WO2021221179A1

WO2021221179A1 - Establishment of mouse model using human pancreatic cancer organoid

Info

Publication number: WO2021221179A1
Application number: PCT/JP2021/017608
Authority: WO
Inventors: 恵介谷内; 英樹谷口
Original assignee: 国立大学法人高知大学; 公立大学法人横浜市立大学
Priority date: 2020-04-28
Filing date: 2021-04-27
Publication date: 2021-11-04
Also published as: JPWO2021221179A1

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

Establishment of mouse model using human pancreatic cancer organoid

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

There is an urgent need to develop new treatments for intractable cancers such as pancreatic cancer. Conventionally, in the development of a new therapeutic method, a search for a therapeutic drug using a cancer mouse model has been carried out. In addition, 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.
However, for 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.
In addition, xenografts (PDX) 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. However, in 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. In addition, 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. Therefore, PDX lacks high throughput and is not sufficiently versatile in drug development and the like.
On the other hand, 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. However, 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.

Japanese Unexamined Patent Publication No. 2018-110575

In view of the above circumstances, it is an object of the present invention to provide a mouse model of pancreatic cancer similar to human pancreatic cancer.
As a result of diligent research to solve the above problems, by transplanting a pancreatic cancer organoid prepared using the human pancreatic cancer cell line S2-013 into a mouse, a tumor having a tissue structure similar to that of human pancreatic cancer tissue can be obtained in the mouse. It was found that it was formed, and the present invention was completed.
That is, 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).
(3) A method for evaluating the efficacy of a pancreatic cancer therapeutic agent using the human pancreatic cancer mouse model described in (1).
(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 described in (1).
(5) A method for screening a pancreatic cancer diagnostic marker using a serum sample collected from the human pancreatic cancer mouse model described in (1).
This specification includes the disclosure content of Japanese Patent Application No. 2020-078771, which is the basis of the priority of the present application.

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). According to 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. A time-lapse photograph of human pancreatic cancer formed by transplanting a human pancreatic cancer organoid (Orga-5 (org5)) subcutaneously to the flank of a nude mouse (tumor No. 3) on Day 1 in Examples and dissection 6 weeks after transplantation. It is a photograph of the tumor tissue of the mouse model at the time. A time-lapse photograph of human pancreatic cancer formed by subcutaneously transplanting a human pancreatic cancer organoid (Orga-8 (org8)) to Day 1 of an example of a nude mouse (tumor No. 4) and dissecting it 6 weeks after transplantation. It is a photograph of the tumor tissue of the mouse model. Hematoxylin and eosin of (A) tumor tissue removed from a human pancreatic cancer mouse model (mouse No. 4) carrying a pancreatic cancer organoid (Orga-8) in Examples and (B) tumor tissue removed from a mouse carrying a conventional Xenograft. It is a dyed photograph. Hematoxylin and eosin of (A) tumor tissue removed from a human pancreatic cancer mouse model (mouse No. 4) carrying a pancreatic cancer organoid (Orga-8) in Examples and (B) tumor tissue removed from a mouse carrying a conventional Xenograft. It is a dyed photograph. Hematoxylin and eosin of (A) tumor tissue removed from a human pancreatic cancer mouse model (mouse No. 3) carrying a pancreatic cancer organoid (Orga-5) in Examples and (B) tumor tissue removed from a mouse carrying a conventional Xenograft. It is a dyed photograph. It is a hematoxylin-eosin-stained photograph of a tumor tissue excised from a human pancreatic cancer mouse model (mouse No. 5) carrying a pancreatic cancer organoid (Orga-9 (org9)) in an example. (A and B) hematoxylin / eosin staining, (C) immunohistochemical staining with anti-CK19 antibody, and (C) immunohistochemical staining of tumor tissue excised from a human pancreatic cancer mouse model (mouse No. 5) carrying a pancreatic cancer organoid (Orga-9) in Examples. (D) It is a photograph of immunohistochemical staining with an anti-vimentin antibody. Photographs of (A) hematoxylin and eosin staining, (B) immunohistochemical staining with anti-CK19 antibody, and (C) immunohistochemical staining with anti-vimentin antibody in tumor tissue removed from conventional Xenograft-carrying mice in Examples. be. It is a photograph showing the dissociation from the clinical pancreatic cancer tissue. It is a photograph of the pancreatic cancer tumor tissue surgically removed from the pancreatic cancer patient in the example, stained with hematoxylin and eosin. It shows an image of infiltration into adipose tissue accompanied by a stromal reaction. It is a photograph of the pancreatic cancer tumor tissue surgically removed from the pancreatic cancer patient in the example, stained with hematoxylin and eosin. Upper right: papillary structure with stromal stalk, lower left: micropapillary structure without stromal stalk. It is a photograph of the pancreatic cancer tumor tissue surgically removed from the pancreatic cancer patient in the example, stained with hematoxylin and eosin. Shows lymph node metastasis. It is a photograph of (A) hematoxylin / eosin staining, (B) immunohistochemical staining with anti-CK19 cytokeratin antibody, and (C) immunohistochemical staining with anti-vimentin antibody of pancreatic cancer tumor tissue surgically removed from a pancreatic cancer patient in Examples. .. A region of poorly differentiated, spindle-shaped cells was found in the tumor. Here, the stainability of cytokeratin (photograph of (B)) decreased, and vimentin (photograph of (C)) became positive. EMT findings. It is a photograph which shows the observation of the antitumor effect by administration of TS-1 to the human pancreatic cancer mouse model in an Example. It is the graph (A) and the table (B) which show the antitumor effect by administration of TS-1 to the human pancreatic cancer mouse model in an Example. 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. It is a graph which shows the antitumor effect by administration of TS-1 to the mouse which carried the conventional Xenograft in an Example. It is a figure which shows the measurement result of the pancreatic cancer diagnostic marker CA19-9 in the time-dependent tumor volume of the mouse which carried (A) the conventional Xenograft in the Example, and (B) the serum collected from the mouse which carried the conventional Xenograft.

