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WO2021221179A1 - Établissement d'un modèle de souris à l'aide d'un organoïde du cancer du pancréas humain - Google Patents

Établissement d'un modèle de souris à l'aide d'un organoïde du cancer du pancréas humain Download PDF

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WO2021221179A1
WO2021221179A1 PCT/JP2021/017608 JP2021017608W WO2021221179A1 WO 2021221179 A1 WO2021221179 A1 WO 2021221179A1 JP 2021017608 W JP2021017608 W JP 2021017608W WO 2021221179 A1 WO2021221179 A1 WO 2021221179A1
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pancreatic cancer
mouse model
human pancreatic
cells
human
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Japanese (ja)
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恵介 谷内
英樹 谷口
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Kochi University NUC
Yokohama City University
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Kochi University NUC
Yokohama City University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood

Definitions

  • the present invention relates to, for example, a human pancreatic cancer mouse model and the use of the mouse model.
  • pancreatic cancer which is a representative cancer with a poor prognosis, is difficult to detect at an early stage, and development of a useful diagnostic marker is desired.
  • pancreatic cancer there is no mouse model capable of forming a tumor having a tissue structure similar to that of human pancreatic cancer tissue at high throughput. For this reason, there was no useful pancreatic cancer mouse model that could be used to experiment with drug efficacy.
  • xenografts prepared by transplanting pancreatic cancer tissue obtained from patients into immunodeficient animals are patients with stromal cells / cancer-related fibroblasts and tumor macrophages in the tumor. Since it contains derived cells, it retains the characteristics of patient-derived tumors and is strongly expected to be used for tumor pathological analysis, tumor marker analysis, drug development, and the like.
  • PDX tumor growth in the mouse body accelerates as the passage progresses, and the gene expression profile changes as compared with the patient-derived sample.
  • PDX establishment usually takes 3-6 months, and at present it is difficult to be directly involved in the personalized care of the patient who donated the tumor.
  • Patent Document 1 describes human pancreatic cancer organoids by co-culturing human pancreatic cancer cell lines (PANC-1, CFPAC-1, SW1990), human vascular endothelial cells (HUVEC) and human mesenchymal cells (hMSC). It is disclosed that a pancreatic cancer xenograft having abundant stroma and ductal structure was formed from this pancreatic cancer organoid.
  • Patent Document 1 states that in mice carrying the human pancreatic cancer organoid, papillary structure, infiltration of pancreatic cancer cells with cancer stroma, lymphovascular invasion, lymph node metastasis, epithelial-mesenchymal transition (EMT), etc. It is not disclosed that tumors that form a tissue structure similar to clinical pancreatic cancer have been formed. Further, Patent Document 1 does not disclose that pancreatic cancer diagnostic markers behave similarly to clinical pancreatic cancer in sera collected from mice carrying human pancreatic cancer organoids. Further, Patent Document 1 does not disclose that a mouse carrying a human pancreatic cancer organoid is a useful model for determining the therapeutic effect of a pancreatic cancer therapeutic agent.
  • the present invention includes the following. (1) A mouse model of human pancreatic cancer carrying a pancreatic cancer organoid 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 described in (1).
  • FIG. 1 It is a photograph of a human pancreatic cancer organoid prepared in a petri dish in an example.
  • This human pancreatic cancer organoid was subcutaneously transplanted into nude mice. The day of transplantation was designated as Day 1.
  • Ten human pancreatic cancer organoids were generated (Orga-1 to Orga-10).
  • the method for producing human pancreatic cancer organoids shown in Examples one researcher can produce 10 to 20 uniform human pancreatic cancer organoids, and the method is for producing high-throughput human pancreatic cancer organoids. The method.
  • pancreatic cancer tumor tissue surgically removed from the pancreatic cancer patient in the example, stained with hematoxylin and eosin.
  • pancreatic cancer tumor tissue surgically removed from the pancreatic cancer patient in the example, stained with hematoxylin and eosin. Shows lymph node metastasis.
  • the graph in (A) shows the tumor volume over time when the tumor size was measured using a caliper.
  • Table (B) shows the pathological findings of the tumor tissue removed 8 weeks after transplantation. It is a figure which shows the measurement result of the pancreatic cancer diagnostic marker CA19-9 in the time-dependent tumor volume of (A) human pancreatic cancer mouse model, and the serum collected from (B and C) human pancreatic cancer mouse model in an Example. It is a photograph which shows the observation of the antitumor effect by administration of TS-1 to the mouse which carried the conventional Xenograft in Example.
