A method for constructing a gastric adenocarcinoma model

Abstract

This invention belongs to the field of biotechnology, specifically relating to a method for constructing a gastric adenocarcinoma model. The method includes the following steps: breeding Anxa10-Cre / + animals or their progeny with LSL-KrasG12D / +;Tp53 R172H / + animals or their progeny to obtain LSL-KrasG12D / +;Tp53 R172H / +;Anxa10-Cre / + animals; administering N-methyl-N-nitrosourea to the LSL-KrasG12D / +;Tp53 R172H / +;Anxa10-Cre / + animals. This method shortens the tumorigenesis period (approximately 2.5 months), increases the tumorigenesis rate (100%), and confirms the pathological type of the obtained gastric adenocarcinoma model as adenocarcinoma.

Description

A method for constructing a gastric adenocarcinoma model Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for constructing a gastric adenocarcinoma model. Background Technology

[0002] Gastric cancer (GC) is one of the most common malignant tumors worldwide, ranking fifth in incidence and fourth in mortality as of 2020. In 2020 alone, over one million new diagnoses and approximately 770,000 deaths were attributed to the disease. With the advent of precision medicine for gastric cancer, targeted therapy and immunotherapy are gradually becoming mainstream treatments, and a large amount of basic and translational research related to precision medicine for gastric cancer is underway. However, precision medicine implies a need for more precise subtyping of gastric cancer, and the lack of animal models that can more accurately characterize the composition and function of the tumor microenvironment is a key issue. Classic gastric cancer models include patient-derived tumor xenograft (PDX) models and cell-derived xenograft (CDX) models, most of which are implanted in the flanks of immunodeficient mice and cannot reflect the microenvironmental characteristics of the orthotopic gastric cancer tumor. While dual-humanized immunized mice, which use severely defective mice to construct mouse models of the humanized immune system (such as NCG-hIL15) (Chinese patent document CN114786479A), can partially mimic the immune microenvironment, they suffer from low NK cell survival rates, affecting the accuracy of drug efficacy evaluation, extremely high costs, and the inability to create a biomarker. Currently, the only commercially available mouse gastric cancer cell line is the MFC gastric squamous cell carcinoma line. Gastric squamous cell carcinoma accounts for only 0.04%-1% of gastric cancer patients, failing to reflect the characteristics of the vast majority of gastric cancer patients' pathological types, namely gastric adenocarcinoma. Therefore, there is an urgent need to establish a gastric adenocarcinoma model and construct a gastric adenocarcinoma cell line. Summary of the Invention

[0003] The first objective of this invention is to provide a method for constructing a gastric adenocarcinoma model.

[0004] The second objective of this invention is to provide a method for preparing gastric adenocarcinoma cells.

[0005] The third aspect of this invention is to provide a gastric adenocarcinoma cell.

[0006] The fourth aspect of this invention aims to provide a method for constructing an animal model of gastric adenocarcinoma.

[0007] The fifth aspect of this invention aims to provide applications of gastric adenocarcinoma models or their offspring obtained by the construction method of the first aspect of this invention, gastric adenocarcinoma cells obtained by the construction method of the third aspect of this invention, and gastric adenocarcinoma animal models or their offspring obtained by the construction method of the fourth aspect of this invention.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides a method for constructing a gastric adenocarcinoma model, comprising the following steps: breeding Anxa10-Cre / + animals or their progeny with LSL-KrasG12D / +;Tp53 R172H / + animals or their progeny to obtain LSL-KrasG12D / +;Tp53 R172H / +;Anxa10-Cre / + animals.

[0010] In some embodiments, the method includes the following steps: breeding offspring of Anxa10-Cre / + animals with offspring of LSL-KrasG12D / +;Tp53 R172H / + animals to obtain LSL-KrasG12D / +;Tp53 R172H / +;Anxa10-Cre / + animals.

[0011] In some embodiments, the method for constructing the LSL-KrasG12D / +;Tp53 R172H / + animal includes the following steps: breeding the LSL-KrasG12D / + animal or its offspring with the Tp53 R172H / + animal or its offspring to obtain the LSL-KrasG12D / +;Tp53 R172H / + animal.

