Mouse-derived gastric cancer organ as well as establishment method and application thereof

Abstract

The invention discloses a mouse-derived gastric cancer organoid as well as an establishment method and application thereof. The mouse-derived gastric cancer organoid is mainly obtained by culturing a mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) in a culture medium. The mouse-derived gastric cancer organ can be used for constructing various types of gastric cancer animal models and screening gastric cancer treatment drugs. The established mouse source gastric cancer organoid is derived from a transgenic mouse, the repeatability of the construction process is high, the passage stability is good, the tumor characteristics of mouse gastric cancer can be well simulated, tumor formation in an immunodeficient mouse body can be achieved, especially tumor formation in a C57BL / 6 mouse body with a sound immune system can be achieved, the metastatic tumor formation capacity is achieved, and the mouse source gastric cancer organoid has a good application prospect. The method is suitable for gastric cancer immune microenvironment and tumor generation and development mechanism research.

Description

Technical Field

[0001] The present invention belongs to the technical field of gastric cancer organoids, and specifically relates to a gastric cancer organoid derived from mice, and a method for establishing the organoid and an application thereof. Background Art

[0002] Tumor cell lines are now widely used as in vitro disease models for studying tumor biology. Tumor cell lines are easy to establish and maintain in culture, and can be genetically modified using various techniques (such as lentiviral transfection, CRISPR-Cas9 genome editing, etc.) to alter the expression of specific genes or perform knock-in or knock-out. The use of tumor cell lines has greatly facilitated the study of specific signaling pathways in various aspects of cancer biology, including tumor invasion, metastasis, drug responsiveness, etc., and can also be used for high-throughput screening of drugs. However, the failure rate of cell line-based drug screening and validation projects is high because tumor cell lines cannot characterize the various characteristics of the original tumor. These characteristics include the genetic composition of the tumor, genetic heterogeneity within the tumor, and the lack of relevant stromal components.

[0003] Organoids are three-dimensional cultures composed of multiple organ-specific cell types that can mimic the structure and function of natural organs, providing a new perspective for the study of tumor physiology and pathology. Compared with two-dimensional cell cultures, organoids can more accurately characterize human tissues, allowing for more reliable and efficient drug screening and functional validation. This feature is particularly valuable in the context of tumor research, where organoids can mimic the tumor microenvironment with higher clinical fidelity than traditional cell line models, which enables rapid functional testing of drugs and improves the efficiency of new drugs from discovery to clinical application. In addition, organoids provide a new platform for in vitro gene editing therapy. By leveraging gene editing tools such as CRISPR-Cas9, researchers can use organoids to model genetic diseases and test treatment strategies to better advance personalized medicine.

[0004] In the past decade, organoids have been widely used to simulate various human malignancies, such as gastric cancer, colorectal cancer, liver cancer, lung cancer, etc. These organoid models have been used in all aspects of tumor research, such as tumor modeling, tumor treatment research, anti-tumor drug discovery, and personalized precision medicine, and organoids can be used to model the occurrence and development of malignant tumors. Tumor organoids provide a new method for anti-tumor drug discovery and testing. Compared with other drug screening models, patient-derived tumor organoids can be cultured and produced at a lower cost and in a shorter time while maintaining intra-tumor heterogeneity. There are now several studies using tumor organoids for drug screening to simulate chemotherapy effectiveness and tumor responsiveness. In addition, organoids can be used as a platform for high-throughput screening and drug development for rare cancers and diseases.

[0005] Compared with tumor organoids from gastric cancer patients, gastric cancer organoids from mice are relatively scarce. The several construction methods disclosed in the literature and patents all involve 3-4 gene knockouts / mutations in mouse gastric epithelial cells or organoids. This construction is difficult, and the overall construction takes more than 6 months to complete, with a high failure rate. Summary of the invention

[0006] Purpose of the invention: In order to solve the problems existing in the prior art, the present invention provides a mouse-derived gastric cancer organoid and its establishment method and application. The gastric cancer organoid established by the present invention has strong reproducibility and good passage stability, can form tumors in C57BL / 6 mice with a sound immune system, and can be used for anti-tumor drug screening, etc. The cell line can be used for research on the immune microenvironment, occurrence and development mechanism, distant metastasis mechanism, drug resistance mechanism and new drug screening of gastric cancer.

[0007] Technical solution: To achieve the above purpose, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for establishing gastric cancer organoids from mice, comprising the following steps:

[0009] The mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is cultured in a culture medium to obtain the mouse-derived gastric cancer organoid.

[0010] As a specific implementation scheme, the mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is deposited in the China Center for Type Culture Collection on June 15, 2024, with a deposit number of CCTCC NO: C2024183.

[0011] As a specific embodiment, the culture medium is: Wnt3a, R-spondin 1, Noggin, FGF10, gastrin, n-Acetylcysteine ​​and EGF are added to RPMI 1640 culture medium containing FBS.

