Mice as a model for microsatellite stable and unstable intestinal cancer research
By using the F1 hybrid mice of C57BL/6×BALB/c and representative colorectal cancer cells CT26 and MC38, MSS and MSI-H type colorectal cancer mouse models with the same genetic background were constructed, which solved the problem of experimental result bias caused by differences in genetic background and achieved more scientific research conditions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG CANCER HOSPITAL
- Filing Date
- 2023-04-27
- Publication Date
- 2026-07-10
Smart Images
Figure CN118844384B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of experimental animals and disease models, and specifically relates to mouse models for the study of microsatellite stable and unstable colorectal cancer. Background Technology
[0002] Colorectal cancer is a common malignant tumor of the digestive tract. According to the 2020 global cancer statistics, colorectal cancer ranks third in incidence (10.0%) and second in mortality (9.4%). Surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy are currently the main treatments for colorectal cancer. Although comprehensive treatment, primarily surgery, is constantly improving, and early screening is being promoted and implemented, the implementation and strategies of colorectal cancer screening vary. Early symptoms are often not obvious, and the cancer is highly invasive and prone to metastasis, leading to most patients being diagnosed at an advanced stage, requiring comprehensive systemic treatment.
[0003] Tumor immunotherapy utilizes the principle of the body's immune system to specifically recognize and kill tumor cells in order to treat tumors. Currently popular tumor immunotherapies mainly include immune checkpoint blockade (ICB) targeting programmed cell death receptor 1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic T lymphocyte associated antigen 4 (CTLA-4). ICB has been leading the treatment of various tumor types with great success, such as melanoma and non-small cell lung cancer.
[0004] In colorectal cancer, PD-1 blockade has been approved for the treatment of tumors with microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) mutations. Clinically, colorectal cancer can be classified into MSI-H / dMMR type colorectal cancer (“hot tumors”) and MSS / pMMR type colorectal cancer (“cold tumors”) based on the status of microsatellite instability or mismatch repair genes.
[0005] Currently, although some MSS and MSI-H colorectal cancer animal models have been constructed, the differences between MSI-H / dMMR and MSS / pMMR colorectal cancers make it impossible to successfully construct corresponding MSS and MSI-H colorectal cancer animal models in non-human models with the same genetic background. Therefore, it is necessary to construct MSS and MSI-H colorectal cancer animal models separately in non-human models with different genetic backgrounds (e.g., different strains of mice). Since different genetic backgrounds can lead to various unpredictable effects, resulting in different influences on experimental results, this can cause certain biases in the experimental findings.
[0006] Therefore, there is an urgent need in this field to develop mouse models of MSS-type and MSI-H-type colorectal cancer based on hybrid mice with the same genetic background, so as to provide a more scientific mouse model for the study of MSS-type and MSI-H-type colorectal cancer. Summary of the Invention
[0007] The purpose of this invention is to provide a mouse model of MSS-type and MSI-H-type colorectal cancer based on a hybrid mouse line with the same genetic background, and its application.
[0008] In a first aspect of the invention, there is provided the use of a hybrid mouse line for preparing a reagent or kit for constructing mouse models of MSS-type and MSI-H-type colorectal cancer, wherein the hybrid mouse line is a F1 generation of C57BL / 6×BALB / c.
[0009] In another preferred embodiment, the hybrid mouse line is a F1 generation of C57BL / 6♀×BALB / c♂.
[0010] In another preferred embodiment, the hybrid mouse line is a F1 generation of C57BL / 6♂×BALB / c♀.
[0011] In another preferred embodiment, the reagent further includes MSS type colorectal cancer cells, MSI-H type colorectal cancer cells, or a combination thereof.
[0012] In another preferred embodiment, the reagent further includes an instruction manual that describes how to use the reagent.
[0013] In another preferred embodiment, the concentration of the MSS-type colon cancer cells is 1 × 10⁻⁶. 4 -5×10 5 / 100 μL of cell suspension.
[0014] In another preferred embodiment, the concentration of the MSI-H type colon cancer cells is 1 × 10⁻⁶. 4 -5×10 5 / 100 μL of cell suspension.
[0015] In another preferred embodiment, the MSS-type colon cancer cells are CT26 cells.
[0016] In another preferred embodiment, the MSI-H type colon cancer cells are MC38 cells.
[0017] In a second aspect of the invention, a kit is provided for constructing mouse models of MSS-type and MSI-H-type colorectal cancer, the kit comprising a F1 generation of C57BL / 6×BALB / c and MSS-type and MSI-H-type colorectal cancer cells.
