Method for producing 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, a method for co-culturing or culturing pancreatic cancer organoids with endothelial cells so as to include cancer initiating cells. Supernatant The present invention relates to pancreatic cancer organoids produced using the method. [Background technology]

[0002] Of the 65,479 people who died from disease in 2005, 26.7% died from cancer. Among these cancers, pancreatic cancer has an especially poor prognosis, with a five-year survival rate of less than 10% according to the Korea National Cancer Center. While surgical resection is possible to treat pancreatic cancer, there is a limit to how much surgery is possible, with only 15% of newly diagnosed patients eligible.

[0003] Meanwhile, the number of cancer patients in Korea and abroad is rapidly increasing due to the aging population, and research is being actively conducted to overcome cancer through anti-cancer treatments tailored to each individual patient. In particular, targeted therapeutic agents are highly clinically useful as they can significantly improve the therapeutic efficacy of anti-cancer drugs in terminal cancer patients, minimize side effects of anti-cancer drugs, and predict the responsiveness of cancer cells to therapeutic drugs through cell signaling.

[0004] However, cancer cell lines derived from primary patient specimens have limitations, such as the inefficiency of establishing cell lines from patient samples, the loss of genetic heterogeneity due to genetic mutations that occur during the adaptation and selection process to 2D culture, and the lack of other matrix components within tissues that can be used as standards.

[0005] Histoculture drug response assay (HRDA), which can test a patient's sensitivity to cancer treatment drugs in the preclinical stage, has limitations in that it uses only a small amount of tissue, making it difficult to confirm sensitivity to various anticancer drugs, and the test can only be performed once, resulting in a large loss of primary cancer tissue.

[0006] In addition, the method of transplanting cancer cell lines or cancer cells derived from cancer patients into animal models (patient-derived tumor xenografts, PDTX) has the advantage of better mimicking the biological characteristics of tumor tissue compared to cell-based models, but it has limitations due to the high cost, time, and resource consumption, as well as bioethical issues. Furthermore, some cancer organoids have limitations in that they lack cancer-supporting cells, including immune components, vascular components, or normal epithelial cells, which prevents sufficient clinical results.

[0007] To overcome these limitations, organoids (in vitro tissue structures) are being developed by culturing or recombining cells isolated from stem cells or organ cells. These organoids have the advantage of being able to administer test treatments to organoids instead of patients first, allowing for the selection of anticancer drugs that show beneficial therapeutic effects for individual patients based on the results. However, existing methods have the limitation of having a very low success rate for producing such structures. Summary of the Invention [Problem to be solved by the invention]

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

[0009] In one embodiment of the present invention, a method for producing pancreatic cancer organoids is provided. The method of the present invention includes the steps of: a) dissociating a biological sample isolated from a pancreatic cancer patient and immobilizing it in growth factor reduced Matrigel®; b) culturing the sample immobilized in 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 the dissociated pancreatic cancer organoids with endothelial cells. Supernatant and culturing the mixture.

[0010] The "pancreatic cancer" of the present invention is divided into exocrine tumors and neuroendocrine tumors, with the majority of cases being exocrine tumors, which correspond to pancreatic ductal adenocarcinomas (PDAC). Cancer cells or tissues from PDAC patients contain subpopulations of cancer-initiating cells with CD44(+)CD24(+) and CD44(+)CD24(+)EpCAM(+) phenotypes, which possess drug resistance, self-renewal, and differentiation capabilities. Therefore, most cancer cells from PDAC patients metastasize and are highly resistant to traditional treatments such as chemotherapy, radiotherapy, and immunotherapy. In particular, patients with cells possessing the CD44(+)CD24(+) phenotype have a significantly poorer prognosis than those without such phenotypes. For purposes of the present invention, the pancreatic cancer may be, but is not limited to, pancreatic ductal adenocarcinoma.