[0012] Preferably, the culture medium is: adding Wnt3a, R-spondin 1, Noggin, FGF10 (fibroblast growth factor 10), gastrin, n-Acetylcysteine ​​(N-acetylcysteine) and EGF (epidermal growth factor) to RPMI 1640 culture medium containing 8-12% FBS (fetal bovine serum); wherein the final concentration of Wnt3a is 0.8-1.2 ng / mL, the final concentration of R-spondin 1 is 8-12 ng / mL, the final concentration of Noggin is 0.8-1.2 ng / mL, the final concentration of FGF10 is 0.8-1.2 ng / mL, the final concentration of gastrin is 0.05-0.15 nM, the final concentration of n-Acetylcysteine ​​is 18-22 uM, and the final concentration of EGF is 0.2-0.3 ng / mL.

[0013] Among them, Wnt3a, R-spondin 1, and Noggin are all recombinant proteins, and the above raw materials can be purchased from the market.

[0014] As a specific embodiment, the culturing is carried out under the conditions of 4-6% carbon dioxide concentration and 37±2°C.

[0015] In a second aspect, the present invention provides a mouse-derived gastric cancer organoid, which is obtained by the above-mentioned establishment method.

[0016] In a third aspect, the present invention provides the use of the mouse-derived gastric cancer organoids as an experimental tool in studying gastric cancer.

[0017] In a fourth aspect, the present invention provides the use of the mouse-derived gastric cancer organoids in preparing a gastric cancer animal model.

[0018] As a specific embodiment, the gastric cancer animal model is a subcutaneous tumor animal model, an orthotopic tumor animal model or an abdominal metastasis tumor animal model.

[0019] In a fifth aspect, the present invention provides the use of the mouse-derived gastric cancer organoids in screening drugs for treating gastric cancer.

[0020] The mouse gastric cancer organoids constructed in the present invention are based on the Myc gene, a spontaneous tumorigenic transgenic mouse model of gastric cancer successfully constructed by the applicant previously. LSL / LSL ;Kras loxP / + ;Anxa10-CreER T2+ / -. After the mouse spontaneously formed a primary gastric tumor, the gastric tumor tissue was removed and dissociated into a single-cell suspension. The mouse gastric cancer cell MKC1 was cultured from the single-cell clone, and the mouse gastric cancer cell line was cultured to construct a mouse gastric cancer organoid. Experimental verification has shown that the mouse gastric cancer organoid successfully constructed by this method can form a three-dimensional morphology, grow well and can be continuously passaged. In addition, this mouse gastric cancer organoid can form tumors in C57BL / 6 mice with a sound immune system, and can be used to construct a mouse subcutaneous tumor model, an orthotopic transplantation model, an abdominal metastasis model, etc., and can be used for anti-tumor drug screening. The application of this mouse organoid can accelerate the study of the molecular mechanisms of the occurrence and development of gastric cancer, thereby promoting the development of future preclinical gastric cancer models for anti-tumor drug screening and personalized precision medicine.

[0021] The method for constructing the transgenic mouse model comprises the following steps:

[0022] (1) Insert 2A-CreERT2-Wpre-pA into the stop codon of mouse Anxa10 gene to obtain Anxa10-CreERT2 recombinase transgenic mouse Anxa10-CreERT2 + / + mice; the CAG promoter-loxp-STOP-loxp-Myc-polyA conditional overexpression structure was inserted into the H11 site to establish the H11-LSL-Myc mouse model, which is a MYC overexpression gene mouse MYC LSL / LSL Mouse; loxp-stop-loxp and exon2 containing G12D replaced exon2 of Kras to establish the Kras gene G12D conditional point mutation mouse strain KRAS loxP / + Mouse.

[0023] (2) MYC LSL / LSL Mice were mated with Anxa10-CreERT2 recombinase transgenic mice to obtain F1 generation mice, and heterozygous mice among the F1 generation mice were selected and named Anxa10-CreERT2 + / - ;MYC LSL / + Mice;

[0024] (3) KRAS loxP / + Mice were mated with Anxa10-CreERT2 recombinase transgenic mice to obtain F1 generation mice, and heterozygous mice among the F1 generation mice were selected and named Anxa10-CreERT2 + / - ;Kras loxP / + Mice;

[0025] (4) Transfect the Anxa10-CreERT2 in the above F1 generation + / - ;MYC LSL / +Mice with Anxa10-CreERT2 + / - ;Kras loxP / + The mice were mated to obtain F2 mice, and the genotype of the F2 mice was selected to be Anxa-CreERT2 heterozygous, MYC heterozygous, and KRAS heterozygous mice, named Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + Mice;

[0026] (5) The Anxa10-CreERT2 with the same genotype in the F2 generation + / - ;MYC LSL / + ;Kras loxP / + The mice were mated to obtain F3 mice, and the genotype of the F3 mice was selected to be Anxa10-CreERT2 + / - ;MYC LSL / LSL ;Kras loxP / + The mouse is the transgenic mouse model MKC.

