-glutamine (glutamax), but is not limited thereto.

[0023] The "B27" of the present invention corresponds to a serum-free supplement optimized to assist cell growth or viability. The "growth factor" of the present invention is a protein or steroid hormone that can stimulate cell growth, proliferation, healing, and cell differentiation. For example, it may be Noggin recombinant protein, Epidermal growth factor, and fibroblast growth factor 10, but is not limited thereto.

[0024] The "cell signaling inhibitor" of the present invention refers to a substance that functions to prevent a growth factor from binding to a receptor or block the activation of a receptor in order to suppress the activation of an intracellular signal transduction pathway. For the purpose of the present invention, the cell signaling inhibitor may be an AK inhibitor and a p38 MAPK inhibitor. For example, it may be A83-01 (CAS No. 909910-43-6) and SB202190 (CAS No. 152121-30-7), but is not limited thereto.

[0025] The "additive" of the present invention may be Wnt3a-conditioned medium, RSPO1-conditioned medium, and nicotinamide, but is not limited thereto. In another embodiment of the present invention, a pancreatic cancer organoid produced by the production method of the present invention is provided. Since the pancreatic cancer organoid of the present invention is produced by the production method of the present invention, it can be very effectively used for studying the development of pancreatic cancer, drug resistance, etc. Furthermore, when using such an organoid, the clinical applicability and reliability of the screened drug can be further significantly improved.

[0026] In the pancreatic cancer organoid of the present invention, the content related to pancreatic cancer, endothelial cells, culture Supernatant , ratio, collagenase, etc. is the same as that described in the production method of the pancreatic cancer organoid, and is omitted to avoid excessive complexity of this specification. In yet another embodiment of the present invention, a method for screening pancreatic cancer treatment agents is provided.

[0027] The present invention provides a method for screening pancreatic cancer therapeutic agents, comprising the steps of: a) treating pancreatic cancer organoids according to the present invention with a candidate substance; and b) observing cancer cells contained in the pancreatic cancer organoids depending on whether or not they have been treated with the candidate substance. In step b) of the screening method of the present invention, if the size of cancer cells decreases or is maintained, or if the number of cancer-initiating cells decreases, the candidate substance can be selected as a pancreatic cancer treatment agent.

[0028] In this invention, "screening" means specifically selecting only those substances that possess the desired specific properties from a diverse group of candidate substances. The "candidate substance" in this invention means an unknown substance that is expected to have inhibitory activity against the growth or metastasis of cancer tissue or to induce the death of cancer-initiating cells. The candidate substance may be, but is not limited to, a compound, peptide, protein, antibody, or natural extract. The candidate substances of the present invention can be obtained from libraries of synthetic or natural compounds, biological libraries, spatially addressable parallel solid-phase or solution-phase libraries, and the like. [Effects of the Invention]

[0029] The present invention's method for producing pancreatic cancer organoids fully reflects the interaction between cancer cells and endothelial cells, i.e., crosstalk of the vascular niche. Therefore, compared to existing cancer organoids, it is possible to exhibit the characteristics of cancer-initiating cells (CICs) present in the organic environment, and the clinical applicability and reliability of screened drugs can be significantly improved. [Brief explanation of the drawing]