  • the human pancreatic cancer mouse model according to the present invention is a human pancreatic cancer mouse model carrying a pancreatic cancer organoid including the human pancreatic cancer cell line S2-013.
  • the mouse model according to the present invention constructs a tissue similar to human pancreatic cancer tissue such as papillary structure, invasion of pancreatic cancer cells with cancer stroma, vascular invasion, lymph node metastasis, and epithelial-mesenchymal transition (EMT).
  • pancreatic cancer organoid in the present invention can be produced according to the method described in Patent Document 1.
  • the "pancreatic cancer organoid” is a cell aggregate composed of pancreatic cancer cells and other cells. It is possible to reproduce cell-cell interactions between multiple cells.
  • the pancreatic cancer organoid in the present invention reproduces the pancreatic cancer microenvironment, and is, for example, rich in stroma.
  • cancer tissue has a part called the stroma in addition to the cancer cells.
  • the stroma in addition to mesenchymal cells such as fibroblasts, cells that make up blood vessels, lymph vessels, nerves, etc. (blood cells, vascular cells, immune cells, etc.), cells that control inflammation (inflammatory cells), etc.
  • mesenchymal cells such as fibroblasts, cells that make up blood vessels, lymph vessels, nerves, etc. (blood cells, vascular cells, immune cells, etc.), cells that control inflammation (inflammatory cells), etc.
  • the pancreatic cancer organoid in the present invention may reproduce the cancer microenvironment including the cancer stroma.
  • S2-013 cells are derived from pancreatic duct adenocarcinoma, and components having a ductal structure are also found in the adenocarcinoma tissue.
  • the pancreatic cancer organoid in the present invention may reproduce the cancer microenvironment as well as the ductal structure.
  • the pancreatic cancer organoid in the present invention can be prepared by co-culturing the human pancreatic cancer cell line S2-013 with mesenchymal cells and vascular endothelial cells.
  • the culture may be a three-dimensional (3D) culture.
  • a 3D culture technique suitable for the reconstruction of pancreatic cancer organoids in the present invention is described in Nature, 25; 499 (7459): 481-4, 2013, Nat Protocol.
  • the "human pancreatic cancer cell line S2-013" used in the present invention is the Tohoku University Institute of Aging Medicine Medical Cell Resource Center / Cell Bank (4-1 Seiryomachi, Aoba-ku, Sendai City, Miyagi Prefecture, Japan 980-8575, Japan Tohoku University) It is retained as ID: TKG0709; cell name: S2-013 at the Medical Cell Resources Center of the Institute of Aging Medicine, and can be obtained from here.
  • the "vascular endothelial cell” refers to a cell constituting the vascular endothelium or a cell capable of differentiating into such a cell.
  • a cell is a vascular endothelial cell can be confirmed by examining whether or not a marker protein such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, or CD41 is expressed (any of the above marker proteins). If one or more of them are expressed, it can be determined that they are vascular endothelial cells).
  • the vascular endothelial cells used in the present invention may be differentiated or undifferentiated. Whether or not the vascular endothelial cells are differentiated cells can be confirmed by CD31 and CD144.
  • endothelial cells are included in the vascular endothelial cells in the present invention.
  • Preferred vascular endothelial cells are vascular endothelial cells derived from the umbilical vein.
  • Vascular endothelial cells can be collected from blood vessels or can be prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to a known method.
  • iPS cells induced pluripotent stem cells
  • ES cells embryonic stem cells
  • the "mesenchymal cell” is a connective tissue cell that exists mainly in the connective tissue derived from the mesodermal cell and forms a support structure of a cell that functions in a tissue, but is destined to differentiate into a mesenchymal cell.
  • the mesenchymal cells used in the present invention may be differentiated or undifferentiated. Whether or not a cell is an undifferentiated mesenchymal cell is confirmed by examining whether or not a marker protein such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, or Nestin is expressed. (If any one or more of the marker proteins are expressed, it can be determined that the cells are undifferentiated mesenchymal cells). In addition, mesenchymal cells that do not express any of the markers in the preceding paragraph can be judged to be differentiated mesenchymal cells.