[0012] In some embodiments, the method for constructing the LSL-KrasG12D / +;Tp53 R172H / + animal includes the following steps: breeding the offspring of the LSL-KrasG12D / + animal with the offspring of the Tp53 R172H / + animal to obtain the LSL-KrasG12D / +;Tp53 R172H / + animal.

[0013] In some embodiments, the method for constructing offspring of the Anxa10-Cre / + animal includes the following steps: breeding Anxa10-Cre / + animals with wild-type animals to obtain offspring of the Anxa10-Cre / + animal.

[0014] In some embodiments, the method for constructing offspring of the LSL-KrasG12D / +;Tp53 R172H / + animals includes the following steps: breeding LSL-KrasG12D / +;Tp53 R172H / + animals with wild-type animals to obtain offspring of LSL-KrasG12D / +;Tp53 R172H / + animals.

[0015] In some embodiments, the method for constructing offspring of the LSL-KrasG12D / + animals includes the following steps: breeding LSL-KrasG12D / + animals with wild-type animals to obtain offspring of LSL-KrasG12D / + animals.

[0016] In some embodiments, the method for constructing offspring of the Tp53 R172H / + animal includes the following steps: breeding Tp53 R172H / + animals with wild-type animals to obtain offspring of the Tp53 R172H / + animals.

[0017] In some embodiments, the Anxa10-Cre / + animal is an Anxa10-CreERT2 / + animal.

[0018] In some embodiments, the construction method further includes the step of administering N-methyl-N-nitrosourea to LSL-KrasG12D / +;Tp53R172H / +;Anxa10-Cre / + animals.

[0019] In some embodiments, the N-methyl-N-nitrosourea is administered via drinking water (preferably free access to drinking water).

[0020] In some embodiments, the N-methyl-N-nitrosourea is applied for 2-7 days per week for 8-18 weeks; further, for 2-4 days per week for 8-10 weeks; and even further, for 3 days per week for 8 weeks.

[0021] In some embodiments, the construction method further includes the step of administering a gene knockout activator to LSL-KrasG12D / +;Tp53R172H / +;Anxa10-Cre / + animals.

[0022] In some implementations, the gene knockout activator is applied before the N-methyl-N-nitrosourea is applied; that is, the gene knockout activator is applied first, followed by the application of N-methyl-N-nitrosourea.

[0023] In some embodiments, the gene knockout activator is tamoxifen.

[0024] In some embodiments, the gene knockout activator is administered over a period of 4-6 days.

[0025] In some embodiments, the gene knockout activator is administered at a dose of 50-100 mg / kg / day.

[0026] In some embodiments, the gene knockout activator is administered by injection (preferably intraperitoneal injection).

[0027] In a second aspect, the present invention provides a method for preparing gastric adenocarcinoma cells, comprising the following steps: isolating gastric adenocarcinoma tissue from a gastric adenocarcinoma model or its progeny obtained by the construction method of the first aspect of the present invention and culturing it in vitro to obtain gastric adenocarcinoma cells.

[0028] In some embodiments, the preparation method further includes the step of immortalizing the gastric adenocarcinoma cells.

[0029] In some embodiments, the immortalization process includes the step of infecting the gastric adenocarcinoma cells with an SV40 overexpressing virus (preferably a lentivirus).

[0030] A third aspect of the present invention provides a gastric adenocarcinoma cell line obtained by the preparation method of the second aspect of the present invention.

[0031] A gastric adenocarcinoma cell line, named ST-YC19 and classified as a mouse gastric adenocarcinoma cell line, was deposited on April 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46342, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0032] In a fourth aspect, the present invention provides a method for constructing an animal model of gastric adenocarcinoma, wherein gastric adenocarcinoma cells from the third aspect of the present invention are inoculated into an animal.

[0033] In some embodiments, the inoculation is performed by injection, such as in situ injection, intravenous injection, intraperitoneal injection, subcutaneous injection, intradermal injection, or intramuscular injection.