[0030] [Figure 1] (A) and (B) are figures showing the results of confirming the external shape and cellular phenotype of a pancreatic cancer organoid according to one embodiment of the present invention by microscopy or flow cytometry analysis. [Figure 2] This figure shows a graph of the proportions of different cell phenotypes present in pancreatic cancer organoids, as confirmed by flow cytometry analysis according to one embodiment of the present invention. [Figure 3] This figure shows the results of confirming the three-dimensional structure of a pancreatic cancer organoid according to one embodiment of the present invention using a confocal microscope. [Figure 4] This figure shows the results of examining the cell morphology and phenotype of cancer-initiating cells present in pancreatic cancer organoids by microscopy or flow cytometry analysis when the growth factor according to one embodiment of the present invention is removed. [Figure 5] This is a schematic diagram illustrating a method for producing an organoid system by isolating phenotypic cell lines present in human-derived pancreatic cancer organoids according to one embodiment of the present invention and culturing them for 11 days. [Figure 6] This figure shows the results of flow cytometry analysis to confirm the organoid formation ability of CD24(+)CD44(-) or CD24(- / low)CD44(-) cells isolated from human pancreatic cancer organoids according to one embodiment of the present invention. [Figure 7]This figure shows the results of confirming the organoid formation ability of CD24(+)CD44(-) or CD24(- / low)CD44(-) cells according to one embodiment of the present invention, as determined by fluorescence microscopy analysis. [Figure 8] This is a schematic diagram illustrating a method for producing a pancreatic cancer organoid system, which is a pancreatic cancer organoid containing human umbilical vein endothelial cells (HUVECs), according to one embodiment of the present invention. [Figure 9] This figure shows the results of confirming the morphology of a pancreatic cancer organoid and an organoid system containing HUVECs according to one embodiment of the present invention using a fluorescence microscope. [Figure 10] This figure shows the results of flow cytometry analysis of the cell morphology of cancer-initiating cells present in an organoid system, which is a pancreatic cancer organoid containing HUVECs according to one embodiment of the present invention. [Figure 11] This figure shows the results of comparing the number of cancer-initiating cells present in a pancreatic cancer organoid according to one embodiment of the present invention and in a pancreatic cancer organoid system containing HUVECs. [Figure 12] This figure shows the results of flow cytometry analysis of the cell morphology of cancer-initiating cells present in an organoid system, which is a pancreatic cancer organoid containing HUVECs according to one embodiment of the present invention. [Figure 13] (A) and (B) are figures showing the results of microscopic confirmation of the suppression of pancreatic cancer organoid formation containing HUVECs when Wnt or Notch cell signaling is inhibited according to one embodiment of the present invention, and the results of flow cytometry analysis of the phenotype of cancer initiation cells. [Figure 14] This figure shows the results of comparing the number of cells exhibiting the phenotype of cancer-initiating cells in pancreatic cancer organoids containing HUVECs when Wnt or Notch cell signaling is suppressed according to one embodiment of the present invention. [Figure 15]This figure shows the results of culturing pancreatic cancer organoids using conditioning medium (CM) equivalent to the culture supernatant of control group cells or HUVECs cells according to one embodiment of the present invention, and confirming the cell morphology and phenotype of the pancreatic cancer organoids by microscopy and flow cytometry analysis. [Figure 16] This figure shows the results of flow cytometry analysis to confirm the ratio of cells having the CD24(+)CD44(+) phenotype in pancreatic cancer organoids cultured using a conditioning medium (CM) equivalent to the culture supernatant of cells according to one embodiment of the present invention or HUVECs cells. [Modes for carrying out the invention]

[0031] One embodiment of the present invention includes the steps of: a) dissociating a biological sample isolated from a pancreatic cancer patient and immobilizing it on growth factor reduced Matrigel®; b) culturing the sample immobilized on growth factor reduced Matrigel® in step a) to form pancreatic cancer organoids; c) dissociating the pancreatic cancer organoids formed in step b); and d) co-culturing the dissociated pancreatic cancer organoids with endothelial cells; or culturing endothelial cells on the dissociated pancreatic cancer organoids. Supernatant The present invention provides a method for producing pancreatic cancer organoids, which includes the step of adding and culturing a substance.

[0032] Another embodiment of the present invention provides a pancreatic cancer organoid produced by the manufacturing method according to the present invention. In yet another embodiment of the present invention, a method for screening pancreatic cancer therapeutic agents is provided, comprising the steps of a) treating the pancreatic cancer organoid according to the present invention with a candidate substance, and b) observing cancer cells contained in the pancreatic cancer organoid, including endothelial cells, with or without treatment with the candidate substance. [Examples]

[0033] The present invention will be described in more detail below through examples. These examples are merely for the purpose of illustrating the present invention more concretely, and the scope of the present invention is not limited to these examples. It will be obvious to anyone with ordinary knowledge in the industry that the embodiments described are not limited thereto.