[0018] In another preferred embodiment, the MSS-type colon cancer cells are CT26 cells.
[0019] In another preferred embodiment, the MSI-H type colon cancer cells are MC38 cells.
[0020] In a third aspect of the invention, there is a use for a combination of MSS-type colon cancer cells and MSI-H-type colon cancer cells, wherein the MSS-type colon cancer cells are CT26 cells and the MSI-H-type colon cancer cells are MC38 cells, and the combination is used to prepare reagents or kits for constructing mouse models of MSS-type colon cancer and MSI-H-type colon cancer based on hybrid mice with the same genetic background, wherein the hybrid mice are F1 generation of C57BL / 6×BALB / c.
[0021] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description
[0022] Figure 1 Image A shows the F1 generation obtained by crossing BALB / c♀×C57BL / 6♂.
[0023] Figure 1 Image B shows the F1 generation obtained by crossbreeding C57BL / 6♀×BALB / c♂.
[0024] Figure 2 Image A is a CT scan of tumor formation on day 12 after BALB / c♀×C57BL / 6♂F1 generation vaccination.
[0025] Figure 2 Image B shows tumor formation at MC38 on day 12 after BALB / c♀×C57BL / 6♂F1 generation vaccination.
[0026] Figure 2 C is a CT scan image of tumor formation on day 12 after C57BL / 6♀×BALB / c♂F1 generation vaccination.
[0027] Figure 2 Image D shows tumor formation in MC38 on day 12 after C57BL / 6♀×BALB / c♂F1 generation vaccination.
[0028] Figure 3 A represents the body weight growth curve of BALB / c♀×C57BL / 6♂F1 generation subcutaneous tumorigenic mice.
[0029] Figure 3 B is the tumor volume change curve of BALB / c♀×C57BL / 6♂F1 generation subcutaneous tumors.
[0030] Figure 4 Image A is an HE staining image of BALB / c♀×C57BL / 6♂F1 generation CT26 subcutaneous tumor tissue.
[0031] Figure 4 Image B is an HE staining image of BALB / c♀×C57BL / 6♂F1 generation MC38 subcutaneous tumor tissue. Detailed Implementation
[0032] Through extensive and in-depth research and numerous screenings, the inventors unexpectedly discovered a specific hybrid mouse line, the F1 generation of C57BL / 6×BALB / c, which is particularly suitable as a hybrid mouse line with the same genetic background for constructing mouse models of MSS and MSI-H colorectal cancer. The F1 generation of C57BL / 6×BALB / c of this invention can not only be used to construct mouse models of MSS and MSI-H colorectal cancer, but also achieves almost 100% success in preparing model animals at lower inoculation doses, thus providing a more scientific mouse model for the study of MSS and MSI-H colorectal cancer. This invention was completed based on this discovery.
[0033] Specifically, the inventors crossed BALB / c and C57BL / 6 mice to obtain the F1 hybrid mouse line. Representative MSS-type colon cancer cells and representative MSI-H-type colon cancer cells (CT26 and MC38) were then introduced at a certain number (e.g., 1 × 10⁻⁶). 4 One or 2×10 5 When cells (per mouse / site) were subcutaneously inoculated into mice, not only could all of them form tumors in the F1 generation, but the tumor formation rate was almost 100%.
[0034] Terminology Explanation
[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0036] Hybrid mice
[0037] In this invention, the term "hybrid mouse of the present invention" refers to the F1 generation of the C57BL / 6×BALB / c cross.
[0038] Preferably, the hybrid mouse line is the F1 generation of C57BL / 6♀×BALB / c♂.
[0039] Preferably, the hybrid mouse line is the F1 generation of C57BL / 6♂×BALB / c♀.
[0040] Preferably, the BALB / c mouse is an MHC haplotype H-2K. d .
[0041] Preferably, the C57BL / 6 mouse described herein is an MHC haplotype H-2K. b .
[0042] colon cancer cells
[0043] In this invention, the colorectal cancer cells used to construct animal models include: MSI-H / dMMR colorectal cancer cells (“hot tumor type”) and MSS / pMMR colorectal cancer cells (“cold tumor type”).
[0044] In this invention, the colon cancer cells are MSS-type colon cancer cells (or MSS / pMMR-type colon cancer cells), MSI-H-type colon cancer cells (MSI-H / dMMR-type colon cancer cells), or a combination thereof.
[0045] Preferably, the MSS type colon cancer cells include (but are not limited to): CT26 cells.
[0046] Preferably, the MSI-H type colon cancer cells include (but are not limited to): MC38 cells.