[0034] A fifth aspect of the invention provides any one of applications a1)-a2):

[0035] a1) The use of gastric adenocarcinoma cells of the third aspect of the present invention in any one of b1)-b6):

[0036] b1) Construct an animal model of gastric adenocarcinoma;

[0037] b2) Screening for drugs to prevent and / or treat gastric adenocarcinoma;

[0038] b3) To study the pathogenesis, development and / or metastasis mechanisms of gastric adenocarcinoma;

[0039] b4) Construct a gastric adenocarcinoma cell model;

[0040] b5) Screen for biomarkers related to gastric adenocarcinoma, or diagnostic and / or therapeutic targets;

[0041] b6) Prepare a product, said product being used in any one of b1)-b5);

[0042] a2) The use of the gastric adenocarcinoma model or its progeny obtained by the construction method of the first aspect of the present invention, and / or the gastric adenocarcinoma animal model or its progeny obtained by the construction method of the fourth aspect of the present invention in any of b2)-b6):

[0043] b2) Screening for drugs to prevent and / or treat gastric adenocarcinoma;

[0044] b3) To study the pathogenesis, development and / or metastasis mechanisms of gastric adenocarcinoma;

[0045] b4) Construct a gastric adenocarcinoma cell model;

[0046] b5) Screen for biomarkers related to gastric adenocarcinoma, or diagnostic and / or therapeutic targets;

[0047] b6) Prepare a product, said product being used in any one of b2)-b5).

[0048] In some embodiments, the product includes reagents or kits.

[0049] In some implementations, the application does not involve the diagnosis or treatment of diseases.

[0050] In this invention, the animal is a non-human animal.

[0051] In some embodiments, the non-human animal includes non-human mammals; further includes at least one of non-human primates, rodents, cattle (e.g., cows, bulls, buffalo), deer, pigs, sheep (e.g., sheep, goats), dogs, rabbits, chickens, cats, ferrets, and horses; and further includes rodents.

[0052] In some embodiments, the non-human primates include at least one of orangutans, apes, and monkeys (e.g., marmosets, rhesus monkeys).

[0053] In some embodiments, the rodent includes at least one of mice, rats, hamsters, and guinea pigs; more specifically, at least one of mice and rats.

[0054] In some embodiments, the rodent is selected from the rat family; further selected from the families Cricetidae (e.g., mouse-like hamster), Cricetidae (e.g., hamster, New World rat and mouse, vole), Muridae (true mouse and rat, gerbil, spiny rat, crested rat), Madagascar Muridae (climbing mouse, rock mouse, tailed rat, Madagascar rat and mouse), Spiny Dormicidae (e.g., spiny dormice), and Mole-like Muridae (e.g., mole rat, bamboo rat, and mole rat).

[0055] In some embodiments, the rodent is a C57BL strain of mouse, wherein the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, and 129 / Sv-ter / +. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant content of which is incorporated herein by reference in its entirety. In some embodiments, the mice are a cross between the 129 strain and the C57BL / 6 strain. In some embodiments, the mice are a cross between the 129 strain or the BL / 6 strain. In some embodiments, the mice are a BALB strain, such as the BALB / c strain. In some embodiments, the mice are a cross between the BALB strain and another strain. In some embodiments, the mice are derived from a hybrid line (e.g., 50% BALB / c - 50% 12954 / Sv; or 50% C57BL / 6 - 50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse with the strains BALB / c, BALB / cHeAn, BALB / cJ, BALB / cRl, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, or CBA / H, and a background of NOD, NOD / SCID, NOD-Prkdcscid IL-2rgnull.

[0056] In this invention, LSL-KrasG12D / + animals represent heterozygous animals possessing both the KrasG12D mutant and wild-type; similarly, Tp53 R172H / + animals represent heterozygous animals possessing both the Tp53 R172H mutant and wild-type; Anxa10-Cre / + animals represent heterozygous animals possessing both a mutant (with inserted Anxa10-Cre) and wild-type; LSL-KrasG12D / +;Tp53 R172H / + animals represent heterozygous animals possessing both the KrasG12D mutant and wild-type, and both the Tp53 R172H mutant and wild-type; LSL-KrasG12D / +;Tp53 R172H / +;Anxa10-Cre / + animals represent animals possessing both the KrasG12D mutant and wild-type, and both the Tp53 R172H mutant and wild-type; Heterozygous animals of the R172H mutant and wild type, and mutant (with insertion of Anxa10-Cre) and wild type.

[0057] In this invention, reproduction is mating.

[0058] In this invention, the gastric adenocarcinoma cells include their daughter cells.