[0034] ●Experimental Method ●[Experimental Method 1] Samples from patients with pancreatic ductal adenocarcinoma Pancreatic ductal adenocarcinoma (PDAC) tissue and endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) samples were obtained from Severance Hospital, Yonsei University College of Medicine, South Korea. All human experiments related to this application were approved by the Severance Hospital Institutional Review Board (IRB), and prior consent was obtained from the pancreatic cancer patients who provided the samples, in accordance with the IRB's prior consent guidelines.

[0035] ●[Experimental Method 2] Creation of human-derived pancreatic cancer organoids The tissue sample obtained in [Experimental Method 1] above was cut, and the cut tissue sample was placed in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 2 mg / ml collagenase D (Sigma Aldrich, catalog number: 11088866001) and 10% FBS (Fetal bovine serum), and cultured at 37°C for 90 minutes to thoroughly pulverize and decompose it. Subsequently, the decomposed tissue was washed with DPBS (Distilled phosphate buffer saline) containing 10% FBS (Fetal Bovine Serum), and embedded in growth factor reduce (GFR) Matrigel® (Corning, catalog number: 356231). In the case of EUS-FNA (endoscopic ultrasound-guided fine needle aspiration) samples, a small amount of cellular material was collected by centrifugation at 400×g for 10 minutes. The EUS-FNA samples were then lysed with RBC lysis buffer (QIAGen, catalog number: 158904) and washed twice with adDMEM / F12 medium (Thermo Fisher Scientific).

[0036] Subsequently, the matrix was polymerized by culturing in an incubator for 10 minutes. Then, pancreatic cancer organoid culture medium (medium 1), which additionally contains the components listed in Table 1 below, was added to adDMEM / F12, and the culture was continued until organoids were formed. After organoid formation occurred, the medium was replaced with medium (medium 2), from which PGE2 had been removed from the pancreatic cancer organoid culture medium. After two passaging cycles, experiments were conducted to confirm the characteristics of the organoids as described below.

[0037] [Table 1]

[0038] For the passage of the aforementioned pancreatic cancer organoids, the organoids were isolated using TrypLE Express (Invitrogen, catalog number: 12604013) and washed with adDMEM / F12 medium to remove any remaining trypsin. Finally, the fragments of the pancreatic cancer organoids were embedded in GFR Matrigel (registered trademark), and 10 μM Y-27632 (Tocris) was added to the pancreatic cancer organoid culture medium (medium 1) described in Table 1. Hereinafter, the organoids prepared in this manner will be referred to as human-derived pancreatic cancer organoids.

[0039] ●[Experimental Method 3] Cell line culture method HUVECs(Human umbilical vein endothelial Cells were dispensed into culture dishes coated with 1% gelatin and cultured in EBM-2-based medium (Lonza, catalog number: CC-3156) containing a 1×EGM-2MV single-quart supplement pack (catalog number: CC-4147). The medium was replaced every 2 or 3 days.

[0040] In addition, we purchased AsPC1, Mia PACA2, and PANC1 cell lines, which correspond to human pancreatic ductal adenocarcinoma (PDAC) cell lines, from the Korea Cell Line Bank. The aforementioned AsPC1 cell line contains 10% FBS. PMI1640 was used for culture, while Mia PACA2 and PANC1 were cultured using DMEM containing 10% FBS.

[0041] ●[Experimental Method 4] Co-culture method for pancreatic cancer organoids and HUVECs As shown in Figure 4, human-derived pancreatic cancer organoids and HUVECs were co-cultured to construct pancreatic cancer organoids containing HUVECs (hereinafter referred to as the "human-derived pancreatic cancer organoid system"). Specifically, the pancreatic cancer organoids produced in [Experimental Method 2] above were dissociated using TypeLE Express, stained with CellTracker Blue CMAC dye, and the HUVECs were stained using CFSE (Carboxyfluorescein succinimidyl ester, Thermo) according to the manufacturer's protocol. Subsequently, the human-derived pancreatic cancer organoids and HUVECs were mixed in a ratio of 1:4 (5 × 10⁻¹⁶). 5 Number of cells: 2 × 10 6 The cells were mixed (in terms of cell count). Subsequently, the cell mixture was placed in round-bottom ultra-low attachment plates (corning), and medium 1 containing 10% growth factor reduced Matrigel® was added. The mixture was then centrifuged at 100 × g for 3 minutes and cultured for an additional day to form aggregates.