  • a marker protein such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, or Nestin is expressed.
  • mesenchymal stem cells mesenchymal progenitor cells, mesenchymal cells (R. Peters, et al. PLos One.30; 5 (12): e15689. (2010)) and the like are of the present invention.
  • mesenchymal cells include bone marrow-derived mesenchymal cells (particularly mesenchymal stem cells).
  • the mesenchymal cells are collected from tissues such as bone marrow, adipose tissue, placenta tissue, umbilical cord tissue, and dental pulp, or are pluripotent such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).
  • iPS cells induced pluripotent stem cells
  • ES cells embryonic stem cells
  • mesenchymal cells are mainly derived from humans, animals used for animals other than humans (for example, experimental animals, pet animals, service animals, race horses, fighting dogs, etc., specifically, mice and rats , Rabbits, pigs, dogs, monkeys, cows, horses, sheep, chickens, sharks, rays, ginseng, salmon, shrimp, crabs, etc.) may be used.
  • the culture ratio of the three types of cells in the co-culture is not particularly limited as long as the pancreatic cancer organoid can be formed, but a suitable cell number ratio is human pancreatic cancer cell line S2-013: vascular endothelial cell: mesenchymal cell.
  • About 200,000 human pancreatic cancer cell lines S2-013, about 140,000 vascular endothelial cells, and about 400,000 mesenchymal cells are co-cultured to produce pancreatic cancer organoids with a size of about 50,000 to 50,000 micrometers.
  • the medium used for culturing may be any medium as long as it forms a pancreatic cancer organoid, but is a mixture of a medium for vascular endothelial cell culture, a medium for cancer cell culture, and the above two media. Etc. are preferred.
  • vascular endothelial cell culture Any medium for vascular endothelial cell culture may be used, but hEGF (recombinant human epidermal growth factor), VEGF (vascular endothelial growth factor), hydrocortisone, bFGF, ascorbic acid, IGF1, FBS. , Antibiotics (eg, gentamicin, amphotericin B, etc.), Heparin, L-Glutamine, Phenolred, BBE.
  • EGM-2 Bullet Kit manufactured by Lonza
  • EGM Bullet Kit manufactured by Lonza
  • VascuLife EnGS Comp Kit manufactured by LCT
  • Human Endothelial-SFM Thermo Fisher Human microvascular endothelial cell growth medium
  • TOYOBO Human microvascular endothelial cell growth medium
  • the support has an appropriate hardness (for example, Young's modulus of 200 kPa or less (for example, when the shape coated with Matrigel is a flat gel), but the appropriate hardness of the support may vary depending on the coating and shape).
  • a gel-like base material having a gel It is preferably a gel-like base material having a gel, and examples of such a base material include hydrogels (for example, acrylamide gel, gelatin, matrigel, etc.), but are not limited thereto.
  • the hardness of the support does not necessarily have to be uniform according to the shape, size, and amount of the target aggregate, and it is possible to set a spatial / temporal gradient for the hardness or to pattern it. It is possible.
  • the hardness of the support is preferably 100 kPa or less, more preferably 1 to 50 kPa.
  • the gel-like support may be flat, or the cross section of the gel-like support on the culture side may be U or V-shaped.
  • the U or V-shaped cross section of the gel-like support on the culture side allows cells to gather on the culture surface of the support, and a cell aggregate is formed with a smaller number of cells and / or tissues. It is advantageous because it is done.
  • the support may be chemically or physically modified. Examples of the modifying substance include matrigel, laminin, entactin, collagen, fibronectin, vitronectin and the like.
  • An example in which the hardness of the gel-like culture support is set to a spatial gradient is a gel-like culture support in which the hardness of the central portion is harder than the hardness of the peripheral portion.
  • the appropriate hardness of the central part is 200 kPa or less, and the hardness of the peripheral part should be softer than that of the central part, but the appropriate hardness of the central part and the peripheral part of the support varies depending on the coating and shape. sell.
  • Another example in which the hardness of the gel-like culture support is set to a spatial gradient is a gel-like culture support in which the hardness of the peripheral portion is harder than the hardness of the central portion.
  • An example of a 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 the hardness of the peripheral portion.
  • the appropriate hardness of the central part is 200 kPa or less, and the hardness of the peripheral part should be softer than that of the central part, but the appropriate hardness of the central part and the peripheral part of the support varies depending on the coating and shape. sell.