[0011] In the case of pancreatic cancer patients according to the present invention, endothelial cells account for an important proportion in cancer tissue, and in the case of the endothelial cells, they can support the self-renewal and maintenance of cancer-initiating cells in the tumor microenvironment through Notch, TGF-β, nitric oxide (NO), sonic hedgehog (SHH), integrin, and Wnt cell signaling pathways.

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

[0013] The biological sample of the present invention refers to 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, buffy coat of leukocytes, etc. buffy coat, blood including 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 For purposes of the present invention, the biological sample may be a tissue or a pancreatic cancer cell line isolated from a pancreatic cancer patient, for example, but is not limited to, a tissue isolated from a pancreatic cancer patient.

[0014] The pancreatic cancer organoids of the present invention do not undergo primary culture using cancer cells dissociated from cancer tissue after obtaining the tissue from the patient, and therefore can very effectively prevent genetic mutations in cancer cells that may occur during such processes and the loss of phenotypic characteristics in vitro.

[0015] For purposes of the present invention, the pancreatic cancer may be, but is not limited to, pancreatic ductal adenocarcinoma. Compared to other types of cancer, tissues obtained from patients with pancreatic ductal adenocarcinoma can effectively maintain histological characteristics similar to those of the original cancer tissue 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, pancreatic cancer organoid and endothelial cells can be mixed in a ratio of 1:1 to 1:6, for example, can be mixed in a ratio of 1:4, but is not limited thereto.When mixed in this ratio of 1:1 to 1:6 to prepare pancreatic cancer organoid containing endothelial cells, it can fully reflect the interaction between endothelial cells and pancreatic cancer cells in tumor microenvironment, and can more effectively show the characteristics of cancer-initiating cells.

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

[0019] The pancreatic cancer organoids of the present invention comprising endothelial cells can also comprise cancer-initiating cells.For the purposes of the present invention, the pancreatic cancer organoids can be characterized as those that interact with endothelial cells or grow in the supernatant of endothelial cells without the addition of other growth factors that can maintain cancer-initiating cells. The substances contained in the fluid allow the self-renewal and maintenance of cancer-initiating cells to continue.

[0020] In the step b) of the present invention, the step of forming organoids can be carried out by culturing the dissociated sample of the step a) in DMEM / F12 medium, which further comprises one or more selected from the group consisting of antibiotics, glutamine, B27, N-acetyl-L-cysteine, growth factors, gastrin, cell signaling inhibitors, PGE2 (Prostaglandin E2) and additives.For the purpose of the present invention, when using such medium, cells can be very effectively transformed into cancer organoids without the process of primary culture of cells dissociated from tissue.

[0021] The "antibiotic" of the present invention is intended to prevent contamination by bacteria, fungi, mycoplasma, enzymes, etc. during the process of culturing 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, alanylglutamine or glutamine dipeptide, such as 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 support 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, such as Noggin recombinant protein, epidermal growth factor (EGF), and the like. growth factor) and fibroblast growth factor 10 Growth factor 10) may be used, but is not limited to this.

[0024] The "cell signaling inhibitor" of the present invention refers to an agent that inhibits the activation of intracellular signaling pathways by preventing growth factors from binding to receptors or blocking receptor activation. For purposes of the present invention, the cell signaling inhibitor may be an AK inhibitor or a p38 MAPK inhibitor, such as, but not limited to, A83-01 (CAS No. 909910-43-6) and SB202190 (CAS No. 152121-30-7).

[0025] The "additive" of the present invention may be, but is not limited to, Wnt3a-conditioned medium, RSPO1-conditioned medium, and nicotinamide. In another embodiment of the present invention, there is provided a pancreatic cancer organoid produced by the production method of the present invention. The pancreatic cancer organoids of the present invention are produced by the production method of the present invention, and therefore can be used very effectively to study pancreatic cancer development, drug resistance, etc. Furthermore, when such organoids are used, the clinical applicability and reliability of the screened drugs can be significantly improved.

