Establishment of a mouse model using human pancreatic cancer organoids

Abstract

The purpose of the present invention is to provide a mouse model for pancreatic cancer similar to human pancreatic cancer. Specifically, the present invention pertains to a human pancreatic cancer mouse model which carries a pancreatic cancer organoid containing a human pancreatic cancer cell line S2-130.

Description

Technical Field

[0001] The present invention relates to, for example, a human pancreatic cancer mouse model and the use of the mouse model.

Background Art

[0002] For intractable cancers such as pancreatic cancer, the development of new treatment methods has become an urgent task. Conventionally, in the development of new treatment methods, the search for therapeutic drugs using cancer mouse models has been carried out. In addition, pancreatic cancer, which is representative of cancers with a poor prognosis, is difficult to detect at an early stage, and the development of useful diagnostic markers is desired. However, for pancreatic cancer, there is no mouse model that can form tumors with a tissue structure similar to human pancreatic cancer tissue with high throughput. For this reason, there has been no useful pancreatic cancer mouse model that can be used for experiments on the efficacy of drugs. In addition, a xenograft (Patient-Derived Xenograft, PDX) prepared by transplanting pancreatic cancer tissue obtained from a patient into an immunodeficient animal contains patient-derived cells such as stromal cells, cancer-related fibroblasts, and tumor macrophages in the tumor, so it retains the characteristics of the patient-derived tumor, and is strongly expected to be used for pathological analysis of tumors, tumor marker analysis, drug development, etc. However, in PDX, as the passage progresses, tumor growth in the mouse body accelerates, and the gene expression profile changes compared to the patient-derived sample. Furthermore, the establishment of PDX usually takes 3 to 6 months, and at present, it is difficult to directly participate in the individual medical treatment of the patient who provided the tumor. For this reason, PDX lacks high throughput and is not sufficiently versatile in pharmaceutical development and the like. On the one hand, Patent Document 1 discloses that by co-culturing human pancreatic cancer cell lines (PANC-1, CFPAC-1, SW1990), human vascular endothelial cells (HUVEC), and human mesenchymal cells (hMSC), human pancreatic cancer organoids can be reconstructed, and pancreatic cancer xenografts with rich stroma and ductal structures can be formed from these pancreatic cancer organoids. However, Patent Document 1 does not disclose that in mice carrying the human pancreatic cancer organoids, tumors that form a tissue structure similar to clinical pancreatic cancer, such as papillary structures, invasion of pancreatic cancer cells accompanied by cancer stroma, vascular invasion, lymph node metastasis, and epithelial-mesenchymal transition (EMT), are formed. In addition, Patent Document 1 does not disclose that in the serum collected from mice carrying human pancreatic cancer organoids, pancreatic cancer diagnostic markers show the same behavior as clinical pancreatic cancer. Furthermore, Patent Document 1 does not disclose that mice carrying human pancreatic cancer organoids are useful models for determining the therapeutic effect of pancreatic cancer therapeutic agents.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

[0004] In view of the above circumstances, an object of the present invention is to provide a mouse model of pancreatic cancer similar to human pancreatic cancer. As a result of intensive research to solve the above problems, it has been found that by transplanting pancreatic cancer organoids prepared using the human pancreatic cancer cell line S2-013 into mice, tumors that form a tissue structure similar to human pancreatic cancer tissue are formed in the mice, and the present invention has been completed. That is, the present invention includes the following. (1) A human pancreatic cancer mouse model carrying pancreatic cancer organoids containing the human pancreatic cancer cell line S2-013. (2) A method for screening a pancreatic cancer therapeutic agent using the human pancreatic cancer mouse model according to (1). (3)(1) A method for evaluating the efficacy of a pancreatic cancer therapeutic agent using the human pancreatic cancer mouse model described. (4)(1) A method for evaluating the usefulness of a pancreatic cancer diagnostic marker using a serum sample collected from the human pancreatic cancer mouse model described. (5)(1) A method for screening a pancreatic cancer diagnostic marker using a serum sample collected from the human pancreatic cancer mouse model described. This specification includes the disclosure of Japanese Patent Application No. 2020-078771, which is the basis of the priority of this application.

Brief Description of the Drawings

[0005]

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Mode for Carrying Out the Invention