[0047] In this invention, MSS-type and MSI-H-type colorectal cancer cells can be generated using existing, commercially available cell lines, or cell lines prepared or isolated using conventional methods. For example, cell lines can be purchased from collections such as ATCC.
[0048] Methods for constructing animal models of MSS-type and MSI-H-type colorectal cancer
[0049] The present invention also provides a method for constructing animal models of MSS-type and MSI-H-type colorectal cancer using the C57BL / 6×BALB / c progeny mice of the present invention.
[0050] Typically, the method of the present invention includes the following steps:
[0051] (1) Cross the BALB / c and C57BL / 6 parents (e.g., BALB / c♀×C57BL / 6♂ or C57BL / 6♀×BALB / c♂) to obtain the F1 hybrid mouse line;
[0052] (2) MSS-type colon cancer cells and MSI-H-type colon cancer cells (such as CT26 and MC38) were subcutaneously inoculated into the F1 hybrid mice to obtain MSS-type colon cancer and MSI-H-type colon cancer model animals.
[0053] Preferably, the seeding concentration of the MSS type colon cancer cells and MSI-H type colon cancer cells is 1×10⁻⁶. 4 -5×10 5 / 100 μL cell suspension, preferably 5 × 10 4 -2×10 5 / 100 μL of cell suspension. The inoculation volume at each inoculation site is 20-100 μL.
[0054] Preferably, both CT26 and MC38 cells are used at a concentration of 1×10⁻⁶. 5 A certain number of cells were inoculated into the hybrid mice of the present invention.
[0055] In this invention, the inoculation is a subcutaneous inoculation.
[0056] Compared with the prior art, the main advantages of the present invention are as follows:
[0057] (1) The mouse model of the present invention provides the same genetic background for MSS type colorectal cancer cells and MSI-H type colorectal cancer cells, making the study of MSS type colorectal cancer and MSI-H type colorectal cancer more scientific.
[0058] (2) In the mouse model of the present invention, intestinal cancer cells can be produced in a smaller number of cells (1×10⁻⁶). 4 A 100 μL cell suspension (inoculated with 100 μL) can induce tumor formation in the hybrid mice of this invention.
[0059] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.
[0060] Material
[0061] CT26 cells: Commonly used MSS type colon cancer cells.
[0062] MC38 cells: Commonly used MSI-H type colon cancer cells.
[0063] Example 1: Crossbreeding of inbred mouse strains C57BL / 6 and BALB / c
[0064] 1.1 Animal husbandry
[0065] Six-week-old female BALB / c and six-week-old male C57BL / 6 were purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd. (SCXK(Zhejiang)2019-0001) and housed at the Laboratory Animal Center of Zhejiang Cancer Hospital (SYXK(Zhejiang)2022-0035).
[0066] 1.2 Mouse hybridization
[0067] BALB / c and C57BL / 6 mice were mated in cages, and pregnancy and parturition were closely observed. The mice were numbered and randomly divided into 6 groups (n=4) according to a numbering table, as follows:
[0068] (1) BALB / c and C57BL / 6 mice were housed in different cages in an SPF environment. After about 5 weeks, BALB / c♀ were put together with C57BL / 6♂, or C57BL / 6♀ were put together with BALB / c♂.
[0069] (2) The F1 generation bred from the cross between parent mice is numbered and marked.
[0070] F1 offspring mice of female BALB / c and male C57BL / 6, such as Figure 1 A and Figure 1 As shown in B, the hybrid is brown.
[0071] The situation was similar for the F1 generation mice of male BALB / c and female C57BL / 6.
[0072] Example 2: Tumorigenesis experiment of CT26 and MC38 cells
[0073] 2.1 Method
[0074] (1) CT26 and MC38 cell culture: The two cell lines were cultured in a cell culture incubator with a concentration of 5% CO2 and a temperature of 37°C. CT26 was cultured in RPMI-1640 medium containing 10% fetal bovine serum, and MC38 was cultured in DMEM high glucose medium containing 10% fetal bovine serum.
[0075] (2) Cell counting: When the cells are in the logarithmic growth stage, digest and collect the cells, and automatically count them using a cell counter. Prepare a cell suspension based on the cell counting results, and suspend the cells in Hanks' solution so that each 100 μL of cell suspension contains approximately 1 × 10⁻⁶ cells. 4 (low dose), 2×10 5 (medium dose), 5×10 5(High dose) cells;
[0076] (3) Subcutaneous inoculation: Shave the hair on the right scapula of the mouse with a shaver, insert a 1 ml syringe obliquely under the skin, move the syringe needle left and right to form a subcutaneous cavity, and inject 100 μL of cell suspension into the cavity.