[0026] In the pancreatic cancer organoid of the present invention, pancreatic cancer, endothelial cells, culture Supernatant The contents relating to the ratio, collagenase, etc. are the same as those described in the method for producing the pancreatic cancer organoids, and will be omitted to avoid overcomplicating this specification. In yet another embodiment of the present invention, a method for screening a therapeutic agent for pancreatic cancer is provided.

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

[0028] The term "screening" as used herein means specifically selecting only substances having specific properties of interest from a group of diverse candidate substances. The term "candidate substance" as used herein refers to an unknown substance that is expected to have inhibitory activity against the growth or metastasis of cancer tissues or to induce the death of cancer-initiating cells. The candidate substance may be, but is not limited to, a compound, a peptide, a protein, an antibody, a natural extract, etc. 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 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. [Brief explanation of the drawings]

[0030] [Figure 1] (A) and (B) are diagrams showing the results of confirming the external shape and cell phenotype of pancreatic cancer organoids according to one embodiment of the present invention by microscopic or flow cytometry analysis. [Figure 2] FIG. 1 is a graph showing the ratio of each cell phenotype in pancreatic cancer organoids confirmed by flow cytometry analysis according to one embodiment of the present invention. [Figure 3] FIG. 1 shows the results of confirming the three-dimensional structure of pancreatic cancer organoids using a confocal microscope according to one embodiment of the present invention. [Figure 4] FIG. 10 shows the results of examining the cell morphology and phenotype of cancer-initiating cells present in pancreatic cancer organoids by microscopic or flow cytometric analysis when growth factors according to one embodiment of the present invention are removed. [Figure 5] This is a schematic diagram showing a method for producing an organoid system by isolating phenotypical cell lines present in human-derived pancreatic cancer organoids and culturing them for 11 days according to one embodiment of the present invention. [Figure 6] FIG. 1 shows the results of flow cytometry analysis confirming the organoid-forming ability of CD24(+)CD44(-) or CD24(- / low)CD44(-) cells isolated from human-derived pancreatic cancer organoids according to one embodiment of the present invention. [Figure 7]FIG. 1 shows the results of fluorescence microscopic analysis of the organoid-forming ability of CD24(+)CD44(-) or CD24(- / low)CD44(-) cells according to one embodiment of the present invention. [Figure 8] FIG. 1 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] FIG. 1 shows the results of observing the morphology of an organoid system, which is a pancreatic cancer organoid according to one embodiment of the present invention and a pancreatic cancer organoid containing HUVECs, using a fluorescence microscope. [Figure 10] FIG. 1 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 system according to one embodiment of the present invention and a pancreatic cancer organoid system containing HUVECs. [Figure 12] FIG. 1 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) show the results of microscopic observation of the inhibition 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-initiating cells. [Figure 14] FIG. 10 shows the results of comparing the number of cells with the phenotype of cancer-initiating cells present in pancreatic cancer organoids containing HUVECs when Wnt or Notch cell signaling is inhibited according to one embodiment of the present invention. [Figure 15]This figure shows the results of culturing pancreatic cancer organoids using a conditioned medium (CM) equivalent to the culture supernatant of control 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] FIG. 1 shows the results of flow cytometry analysis of the proportion of cells with the CD24(+)CD44(+) phenotype in pancreatic cancer organoids cultured using a conditioned medium (CM) corresponding to the culture supernatant of cells according to one embodiment of the present invention or HUVECs cells. DETAILED DESCRIPTION OF THE INVENTION

[0031] In one embodiment of the present invention, a) a biological sample isolated from a pancreatic cancer patient is dissociated and immobilized in growth factor reduced Matrigel®; b) a sample immobilized in growth factor reduced Matrigel® in step a) is cultured to form pancreatic cancer organoids; c) a step of dissociating the pancreatic cancer organoids formed in step b); and d) co-culturing the dissociated pancreatic cancer organoids with endothelial cells; or culturing the dissociated pancreatic cancer organoids with endothelial cells. Supernatant and culturing the organoids.