[0042] The aggregates were embedded in GFR Matrigel (registered trademark), and after sufficient coagulation, the aggregates were washed with basal culture medium. The first human-derived pancreatic cancer organoid system was then constructed by culturing the aggregates in adDMEM / F12 medium containing glutamax and 5% FBS for 3 days.

[0043] In the case of experiments to confirm the suppression of the Wnt and Notch cell signaling systems, the aggregates are prepared using GFR Matrigel. (Registered trademark) When implanted, the cells were cultured for 3 days with an additional 100 nM Wnt-C59 or 200 μM DAPT, which are cell signaling inhibitors. To confirm the phenotype of the cells, the aggregates of the human-derived pancreatic cancer organoid system were thoroughly separated into single cells using TrypLE Express, and then analyzed by flow cytometry as described in [5-1] below. ●[Experimental Method 5] HUVECs Culture Supernatant Culture of human-derived pancreatic cancer organoids using

[0044] HUVECs were dispensed into GM-2MV and cultured until 95% confluence was reached. The HUVECs were washed twice, the culture medium was replaced with adDMEM / F12 containing 5% fetal bovine serum and glutamax, and the cultures were incubated at 37°C for 3 days.

[0045] In contrast, in the control group, adDMEM / F12 containing 5% fetal bovine serum and glutamax was placed in a 37°C incubator for 3 days on plates that did not contain cells (HUVECs). The culture medium obtained in this way was centrifuged at 4°C, and the upper layer was collected and filtered through a 0.22 μm filter to obtain the culture medium equivalent to the supernatant. Supernatant The following was obtained. In the case of HUVEC conditioning medium (CM) pretreated with Wnt-C59 and DAPT, HUVEC cells were cultured for 1 day after Wnt-C59 and DAPT treatment, washed twice, and then replaced with adDMEM / F12 containing 5% fetal bovine serum and glutamax, and cultured for 3 days. Subsequently, the culture of the supernatant was performed in the same manner as described above. Supernatant I obtained it. The culture obtained above Supernatant A second human-derived pancreatic cancer organoid system was manufactured by inserting the substance into the pancreatic cancer organoid produced in [Experimental Method 2].

[0046] ●[Experimental Method 6] Measurement, staining, and analysis methods ●[6-1] Flow cytometry analysis For cell surface staining, single cells extracted from the aggregates in [Experimental Method 4] above were stained with 1% F After collecting the cells in FACS buffer (dPBS; Gibco) containing BS and 0.1% BSA (MP bio), the cells were blocked with an Fc receptor blocker (Milteni Biotec). Subsequently, the cells were stained with CD44-APCcy7 (Biolegend), CD24-Pecy7, and EpCAM-PerCPcy5.5, and then flow cytometry analysis was performed using LSR II, followed by analysis using FlowJo and DIVA software.

[0047] ●[6-2] Immunofluorescence staining The aforementioned human-derived pancreatic cancer organoids or human-derived or cell line-derived pancreatic cancer organoid systems were immobilized by placing them in PBS (1% paraformaldehyde containing 0.1% glutaraldehyde) and culturing for 10 minutes. They were then washed at least three times with PBS containing 10 mM NaBH4. All samples were blocked with 1% BSA (Bovine serum albumin) at room temperature for 1 hour, and pancreatic cancer organoids or pancreatic cancer organoid systems stained with CD44 antibody were washed three times with PBS.

[0048] Subsequently, the human-derived pancreatic cancer organoids or human-derived or cell line-derived pancreatic cancer organoid systems were cultured for 1 hour with a secondary antibody and DAPI (4,6-diamidino-2-phenylindole), washed three times with PBS, and then observed using a Zeiss LSM710 laser-scan confocal local microscope.

[0049] ●[6-3] Statistical analysis Experimental results were calculated to be presented as mean ± SEM values. The Mann-Whitney U test was used for statistical comparisons between two distinct samples, and Dunnett's multiple comparison test was used to assess statistical significance when comparing multiple samples. A statistically significant value was set to less than 0.05, and statistical tests were performed using the statistical package SPSS version 25 (SPSS Inc., Chicago, IL) and GraphPad Prism 8.