  • 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 the hardness of the central portion.
  • the appropriate hardness of the peripheral portion is 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 portion and the peripheral portion of the support varies depending on the coating and shape. sell.
  • the temperature at the time of culturing is not particularly limited, but is preferably 30 to 40 ° C, more preferably 37 ° C.
  • the culture period is not particularly limited, but is preferably 1 to 60 days, and more preferably 1 to 7 days.
  • 1-2. Preparation of mouse model A human pancreatic cancer mouse model (Xenograft model) that reproduces the ductal structure and pancreatic cancer microenvironment by transplanting a reconstructed pancreatic cancer organoid that reproduces the above-mentioned pancreatic cancer microenvironment into a mouse is prepared. be able to.
  • Examples of the mouse used in the present invention include BALB / cSlc-nu / nu mice.
  • pancreatic cancer therapeutic agents anticancer agents
  • pancreatic cancer diagnostic markers screening of pancreatic cancer therapeutic agents
  • a test substance which is a therapeutic agent candidate compound is administered to a mouse model according to the present invention by an appropriate administration route such as oral administration, and the size, invasion, and distant metastasis of pancreatic cancer are determined in the mouse model.
  • the effect of the test substance can be determined by observing the number, death of the mouse, and the like.
  • 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, invasion, and distant metastasis of pancreatic cancer in the mouse model.
  • the efficacy of the pancreatic cancer therapeutic agent can be evaluated by observing the number of patients, the death of the mouse, and the like.
  • the pancreatic cancer diagnostic marker behaves similarly to clinical pancreatic cancer.
  • the mouse model according to the present invention can be used for evaluation of the usefulness of a pancreatic cancer diagnostic marker or screening of a pancreatic cancer diagnostic marker in a biological sample such as serum collected from the mouse model.
  • a biological sample such as serum collected from the mouse model according to the present invention is used for an immunological measurement method using an antibody against the pancreatic cancer diagnostic marker to be evaluated.
  • the pancreatic cancer diagnostic marker is measured at the protein level.
  • the measured pancreatic cancer diagnostic marker level concentration in a biological sample such as serum
  • pancreatic cancer diagnostic marker can be evaluated by determining whether or not a significant change (increase or decrease) according to the pancreatic cancer diagnostic marker to be evaluated is observed as compared with the target sample).
  • 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, and the screening is performed at the protein level. Measure pancreatic cancer diagnostic marker candidates.
  • pancreatic cancer diagnostic marker candidate can be identified as a pancreatic cancer diagnostic marker by significantly fluctuating (increasing or decreasing) as compared with the scientific sample).
  • the immunological measurement method is not particularly limited, and examples thereof include ELISA, flow cytometry, and Western blotting.
  • MSCs were cultured in MSCGM medium.
  • SIGMA trypsin
  • sigco trypsin
  • each cell After suspending in the culture medium suitable for each cell, trypan blue and the cell suspension were mixed in an equivalent amount in 1.5 mL assist tube, and 10 ⁇ L of the mixture was used for cell counting with a hemocytometer. After confirming the viable cell rate and the number of cells, each cell was dispensed in the required amount using one 15 mL falcon tube for each organoid. After dispensing, it was stored on ice. The number of cells required for one organoid was S2-013: 20 ⁇ 10 4 cells, MSC: 40 ⁇ 10 4 cells, and HUVEC: 14 ⁇ 10 4 cells. On the other hand, an equivalent amount of Matrigel was added to DMEM which had been cooled in advance.
  • a pipette tip wet with well-chilled PBS was used to prevent the matrigel from solidifying.
  • the DMEM / Matrigel mixture was mixed well and placed on ice.
  • the 48-well plate was then wetted with 200 ⁇ L / well of well-chilled PBS, the PBS was removed from the well, and then the DMEM / Matrigel mixed solution was applied at 160 ⁇ L / well.
  • the bubbles were crushed with the tip of a chip or the like, and after confirming that the liquid level was flat, they were incubated at 37 ° C. for 1 hour in a CO 2 incubator.