[0006] Hereinafter, the present invention will be described in detail. The human pancreatic cancer mouse model according to the present invention (hereinafter referred to as "the mouse model according to the present invention") is a human pancreatic cancer mouse model carrying a pancreatic cancer organoid containing the human pancreatic cancer cell line S2-013. The mouse model according to the present invention has a tumor with a tissue structure similar to that of human pancreatic cancer tissue, such as a papillary structure, invasion of pancreatic cancer cells with cancer stroma, vascular invasion, lymph node metastasis, and epithelial-mesenchymal transition (EMT), and is a useful mouse model that can be used in experiments for evaluating the efficacy of drugs such as pancreatic cancer therapeutic agents and in experiments for evaluating the usefulness of pancreatic cancer diagnostic markers. In the present invention, by using the human pancreatic cancer cell line S2-013 and conducting a detailed examination of the production conditions of its organoids, the individual-to-individual homogenization of the pancreatic cancer cell growth speed (= increase in tumor volume) was successfully achieved, enabling the use of a human pancreatic cancer mouse model having the following characteristics: (1) Tumors of almost the same size can be formed with a success rate of almost 100%. It can cope with high throughput. Since the variation in tumor volume is extremely small, accurate efficacy evaluation is possible (a model with stable tumor volume was established for the first time among organoid transplantation models where the problem was that the tumor volumes were not uniform); (2) Since there is no bleeding from the tumor, the tumor diameter can be accurately measured; (3) By using serum CA19-9 as a marker for efficacy evaluation, the efficacy evaluation can be enhanced. 1. Preparation of the mouse model according to the present invention 1-1. Preparation of pancreatic cancer organoids The pancreatic cancer organoids in the present invention can be prepared according to the method described in Patent Document 1. Here, the "pancreatic cancer organoid" is a cell aggregate composed of pancreatic cancer cells and other cells. It is possible to reproduce the cell-cell interaction among multiple cells. The pancreatic cancer organoids in the present invention reproduce the pancreatic cancer microenvironment and, for example, are rich in stroma. In many cases, cancer tissue has a part called stroma in addition to cancer cells. In the stroma, in addition to mesenchymal cells such as fibroblasts, there are various types of cells that make up blood vessels, lymphatic vessels, nerves, etc. (blood cells, vascular cells, immune cells, etc.), cells that control inflammation (inflammatory cells), etc., and connective tissue composed of collagen, etc. existing between these cells forms a characteristic structure. This is called the "cancer microenvironment." The pancreatic cancer organoid in the present invention preferably reproduces the cancer microenvironment including the cancer stroma. The S2-013 cells are derived from pancreatic ductal adenocarcinoma, and components having a ductal structure are also recognized in the adenocarcinoma tissue. The pancreatic cancer organoid in the present invention preferably reproduces the ductal structure in addition to the cancer microenvironment. The pancreatic cancer organoid in the present invention can be produced by co-culturing the human pancreatic cancer cell line S2-013 with mesenchymal cells and vascular endothelial cells. The culture is preferably three-dimensional (3D) culture. 3D culture techniques suitable for the reconstruction of the pancreatic cancer organoid in the present invention have been reported in Nature, 25; 499(7459):481-4, 2013, Nat Protoc. 9(2):396-409, 2014, Cell Stem Cell, 7; 16(5):556-65, 2015, etc. The "human pancreatic cancer cell line S2-013" used in the present invention is held at the Medical Cell Resource Center, Cell Bank, Tohoku University Institute of Aging Medicine (4-1 Seiryo-machi, Aoba-ku, Sendai-shi, Miyagi-ken 980-8575, Japan, Tohoku University Institute of Aging Medicine, Medical Cell Resource Center) under the ID: TKG0709; cell name: S2-013, and can be obtained therefrom. In the present invention, the term "vascular endothelial cells" refers to cells that constitute vascular endothelium or cells that can differentiate into such cells. Whether a certain cell is a vascular endothelial cell can be confirmed by examining whether marker proteins such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, and CD41 are expressed (if any one or more of the marker proteins are expressed, it can be determined that the cell is a vascular endothelial cell). The vascular endothelial cells used in the present invention may be differentiated or undifferentiated. Whether the vascular endothelial cells are differentiated cells can be confirmed by CD31 and CD144. Among the terms used among those skilled in the art, endothelial cells, umbilical vein endothelial cells, endothelial progenitor cells, endothelial precursor cells, vasculogenic progenitors, hemangioblast (HJ.joo, et al. Blood. 25;118(8):2094-104.