[0059] The beneficial effects of this invention are:

[0060] This invention provides a method for constructing a gastric adenocarcinoma model, comprising the following steps: breeding Anxa10-Cre / + animals or their progeny with LSL-KrasG12D / +;Tp53 R172H / + animals or their progeny to obtain LSL-KrasG12D / +;Tp53 R172H / +;Anxa10-Cre / + animals, wherein the pathological type of the gastric adenocarcinoma model obtained by this method is determined to be adenocarcinoma.

[0061] Furthermore, the construction method also includes the step of applying N-methyl-N-nitrosourea, thereby further shortening the tumorigenesis cycle (approximately 2.5 months) and increasing the tumorigenesis rate (21 / 21, or 100%).

[0062] This invention provides a gastric adenocarcinoma cell line that can form tumors in vivo and is an adenocarcinoma. Furthermore, it exhibits strong proliferative, invasive, and / or migratory abilities. Attached Figure Description

[0063] Figure 1 shows a schematic diagram of the method for constructing mice with the genotypes Kras G12D / +; Tp53 R172H / +; Anxa10-CreERT2 / +.

[0064] Figure 2 shows the successful construction of mice with the genotypes Kras G12D / +; Tp53 R172H / +; Anxa10-CreERT2 / +: A shows the results of tail DNA electrophoresis gel identification of mouse Kras and Anxa10 genotypes; B shows the results of first-generation sequencing identification of mouse Tp53 and Kras genotypes.

[0065] Figure 3 shows the successful induction of a mouse model of gastric adenocarcinoma: A shows a schematic diagram of the induction process of the mouse model of gastric adenocarcinoma; B shows an anatomical diagram of the mouse in situ carcinoma (yellow arrow); C shows CT imaging confirming the occurrence of in situ gastric carcinoma in mice; D shows 18F-FDG-PET imaging confirming the occurrence of in situ gastric carcinoma in mice.

[0066] Figure 4 shows the induced mouse gastric orthotopic tumor model as adenocarcinoma, with a scale bar of 100 µm.

[0067] Figure 5 shows the immunofluorescence staining pattern of primary cells, with a scale bar of 100 µm.

[0068] Figure 6 shows that ST-YC19 can induce tumor formation in mice, specifically adenocarcinoma, with a scale bar of 100 µm.

[0069] Figure 7 shows the malignant phenotypes of ST-YC19 and common gastric cancer cells: A shows the cell morphology of ST-YC19 under bright field, with a scale bar of 100µm; B shows the migration and invasion abilities of ST-YC19, human gastric adenocarcinoma cell line HGC-27, and mouse gastric squamous cell carcinoma cell line MFC, with a scale bar of 200µm; C shows the proliferation ability of ST-YC19, human gastric adenocarcinoma cell line HGC-27, mouse gastric squamous cell carcinoma cell line MFC, and normal mouse gastric mucosal epithelial cells (**** indicates p < 0.0001 compared to ST-YC19). Detailed Implementation

[0070] The present invention will be further described in detail below through specific embodiments.

[0071] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0072] the term

[0073] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0074] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0075] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted. Examples

[0076] I. Construction of a mouse model of gastric adenocarcinoma

[0077] The inventors discovered key gene mutations in the development of gastric adenocarcinoma: TP53 mutation and RTK-KRAS pathway activation are the most critical alterations in gastric adenocarcinoma, characteristic of the most common molecular subtype of gastric cancer—chromosomal instability (CIN) gastric adenocarcinoma. TP53 (Trp53) and KRAS mutations are key events driving malignant transformation, enabling cells to proliferate rapidly and without restriction. Annexin A10 (ANXA10) belongs to the annexin (ANX) family and is specifically expressed in the gastric mucosa but absent in other normal organs. Therefore, LSL-KrasG12D / +Tp53R172H / + alleles and Anxa10-CreERT2 / + mice were first constructed, demonstrating that KrasG12D / + specifically occurs in the mouse gastric mucosa under tamoxifen induction. Specific operational details:

[0078] (1) Construction of Anxa10-CreERT2 / + mice:

[0079] C57BL / 6Smoc-Anxa10 em1(2A-CreERT2-Wpre-pA)Smoc Mice (purchased from Nanmo Biotechnology, catalog number NM-KI-200312) were mated with C57BL / 6J mice to obtain positive offspring mice, namely Anxa10-CreERT2 / + mice (Anxa10 CreERT2 Mice).