[0077] (4) Observe tumor formation: Closely observe the tumor formation of mice every day after inoculation. The day of inoculation is recorded as day 0. Starting from day 6, weigh and measure the tumor diameter every 2 to 3 days. Euthanize the mice on day 15 after inoculation.
[0078] (5) HE staining of mouse tumors: The tumors were removed and fixed in 4% paraformaldehyde, and then embedded in paraffin and sectioned for HE staining.
[0079] 2.2 Results
[0080] The tumorigenesis results are shown in Table 1. Tumors formed from low, medium, and high doses of CT26 and MC38 cells. Tumors appeared approximately 11 days after inoculation in the low-dose group, and approximately 6 days after inoculation in the medium and high-dose groups.
[0081] 2×10 5 Tumor formation on day 12 after cell inoculation: Figure 2 A, Figure 2 B Figure 2 C and Figure 2 As shown in D.
[0082] Table 1. Tumor formation at different cell doses
[0083]
[0084] The formula for calculating the volume of the tumor is V = ab. 2 / 2, (a is the major axis, b is the minor axis, in mm).
[0085] F1 generation offspring of tumors C57BL / 6×BALB / c inoculated with the low-dose group can form tumors, but tumor growth is relatively slow. 2×10 5 The cell seeding dose weight gain curve and tumor volume change curve are shown in the figure. Figure 3 A, Figure 3 As shown in B, the tumorigenicity trends of the CT26 and MC38 cell lines are roughly the same. Subcutaneous tumor tissue from the CT26 cell line grows slightly faster than that from the MC38 cell line, which is consistent with the culture trend of the cell lines.
[0086] HE staining results of mouse tumors are as follows Figure 4 A, Figure 4 The results shown in B indicate that the tumor cells in both types of tumor tissues are closely arranged, have diverse cell morphologies, and have large, deeply stained nuclei.
[0087] Example 3: Tumorigenesis experiment of CT26 cells in BALB / c and C57BL / 6 mice.
[0088] The experiment in this embodiment is the same as in embodiment 2, except that MSS-type colon cancer cells CT26 were inoculated into BALB / c mice and C57BL / 6 mice to induce tumor formation.
[0089] Experimental results show that when the quantity is 1×10 4 3×10 4 5×10 4 1×10 5 When CT26 cells were subcutaneously inoculated into BALB / c mice (n=3), only the tumor cells CT26 reached a quantity of 1×10⁻⁶. 5 Only in the subcutaneous group can 100% be achieved. The quantity is 1×10 5 and 4×10 6 CT26 cells were injected subcutaneously into C57BL / 6 mice, but no tumors formed.
[0090] This indicates that when tumor cells match the mouse MHC I phenotype, the number of cells required for tumor formation is much smaller than the number of cells required when the MHC I phenotype does not match. When tumor cells do not match the mouse MHC I phenotype, even an increase in the number of cells cannot achieve a good tumor formation effect.
[0091] discuss
[0092] In colorectal cancer patients, over 95% are pMMR or MSS, and these patients do not benefit from single-agent immune checkpoint inhibitors. Current treatments for MSS-type colorectal cancer mainly include immunotherapy combined with chemotherapy, immunotherapy combined with immunotherapy, and immunotherapy combined with radiotherapy. However, most treatment regimens are still in clinical trials, such as the CheckMate 142 trial and the KEYNOTE-177 trial. The mechanism of resistance to immune checkpoint blockers (ICBs) in MSS-type colorectal cancer is unclear. Lack of tumor mutations and immune cell infiltration are considered among the most likely mechanisms of resistance.
[0093] In in vitro studies, for the two microsatellite stable and unstable colorectal cancer subtypes, CT26 is generally used as the MSS-type colorectal cancer cell model in BALB / c mice, and MC38 is used as the MSI-H-type colorectal cancer cell model in C57BL / 6 mice. The MHC haplotype of BALB / c mice is H-2K. d The MHC haplotype of C57BL / 6 mice is H-2K. bThe construction of in vitro mouse models is based on MHC matching. Comparative analysis on two types of mice with different genetic backgrounds may have adverse or unknown effects on the experimental results due to differences in mouse strains.