(2011)), etc. are included in the vascular endothelial cells in the present invention. Preferred vascular endothelial cells are vascular endothelial cells derived from umbilical veins. Vascular endothelial cells can be collected from blood vessels or prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods. Although vascular endothelial cells mainly derived from humans are used, vascular endothelial cells derived from animals other than humans (for example, animals used in experimental animals, pets, working animals, racehorses, fighting dogs, etc., specifically, mice, rats, rabbits, pigs, dogs, monkeys, cows, horses, sheep, chickens, sharks, rays, hammerhead sharks, salmon, shrimps, crabs, etc.) may also be used. In the present invention, the term "mesenchymal cell" refers to connective tissue cells that mainly exist in connective tissues derived from the mesoderm and form a support structure for cells functioning in tissues. However, it also includes cells whose differentiation fate into mesenchymal cells has been determined but have not yet differentiated into mesenchymal cells. The mesenchymal cells used in the present invention may be either differentiated or undifferentiated. Whether a certain cell is an undifferentiated mesenchymal cell can be confirmed by examining whether marker proteins, such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, and Nestin, are expressed (if any one or more of the above marker proteins are expressed, it can be determined that the cell is an undifferentiated mesenchymal cell). Also, mesenchymal cells that do not express any of the above markers can be determined to be differentiated mesenchymal cells. Among the terms used among those skilled in the art, mesenchymal stem cells, mesenchymal progenitor cells, mesenchymal cells (R. Peters, et al. PLoS One. 30; 5(12): e15689. (2010)), etc. are included in the mesenchymal cells in the present invention. Preferred mesenchymal cells are mesenchymal cells derived from bone marrow (particularly, mesenchymal stem cells). Mesenchymal cells can be collected from tissues such as bone marrow, adipose tissue, placental tissue, umbilical cord tissue, dental pulp, etc., or can be prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods. Mesenchymal cells are mainly derived from humans, but undifferentiated mesenchymal cells derived from animals other than humans (for example, animals used in experimental animals, pets, working animals, racehorses, fighting dogs, etc., specifically, mice, rats, rabbits, pigs, dogs, monkeys, cows, horses, sheep, chickens, sharks, rays, dogfish, salmon, shrimps, crabs, etc.) may also be used. The culture ratio of the three types of cells in co - culture is not particularly limited as long as it is within the range where pancreatic cancer organoids can be formed. However, a preferred cell number ratio is human pancreatic cancer cell line S2 - 013:vascular endothelial cells:mesenchymal cells = 10:1 - 100:1 - 100. More preferably, human pancreatic cancer cell line S2 - 013:vascular endothelial cells:mesenchymal cells = 10:1 - 100:5 - 100. Co - culture of about 200,000 human pancreatic cancer cell line S2 - 013, about 140,000 vascular endothelial cells, and about 400,000 mesenchymal cells can form pancreatic cancer organoids with a size of about 50 - 50,000 micrometers. The medium used for culturing can be any medium as long as pancreatic cancer organoids can be formed. However, it is preferable to use a medium for culturing vascular endothelial cells, a medium for culturing cancer cells, a mixture of the above two media, etc. Any medium can be used for culturing vascular endothelial cells, but it is preferable to use a medium containing at least one of hEGF (recombinant human epidermal growth factor), VEGF (vascular endothelial growth factor), hydrocortisone, bFGF, ascorbic acid, IGF1, FBS, Antibiotics (e.g., gentamicin, amphotericin B, etc.), Heparin, L - Glutamine, Phenolred, BBE. As the medium for culturing vascular endothelial cells, EGM - 2 BulletKit (manufactured by Lonza), EGM BulletKit (manufactured by Lonza), VascuLife EnGS Comp Kit (manufactured by LCT), Human Endothelial - SFM Medium (manufactured by Thermo Fisher Scientific), human microvascular endothelial cell growth medium (manufactured by TOYOBO), etc. can be used. Any medium can be used for culturing cancer cells, for example, DMEM medium. In particular, for the preparation of pancreatic cancer organoids, a medium with EGM:DMEM = 1:1 is suitable. When culturing cells, it is not necessary to use a scaffold material, but it is advisable to culture a mixture of the three types of cells on a gel - like support that allows mesenchymal cells to contract. The contraction of mesenchymal cells can be confirmed in the following ways, such as morphologically observing the formation of three-dimensional tissues (using a microscope or the naked eye), or indicating that the shape of the tissue is maintained with a certain strength when collected with a medicine spoon, etc. (Takebe et al. Nature 499(7459), 481-484, 2013). The support is preferably a gel-like substrate having an appropriate hardness (for example, a Young's modulus of 200 kPa or less (such as in the case of a flat gel coated with Matrigel), but the appropriate hardness of the support may vary depending on the coating and shape). Examples of such substrates include hydrogels (such as acrylamide gels, gelatin, Matrigel, etc.), but are not limited thereto. Note that depending on the shape, size, and amount of the target aggregate, there is no necessity for the hardness of the support to be uniform, and it is possible to set a spatial or temporal gradient in hardness or to pattern it. When the hardness of the support is uniform, the hardness of the support is preferably 100 kPa or less, more preferably 1-50 kPa. The gel-like support may be planar, or the cross-section of the side on which the gel-like support is cultured may be in a U or V shape. By having the cross-section of the side on which the gel-like support is cultured in a U or V shape, cells gather on the culture surface of the support, and it is advantageous because a cell aggregate is formed with a smaller number of cells and / or tissues. Also, the support may be chemically or physically modified. Examples of the modifying substance include Matrigel, laminin, entactin, collagen, fibronectin, vitronectin, etc. An example of setting a spatial gradient in the hardness of the gel-like culture support is a gel-like culture support in which the hardness of the central part is harder than that of the peripheral part. The hardness of the central part is appropriately 200 kPa or less, and the hardness of the peripheral part may be softer than that of the central part, but the appropriate hardness of the central and peripheral parts of the support may vary depending on the coating and shape. Another example of setting a spatial gradient in the hardness of the gel-like culture support is a gel-like culture support in which the hardness of the peripheral part is harder than that