[0027] Beneficial effects: Compared with the prior art, the mouse gastric cancer organoids established by the present invention are derived from transgenic mice, the construction process is highly repeatable, the passage stability is good, and it can better simulate the tumor characteristics of mouse gastric cancer. It can not only form tumors in immunodeficient mice, but also in C57BL / 6 mice with a sound immune system, and has the ability to form metastatic tumors. It is suitable for the study of the immune microenvironment of gastric cancer and the mechanism of tumor occurrence and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the electrophoresis diagram of the product amplified using the Anxa10-CreERT2 primer, where lanes 1, 2 and 3 represent three different samples.

[0029] Figure 2 This is the electrophoresis diagram of the product amplified using MYC primers, where lanes 1, 2 and 3 represent three different samples.

[0030] Figure 3 The electrophoresis diagram of the product amplified using KRAS primers, where lanes 1, 2 and 3 represent three different samples.

[0031] Figure 4 This is a picture of tumor formation in transgenic MKC mice.

[0032] Figure 5 This is the microscopic morphology of MKC1 cells.

[0033] Figure 6 These are microscopic photos of mouse gastric cancer organoids.

[0034] Figure 7HE staining of mouse gastric cancer organoids

[0035] Figure 8 Genotyping Mouse Gastric Cancer Organoids

[0036] Fig. 9 Establishment of a subcutaneous transplantation model of mouse gastric cancer organoids in C57BL / 6 mice

[0037] Fig.10 Establishment of an orthotopic gastric transplantation model for mouse gastric cancer organoids in C57BL / 6 mice

[0038] Fig.11 Establishment of a C57BL / 6 mouse peritoneal metastasis model for mouse gastric cancer organoids

[0039] Fig.12 IC50 curve of 5-Fu and oxaliplatin detected in mouse gastric cancer organoids. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The experimental methods used in the examples are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified.

[0041] Example 1 Construction of MKC gastric cancer spontaneous tumorigenesis transgenic mouse model

[0042] The Anxa10-CreERT2 transgenic mice, which specifically expressed CreERT2 recombinase in gastric mucosal epithelial cells, were mixed with MYC overexpressing transgenic mice. LSL / LSL and mice with KRAS activating point mutations loxP / + The mice were crossed to obtain the F1 generation heterozygous mice, Anxa10-CreERT2 + / - ;MYC LSL / + Mice with Anxa10-CreERT2 + / - ;Kras loxP / + Mice. Two F1 generation mice were mated to obtain F2 mice. The genotype of the F2 generation mice was selected to be Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + The mice were mated and the F3 generation had the genotype of Anxa10-CreERT2 + / - ;MYC LSL / LSL ;Kras loxP / + Mice (MKC mouse model), genotype identification as Figure 1-3As shown. This genotype of mice was selected and injected with tamoxifen to induce Cre expression starting from 6 weeks of age. The vital signs and body weight of the mice were monitored. The transgenic mice formed primary gastric tumors after induction (as shown in Figure 2A). Figure 4 As shown in the figure, spontaneous tumors formed in the stomach of mice, and the mice were killed at week 8 of induction. The mice were dissected and the stomach was removed, the stomach wall was cut open, the stomach contents were taken out and the stomach cavity was flushed with PBS for subsequent processing.

[0043] The specific method for constructing the MKC mouse model is as follows:

[0044] 1. Gene sources and construction principles of each transgenic mouse

[0045] 1. Obtaining Anxa10-CreERT2 mice

[0046] 2A-CreERT2-Wpre-pA was inserted into the stop codon of mouse Anxa10 gene to obtain Anxa10-CreERT2 recombinase transgenic mouse Anxa10-CreERT2 + / + Mice. The mice were purchased from Shanghai Model Organisms Technology Co., Ltd.

[0047] Anxa10-2A-CreERT2 gene:

[0048] NCBI ID: 26359

[0049] MGI ID: 1347090

[0050] Ensembl ID: ENSMUSG00000031635

[0051] Pubmed: Anxa10

[0052] Anxa10 gene:

[0053] Anxa10 annexin A10[Mus musculus(house mouse)]

[0054] NCBI Gene ID:26359, updated on 11-Sep-2023

[0055] Examples of gene-marked cell types: gastric epithelial cells, skin hair follicle cells

[0056] This mouse expresses CreERT2 recombinase at the Anxa10 locus. When Anxa10-2A-CreERT2 is bred with reporter mice, the reporter gene is expressed in the gastric epithelial cells of the adult after Tamoxifen induction; when bred with mice containing loxP flanking sequences, Tamoxifen-induced recombination will lead to the deletion of the floxed sequence in the gastric epithelial cells. It can be used for gastric cancer research.