Hereinafter, the present invention will be described in detail.
The human pancreatic cancer mouse model according to the present invention (hereinafter referred to as “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). It is a useful mouse model that has a tumor and can be used in an experiment for evaluating the efficacy of a drug such as a pancreatic cancer therapeutic agent and an experiment for evaluating the usefulness of a pancreatic cancer diagnostic marker.
In the present invention, the human pancreatic cancer cell line S2-013 was used, and the conditions for producing the organoid thereof were examined in detail, thereby succeeding in homogenizing the pancreatic cancer cell proliferation speed (= increase in tumor volume) among individuals. Made it possible to use a mouse model of human pancreatic cancer with the following characteristics:
(1) Tumors of uniform size can be formed with a success rate of almost 100%. It supports high throughput. Since the variation in tumor volume is extremely small, accurate drug efficacy evaluation is possible (a model with stable tumor volume was established for the first time among organoid transplantation models, which had the problem of inconsistent tumor volume);
(2) Since the tumor does not bleed, the tumor diameter can be measured accurately;
(3) By using serum CA19-9 as a marker for drug efficacy evaluation, the drug efficacy evaluation can be enhanced.
1. 1. Preparation of mouse model according to the present invention 1-1. Production of Pancreatic Cancer Organoid The pancreatic cancer organoid in the present invention can be produced 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 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.
In many cases, cancer tissue has a part called the stroma in addition to the cancer cells. In 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. There are many types of cells, connective tissue composed of collagen and the like existing between these cells, and they form a characteristic structure. This is called the "cancer microenvironment".
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. 9 (2): 396-409, 2014, Cell Stem Cell, 7; 16 (5): 556-65, 2015 and the like.
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.
In the present invention, the "vascular endothelial cell" refers to a cell constituting the vascular endothelium or a cell capable of differentiating into such a cell. Whether or not 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. Among the terms used among those skilled in the art, endothelial cells, umbilical vein endothelial cells, endothelial progenitor cells, endothelial progenitor cells, endothelial progenitor cells, endothelium precursor cells, vasculogenic project. −104. (2011)) and the like 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. Although human-derived vascular endothelial cells are mainly used, animals used for animals other than humans (for example, laboratory animals, pet animals, service animals, race horses, fighting dogs, etc., specifically, mice, rats, etc. Vascular endothelial cells derived from rabbits, pigs, dogs, monkeys, cows, horses, sheep, chickens, sharks, rays, ginseng, salmon, shrimp, crabs, etc.) may be used.
In the present invention, 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. However, it is a concept that includes cells that have not yet differentiated into mesenchymal cells. 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. 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)) and the like are of the present invention. Included in mesenchymal cells in. Preferred mesenchymal cells are 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). It can be prepared from stem cells according to a known method. Although 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. = 10: 1 to 100: 1 to 100, and more preferably, human pancreatic cancer cell line S2-013: vascular endothelial cell: mesenchymal cell = 10: 1 to 100: 5 to 100. 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. Can be formed.
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. 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. As a medium for culturing vascular endothelial cells, 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 (Ther) , Human microvascular endothelial cell growth medium (manufactured by TOYOBO) and the like can be used. Any medium may be used for culturing cancer cells, and examples thereof include DMEM medium. In particular, a medium of EGM: DMEM = 1: 1 is suitable for producing pancreatic cancer organoids.
It is not necessary to use a scaffolding material for culturing cells, but it is preferable to cultivate a mixture of three types of cells on a gel-like support in which mesenchymal cells can contract.