of the central part. An example of the patterned gel-like culture support is a gel-like culture support having one or more patterns in which the hardness of the central portion is harder than that of the peripheral portion. The hardness of the central portion is preferably 200 kPa or less, and the hardness of the peripheral portion may be softer than that of the central portion, but the appropriate hardness of the central and peripheral portions of the support may vary depending on the coating and shape. Another example of the patterned gel-like culture support is a gel-like culture support having one or more patterns in which the hardness of the peripheral portion is harder than that of the central portion. The hardness of the peripheral portion is preferably 200 kPa or less, and the hardness of the central portion may be softer than that of the peripheral portion, but the appropriate hardness of the central and peripheral portions of the support may vary depending on the coating and shape. The temperature during culturing is not particularly limited, but it is preferably 30 to 40 °C, and more preferably 37 °C. The culturing period is not particularly limited, but it is preferably 1 to 60 days, and more preferably 1 to 7 days. 1-2. Preparation of mouse model By transplanting the reconstituted pancreatic cancer organoids that reproduce the above-described pancreatic cancer microenvironment into a mouse, a human pancreatic cancer mouse model (xenograft model) that reproduces the ductal structure and the pancreatic cancer microenvironment can be prepared. Examples of the mouse used in the present invention include BALB / cSlc-nu / nu mice. As prepared above, it is confirmed by stereomicroscopic observation that circular pancreatic cancer organoids are formed, and the circular pancreatic cancer organoids are transplanted subcutaneously into the mouse. The transplantation can be performed according to the conventional xenograft transplantation method. For example, 2 to 10 weeks after transplantation, tumor tissue is sufficiently formed in the mouse, and it can be used as a human pancreatic cancer mouse model. 2. Use of human pancreatic cancer mouse model Since the mouse model according to the present invention described above has pancreatic cancer similar to human pancreatic cancer, it can be used as a pathological model mouse for human pancreatic cancer. For example, it can be used for elucidating the onset mechanism or pathology of human pancreatic cancer, screening of pancreatic cancer therapeutic agents (anticancer agents) and evaluation of the drug efficacy of pancreatic cancer therapeutic agents, and screening of pancreatic cancer diagnostic markers and evaluation of their usefulness. For example, in the screening of a pancreatic cancer therapeutic agent, a test substance, which is a candidate compound for the therapeutic agent, is administered to the mouse model according to the present invention by an appropriate administration route such as oral administration, and the size of pancreatic cancer, invasion, the number of distant metastases, death of the mouse, etc. are observed in the mouse model to determine the effect of the test substance. Similarly, in the evaluation of the efficacy of a pancreatic cancer therapeutic agent, the pancreatic cancer therapeutic agent to be evaluated is administered to the mouse model according to the present invention by an appropriate administration route such as oral administration, and the size of pancreatic cancer, invasion, the number of distant metastases, death of the mouse, etc. are observed in the mouse model to evaluate the efficacy of the pancreatic cancer therapeutic agent. In addition, in the serum collected from the mouse model according to the present invention, the pancreatic cancer diagnostic marker shows the same behavior as clinical pancreatic cancer. Therefore, the mouse model according to the present invention can be used for the evaluation of the usefulness of a pancreatic cancer diagnostic marker or the screening of a pancreatic cancer diagnostic marker in a biological sample such as serum collected from the mouse model. For example, in the evaluation of the usefulness of a pancreatic cancer diagnostic marker, a biological sample such as serum collected from the mouse model according to the present invention is subjected to an immunological measurement method using an antibody against the pancreatic cancer diagnostic marker to be evaluated, etc., and the pancreatic cancer diagnostic marker is measured at the protein level. By determining whether a significant variation (increase or decrease) according to the pancreatic cancer diagnostic marker to be evaluated is observed when the measured pancreatic cancer diagnostic marker level (concentration in a biological sample such as serum) is compared with a negative control (for example, a biological sample such as serum derived from a mouse identical to the mouse model according to the present invention except that it does not carry a pancreatic cancer organoid), the usefulness of the pancreatic cancer diagnostic marker can be evaluated. Similarly, for screening pancreatic cancer diagnostic markers, biological samples such as serum collected from the mouse model according to the present invention are subjected to an immunological measurement method using an antibody against a pancreatic cancer diagnostic marker candidate, etc., to measure the pancreatic cancer diagnostic marker candidate at the protein level. When the measured level of the pancreatic cancer diagnostic marker candidate (concentration in a biological sample such as serum) significantly varies (increases or decreases) compared to a negative control (for example, a biological sample such as serum derived from a mouse identical to the mouse model according to the present invention except that it does not carry pancreatic cancer organoids), the pancreatic cancer diagnostic marker candidate can be identified as a pancreatic cancer diagnostic marker. Here, the immunological measurement method is not particularly limited, and examples include ELISA, flow cytometry, Western blotting, etc.