[0057] 2. Obtaining MYC overexpressing mice

[0058] The CAG promoter-loxp-STOP-loxp-Myc-polyA conditional overexpression structure was inserted into the H11 locus to establish the H11-LSL-Myc mouse model, which is a MYC overexpression gene mouse MYC LSL / LSL Homozygous mice were purchased from Shanghai Model Organisms Technology Co., Ltd.

[0059] MYC gene:

[0060] MGI ID: 5461148

[0061] https: / / www.modelorg.com / index.php / portal / article / index / id / 858 / post_type / 3.html

[0062] The c-Myc gene (also called Myc) is abnormally expressed in many tumors, affecting cell proliferation, growth metabolism, gene instability, stimulating angiogenesis, cell malignant transformation, differentiation and apoptosis. The MYC proto-oncogene encodes a family of transcription factors and is one of the most common activated oncoproteins in human tumors. In fact, in most cancers, MYC aberrations or MYC-related pathways are upregulated through alternative mechanisms. MYC protein is a major regulator of cellular programs. The c-Myc gene (also called Myc) is abnormally expressed in many tumors, affecting cell proliferation, growth metabolism, gene instability, stimulating angiogenesis, cell malignant transformation, differentiation and apoptosis. The CAGpromoter-loxp-STOP-loxp-Myc-polyA conditional overexpression structure was inserted into the H11 site to establish the H11-LSL-Myc mouse model. The H11 site is located on mouse chromosome 11. This site has been shown to be similar to Rosa26 and can be used to express exogenous genes more widely. H11-LSL-Myc can highly express the Myc gene in Cre-expressing tissues after mating with Cre-expressing mice. It can be used to establish tumor models and conduct tumor research.

[0063] 3.Kras gene G12D conditional point mutation mouse strain KRAS loxP / + Acquisition of mice

[0064] The loxp-stop-loxp and exon2 containing G12D replaced the exon2 of Kras to establish the Kras gene G12D conditional point mutation mouse strain KRAS loxP / + Mice. The mice were purchased from Jackson lab.

[0065] Kras gene:

[0066] Kras Kirsten rat sarcoma viral oncogene homolog[Mus musculus(housemouse)];

[0067] NCBI Gene ID:16653, updated on 11-Oct-2023

[0068] KRAS mutations can lead to uncontrolled activation of RAS protein. RAS protein is located in the cell membrane and regulates cell growth, proliferation, movement, migration and angiogenesis. KRAS mutations will significantly increase the invasiveness of gastric cancer. KRAS expression is high in the Lauren classification of intestinal type. The most common model is to use K19-CreERT mice and Loxp-STOP-Loxp-K-Ras (G12D) mice to obtain mice that conditionally express K-Ras (G12D). These mice also have multiple dysplasia, metaplasia and adenocarcinoma in the stomach. The activation / inactivation effect of wild-type Kras is controlled, while the mutant Kras protein is dysfunctional and continuously in an activated state, leading to the continuous proliferation of tumor cells. The loxp-stop-loxp and exon2 containing G12D replace the Kras exon2 to establish a Kras gene G12D conditional point mutation mouse strain. After mating with Cre mice, G12D point mutation mice can be obtained.

[0069] 2. Specific process of constructing gastric adenocarcinoma transgenic mouse model

[0070] (1) MYC overexpression gene mice LSL / LSL The mice were mated with Anxa10-CreERT2 recombinase transgenic mice that specifically expressed CreERT2 recombinase in gastric epithelial cells to obtain F1 mice; heterozygous mice among the F1 mice were selected and named Anxa10-CreERT2 + / - ;MYC LSL / + Mouse.

[0071] (2) KRAS conditional point mutation activated mice - KRAS loxP / + The mice were mated with Anxa10-CreERT2 recombinase transgenic mice that specifically expressed CreERT2 recombinase in gastric epithelial cells to obtain F1 mice; heterozygous mice among the F1 mice were selected and named Anxa10-CreERT2 + / - ;Kras loxP / + Mouse.

[0072] (3) Transfecting the Anxa10-CreERT2 in the F1 generation + / - ;MYC LSL / + Anxa10-CreERT2 in mice and the F1 generation + / - ;KRAS loxP / + The mice were mated to obtain F2 mice; the F2 mice were selected to have the genotype of Anxa-CreERT2 heterozygous, MYC heterozygous, and KRAS heterozygous, and their genotype was named Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + Mice;

[0073] (4) Select the genotype of F2 mice to be Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + The mice were mated to obtain F3 mice, and the genotype of the F3 mice was selected to be Anxa10-CreERT2

[0074] + / - ;MYC LSL / LSL ;Kras loxP / + The mouse is a gastric epithelial cell-specific MYC overexpression and Kras activation mutation mouse, which is the gastric adenocarcinoma transgenic mouse model;

[0075] Anxa10-CreERT2 + / + The mice were homozygous for the Anxa10-CreERT2 recombinase transgenic mice. + / - The mice were Anxa10-CreERT2 recombinase transgenic heterozygous mice; MYC LSL / LSL Mice are homozygous for MYC overexpression. LSL / + MYC overexpression heterozygous mice; KRAS loxP / + The mice were heterozygous for the Kras gene G12D conditional point mutation.