[0080] (2) Construction of Tp53R172H / + (Tp53em4) mice:

[0081] C57BL / 6Smoc-Trp53 em4(R172H)Smoc Mice (purchased from Nanmo Biotechnology, catalog number NM-KI-18028) were mated with C57BL / 6J mice to obtain positive offspring mice, namely Tp53R172H / + (Tp53em4) mice (P53). R172H Mice).

[0082] (3) Construction of LSL-KrasG12D / + (Krasem4) mice:

[0083] C57BL / 6Smoc-Kras em4(LSL-G12D)Smoc Mice (purchased from Nanmo Biotechnology, catalog number NM-KI-190003) were mated with C57BL / 6J mice to obtain positive offspring mice, namely LSL-KrasG12D / + (Krasem4) mice (LSL-Kras). G12D Mice).

[0084] (4) Construct mice with the genotype Kras G12D / +; Tp53 R172H / +; Anxa10-CreERT2 / +:

[0085] Figure 1 shows a schematic diagram of the method for constructing mice with the genotype Kras G12D / +; Tp53 R172H / +; Anxa10-CreERT2 / +. Specifically, Tp53R172H / + (Tp53em4) mice (P53...) were used... R172H Mice) and LSL-KrasG12D / + (Krasem4) mice (LSL-Kras G12D Mice (LSL-Kras) carrying the KrasG12D / + (Krasem4) and Tp53R172H / + (Tp53em4) alleles were obtained by crossing mice. G12D P53 R172H Mice) and Anxa10-CreERT2 / + mice (Anxa10 CreERT2 Mice were hybridized with mice, and mice with the genotype Kras G12D / +; Tp53 R172H / +; Anxa10-CreERT2 / + were selected from the hybrid offspring.

[0086] The following method was used to screen and obtain mice with the genotype Kras G12D / +; Tp53 R172H / +; Anxa10-CreERT2 / + from the hybrid progeny: DNA was extracted from the tail tissue of the hybrid progeny mice using a DNA extraction kit (TIANGEN, Beijing, China). Primer sequences for genotyping of the transgenic mice are shown in Tables 1 and 2. PCR amplification was performed using a 2x TransFast TaqPCR SuperMix (TransGen, Beijing, China). The thermal cycling conditions included pre-denaturation at 94°C for 3 minutes, followed by 35 cycles, each consisting of denaturation at 95°C for 5 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 35 seconds. A final extension at 72°C for 5 minutes was performed. PCR products were separated on a 1.2% agarose gel and visualized using nucleic acid staining (Gene-Protein Link, Beijing, China) to identify the mouse KRAS and Anax10 genotypes. Meanwhile, the P53 and KRAS genotypes were identified using first-generation sequencing, and the results are shown in Figure 2. Mice with the genotypes Kras G12D / +; Tp53 R172H / +; Anxa10-CreERT2 / + were selected from the results.

[0087] Table 1. PCR primers used for KrasG12D and Anxa10-CreERT2 genotyping.

[0088] Gene primer sequences: KrasG12D universal (forward primer) 5′-CTGCATAGTACGCTATACCCTGT-3′, SEQ ID NO:1 wild-type (reverse primer) 5′-TGTCTTTCCCCAGCACAGT-3′, SEQ ID NO:2 mutant (reverse primer) 5′-GCAGGTCGAGGGACCTAATA-3′, SEQ ID NO:3 Anxa10-CreERT2 universal (forward primer) 5′-ATCTCCCATCCTCCCTAACTT-3′, SEQ ID NO:4 wild-type (reverse primer) 5′-ATCGCATCACCTTCAAACTCT-3′, SEQ ID NO:5 mutant (reverse primer) 5′-AGCCCGGACCGACGATGAAGC-3′, SEQ ID NO:6 surface

[0089] Table 2 Sequencing primers used for KrasG12D and Tp53R172H genotyping

[0090] Gene primer sequences: KrasG12D forward primer 5′-CCATTAGCTGCTACAAAACA-3′, SEQ ID NO:7; reverse primer 5′-CTCTATCGTAGGGTCGTACT-3′, SEQ ID NO:8; Tp53R172H forward primer 5′-GAGGGCGTCCAATGGTGCTT-3′, SEQ ID NO:9; reverse primer 5′-CTAGGCTGGAGTCAACTGTC-3′, SEQ ID NO:10 surface