Example

[0007] Hereinafter, the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to these examples. [Preparation and Evaluation of Human Pancreatic Cancer Mouse Model] 1. Materials and Methods 1-1. Cells The cells used were the human pancreatic cancer cell line S2-013 (hereinafter sometimes referred to as "S2-013"), human umbilical vein endothelial cells HUVEC (LONZA) (hereinafter sometimes referred to as "HUVEC"), and human mesenchymal stem cells MSC (LONZA) (hereinafter sometimes referred to as "MSC"). S2-013 was cultured in DMEM medium. HUVEC was cultured in EGM-2·HUVEC medium. MSC was cultured in MSCGM medium. 1-2. Preparation of Pancreatic Cancer Organoids S2-013, MSC, and HUVEC during culture were sequentially collected one by one using trypsin. S2-013 was detached using 0.5% trypsin (SIGMA), while MSC and HUVEC were detached using 0.05% trypsin (gibco). The trypsin treatment time was adjusted according to the state of each cell. Subsequently, trypsin was neutralized with DMEM containing FCS, and after centrifugation, the culture medium was removed as much as possible. After suspending in the culture medium appropriate for each cell, trypan blue and the cell suspension were mixed in equal amounts in a 1.5 mL assist tube, and 10 μL of it was used to count cells using a hemocytometer. After confirming the viable cell rate and cell number, for each organoid, the required amount of each cell was dispensed using one 15 mL Falcon tube per organoid. After dispensing, it was stored on ice. The number of cells required per organoid was S2-013: 20×10 4 cells, MSC: 40×10 4 cells, and HUVEC: 14×10 4 cells. On the other hand, Matrigel was added in an equal amount to pre-cooled DMEM. To prevent Matrigel from solidifying, a pipette tip wetted with well-cooled PBS was used. The DMEM / Matrigel mixed solution was mixed well and placed on ice. Next, a 48-well plate was wetted with 200 μL / well of well-cooled PBS, and after removing the PBS from the wells, the DMEM / Matrigel mixed solution was applied at 160 μL / well. At this time, bubbles were crushed at the tip of the pipette, etc., and after confirming that the liquid surface was flat, it was incubated at 37°C for 1 hour in a CO2 incubator. The mixed solution of the three types of cells prepared by the above dispensing was mixed well, centrifuged at 600 g for 5 minutes at room temperature, and the supernatant was removed as much as possible using a Pasteur pipette with a 10 μL tip. Subsequently, one Falcon tube containing the cell mixed solution was applied to each well of the 48-well plate after incubation. Care was taken not to allow bubbles to enter or the wells to crack during this process. The 48-well plate applied with the cell mixture was incubated in a CO2 incubator at 37 °C for 30 minutes. During the waiting time of incubation, a DMEM / EGM mixed solution was prepared by mixing DMEM and EGM in equivalent amounts. After the incubation was completed, 300 μL / well of the DMEM / EGM mixed solution was applied, and it was confirmed that there were no bubbles between the solution and Matrigel. Then, the plate was placed back into the CO2 incubator and incubated at 37 °C for 24 hours. In this way, one pancreatic cancer organoid was prepared in each well of the 48-well plate. 1-3. Preparation of human pancreatic cancer mouse model In Sections 1-2, the pancreatic cancer organoids prepared the previous day were observed under a microscope to confirm their usable shape, such as whether they were shrunk or not cracked. A Matrigel / DMEM mixed solution was prepared and dispensed into 1.5 mL tubes at 50 μL / tube. The same number of tubes as the number of pancreatic cancer organoids was prepared and placed on ice. Six-week-old nude mice (BALB / cSlc-nu / nu) were purchased from Japan SLC Inc. and treated according to the guidelines for animal management and use within the Kochi University research institution. The skin on the flank of the anesthetized nude mice was incised about 5-8 mm, and the subcutaneous tissue was bluntly dissected with hemostatic forceps to create a pocket large enough to accommodate human pancreatic cancer organoids. A 1000 μL tip was wetted with chilled PBS, and the medium and gel were removed from the 48-well plate in which the pancreatic cancer organoids were cultured. Then, a 200 μL tip with the tip cut off in advance and sterilized was wetted with chilled PBS, and the pancreatic cancer organoids were aspirated and placed into the tube containing the previously prepared DMEM / Matrigel mixed solution. The entire mixed solution containing the pancreatic cancer organoids was applied to the subcutaneous pocket on the flank of the prepared nude mice and sutured. In this way, a human pancreatic cancer mouse model was prepared. 1-4. Administration of TS-1 to the human pancreatic cancer mouse model As described in Sections 1-3, human pancreatic cancer organoids derived from the human pancreatic cancer cell line S2-013 were transplanted subcutaneously into the flanks of nude mice. The human pancreatic cancer mouse model was divided into two groups. From the week following transplantation, TS-1 (10 mg / kg), a standard chemotherapeutic drug for pancreatic cancer, was orally administered to six mice at a frequency of 5 days / week. After 4 weeks of drug administration, the drug was discontinued for 2 weeks and then administered again for 2 weeks. In addition, as a control group, six mice were only observed without drug administration. From 2 weeks after transplantation, the tumor diameter of the pancreatic cancer tissue was measured and photographed weekly. Photographs of each mouse 8 weeks after transplantation are shown in Fig. 14, and the changes in tumor diameter over time for each group of mice are shown in Fig. 15. In Fig. 15, "*" indicates a significant difference with p < 0.05 compared to the control group in the t-test. The table shows the histopathological findings of the excised human pancreatic cancer. 1-5. Measurement of pancreatic cancer diagnostic markers in serum collected from a human pancreatic cancer mouse model As described in Sections 1-3, human pancreatic cancer organoids derived from the human pancreatic cancer cell line S2-013 were transplanted subcutaneously into the flanks of nude mice. The human pancreatic cancer mouse model was divided into two groups. In the first group of five human pancreatic cancer mouse models, whole blood was collected 4 weeks after transplantation of pancreatic cancer organoids with an average tumor diameter of 5 mm. In the second group of five human pancreatic cancer mouse models, whole blood was collected 8 weeks after transplantation of pancreatic cancer organoids with an average tumor diameter of 20 mm. As a control group, whole blood was collected from five nude mice without transplanted human pancreatic cancer organoids. Serum was separated from all the blood and stored frozen. Subsequently, the CA19-9 concentration in all the sera was measured using a commercially available ELISA kit (EIA-5069, DRG). 