[0032] In another embodiment of the present invention, there is provided a pancreatic cancer organoid produced by the production method of the present invention. In yet another embodiment of the present invention, a method for screening a pancreatic cancer therapeutic agent is provided, comprising the steps of: a) treating the pancreatic cancer organoids according to the present invention with a candidate substance; and b) observing the cancer cells contained in the pancreatic cancer organoids containing endothelial cells depending on whether or not the candidate substance has been treated. [Example]

[0033] The present invention will be described in more detail with reference to the following examples. These examples are merely for the purpose of explaining the present invention in more detail, and the scope of the present invention will not be limited by these examples. It will be obvious to those skilled in the art that the present invention is not limited by the following examples.

[0034] Experimental method ●[Experimental Method 1] Pancreatic ductal adenocarcinoma patient samples Pancreatic ductal adenocarcinoma (PDAC) tissues 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 consent was obtained in advance from the pancreatic cancer patients who served as donors in accordance with the IRB's prior consent guidelines.

[0035] ●[Experimental Method 2] Creation of human-derived pancreatic cancer organoids The tissue samples obtained in [Experimental Method 1] were cut and thoroughly pulverized and degraded by placing them in Dulbecco's Modified Eagle's Medium (DMEM) containing 2 mg / ml collagenase D (Sigma Aldrich, Catalog No. 11088866001) and 10% Fetal Bovine Serum (FBS) and incubating at 37°C for 90 minutes. The degraded tissues were then washed with Distilled Phosphate Buffered Saline (DPBS) containing 10% Fetal Bovine Serum (FBS) and embedded in growth factor reduced (GFR) Matrigel® (Corning, Catalog No. 356231). For 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 lysed using RBC lysis buffer (QIAGen, catalog number 158904) and washed twice with adDMEM / F12 medium (Thermo Fisher Scientific).

[0036] After 10 minutes of incubation in an incubator to polymerize the matrix, a pancreatic cancer organoid culture medium (medium 1) containing the ingredients listed in Table 1 below in adDMEM / F12 was added and cultured until organoids were formed. When organoid formation began, the pancreatic cancer organoid culture medium was replaced with a medium (medium 2) lacking PGE2, and after two passages, experiments were conducted to confirm the characteristics of the organoids, as described below.

[0037] [Table 1]

[0038] For the passage of the pancreatic cancer organoids, the organoids were isolated using TrypLE Express (Invitrogen, Catalog No. 12604013) and washed with adDMEM / F12 medium to remove any trypsin. Finally, the pancreatic cancer organoid fragments were embedded in GFR Matrigel (registered trademark), and 10 μM Y-27632 (Tocris) was added to the pancreatic cancer organoid culture medium (Medium 1) listed in Table 1. Hereinafter, the organoids prepared in this manner are referred to as human-derived pancreatic cancer organoids.

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

[0040] In addition, AsPC1, Mia PACA2, and PANC1 cell lines corresponding to human pancreatic ductal adenocarcinoma (PDAC) cell lines were purchased from the Korea Cell Line Bank. The AsPC1 cell line was cultured in R 1000 ml containing 10% FBS. Mia PACA2 and PANC1 were cultured in DMEM containing 10% FBS.

[0041] ● [Experimental Method 4] Co-culture method of 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 and then stained with CellTracker Blue CMAC dye. HUVECs were stained with CFSE (Carboxyfluorescein succinimidyl ester, Thermo Scientific) according to the manufacturer's protocol. The human-derived pancreatic cancer organoids and HUVECs were then co-cultured at a 1:4 ratio (5 x 10 5 Number of cells: 2 x 10 6 The cell mixture was then 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 further cultured for 1 day to form aggregates.

[0042] The aggregates were embedded in GFR Matrigel®, and when fully solidified, the aggregates were washed with basal medium and then cultured for 3 days in adDMEM / F12 medium containing GlutaMAX and 5% FBS to construct the first human-derived pancreatic cancer organoid system.