[0091] (5) Induction of a mouse model of gastric adenocarcinoma:

[0092] A schematic diagram of the induction process of a mouse model of gastric adenocarcinoma is shown in Figure 3A, as follows:

[0093] Six- to eight-week-old Kras G12D / +; Tp53 R172H / +; Anxa10-CreERT2 / + mice were transferred to the Laboratory Animal Center of Peking University Cancer Hospital and housed in a specific pathogen-free environment. The facility maintained a 12-hour light / dark cycle at 24°C. Mice food, water, and bedding were sterilized. Tamoxifen (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in corn oil (Aladdin, Shanghai, China) to a concentration of 20 mg / ml and incubated overnight at 37°C with shaking. The dissolved tamoxifen solution was stored at 4°C during injection. Tamoxifen was administered intraperitoneally once daily at a dose of 75 mg / kg for 5 days (days 1-5), based on the mice's body weight. Injection sites were disinfected with 70% ethanol before each injection. The experimental procedures followed protocols approved by the Ethics Committee of Peking University Cancer Hospital.

[0094] Following tamoxifen administration (day 6), N-methyl-N-nitrosourea (MNU) was used as an adjuvant inducer. The specific method was as follows: MNU solution was placed in a light-proof bottle and left as the long-term drinking water for mice on three days each week (Monday, Wednesday, and Friday). This treatment continued for eight weeks. The MNU solution (Aladdin, Shanghai, China) was freshly prepared with distilled water at a concentration of 120 ppm. During the tamoxifen injection, MNU administration, and observation periods, the mice were closely monitored to observe for tumor formation and any adverse reactions. After eight weeks of MNU treatment, a mass was palpable in the left upper abdomen of the mice. 18F-FDG-PET / CT confirmed the in situ tumor formation (Figure 3, C and D). Dissection of the mice revealed an in situ gastric tumor (Figure 3, B), which was identified as gastric adenocarcinoma by HE staining (Figure 4), indicating in situ gastric adenocarcinoma in mice.

[0095] It is evident that the tumorigenesis cycle of the mouse gastric adenocarcinoma model provided by this invention is shortened (approximately 2.5 months), which is lower than other model construction methods: 13 months (MNU, Cancer Letters. 2002 May 28;179(2):121-32); 12 months (Tp53 R270H / + combined with MNU, Nature Genetics. 2020 Feb 5;52(2):219–230); 5 months (Tp53R172H / +; KrasG12D / +, Gastroenterology. 2023 Feb 4;164(7):1119–1136.e12); and the tumorigenesis rate is significantly improved (21 / 21, i.e., 100%), which is higher than other model construction methods: 31.8% (MNU, Cancer Letters. 2002 May 28;179(2):121-32); 63% (Tp53 R270H / + in combination with MNU, Nature Genetics. 2020 Feb5;52(2):219–230.

[0096] II. Construction of an immortalized mouse gastric adenocarcinoma cell line capable of in vivo tumor formation