2. Results 2-1. Histopathological examination of tumor tissue excised from a human pancreatic cancer mouse model As described above, pancreatic cancer organoids were prepared using the human pancreatic cancer cell line S2-013 and transplanted subcutaneously into mice. As shown in Figs. 1, 2, and 3, 6 weeks of observation was carried out after transplantation of the pancreatic cancer organoids subcutaneously into mice, and the tumor tissue was excised. The tumor tissue increased over time. Tissue specimens of the extracted tumor tissues were prepared for histopathological examination. The most prominent feature of human pancreatic cancer tissue is the abundant cancer stroma. Among the cancer stroma, pancreatic cancer cells actively move around and infiltrate the surrounding tissues. As shown in Fig. 4, in the tumor tissues extracted from the human pancreatic cancer mouse model carrying the pancreatic cancer organoids prepared as described above, cancer stroma derived from the human pancreatic cancer organoids was abundantly present, and parts presenting acinar structures and micro-papillary structures were intermixed. An infiltration image of pancreatic cancer cells accompanied by cancer stroma was observed. On the other hand, in the tumor tissues extracted from the mice carrying the conventional xenograft, most parts were solid, lacking acinar structures and cancer stroma, and could not be said to be adenocarcinoma tissue. In this example, the "mice carrying the conventional xenograft" were heterotopic transplantation models in which a suspension of S2-013 cells was injected subcutaneously into the flanks of nude mice to form tumors. Mice carrying the conventional xenograft: Six-week-old nude mice (BALB / cSlc-nu / nu) were used. 2.0×10 6 individual S2-013 cells suspended in PBS were subcutaneously injected into the flanks of the anesthetized nude mice to form tumors subcutaneously in the mice. As shown in Fig. 5, in the tumor tissues extracted from the human pancreatic cancer mouse model carrying the pancreatic cancer organoids prepared as described above, it was clear that they were adenocarcinoma from the acini (left photo in (A)) and micro-papillary structures (right photo in (A)). On the other hand, in the tumor tissues extracted from the mice carrying the conventional xenograft, there were almost no ductal structures or papillary structures and they showed poor differentiation into adenocarcinoma (photo in (B)). As shown in Fig. 6, in the tumor tissues extracted from the human pancreatic cancer mouse model carrying the pancreatic cancer organoids prepared as described above, infiltration of cells into the muscular layer and subcutaneous tissue accompanied by abundant cancer stroma was observed (photo in (A)). On the other hand, in the tumor tissues extracted from the mice carrying the conventional xenograft, infiltration of cells into the muscular layer was observed, but the cancer stroma was scarce (photo in (B)). As shown in Fig. 7, in the tumor tissues excised from the human pancreatic cancer mouse model carrying the pancreatic cancer organoids prepared as described above, vascular invasion near the tumor (left in the figure) and metastasis to the subcutaneous lymph nodes (right in the figure) were observed. On the other hand, in the tumor tissues excised from the mice carrying the conventional xenograft, vascular invasion and lymph node metastasis were not observed. As shown in Fig. 8, in the tumor tissues excised from the human pancreatic cancer mouse model carrying the pancreatic cancer organoids prepared as described above, epithelial-mesenchymal transition (EMT) was observed (photo in (A): within the dotted frame). The staining property of CK19, which is frequently expressed in pancreatic cancer, was also low in the same part (photo in (C)), and conversely, vimentin was expressed (photo in (D)). The tumor cells were CK19(+), vimentin(-). EMT is involved in invasion and metastasis. Papillary structures with stromal stalks were also partly observed (photo in (B)). As shown in Fig. 9, in the tumor tissues excised from the mice carrying the conventional xenograft, the differentiation of the pancreatic cancer tissues was uniform overall, and no variation in differentiation was observed among different sites. There were also no EMT images or papillary structures with stromal stalks. The tumor cells were CK19(+), vimentin(-), which was the same finding as in the pancreatic cancer organoid tumors. However, the expression of vimentin was generally stronger than that in the pancreatic cancer organoid tumors. The most significant feature was the lack of variation in differentiation and the almost uniform nature, which deviated from the clinical pancreatic cancer tissues with rich variation in differentiation and abundant cancer stroma. As shown in Fig. 10, in the pancreatic cancer tissues surgically excised from pancreatic cancer patients, pancreatic cancer cells with abundant cancer stroma formed glandular structures and infiltration into the pancreatic adipose tissue was observed. As shown in Fig. 4, the tumor tissues excised from the human pancreatic cancer mouse model were similar to clinical pancreatic cancer, and the human pancreatic cancer mouse model was a model mouse that did not deviate from clinical pancreatic cancer. As shown in Fig. 11, papillary structures were observed in the pancreatic cancer tissues surgically excised from pancreatic cancer patients. As shown in Fig. 5, papillary structures were also observed in the tumor tissues excised from the human pancreatic cancer mouse model. As shown in Fig. 12, lymph node metastasis in the abdominal cavity was observed in the pancreatic cancer tissue surgically removed from a pancreatic cancer patient. As shown in Fig. 7, lymph node metastasis was also observed in the tumor tissue removed from a human pancreatic cancer mouse model. As shown in Fig. 13, EMT was observed in the pancreatic cancer tissue surgically removed from a pancreatic cancer patient. As shown in Fig. 8, EMT was also observed in the tumor tissue removed from a human pancreatic cancer mouse model. On the other hand, in the tumor tissue removed from a mouse bearing a conventional xenograft, none of the findings of (1) infiltration of pancreatic cancer cells with abundant cancer stroma, (2) ductal structure and papillary structure characteristic of adenocarcinoma, (3) vascular invasion, lymph node metastasis, and (4) EMT, which are clinical findings of pancreatic cancer, were observed. 