[0043] In the case of an experiment to confirm the inhibition of Wnt and Notch cell signaling pathways, the aggregates were placed in a GFR Matrigel (registered trademark) The cells were embedded in the cytoplasm and cultured for 3 days in the presence of the cell signaling inhibitors 100 nM Wnt-C59 or 200 μM DAPT. To confirm the cell phenotype, TrypLE Express was used to thoroughly disaggregate the aggregates of the human-derived pancreatic cancer organoid system, followed by flow cytometry analysis as described in [5-1] below. ●[Experimental Method 5] HUVECs culture Supernatant Culture of human-derived pancreatic cancer organoids using

[0044] HUVECs were plated onto GM-2MV and cultured until they reached 95% confluence. After washing twice, the medium was replaced with adDMEM / F12 containing 5% fetal bovine serum and Glutamax, and the cells were cultured in an incubator at 37°C for 3 days.

[0045] On the other hand, for the control group, a plate containing no cells (HUVECs) was placed in adDMEM / F12 containing 5% fetal bovine serum and Glutamax in an incubator at 37°C for 3 days. The culture solution thus obtained was centrifuged at 4°C, and the supernatant was collected and filtered through a 0.22 μm filter. Supernatant For HUVEC conditioned 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 for 3 days. The supernatant was then cultured in the same manner as previously described. Supernatant obtained. The obtained culture Supernatant A second human-derived pancreatic cancer organoid system was prepared by adding the above to the pancreatic cancer organoids prepared in [Experimental Method 2].

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

[0047] ●[6-2] Immunofluorescence staining The human-derived pancreatic cancer organoids or human-derived or cell line-derived pancreatic cancer organoid systems were fixed by incubating them in 1% paraformaldehyde containing PBS (0.1% glutaraldehyde) for 10 minutes. They were then washed at least three times with PBS containing 10 mM NaBH4. All samples were blocked with 1% bovine serum albumin (BSA) at room temperature for one hour. In the case of pancreatic cancer organoids or pancreatic cancer organoid systems stained with CD44 antibody, they 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 with secondary antibodies and DAPI (4,6-diamidino-2-phenylindole) for 1 hour, washed three times with PBS, and then observed using a Zeiss LSM710 laser scanning confocal local microscope.

[0049] ●[6-3] Statistical analysis Experimental results were calculated and presented as mean ± SEM values. The Mann-Whitney U test was used for statistical comparisons between two different samples, and Dunnett's multiple comparison test was used to assess statistical significance when comparing multiple samples. Statistically significant values ​​were set at 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 plated in white 96-well plates and cultured in DMEM supplemented with 10% FBS and 200 μg / ml G-418, followed by incubation with 50% conditioned medium or control medium for 48 hours.

[0051] Luciferase signals were measured using the Bright-Glo Luciferase Assay System (Promega) according to the guidelines provided by the manufacturer.

[0052] ●Experimental results ●[Experimental Result 1] Confirmation of enrichment of cancer-initiating cells ●[1-1] Confirmation of the phenotype and appearance of human-derived pancreatic cancer organoids The phenotypes corresponding to CD24(+)CD44(+) and CD24(+)CD44(+)ESA(+) were confirmed in the human-derived pancreatic cancer organoids described in [Experimental Method 2] above, and the results are shown in Figures 1(A) and (B), 2 and 3. As shown in Figure 1 (A) and (B), both microscopic observation (A) and flow cytometry analysis (B) revealed that cells with the CD24(+)CD44(+) phenotype accounted for 73.3% of the human-derived pancreatic cancer organoids. Among the cells bearing the ESA(+) phenotype, 99.5% were ESA(+) cells.

[0053] As shown in Figure 2, in human-derived pancreatic cancer organoids, the total number of cancer-initiating cells (CICs) corresponding to CD24(+)CD44(+) was 69.2±3.5%, and 99.5±0.3% of CD24(+)CD44(+) cells were confirmed to be ESA(+) cells. As shown in Figure 3, CD44 was expressed at very high levels in human-derived pancreatic cancer organoids, and the cells constituting the organoids were confirmed to be composed of three-dimensional circular (lumen structure) morphologies with an open center. These results demonstrate that CD24(+)CD44(+) and CD24(+)CD44(+)ESA(+) cells are enriched at higher levels than CD24(-)CD44(-) cells in human-derived pancreatic cancer organoids.