  • the mixed solution of the three types of cells prepared by the above-mentioned dispensing was mixed well, centrifuged at 600 g for 5 minutes at room temperature, and the supernatant was removed as much as possible using Pasteur with a 10 ⁇ L tip. Then, one falcon tube containing the cell mixture was applied to each well of the 48 wells that had been incubated. At this time, care was taken not to allow bubbles to enter or crack. A 48-well plate to which the cell mixture was applied was incubated in a CO 2 incubator at 37 ° C. for 30 minutes. During the incubation waiting time, DMEM / EGM mixed solution was prepared by mixing DMEM and EGM in equivalent amounts.
  • pancreatic cancer organoid was prepared for each well of the 48-well plate.
  • 1-3 Preparation of human pancreatic cancer mouse model In Section 1-2, the pancreatic cancer organoid prepared the day before was observed under a microscope to confirm whether it was contracted or not cracked and whether it had a usable shape.
  • a Matrigel / DMEM mixed solution was prepared and dispensed into 1.5 mL tube at 50 ⁇ L / tube. The same number as the number of pancreatic cancer organoids was prepared and placed on ice.
  • a 6-week-old nude mouse (BALB / cSlc-nu / nu) was purchased from Nippon SLC Co., Ltd. and handled according to the animal management and use guidelines in the Kochi University Research Institute. The skin on the flank of the anesthetized nude mouse was incised about 5-8 mm, and the skin was bluntly peeled off with hemostatic forceps to make a pocket large enough to contain a human pancreatic carcinoma organoid.
  • the 1000 ⁇ L chip was wetted with cold PBS and the medium and gel were removed from the 48 well plate in which the pancreatic cancer organoids were cultured.
  • the 200 ⁇ L chip which had been pre-cut and sterilized, was then wetted with cold PBS, the pancreatic cancer organoids were aspirated, and placed in the tube containing the DMEM / Matrigel mixture solution prepared first.
  • the entire mixed solution containing the pancreatic cancer organoid was applied to the subcutaneous pocket on the flank of the prepared nude mouse and sutured. In this way, a human pancreatic cancer mouse model was prepared. 1-4.
  • TS-1 a human pancreatic cancer organoid derived from the human pancreatic cancer cell line S2-013 was implanted subcutaneously in the flank of a nude mouse.
  • the human pancreatic cancer mouse model was divided into two groups, and from the week following the transplantation, TS-1 (10 mg / kg), which is a standard chemotherapeutic agent for pancreatic cancer, was orally administered to 6 animals at a frequency of 5 days / week. After administration of the drug for 4 weeks, the drug was withdrawn for 2 weeks, and the drug was administered again for 2 weeks. In addition, as a control group, 6 animals were observed without administration of the drug.
  • the tumor diameter of the pancreatic cancer tissue was measured and photographed every week from 2 weeks after the transplantation.
  • a photograph of each mouse 8 weeks after transplantation is shown in FIG. 14, and a change over time in tumor diameter of each group of mice is shown in FIG.
  • “*” indicates that there is a significant difference in p ⁇ 0.05 with respect to the control group in the t-test.
  • the table shows the histopathological findings of the resected human pancreatic cancer. 1-5.
  • Measurement of Pancreatic Cancer Diagnostic Markers in Serum Collected from Human Pancreatic Cancer Mouse Model As described in Section 1-3, human pancreatic cancer organoids derived from human pancreatic cancer cell line S2-013 were implanted subcutaneously in the flanks of nude mice.
  • the human pancreatic cancer mouse model was divided into two groups, and in the five human pancreatic cancer mouse models in the first group, pancreatic cancer organoids having an average tumor diameter of 5 mm were collected, and whole blood was collected 4 weeks after transplantation. In the 5 human pancreatic cancer mouse models in the second group, pancreatic cancer organoids having an average tumor diameter of 20 mm were collected from whole blood 8 weeks after transplantation. Whole blood was collected from 5 nude mice that had not been transplanted with human pancreatic cancer organoids as a control group. Serum was separated from all blood and stored frozen. All serum CA19-9 concentrations were then measured using a commercially available ELISA kit (EIA-5069, DRG). 2. Result 2-1.
  • pancreatic cancer organoid was prepared using the human pancreatic cancer cell line S2-013 and transplanted subcutaneously into the mouse. As shown in FIGS. 1, 2 and 3, pancreatic cancer organoids were subcutaneously transplanted into mice, followed up for 6 weeks, and tumor tissue was removed. Tumor tissue grew over time. A histopathological examination was performed by preparing a histological specimen of the excised tumor tissue. The most characteristic feature of human pancreatic cancer tissue is abundant cancer stroma. Pancreatic cancer cells move around actively in the cancer stroma and invade surrounding tissues. As shown in FIG.