2-2. Observation of antitumor effect by administration of TS-1 to a human pancreatic cancer mouse model The human pancreatic cancer mouse model prepared as described above was administered TS-1, which is the first-choice drug for postoperative pancreatic cancer, to observe the antitumor effect. After administration to the human pancreatic cancer model mice, the tumor diameter was measured over time and histopathological examination was performed. The method of administering TS-1 to the human pancreatic cancer mouse model was to administer 10 mg / kg of TS-1 per mouse body weight 5 days a week for 4 weeks, followed by a 2-week drug holiday, and this cycle was repeated during the experimental period. The results are shown in Figs. 14 and 15. As shown in Figs. 14 and 15, the volume of the pancreatic cancer tumors formed subcutaneously in the human pancreatic cancer mouse model group administered TS-1 for 8 weeks was significantly suppressed from the 7th week after transplantation compared with the control group. Furthermore, at the 8th week after subcutaneous transplantation of human pancreatic cancer organoids, each mouse was dissected, and the tumor tissue formed in the mouse flanks was fixed with formalin and subjected to hematoxylin-eosin staining. The results of the histopathological examination are shown in Table (B) of Fig. 15. As shown in Table (B) of Fig. 15, no difference was observed between TS-1 and the control group in the inhibitory effect on vascular invasion and muscular layer invasion. In the human pancreatic cancer mouse model group administered TS-1 orally, cyst formation due to necrosis was remarkable compared to the control group. These results were consistent with the pharmacological effects obtained in clinical practice that TS-1 suppresses tumor growth by inhibiting cell proliferation. It was shown that the human pancreatic cancer mouse model can solve the deviation from clinical pancreatic cancer tissue, which was a problem in the conventional Xenograft model, and can provide accurate information when evaluating the pharmacological effects of new drugs for pancreatic cancer. 2-3. Measurement of Pancreatic Cancer Diagnostic Markers in Serum Collected from Human Pancreatic Cancer Mouse Models The serum concentration of the pancreatic cancer tumor marker CA19-9 was measured in the serum collected from the human pancreatic cancer mouse models prepared as described above. Blood was collected from the human pancreatic cancer mouse models at the 4th, 8th, and 10th weeks after transplantation, and the serum concentration of the pancreatic cancer diagnostic marker was measured. As shown in Fig. 16, the CA19-9 concentration in the serum collected from the human pancreatic cancer mouse models increased over time. CA19-9 leaked from the tumor tissue into the blood could be detected, and the concentration increased as the tumor grew. Thus, in research aiming to identify new pancreatic cancer diagnostic markers, measuring the serum concentration of the human pancreatic cancer mouse model over time can provide information useful for evaluating whether it is a promising diagnostic marker. 2-4. Observation of Antitumor Effect by Administration of TS-1 to Mice Bearing Conventional Xenografts TS-1 was administered to mice bearing conventional Xenografts for 8 weeks to observe the antitumor effect. After administration to mice bearing Xenografts, the tumor diameter was measured over time. As shown in Fig. 17, the volume of pancreatic cancer tumors formed subcutaneously in the mouse group bearing Xenografts administered with TS-1 was not suppressed compared to the control group. Also, it was considered unqualified as a model for observing tumor growth as the subcutaneous tumor tissue was bleeding and excavated. On the other hand, as shown in Fig. 14, no bleeding was observed in the pancreatic cancer tumors formed subcutaneously in the human pancreatic cancer mouse model group. As shown in Fig. 18, in mice bearing conventional Xenografts, the effect of TS-1 on suppressing tumor growth could not be detected. 2-5. Measurement of Pancreatic Cancer Diagnostic Markers in Serum Collected from Mice Bearing Conventional Xenografts The serum concentration of the pancreatic cancer tumor marker CA19-9 was measured in serum collected from mice bearing conventional Xenografts. Blood was collected from mice bearing conventional Xenografts at the 4th and 8th weeks after transplantation, and the serum concentration of pancreatic cancer diagnostic markers was measured. As shown in Fig. 19, the CA19-9 concentration in serum collected from mice bearing conventional Xenografts did not show a time-dependent increase. Although bleeding was observed from the tumor, despite the tumor growing larger over time, the CA19-9 leaked from the tumor tissue into the blood and its concentration decreased over time. 3. Summary The human pancreatic cancer tissue of the human pancreatic cancer mouse model bearing pancreatic cancer organoids was extremely similar in tissue structure to clinical pancreatic cancer tissue, and a rich cancer stroma derived from human pancreatic cancer was present. Also, when TS-1, the first-choice drug for postoperative treatment of pancreatic cancer, was administered to the human pancreatic cancer mouse model bearing pancreatic cancer organoids, it was revealed that TS-1 suppressed the growth of pancreatic cancer tumors but had a low effect on suppressing invasion into surrounding tissues. It was shown that TS-1 could not sufficiently suppress the "strong invasion with cancer stroma," which is a characteristic of pancreatic cancer, indicating a limitation in its drug efficacy. Furthermore, the human pancreatic cancer mouse model bearing pancreatic cancer organoids could solve the divergence from clinical pancreatic cancer tissue, which was a problem in conventional models, and could provide accurate information when evaluating the drug efficacy of new pancreatic cancer drugs.