[0054] ●[1-2] Confirmation of maintenance of cancer-initiating cells in pancreatic cancer organoids To confirm whether artificial stem cell support factors can maintain cancer-initiating cells, the human-derived pancreatic cancer organoids from [Experimental Method 2] were cultured in a basal medium, 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 when cultured in basal medium for 3 days.

[0055] These results demonstrate 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 potential The organoid-forming ability of CD24(+)CD44(-) or CD24(- / low)CD44(-) cells isolated from human-derived pancreatic cancer organoids was evaluated. Specifically, as shown 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 using the media 1 and 2. Then, flow cytometry and fluorescence microscopy 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 reconstituted into round and centrally open lumen structures (Figure 7).

[0058] These results demonstrate that organoid culture medium containing Wnt, EGF, and FGF can induce the expansion of CD24(+)CD44(+) cancer-initiating cells and CD24(+)CD44(+)ESA(+) cancer-initiating cells, and can reprogram CD24(+)CD44(-) and CD24(- / low)CD44(-) cells 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-renewal and maintenance of cancer-initiating cells, the first human-derived pancreatic cancer organoid system and human-derived pancreatic cancer organoids from [Experimental Method 4] were cultured in a growth factor-free environment, 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 size of the organoids themselves was significantly larger in the first human-derived pancreatic cancer organoid system than in 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 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 observed, and the results are shown in Figure 13 (A) and (B) and Figure 14. As shown in Figures 13(A) and 13(B) and 14, inhibition of Wnt or Notch cell signaling suppressed organoid formation in the organoid system (Figure 13(A)). Furthermore, we confirmed that the number of cancer-initiating cells contained in the organoid system was significantly reduced (Figure 13(B) and 14).

[0062] These results suggest that cancer-initiating cells present in the pancreatic cancer organoid system undergo self-renewal and maintenance through interactions with endothelial cells, and that this phenomenon is mediated by Wint and Notch cell signaling.

[0063] ●[Experimental Result 5] Confirmation of the characteristics of the human-derived organoid system using the supernatant of HUVECs To evaluate the role of secreted proteins such as endothelial cells' Wint and Notch ligands in the maintenance of cancer-initiating cells, pancreatic cancer organoids were cultured in adDMEM / F12 with HUVEC conditioned medium (CM) containing 5% fetal bovine serum. The morphology was confirmed microscopically, and the cell phenotype was confirmed by flow cytometry analysis. The results are shown in Figures 15 and 16. As described above in [Experimental Method 5], a control group was used in which only culture medium without cells (HUVECs) was cultured in an incubator at 37°C.

[0064] As shown in Figures 15 and 16, CD44+ cells with the phenotype of cancer-initiating cells were significantly increased when cultured in HUVEC conditioned medium (HEVEC CM) compared to when cultured in control conditioned medium (control CM).

[0065] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents. [Industrial Applicability]

[0066] 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.

Claims

1. a) Biological samples isolated from pancreatic cancer patients were dissociated and growth factor depleted. immobilization in reduced factor Matrigel®; b) culturing the sample immobilized in growth factor-reduced Matrigel® in step a) to form pancreatic cancer organoids; c) dissociating the pancreatic cancer organoids formed in step b); d) In an environment where growth factors are removed, the dissociated pancreatic cancer organoids are cultured in a vascular endothelial cell culture supernatant. A method for producing CD24(+)CD44(+) pancreatic cancer organoids.

2. The method for producing pancreatic cancer organoids according to claim 1, wherein in step a), the dissociation step uses collagenase.

3. The method for producing pancreatic cancer organoids according to claim 1, wherein the pancreatic cancer organoids containing vascular endothelial cells contain cancer initiating cells (CICs).

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

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