[0050] ●[Experimental Method 7] Wnt activity assay HEK 293 STF cells (ATCC, CRL-3249) were aliquoted into white 96-well plates and cultured in DMEM containing 10% FBS and 200 μg / ml G-418. They were then cultured for 48 hours in 50% conditioned medium and control medium.

[0051] The luciferase signal was measured using the Bright-Glo Luciferase Assay System (promega) according to the manufacturer's guidelines.

[0052] ● Experimental results ●[Experimental Result 1] Confirmation of concentration of cancer-initiating cells ●[1-1] Confirmation of the phenotype and external appearance of human-derived pancreatic cancer organoids In the human-derived pancreatic cancer organoids described in [Experimental Method 2], phenotypes corresponding to CD24(+)CD44(+) and CD24(+)CD44(+)ESA(+) were identified, and the results are shown in Figures 1(A) and (B), 2 and 3. As shown in Figures 1(A) and (B), both microscopic observation (A) and flow cytometry analysis (B) revealed that 73.3% of human-derived pancreatic cancer organoids possessed the CD24(+)CD44(+) phenotype. 99.5% of the cells possessed were ESA(+) phenotype.

[0053] As shown in Figure 2, in human-derived pancreatic cancer organoids, cancer-initiating cells (CICs) corresponding to CD24(+)CD44(+) accounted for a total of 69.2±3.5%, and it was confirmed that 99.5±0.3% of the CD24(+)CD44(+) cells were ESA(+) cells. As shown in Figure 3, CD44 was expressed at a very high level in human-derived pancreatic cancer organoids, and it was confirmed that the cells constituting the organoids were composed of a three-dimensional circular (lumen structure) morphology with a central cavity. The results above show that in human-derived pancreatic cancer organoids, CD24(+)CD44(+) and CD24(+)CD44(+)ESA(+) cells are enriched to a higher level than CD24(-)CD44(-) cells.

[0054] ●[1-2] Confirmation of the maintenance of cancer-initiating cells in pancreatic cancer organoids To confirm whether cancer-initiating cells are maintained by artificial stem cell support factors, human-derived pancreatic cancer organoids from [Experimental Method 2] were cultured in a basal medium consisting of adDMEM / F12 medium containing 5% FBS and glutamax, and the cell number and phenotype were observed. The results are shown in Figure 4. As shown in Figure 4, the number of cancer-initiating cells decreased after being cultured in basal medium for 3 days.

[0055] The results described above indicate that factors such as Wnt, EGF, and FGF can expand cancer-initiating cells in pancreatic cancer organoids.

[0056] ●[Experimental Result 2] Evaluation of organoid formation possibility The organoid-forming ability of CD24(+)CD44(-) or CD24(- / low)CD44(-) cells isolated from human-derived pancreatic cancer organoids was evaluated. Specifically, as illustrated in Figure 5, CD24(-low)CD44(-), CD24(+)CD44(-), and CD24(+)CD44(+) cells were isolated from the human-derived pancreatic cancer organoids by flow cytometry (FACS), and cultured for 11 days in media 1 and 2. Subsequently, flow cytometry and fluorescence microscopy analyses were performed, and the results are shown in Figures 6 and 7.

[0057] As shown in Figures 6 and 7, organoids produced by culturing CD24(+)CD44(-) and CD24(- / low)CD44(-) cells also expressed CD44 (Figure 6), and not only CD24(+)CD44(-) and CD24(- / low)CD44(-) cells, but also CD24(+)CD44(+) cells were reconfigured into a circular lumen structure with a central opening (Figure 7).

[0058] Through these results, it can be seen that organoid culture media containing Wnt, EGF, and FGF can induce expansion of CD24(+)CD44(+) cancer-initiating cells and CD24(+)CD44(+)ESA(+) cancer-initiating cells, and that CD24(+)CD44(-) and CD24(- / low)CD44(-) cells can be reprogrammed into CD44(+) cancer-initiating cells.