[0097] After confirming in situ tumor formation in mice via 18F-FDG-PET / CT imaging, the mice were euthanized by cervical dislocation and disinfected by immersion in 75% alcohol. The mice were then dissected in a sterile operating table, and tumors were harvested. A portion of the tumor was fixed in 4% paraformaldehyde, embedded, and histopathologically identified by HE staining. The results are shown in Figure 4: the induced in situ gastric tumor model in mice was adenocarcinoma; a portion was cut into 1mm pieces. 3 Small pieces were implanted subcutaneously in C57 mice. When the tumor grew to 500 mm... 3 Mice were euthanized by cervical dislocation. Tumor tissue was excised in a sterile operating table, placed in phosphate-buffered saline (PBS), and washed twice to carefully remove any connective tissue. One portion of the tissue was fixed in 4% paraformaldehyde, embedded, and hematoxylin and eosin (HE) stained to identify the pathological type of the tumor tissue. Another portion of the tumor tissue was cut into small pieces of approximately 1 mm³, washed twice with PBS, and 1% type IV collagenase was added. The mixture was gently mixed with a pipette and incubated in a 37°C shaker water bath for 1 hour. Digestion was terminated by adding twice the volume of complete culture medium containing 10% fetal bovine serum (FBS). The sample was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in epithelial cell-specific complete culture medium to prepare a cell suspension. The suspension was centrifuged at 2 × 10⁻⁶... 5Cells were seeded at a density of 5 mL / mL into T25 culture flasks coated with Matrigel. The flasks were incubated at 37°C with 5% CO2, with the culture medium changed every 2-3 days. Once the cells reached >80% confluence, they were passaged. The immunofluorescence staining results of the primary cells are shown in Figure 5: the cells expressed CK-19, indicating they were gastric adenocarcinoma cells. To achieve cell immortalization, the cells were transfected with SV40-overexpressing lentivirus: approximately 1 × 10⁶ cells were seeded into 6-well plates, with each well containing approximately 1 × 10⁶ cells. 5 Cells were collected. The next day, after cell attachment, the culture medium was changed. Then, 1 mL of complete culture medium and 20 μL of SV40 overexpressing lentivirus (Mason cells CTCC-DZ-0120, infection concentration MOI=10) were added and gently mixed. The cells were incubated. After 12 hours, the cell status was checked, and the culture medium was replaced with fresh complete culture medium. When the cells reached confluence at the bottom of the well, they were passaged into T25 culture flasks. After 6 passages, the cell line was confirmed and named ST-YC19. It was classified as a mouse gastric adenocarcinoma cell line and deposited on April 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46342, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The bright-field image of ST-YC19 is shown in Figure 7A.

[0098] To verify that ST-YC19 can induce tumor formation in mice, ST-YC19 cells were cultured in vitro, counted, and resuspended in PBS solution at a concentration of 1×10⁻⁶ cells / mL. 6 Cells were injected into the rib and abdomen of mice at a dose of 1 cell per mouse, and tumor formation was observed regularly. The tumor grew to 500 mm. 3 HE staining was then performed to observe the pathological type. The results are shown in Figure 6: ST-YC19 can induce tumor formation in mice, and the tumor is adenocarcinoma.

[0099] III. Comparison of malignant phenotypes between the immortalized mouse gastric adenocarcinoma cell line ST-YC19 and common gastric cancer cells.

[0100] The CCK-8 assay (Dongren, Japan) was used to compare ST-YC19 (experimental group), human gastric adenocarcinoma cell line (human gastric cancer cells, HGC-27, purchased from the National Experimental Cell Resource Sharing Service Platform, number 3101HUMTCHu22, control group), mouse gastric squamous cell carcinoma cell line (mouse pregastric cancer cells, MFC, purchased from the National Experimental Cell Resource Sharing Service Platform, number 1101MOU-PUMC000143, control group), and normal mouse gastric epithelial cells (mouse gastric mucosal epithelial cells, Epithelial, purchased from Shanghai...). The proliferation rate of cells (Haifuheng Biotechnology, catalog number: FH-M110, blank group) was measured as follows: Cells in the proliferation phase (ST-YC19, HGC-27, MFC, Epithelial) were digested with trypsin and counted. They were divided into blank, control, and experimental groups, with 6 replicates per group. Time points of 24h, 48h, 72h, and 96h were also set. The total cell count and total culture medium volume were calculated based on the requirement of 3000 cells and 100μL of culture medium per well. Cell suspension was prepared in centrifuge tubes and gently pipetted to mix. The cell suspension was transferred to a sample well, and 100μL of cell suspension was pipetted into the center of each well of a 96-well plate. 100μL of PBS was added to the remaining wells. The plates were incubated at 37℃. Eight hours later, cell adhesion was observed under a microscope. With most cells adhered, CCK-8 reagent was diluted with serum-free medium (1 mL of medium to 100 μL of CCK-8). The medium was removed from the 96-well plate, and 100 μL of diluted CCK-8 was added. The plate was then incubated in a cell culture incubator for 1 hour, and absorbance was measured at 450 nm using a microplate reader. Simultaneously, cell status at other time points was observed periodically under a microscope, and the medium was changed accordingly. Absorbance was measured using a microplate reader as planned. Cell proliferation curves at each time point were plotted using Graphpad Prism 8.0 software. The results are shown in Figure 7C: ST-YC19 showed significantly better proliferation ability than human gastric adenocarcinoma cell line HGC-27, mouse gastric squamous cell carcinoma cell line (MFC), and normal mouse gastric epithelial cells.