[0076] The constructed Anxa10-CreERT2 + / - ;MYCLSL / LSL ;Kras loxP / + Mice, for adult mice, tamoxifen is injected intraperitoneally at 6 weeks of age. Dissolve tamoxifen in corn oil at a concentration of 20 mg / ml and shake overnight at 37 degrees Celsius. Store it at 4°C during injection. The injection dose is determined by weight, approximately 200 mg / kg body weight. Administer tamoxifen by intraperitoneal injection (using an ACUC-approved injection procedure) once every 24 hours for 3-5 consecutive days. For safety, disinfect the injection site with 70% ethanol before injection. After the last injection, mice should be isolated for 24 hours before returning to their normal animal room.

[0077] 3. Mouse genotype identification method

[0078] The genotype identification of gastric mucosal epithelial cell-specific Anxa10-CreERT2; MYC overexpression and Kras activation point mutation mice and the PCR identification method for obtaining heterozygous mice are as follows:

[0079] Take 2-3 mm of the tail of the offspring mouse and extract the genomic DNA of the tail using standard procedures. Use the genomic DNA of the tail as a template and use gene-specific primers to perform polymerase chain reaction (PCR) to identify the mouse genotype. Use the genomic DNA of the tail of wild-type mice C57 mice as a control.

[0080] 1) The sequence of the wide type primer for identifying the Anxa10-CreERT2 recombinase gene is:

[0081] 5'-ATCTCCCATCCTCCCTAACTT-3' and 5'-ATCGCATCACCTTCAAACTCT-3'.

[0082] The mutant type primer sequences are:

[0083] 5'-CAGGGCACTATGAGAAAGCACTAC-3' and

[0084] 5'-AGCCCGGACCGACGATGAAGC-3'.

[0085] 2) The Wide type primer sequence for identifying the MYC gene is:

[0086] 5'-ATAAGCCATTCTCCATTTCATAA-3' and 5'-CCCCTTGTTCCCTTTCTGC-3'.

[0087] The mutant type primer sequences are:

[0088] 5'-TTTGCCTTTGTTACCTGTTCCATC-3' and 5'-CTCCCCCGTGCCTTCCTTGAC-3'.

[0089] 3) The sequence of the wide type primer for identifying the KRAS point mutation gene is:

[0090] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-TGTCTTTCCCCAGCACAGT-3'.

[0091] The mutant type primer sequence is

[0092] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-GCAGGTCGAGGGACCTAATA-3'.

[0093] Genotype identification system:

[0094] F (upstream primer): 0.25ul, R (downstream primer): 0.25ul, 2xTaq (buffer): 7.5ul, H2O: 5.5ul, mouse tail DNA: 1.5ul, Total: 15ul.

[0095] PCR instrument loading procedure:

[0096] 1) ANXA identification:

[0097] Volume: 15ul, Step1 94℃00:05:00(5min), Step2 94℃00:00:30(30s), Step3 60℃00:00:30(30s), Step4 72℃00:01:00(1min), Step 5Goto2 x35, Step6 72℃00:05:00(5min), Step7 12℃Forever.

[0098] 2) Kras identification:

[0099] Volume: 15ul, Step1 94℃00:03:00(3min), Step2 94℃00:00:15(15s), Step358'C00:00:15(15s), Step4 72℃00:00:40(40s), Step 5Goto2 x30, Step6 72℃00:05:00(5min), Step7 10'℃Forever.

[0100] 3) MYC identification:

[0101] Volume: 15ul, Step1 94℃00:05:00(5min), Step2 94℃00:00:30(30s), Step3 65℃00:00:30.(30s), Step4 72℃00:01:00(1min), Step5 Goto2 x34, Step6 72℃00:05:00(5min), Step7 12℃Forever

[0102] The genotype identification results of mice are shown in Figure 1-3 , the three lanes in the figure represent three samples, Figure 1 The electrophoresis of the product amplified by Anxa10-CreERT2 primers was performed. If a 436bp fragment was specifically amplified, it was Anxa10-CreERT2 positive; if an 823bp fragment was specifically amplified, it was Anxa10-CreERT2 negative.

[0103] Figure 2 For the electrophoresis of the product amplified with MYC primers, if a 434bp fragment can be specifically amplified, it is MYC LSL / LSL homozygous, if a 428bp fragment can be specifically amplified, it is MYC negative, and if a negative-positive band is amplified, it is heterozygous MYC LSL / +.