  • the "conventional Xenograft-carrying mouse” was an ectopic transplantation model in which a suspension of S2-013 cells was injected subcutaneously into the flank of a nude mouse to form a tumor.
  • Conventional Xenograft-carrying mice 6-week-old nude mice (BALB / cSlc-nu / nu) were used. Tumors were formed subcutaneously by subcutaneously injecting 2.0 ⁇ 10 6 S2-013 cells suspended in PBS into the flank of the anesthetized nude mouse. As shown in FIG.
  • EMT epithelial-mesenchymal transition
  • Papillary structures with stromal stalks were also seen in some areas (photo (B)).
  • FIG. 9 in the tumor tissue excised from the conventional Xenograft-carrying mouse, the differentiation of the pancreatic cancer tissue was uniform throughout, and no variation in differentiation was observed for each site. There were no EMT images or papillary structures with stromal stalks.
  • the tumor cells were CK19 (+) and vimentin (-), which were similar to those of pancreatic cancer organoid tumors. However, Vimentin expression was generally stronger than pancreatic organoid tumors. The most distinctive feature was that there was no variation in differentiation and it was almost uniform, and it was separated from the clinical pancreatic cancer tissue, which was rich in variation in differentiation and cancer stroma.
  • pancreatic cancer cells with abundant cancer stroma formed a ductal structure and infiltrated into the adipose tissue in the pancreas.
  • the tumor tissue removed from the human pancreatic cancer mouse model was similar to clinical pancreatic cancer, and the human pancreatic cancer mouse model was a model mouse that did not dissociate from clinical pancreatic cancer.
  • FIG. 11 a papillary structure was observed in the pancreatic cancer tissue surgically removed from the pancreatic cancer patient.
  • papillary structures were also observed in the tumor tissue excised from the human pancreatic cancer mouse model.
  • FIG. 10 papillary structures were also observed in the tumor tissue excised from the human pancreatic cancer mouse model.
  • pancreatic cancer tissue surgically removed from a pancreatic cancer patient.
  • lymph node metastasis was also observed in the tumor tissue excised from the human pancreatic cancer mouse model.
  • EMT was occasionally found in pancreatic cancer tissue surgically removed from a pancreatic cancer patient.
  • EMT was also observed in the tumor tissue excised from the human pancreatic cancer mouse model.
  • clinical findings of pancreatic cancer are (1) infiltration of pancreatic cancer cells with abundant cancer stroma, and (2) adenocarcinoma property.
  • FIGS. 14 and 15 As shown in FIGS. 14 and 15, the volume of pancreatic cancer tumor formed subcutaneously in the human pancreatic cancer mouse model group to which TS-1 was administered for 8 weeks was significantly suppressed from 7 weeks after transplantation as compared with the control group. rice field.
  • pancreatic cancer mouse model can solve the divergence from the clinical pancreatic cancer tissue, which has been a problem in the conventional Xenograft model, and can provide accurate information when evaluating the efficacy of a new pancreatic cancer drug.
  • pancreatic Cancer Diagnostic Marker The serum concentration of pancreatic cancer tumor marker CA19-9 was measured in serum collected from the human pancreatic cancer mouse model prepared as described above. Blood was collected from a human pancreatic cancer mouse model at 4, 8 and 10 weeks after transplantation, and the serum concentration of a pancreatic cancer diagnostic marker was measured. As shown in FIG. 16, the CA19-9 concentration in serum collected from a mouse model of human pancreatic cancer increased with time.
  • pancreatic Cancer Diagnostic Marker in Serum Collected from Conventional Xenograft-Supported Mice
  • the serum concentration of pancreatic cancer tumor marker CA19-9 was measured in serum collected from conventional Xenograft-bearing mice. Blood was collected from the conventional Xenograft-carrying mice 4 and 8 weeks after transplantation, and the serum concentration of the pancreatic cancer diagnostic marker was measured. As shown in FIG. 19, the CA19-9 concentration in the serum collected from the conventional Xenograft-carrying mice did not show an increase with time. Bleeding was observed from the tumor, but the concentration of CA19-9 leaked from the tumor tissue into the blood decreased over time, even though the tumor had grown over time. 3. 3.