Industrial Applicability

[0008] According to the present invention, a pancreatic cancer mouse model showing the characteristics of human pancreatic cancer is provided. Further, using the mouse model according to the present invention, it is possible to screen a therapeutic agent effective for human pancreatic cancer and evaluate the efficacy of the therapeutic agent. Furthermore, using a serum sample of the mouse model according to the present invention, it is possible to screen a pancreatic cancer diagnostic marker and evaluate the usefulness of the marker. In addition, the mouse model according to the present invention has the following advantages: (1) By using a commercially available human pancreatic cancer cell line, it can cope with high throughput; (2) A mouse model can be supplied at the 6th week from the day when three types of cell cultures are started; (3) Since a commercially available human pancreatic cancer cell line is used, pancreatic cancer tissue derived from a surgically removed patient is not required; (4) As described in detail above, the techniques for producing human pancreatic cancer organoids and creating mice have been established; (5) The human pancreatic cancer tissue in the mouse model according to the present invention is extremely similar in tissue structure to clinical pancreatic cancer tissue, has a rich cancer stroma, and shows a high level of serum CA19-9; (6) Although there is no clinical data or gene expression profile of patients, tissue sections of human pancreatic cancer organoid tumors can be sold; (7) When a gene expression profile is required, it is possible to prepare tissue sections of human pancreatic cancer organoid tumors; (8) It is a model useful for evaluating the efficacy of a novel therapeutic agent for pancreatic cancer in non-clinical pharmacological tests. All publications, patents, and patent applications cited in this specification are hereby incorporated by reference as they are.

Claims

**Claim 1** A human pancreatic cancer mouse model carrying pancreatic cancer organoids containing the human pancreatic cancer cell line S2-013. **Claim 2** A method for screening a therapeutic agent for pancreatic cancer using the human pancreatic cancer mouse model according to Claim 1. **Claim 3** A method for evaluating the efficacy of a therapeutic agent for pancreatic cancer using the human pancreatic cancer mouse model according to Claim 1. **Claim 4** A method for evaluating the usefulness of a pancreatic cancer diagnostic marker using a serum sample collected from the human pancreatic cancer mouse model according to Claim 1. **Claim 5** A method for screening a pancreatic cancer diagnostic marker using a serum sample collected from the human pancreatic cancer mouse model according to Claim 1.

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