[0104] Figure 3 The product amplified by KRAS primers is electrophoresed. If a 100bp fragment can be specifically amplified and a 200bp fragment can be amplified, it is a KRAS loxP / + heterozygote. If a 200bp fragment can be specifically amplified, it is KRAS negative and wild type.

[0105] Figure 1-3 The genotype of sample 1 is Anxa10-CreERT2+ / -; MYC LSL / +, the genotype of sample 2 is Anxa10-CreERT2+ / -; MYC LSL / LSL; KrasloxP / +, and the genotype of sample 3 is Anxa10-CreERT2+ / +; MYCLSL / +. Therefore, sample 2 is the required gastric epithelial cell-specific MYC overexpression and Kras activation point mutation mouse, that is, the MKC mouse model.

[0106] Example 2 Isolation and culture of gastric adenocarcinoma cell line MKC1

[0107] 1. The experimental steps for constructing primary cell lines are as follows:

[0108] (1) Place a clean cell culture dish on ice, and use sterile forceps to place the MKC mouse stomach tissue in Example 1 into the cell culture dish; rinse the stomach tissue three times with PBS containing penicillin-streptomycin;

[0109] (2) Use sterile surgical instruments to carefully remove non-target tissue parts such as blood vessels, fibers, mucosa, fat or necrotic tissue, and cut the target tissue into small pieces of about 2 mm;

[0110] (3) Transfer the tissue block to a 5 ml centrifuge tube, add RPMI 1640 digestion solution (1 mg / mL collagenase A, 0.1 mg / mL ribonuclease), and digest in a 37°C water bath for 20 min, pipetting every 5-10 min;

[0111] (4) Place a 70-micron cell sieve in a 15 ml centrifuge tube, rinse the cell sieve with RPMI 1640 + 2% FBS, pipette the sample through the sieve, add 2 ml RPMI 1640 + 2% FBS to rinse the cell sieve, and centrifuge at 4°C, 500 g for 5 min.

[0112] (5) Resuspend the cells in 1 ml RPMI 1640 + 2% FBS, add 3 ml red blood cell lysis buffer, lyse for 5 min, and centrifuge at 500 g at 4°C for 5 min.

[0113] (6) Resuspend the cells in RPMI 1640 medium containing 10% FBS and culture and subculture in a cell culture incubator (37° C., 5% CO 2 ).

[0114] (7) After 2-3 generations of cell culture, the cells were collected, counted, and added to a 96-well plate at 1 cell / 100 μl / well for monoclonal screening to obtain the mouse gastric cancer primary cell line MKC1 ( Figure 5 As shown, the primary cells are photographed under a microscope, and the cells are of uniform morphology and size).

[0115] The above-mentioned mouse gastric cancer cell line MKC1 (Mus musculus) was deposited in the China Center for Type Culture Collection (CCTCC) on June 15, 2024, with the deposit number CCTCC NO: C2024183, and the deposit address is Wuhan University, Wuhan City, Hubei Province, People's Republic of China.

[0116] Example 3 Cultivation and construction of mouse gastric cancer organoids;

[0117] 1) Prepare mouse gastric cancer organoid culture medium: add Wnt3a (final concentration 1 ng / mL), R-spondin 1 (final concentration 10 ng / mL), Noggin (final concentration 1 ng / mL), FGF10 (final concentration 1 ng / mL), gastrin (final concentration 0.1 nM), n-Acetylcysteine ​​(final concentration 20 uM), and EGF (final concentration 0.25 ng / mL) to RPMI 1640 culture medium containing 10% FBS.

[0118] 2) Count the mouse gastric cancer cell line MKC1, adjust the cell concentration to 200 cells / 20ul, resuspend the organoids with Matrigel, spot into a 24-well plate, and place in a cell culture incubator for 15 minutes to wait for the Matrigel to solidify.

[0119] 3) Add 500ul of the above organoid culture medium and culture in a cell culture incubator at 37°C and 5% carbon dioxide concentration. Take pictures under a microscope after 3 days ( Figure 6 ), obtained mouse gastric cancer organoid QLGO1, and the photo results showed that the organoid was in good condition.