The contraction of mesenchymal cells indicates that morphologically three-dimensional tissue formation is observed (microscopically or with the naked eye), and that the tissue shape is maintained by recovery with a spatula or the like (Takebe). It can be confirmed as in et al. Nature 499 (7459), 481-484, 2013)).
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). 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. It should be noted that 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. When the hardness of the support is uniform, 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. Further, 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.
It is prepared as described above, and it is confirmed by stereomicroscopic observation that a circular pancreatic cancer organoid has been formed, and the circular pancreatic cancer organoid is subcutaneously transplanted into a mouse. 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 mice and 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 elucidation of the onset mechanism or pathophysiology of human pancreatic cancer, screening of pancreatic cancer therapeutic agents (anticancer agents), evaluation of drug efficacy of pancreatic cancer therapeutic agents, screening of pancreatic cancer diagnostic markers, and evaluation of usefulness.
For example, in the screening of a pancreatic cancer therapeutic agent, 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. 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, 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.
Moreover, in the serum collected from the mouse model according to the present invention, the pancreatic cancer diagnostic marker behaves similarly to clinical pancreatic cancer. Therefore, 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.
For example, in order to evaluate 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 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) is the same as 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 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).
Similarly, in the screening of 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, and the screening is performed at the protein level. Measure pancreatic cancer diagnostic marker candidates. An organism such as serum derived from a mouse that has the same measured pancreatic cancer diagnostic marker candidate level (concentration in a biological sample such as serum) as a negative control (for example, does not carry a pancreatic cancer organoid) and is the same as the mouse model according to the present invention. The 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).
Here, the immunological measurement method is not particularly limited, and examples thereof include ELISA, flow cytometry, and Western blotting.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these Examples.
[Preparation of mouse model of human pancreatic cancer and its evaluation]
1. 1. Materials and methods 1-1. Cells The cells used are human pancreatic cancer cell line S2-013 (hereinafter, may be referred to as "S2-013"), human umbilical vein endothelial cell HUVEC (LONZA) (hereinafter, may be referred to as "HUVEC") and It was a human mesenchymal stem cell MSC (LONZA) (hereinafter, may be referred to as "MSC").
S2-013 was cultured in DMEM medium. HUVEC was cultured in EGM-2 / HUVEC medium. MSCs were cultured in MSCGM medium.
1-2. Preparation of Pancreatic Cancer Organoids S2-013, MSC and HUVEC in culture were collected one by one using trypsin. S2-013 was stripped with 0.5% trypsin (SIGMA) and MSC and HUVEC were stripped with 0.05% trypsin (sigco). The time of trypsin treatment was adjusted according to the state of each cell. Then, trypsin was neutralized in DMEM containing FCS, and the culture solution was withdrawn as much as possible after centrifugation. 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 μL 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 μL / well of well-chilled PBS, the PBS was removed from the well, and then the DMEM / Matrigel mixed solution was applied at 160 μL / well. At that time, 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 μL 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. After completion of the incubation, 300 μL / well of the DMEM / EGM mixed solution was applied to confirm that there were no bubbles between the DMEM / EGM mixture and the matrigel, and then the plate was _{put into the CO 2} incubator again and incubated at 37 ° C. for 24 hours.