  • the human pancreatic cancer tissue of the human pancreatic cancer mouse model carrying the pancreatic cancer organoid was very similar in tissue structure to the clinical pancreatic cancer tissue, and the cancer stroma derived from human pancreatic cancer was abundantly present.
  • TS-1 which is a first-line drug for postoperative treatment of pancreatic cancer
  • TS-1 suppressed the growth of pancreatic cancer tumor, but to the surrounding tissues. It was clarified that the effect of suppressing infiltration was low.
  • pancreatic cancer mouse model carrying pancreatic cancer organoids can solve the divergence from clinical pancreatic cancer tissue, which has been a problem in the conventional model, and provides accurate information when evaluating the efficacy of new pancreatic cancer drugs. can.
  • a mouse model of pancreatic cancer showing the characteristics of human pancreatic cancer is provided.
  • 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.
  • the 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.
  • the mouse model according to the present invention has the following advantages: (1) High throughput is supported by using a commercially available human pancreatic cancer cell line; (2) A mouse model can be supplied 6 weeks after the start of 3 types of cell culture; (3) Since a available human pancreatic cancer cell line is used, no pancreatic cancer tissue derived from a surgically resected patient is required; (4) As described in detail above, 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 very similar in tissue structure to the clinical pancreatic cancer tissue, has abundant cancer stroma, and shows a high serum CA19-9 value; (6) Although there are no clinical data or gene expression profiles of patients, tissue sections of human pancreatic cancer organoid tumors can be sold; (7) If a gene expression profile is required, tissue sections of human pancreatic cancer organoid tumors can be prepared; (8) This model is useful for evaluating the efficacy of new therapeutic agents for pancreatic cancer in non-

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Abstract

L'objectif de la présente invention est de produire un modèle de souris pour le cancer du pancréas similaire au cancer du pancréas humain. Plus particulièrement, la présente invention concerne un modèle de souris pour le cancer du pancréas humain qui porte un organoïde du cancer du pancréas contenant une lignée cellulaire du cancer du pancréas humain S2-130.
PCT/JP2021/017608 2020-04-28 2021-04-27 Établissement d'un modèle de souris à l'aide d'un organoïde du cancer du pancréas humain Ceased WO2021221179A1 (fr)

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WO2024034559A1 (fr) * 2022-08-08 2024-02-15 株式会社ヘリオス Procédé de production d'agrégats cellulaires

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WO2009088022A1 (fr) * 2008-01-07 2009-07-16 Kagoshima University Nouveau marqueur de cancer et diagnostic l'utilisant
JP2012526148A (ja) * 2009-05-07 2012-10-25 リージェンツ オブ ザ ユニバーシティ オブ ミネソタ トリプトリド製品
WO2013172105A1 (fr) * 2012-05-18 2013-11-21 日東紡績株式会社 Marqueur de détection du cancer du pancréas
WO2016002844A1 (fr) * 2014-07-04 2016-01-07 国立大学法人高知大学 Inhibiteur d'infiltration cellulaire et de métastase du cancer du pancréas
JP2019516384A (ja) * 2016-05-25 2019-06-20 ソーク インスティチュート フォー バイオロジカル スタディーズ オルガノイド作製および疾患モデル化のための組成物および方法
WO2019203255A1 (fr) * 2018-04-19 2019-10-24 公立大学法人横浜市立大学 Procédé d'évaluation de médicament à l'aide d'un tissu cancéreux reconstruit
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JP2012526148A (ja) * 2009-05-07 2012-10-25 リージェンツ オブ ザ ユニバーシティ オブ ミネソタ トリプトリド製品
WO2013172105A1 (fr) * 2012-05-18 2013-11-21 日東紡績株式会社 Marqueur de détection du cancer du pancréas
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JP2019516384A (ja) * 2016-05-25 2019-06-20 ソーク インスティチュート フォー バイオロジカル スタディーズ オルガノイド作製および疾患モデル化のための組成物および方法
WO2019203255A1 (fr) * 2018-04-19 2019-10-24 公立大学法人横浜市立大学 Procédé d'évaluation de médicament à l'aide d'un tissu cancéreux reconstruit
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024034559A1 (fr) * 2022-08-08 2024-02-15 株式会社ヘリオス Procédé de production d'agrégats cellulaires

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