[0094] Existing studies have found that in doxorubicin-induced nephropathy animal models, BALB / c mice are sensitive to doxorubicin while C57BL / 6 mice are resistant. Mouse strains with a B6 genetic background cannot be used in doxorubicin nephropathy models. Therefore, research on treatment regimens for MSS and MSI-H colorectal cancer cells with different genetic backgrounds cannot easily exclude genetic background dependence, meaning the influence of the mouse's own genetic background on drug efficacy. Thus, mice with similar genetic backgrounds are essential.
[0095] In existing technologies, whether exploring combined treatment regimens for MSS and MSI subtypes of colorectal cancer or studying immune checkpoint inhibitors, MC38 cells are typically used as an MSI colorectal cancer model and CT26 cells as an MSS colorectal cancer model, respectively inoculated into C57BL / 6 mice and BALB / c mice. However, C57BL / 6 and BALB / c mice have different genetic backgrounds, and using one model as a control may have adverse or unknown effects on the experimental results due to differences in mouse strains.
[0096] The F1 hybrid mouse line C57BL / 6×BALB / c of this invention overcomes the problem of inconsistent genetic background. It can not only be used to construct animal models of MSS-type and MSI-H-type colorectal cancer, but also can successfully prepare animal models with almost 100% success rate at lower inoculation doses, thus providing a more scientific mouse model for the study of MSS-type and MSI-H-type colorectal cancer.
[0097] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.
Claims
1. The use of a hybrid mouse strain, characterized in that, Reagents or kits for preparing mouse models of MSS-type and MSI-H-type colorectal cancer, wherein the hybrid mice are F1 generation of C57BL / 6×BALB / c; The reagents or kits include the F1 hybrid mice of the C57BL / 6×BALB / c line, MSS type colon cancer cells and MSI-H type colon cancer cells; The method for constructing mouse models of MSS-type and MSI-H-type colorectal cancer includes the following steps: inoculating MSS-type and MSI-H-type colorectal cancer cells into the hybrid mice, respectively, to obtain MSS-type and MSI-H-type colorectal cancer models.
2. The use as described in claim 1, characterized in that, The hybrid mouse line is the F1 generation of C57BL / 6♀×BALB / c♂.
3. The use as described in claim 1, characterized in that, The hybrid mouse line is the F1 generation of C57BL / 6♂×BALB / c♀.
4. The use as described in claim 1, characterized in that, The concentration of the MSS-type colon cancer cells was 1×10⁻⁶. 4 -5×10 5 / 100 μL of cell suspension.
5. The use as described in claim 1, characterized in that, The concentration of the MSI-H type colon cancer cells was 1×10⁻⁶. 4 -5×10 5 / 100 μL of cell suspension.
6. The use as described in claim 1, characterized in that, The MSS-type colon cancer cells were CT26 cells.
7. The use as described in claim 1, characterized in that, The MSI-H type colon cancer cells were MC38 cells.
8. The use as described in claim 1, characterized in that, The amount of vaccine administered at each vaccination site is 20-100 microliters.
9. The use as described in claim 1, characterized in that, The vaccination described is a subcutaneous injection.
10. A kit for constructing mouse models of MSS-type and MSI-H-type colorectal cancer, characterized in that, The kit includes a F1 hybrid mouse line of C57BL / 6×BALB / c and MSS-type and MSI-H-type colon cancer cells; The method for constructing mouse models of MSS-type colorectal cancer and MSI-H-type colorectal cancer includes the following steps: inoculating the MSS-type colorectal cancer cells and MSI-H-type colorectal cancer cells into the hybrid mice, respectively, to obtain MSS-type colorectal cancer and MSI-H-type colorectal cancer models.
11. The kit according to claim 10, characterized in that, The MSS-type colon cancer cells were CT26 cells.
12. The kit according to claim 10, characterized in that, The MSI-H type colon cancer cells were MC38 cells.
13. The use of a combination of MSS-type colon cancer cells and MSI-H-type colon cancer cells, characterized in that, The MSS type colorectal cancer cells are CT26 cells, and the MSI-H type colorectal cancer cells are MC38 cells. The combination is used to prepare reagents or kits for constructing mouse models of MSS type colorectal cancer and MSI-H type colorectal cancer based on hybrid mice with the same genetic background, wherein the hybrid mice are F1 generation of C57BL / 6×BALB / c. The reagents or kits include F1 hybrid mice of C57BL / 6×BALB / c and MSS-type and MSI-H-type colon cancer cells; The method for constructing mouse models of MSS-type colorectal cancer and MSI-H-type colorectal cancer includes the following steps: inoculating the MSS-type colorectal cancer cells and MSI-H-type colorectal cancer cells into the hybrid mice, respectively, to obtain MSS-type colorectal cancer and MSI-H-type colorectal cancer models.