In this way, one 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 μL / 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 μL 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 μL 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. Administration of TS-1 to a Human Pancreatic Cancer Mouse Model As described in Section 1-3, 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. In FIG. 15, “*” 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. Histopathological examination of tumor tissue excised from a human pancreatic cancer mouse model As described above, a 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. 4, in the tumor tissue excised from the human pancreatic cancer mouse model carrying the pancreatic cancer organoid prepared as described above, cancer stroma derived from the human pancreatic cancer organoid is abundantly present, and the glandular structure and micropapillary shape are present. The parts exhibiting the structure were mixed. An infiltration image of pancreatic cancer cells with cancer stroma was observed. On the other hand, in the tumor tissue excised from the conventional Xenograft-carrying mouse, most of the tumor tissue was solid, and the glandular cavity structure and cancer stroma were poor, so that it could not be said to be an adenocarcinoma tissue.
In this example, 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. 5, in the tumor tissue excised from the human pancreatic cancer mouse model carrying the pancreatic cancer organoid prepared as described above, the glandular cavity (left photograph of (A)) and the micropapillary structure ((A)) From the photo on the right), it was clear that it was adenocarcinoma. On the other hand, the tumor tissue excised from the conventional Xenograft-carrying mouse had almost no ductal structure or papillary structure, and was poorly differentiated into adenocarcinoma (photo of (B)).
As shown in FIG. 6, in the tumor tissue excised from the human pancreatic cancer mouse model carrying the pancreatic cancer organoid prepared as described above, muscular layer and subcutaneous cell infiltration with abundant cancer stroma were observed (((() A) Photo). On the other hand, in the tumor tissue excised from the conventional Xenograft-carrying mouse, cell infiltration into the muscular layer was observed, but the cancer stroma was poor (photo of (B)).
As shown in FIG. 7, in the tumor tissue excised from the human pancreatic cancer mouse model carrying the pancreatic cancer organoid prepared as described above, vascular invasion in the vicinity of the tumor (left in the figure) and metastasis to the subcutaneous lymph node (right in the figure). Was admitted. On the other hand, no vascular invasion or lymph node metastasis was observed in the tumor tissue removed from the conventional Xenograft-carrying mouse.
As shown in FIG. 8, epithelial-mesenchymal transition (EMT) was observed in the tumor tissue removed from the human pancreatic cancer mouse model carrying the pancreatic cancer organoid prepared as described above (Photo (A): dotted frame. Inside). The stainability of CK19, which is often expressed in pancreatic cancer, was also low in the same region (photo (C)), and conversely vimentin was expressed (photo (D)). Tumor cells are CK19 (+) and vimentin (-). EMT is involved in infiltration and metastasis. Papillary structures with stromal stalks were also seen in some areas (photo (B)).
As shown in 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.
As shown in FIG. 10, in pancreatic cancer tissue surgically removed from a pancreatic cancer patient, pancreatic cancer cells with abundant cancer stroma formed a ductal structure and infiltrated into the adipose tissue in the pancreas. As shown in FIG. 4, 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.
As shown in FIG. 11, a papillary structure was observed in the pancreatic cancer tissue surgically removed from the pancreatic cancer patient. As shown in FIG. 5, papillary structures were also observed in the tumor tissue excised from the human pancreatic cancer mouse model.
As shown in FIG. 12, intraperitoneal lymph node metastasis was occasionally found in 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 excised from the human pancreatic cancer mouse model.
As shown in FIG. 13, EMT was occasionally found in pancreatic cancer tissue surgically removed from a pancreatic cancer patient. As shown in FIG. 8, EMT was also observed in the tumor tissue excised from the human pancreatic cancer mouse model. On the other hand, in the tumor tissue excised from the conventional Xenograft-carrying mouse, clinical findings of pancreatic cancer are (1) infiltration of pancreatic cancer cells with abundant cancer stroma, and (2) adenocarcinoma property. No findings of structure / papillary structure, (3) lymphovascular invasion, lymph node metastasis, or (4) EMT were observed.