[0120] Example 4 HE staining of organoids

[0121] Collect 2000 organoids QLGO1, add 1ml PBS to resuspend, transfer to 1.5ml centrifuge tube, 300g, 4℃, centrifuge for 5min and discard the liquid. Add 1ml 4% paraformaldehyde to the centrifuge tube, gently pipette to mix, and fix at 4℃ for 1h. After fixation, centrifuge at 300g for 5min at 4℃ and discard the supernatant. Add 1ml PBS to resuspend and transfer to 1.5ml centrifuge tube, 300g for 5min at 4℃ and discard the liquid. Prepare 2% agarose solution, melt it in a microwave oven on high heat, wait for the agarose solution to cool to 50-60℃, take 20-50uL of melted agarose solution, add it to a 1.5ml EP tube to resuspend the organoid precipitate, put it on ice for 30min, and wait for it to solidify. Take out the solidified agarose block containing organoid precipitation, dehydrate it in gradient ethanol, 70%, 80%, 90% and 95% ethanol for 1 hour each, 100% ethanol for 30 minutes, and wait for it to solidify; transfer the dehydrated agarose block to the tissue embedding box, and transfer the embedding box to xylene for 5 minutes twice. After the transparent treatment is completed, immediately transfer it to the melted wax cylinder, soak it in wax at 60℃ for 2 hours, and then replace the new wax cylinder to soak it in wax again for 2 hours; embedding is carried out on an ice box, pour the melted pure wax into one-third of the wax box, place the agar block in the middle, and then drip wax embedding until it is full. After the wax block is completely solidified, carefully remove the mold. Fix the embedded wax block on the slicer, cut it into thin slices 5um thick, and use tweezers to place the slices containing complete tissues in 40℃ warm water. Pick up the slices and put them on the slides in a 37℃ incubator to dry. Put the slices into xylene I for 10 minutes; xylene II for 10 minutes; anhydrous ethanol I for 5 minutes; anhydrous ethanol II for 5 minutes; 95% alcohol for 5 minutes; 90% alcohol for 5 minutes; 80% alcohol for 5 minutes; 70% alcohol for 5 minutes; and wash with distilled water. Stain the slices with hematoxylin for 3 minutes and wash with tap water. Stain the slices with eosin stain for 3 minutes and wash with tap water. Dehydrate and make transparent by putting the slices into 95% alcohol I for 5 minutes; 95% alcohol II for 5 minutes; anhydrous ethanol I for 5 minutes; anhydrous ethanol II for 5 minutes; xylene I for 5 minutes; xylene II for 5 minutes. Take the slices out of xylene and dry them slightly. Seal the slices with neutral gum and observe and take pictures under a microscope. The results are as follows Figure 7 shown.

[0122] Example 5 Organoid genotype identification

[0123] 100,000 organoids QLGO1 were collected, and organoid genomic DNA was extracted using a genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat. No. DP304). DNA polymerase and corresponding primers were added for PCR amplification.

[0124] The sequences of the wide type (WT) primers for identifying the Anxa10-CreERT2 recombinase gene were 5′-ATCTCCCATCCTCCCTAACTT-3′ and 5′-ATCGCATCACCTTCAAACTCT-3′.

[0125] The sequence of the Mutant type (MUT) primer is:

[0126] 5'-CAGGGCACTATGAGAAAGCACTAC-3' and 5'-AGCCCGGACCGACGATGAAGC-3'.

[0127] The Wide type primer sequence for identifying the MYC gene is:

[0128] 5'-ATAAGCCATTCTCCATTTCATAA-3' and 5'-CCCCTTGTTCCCTTTCTGC-3'.

[0129] The mutant type primer sequences are:

[0130] 5'-TTTGCCTTTGTTACCTGTTCCATC-3' and 5'-CTCCCCCGTGCCTTCCTTGAC-3'.

[0131] The sequence of the wide type primer for identifying KRAS point mutation gene is:

[0132] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-TGTCTTTCCCCAGCACAGT-3'.

[0133] The mutant type primer sequences are:

[0134] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-GCAGGTCGAGGGACCTAATA-3'.

[0135] The PCR reaction procedure is as follows

[0136] ①Anxa10-CreERT2

[0137] Step1 94℃5min; Step2 94℃30s; Step3 60℃30s; Step4 72℃1min;

[0138] Step5 Go to 2×35; Step6 72℃5min; Step7 12℃∞.

[0139] ②KRAS

[0140] Step1 94℃3min; Step2 94℃15s; Step3 60℃15s; Step4 72℃40s;

[0141] Step5 Go to 2×30; Step6 72℃5min; Step7 10℃∞;

[0142] ③MYC

[0143] Step1 94℃5min; Step2 94℃30s; Step3 65℃30s; -0.5℃ / Cyc; 1Cyc-T;

[0144] Step4 72℃1min; Step5 Go to 2×34; Step6 72℃5min; Step7 12℃∞.

[0145] Prepare 2% agarose gel, add Ultra GelRed nucleic acid dye at 1:10000, mix well and pour into gel mold. Place agarose gel in electrophoresis tank, use 1×TAE as electrophoresis liquid, add DNA marker, sample to be tested, negative control and positive control respectively, set voltage to 135V, and run electrophoresis for 35min. Finally, use gel imager to develop and take pictures. The results are as follows Figure 8 As shown, the genotype is correct.