[0059] ●[Experimental Result 3] Confirmation of interaction between endothelial cells and cancer-initiating cells To confirm whether endothelial cells maintain the self-regeneration and maintenance of cancer-initiating cells, the first human-derived pancreatic cancer organoid system and human-derived pancreatic cancer organoids described in [Experimental Method 4] were cultured in an environment devoid of growth factors, and then the cell morphology and phenotype were observed. The results are shown in Figures 9 to 12. As shown in Figure 9, we confirmed that the organoids themselves were significantly larger in the first human-derived pancreatic cancer organoid system compared to human-derived pancreatic cancer organoids.

[0060] Furthermore, as shown in Figures 10-12, compared to human-derived pancreatic cancer organoids, the first human-derived pancreatic cancer organoid system contained a large number of CD24(+)CD44(+) cells, which correspond to the phenotype of cancer-initiating cells (Figures 10 and 11), and it was confirmed that 100% of the cells with this phenotype were ESA-positive cells (Figure 12).

[0061] ●[Experimental Result 4] Confirmation of cell signaling involved in the interaction between endothelial cells and cancer-initiating cells. When cell signaling was suppressed in the first human-derived pancreatic cancer organoid system, the organoid formation process was examined, and the results are shown in Figures 13(A) and (B) and Figure 14. As shown in Figures 13(A) and (B), and Figure 14, suppressing Wnt or Notch cell signaling inhibited organoid formation in the organoid system (Figure 13(A)). Furthermore, we confirmed that the number of cancer-initiating cells included in the organoid system was also significantly reduced (Figures 13(B) and 14).

[0062] The results described above indicate that cancer-initiating cells present in the pancreatic cancer organoid system are regenerated and maintained through interaction with endothelial cells, and that this phenomenon is mediated by Wint and Notch cell signaling.

[0063] ●[Experimental Result 5] Confirmation of the characteristics of human-derived organoid systems using HUVEC supernatant. To evaluate the role of secreted proteins such as winth and Notch ligands in endothelial cells in the maintenance of cancer-initiating cells, pancreatic cancer organoids were cultured in adDMEM / F12 with HUVECs cell conditioning medium (CM) containing 5% fetal bovine serum. Their morphology was observed under a microscope, and their phenotype was confirmed by flow cytometry analysis. The results are shown in Figures 15 and 16. As described in [Experimental Method 5] above, the control group consisted of cells cultured in culture medium alone at 37°C without any cells (HUVECs).

[0064] As shown in Figures 15 and 16, we confirmed that the number of CD44+ cells exhibiting the phenotype of cancer-initiating cells was significantly increased when cultured in HUVEC cell conditioning medium (HUVEC CM) compared to when cultured in control conditioning medium (control CM).

[0065] Although specific parts of the present invention have been described in detail above, it will be clear to those with ordinary skill in the art that such specific techniques are merely preferred embodiments and therefore do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention is defined by the appended claims and their equivalents. [Industrial applicability]

[0066] The present invention's method for producing pancreatic cancer organoids fully reflects the interaction between cancer cells and endothelial cells, i.e., crosstalk of the vascular niche. Therefore, compared to existing cancer organoids, it is possible to exhibit the characteristics of cancer-initiating cells (CICs) present in the organic environment, and the clinical applicability and reliability of screened drugs can be significantly improved.

Claims

1. a) Dissociate biological samples isolated from pancreatic cancer patients and reduce growth factors (growth The steps include immobilizing the material in factor-reduced Matrigel (registered trademark), b) A step of culturing the sample immobilized on growth factor-reduced Matrigel (registered trademark) in step a) above to form pancreatic cancer organoids, c) A step of dissociating the pancreatic cancer organoid formed in step b), d) A method for producing CD24(+)CD44(+) pancreatic cancer organoids, comprising the step of culturing the dissociated pancreatic cancer organoids in an environment from which growth factors have been removed by adding the supernatant of vascular endothelial cell culture.

2. The method for producing a pancreatic cancer organoid according to claim 1, wherein in step a) above, the step of dissociation is performed using collagenase.

3. A method for producing a pancreatic cancer organoid according to claim 1, wherein the pancreatic cancer organoid containing vascular endothelial cells contains cancer initiating cells (CICs).

4. The method for producing a pancreatic cancer organoid according to claim 1, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

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