2-2. Observation of antitumor effect by administration of TS-1 to human pancreatic cancer mouse model TS-1 which is the first-line drug for postoperative pancreatic cancer is administered to the human pancreatic cancer mouse model prepared as described above to obtain antitumor effect. Observed. After administration to human pancreatic cancer model mice, the tumor diameter was measured over time and histopathological examination was performed. As for the method of administering TS-1 to a human pancreatic cancer mouse model, 10 mg / kg of TS-1 per mouse body weight was administered 5 days a week for 4 weeks, with a 2-week rest period as one course, and administration 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 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.
Eight weeks after the subcutaneous transplantation of human pancreatic carcinoma organoids, each mouse was dissected, the tumor tissue formed on the flank of the mouse was formalin-fixed, and hematoxylin-eosin staining was performed. The results of histopathological examination are shown in Table (B) of FIG. As shown in Table (B) of FIG. 15, TS-1 did not show any difference from the control group in the effect of suppressing vascular invasion and muscular layer infiltration. In the human pancreatic cancer mouse model group to which TS-1 was orally administered, cyst formation due to necrosis was remarkable as compared with the control group. These results were consistent with the drug efficacy obtained in clinical practice in which TS-1 suppresses tumor growth by suppressing cell proliferation. It was shown that the human 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.
2-3. Measurement of Pancreatic Cancer Diagnostic Marker in Serum Collected from Human Pancreatic Cancer Mouse Model 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. CA19-9 leaked from the tumor tissue into the blood could be detected, and the concentration increased as the tumor grew.
As described above, in the research aiming at the identification of a novel pancreatic cancer diagnostic marker, by measuring the serum concentration of the human pancreatic cancer mouse model over time, it is possible to provide information useful for evaluation of being a promising diagnostic marker.
2-4. Observation of antitumor effect by administration of TS-1 to mice carrying conventional Xenograft TS-1 was administered to mice carrying conventional Xenograft for 8 weeks and the antitumor effect was observed. After administration to mice carrying Xenograft, the tumor diameter was measured over time.
As shown in FIG. 17, the volume of the pancreatic cancer tumor formed under the skin of the mouse group carrying Xenograft to which TS-1 was administered was not suppressed as compared with the control group. In addition, bleeding and digging from the subcutaneous tumor tissue seemed to be unsuitable as a model for observing tumor growth. On the other hand, as shown in FIG. 14, no bleeding was observed in the pancreatic cancer tumor formed under the skin of the human pancreatic cancer mouse model group.
As shown in FIG. 18, the effect of suppressing the tumor growth of TS-1 could not be detected in the conventional Xenograft-carrying mice.
2-5. Measurement of 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. Summary 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.
In addition, when TS-1, which is a first-line drug for postoperative treatment of pancreatic cancer, was administered to a human pancreatic cancer mouse model carrying a pancreatic cancer organoid, 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. It was possible to show the rationale that TS-1 could not sufficiently suppress the "strong infiltration with cancer stroma" which is a characteristic of pancreatic cancer, and the drug efficacy was limited.
Furthermore, the human 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.

According to the present invention, a mouse model of pancreatic cancer showing the characteristics of human pancreatic cancer is provided. In addition, 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 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.
In addition, 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-clinical pharmacology studies.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

A human pancreatic cancer mouse model carrying a pancreatic cancer organoid containing the human pancreatic cancer cell line S2-013.
A method for screening a pancreatic cancer therapeutic agent using the human pancreatic cancer mouse model according to claim 1.
A method for evaluating the efficacy of a pancreatic cancer therapeutic agent using the human pancreatic cancer mouse model according to claim 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 according to claim 1.
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.