[0146] Example 6 Experiments on subcutaneous tumor formation, orthotopic tumor formation, and abdominal metastasis tumor formation in mouse gastric cancer organoids

[0147] 1) Subcutaneous tumor formation experiment: Collect organoids QLGO1, count, and adjust the organoid concentration to 1 million / 100ul. Select 6-week-old C57 / 6J mice, and use a syringe to inject 100ul of organoid suspension into the right armpit of the mouse. The mouse was killed 4 weeks later, and the subcutaneous tumor tissue of the mouse was photographed. The results are as follows Fig. 9 As shown, subcutaneous neoplasia occurs.

[0148] 2) In situ tumor formation experiment: Collect organoids QLGO1, count, and adjust the organoid concentration to 1 million / 10ul. Select 6-week-old C57 / 6J mice, anesthetize the mice, make a left subcostal incision, cut the mouse skin layer by layer, and expose the mouse stomach. Inject 10ul of organoid suspension under the gastric serosa. Suture the incision. After 4 weeks, the mice were killed and the mice were cut open to observe whether there was tumor formation in the stomach. Take pictures of the tumor tissue, and the results are as follows Fig.10 Shown, showing gastric tumor formation.

[0149] 3) Peritoneal metastasis tumorigenesis experiment: Organoids were collected and counted, and the concentration of organoids was adjusted to 5 million / 500ul. Six-week-old C57 / 6J mice were selected, and 500ul of organoid suspension was injected into the peritoneal cavity of the mice using a syringe. The mice were killed after 4 weeks, and the mice were cut open to observe whether there were gastric cancer metastasis lesions in the peritoneal cavity. The peritoneal metastasis lesions were taken and photographed. The results are as follows: Fig.11 As shown, gastric cancer metastases have formed in the abdominal cavity.

[0150] Example 7 Anti-tumor drug screening experiment using mouse gastric cancer organoids

[0151] Organoids were planted on a 96-well plate at 1000 cells / well, and 100ul of organoid culture medium was added to each well. Oxaliplatin / 5-Fluorouracil was added to each well at concentrations of 1, 10, 100, 1000, and 2000μg / ml, respectively. The control group was added with an equal amount of PBS, and 3 replicates were set up for each group. After 48 hours of culture, 10ul of CCK-8 reagent was added to each well. After 2 hours, the absorbance was detected at 450nm, and the IC50 curve was drawn. The effects of different concentrations of oxaliplatin / 5-Fluorouracil on the activity of organoids were shown in the figure. Fig.12 shown.

[0152] The above embodiments are the best implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for establishing gastric cancer organoids from mice, characterized in that: The following steps are involved: The mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is cultured in a culture medium to obtain the mouse-derived gastric cancer organoid.

2. The method for establishing gastric cancer organoids from mice according to claim 1, characterized in that: The mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is deposited in the China Center for Type Culture Collection on June 15, 2024, with a deposit number of CCTCC NO: C2024183.

3. The method for establishing mouse-derived gastric cancer organoids according to claim 1, characterized in that: The culture medium comprises: adding Wnt3a, R-spondin 1, Noggin, FGF10, gastrin, n-Acetylcysteine ​​and EGF to RPMI 1640 culture medium containing FBS.

4. The method for establishing gastric cancer organoids from mice according to claim 3, characterized in that: The culture medium comprises: adding Wnt3a, R-spondin 1, Noggin, FGF10, gastrin, n-Acetylcysteine ​​and EGF to RPMI 1640 culture medium containing 8-12% FBS; wherein the final concentration of Wnt3a is 0.8-1.2 ng / mL, the final concentration of R-spondin1 is 8-12 ng / mL, the final concentration of Noggin is 0.8-1.2 ng / mL, the final concentration of FGF10 is 0.8-1.2 ng / mL, the final concentration of gastrin is 0.05-0.15 nM, the final concentration of n-Acetylcysteine ​​is 18-22 uM, and the final concentration of EGF is 0.2-0.3 ng / mL.

5. The method for establishing gastric cancer organoids from mice according to claim 1, characterized in that: The culture is carried out under the conditions of 4-6% carbon dioxide concentration and 37±2°C.

6. A gastric cancer organoid derived from mice, characterized in that: The gastric cancer organoid is obtained by the establishment method described in any one of claims 1-5.

7. Use of the mouse-derived gastric cancer organoids described in claim 6 as an experimental tool in studying gastric cancer.

8. Use of the mouse-derived gastric cancer organoids according to claim 6 in preparing a gastric cancer animal model.

9. The use according to claim 8, characterized in that: The gastric cancer animal model is a subcutaneous tumor animal model, an orthotopic tumor animal model or an abdominal metastasis tumor animal model.

10. Use of the mouse-derived gastric cancer organoids according to claim 6 in screening drugs for treating gastric cancer.

Images