[0101] The migration and invasion abilities of ST-YC19 (experimental group), human gastric adenocarcinoma cell line HGC-27 (control group), and mouse gastric squamous cell carcinoma cell line (control group) were compared using Transwell technology, as follows:

[0102] Migration assay: Transwell chambers were placed in 24-well plates. 200 μL and 500 μL of serum-free medium were added to the upper and lower chambers, respectively. The plates were then incubated for 30 minutes to equilibrate, after which the equilibration medium was discarded. Cells were digested with trypsin and centrifuged. Cells were then resuspended in serum-free medium and counted using a cell counting chamber. Experimental and control groups were established, with the following time points: 24 hours, three chambers at each time point. The required cell density for each chamber was 5 × 10⁶ cells / well. 4 Calculate the required total number of cells and total volume of serum-free medium for each chamber, using 200 μL of serum-free medium per chamber. Prepare the cell suspension in a centrifuge tube and gently pipette to mix. Add 500 μL of complete medium to the lower chamber and slowly add 200 μL of cell suspension to the upper chamber. Incubate at 37°C. At the corresponding time point after 24 hours, remove the chambers, wash gently with PBS, and fix with ice-cold methanol for 10 minutes; wash again with PBS and air dry, stain with crystal violet for 5 minutes, wash away the crystal violet with PBS, and air dry. Carefully cut the membrane with a blade, place it on a glass slide, and mount with mounting adhesive. After air drying, photograph the membrane. Photograph three fields of view from each chamber. Use ImageJ software to count the number of migrating cells and use Graphpad software to create a bar chart.

[0103] Invasion Assay: Dilute Matrigel 1:8 with PBS buffer. Take 60 μL of the diluted Matrigel and quickly and evenly spread it on the upper surface of the bottom of a Transwell chamber. Incubate the Transwell chamber at 37°C overnight to allow the Matrigel to solidify into a thin film. The next day, aspirate the liquid from the upper chamber and add 100 μL of serum-free culture medium to each well. Return the Transwell chamber to the incubator and incubate for another 30 minutes to allow for basement membrane hydration. The remaining steps are the same as the cell migration assay, except that the required cell density for each chamber is 8 × 10⁶ cells / well. 4 .

[0104] The results are shown in Figure 7B: ST-YC19 showed significantly better migration ability than human gastric adenocarcinoma cell line HGC-27 and mouse gastric squamous cell carcinoma cell line. ST-YC19 also showed significantly better invasion ability than mouse gastric squamous cell carcinoma cell line. Although ST-YC19 did not show significantly better invasion ability than human gastric adenocarcinoma cell line HGC-27, it was still better than mouse gastric squamous cell carcinoma cell line.

[0105] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for constructing a mouse model of gastric adenocarcinoma, comprising the following steps: breeding Anxa10-Cre / + mice with LSL-KrasG12D / +;Tp53 R172H / + mice to obtain LSL-KrasG12D / +;Tp53 R172H / +;Anxa10-Cre / + mice; administering a gene knockout activator and N-methyl-N-nitrosourea to the LSL-KrasG12D / +;Tp53 R172H / +;Anxa10-Cre / + mice; wherein, The gene knockout activator is applied before the application of N-methyl-N-nitrosourea; the gene knockout activator is tamoxifen; the application time of the gene knockout activator is 4-6 days; the application method of N-methyl-N-nitrosourea is: apply 2-7 days a week for 8-18 weeks.

2. The construction method according to claim 1, characterized in that, The method for constructing the LSL-KrasG12D / +;Tp53 R172H / + mouse includes the following steps: breeding LSL-KrasG12D / + mice with Tp53 R172H / + mice to obtain LSL-KrasG12D / +;Tp53 R172H / + mice.

3. A method for preparing gastric adenocarcinoma cells, comprising the following steps: isolating gastric adenocarcinoma tumor tissue from a mouse model of gastric adenocarcinoma obtained by the construction method according to any one of claims 1-2 and culturing it in vitro to obtain gastric adenocarcinoma cells.

4. The preparation method according to claim 3, characterized in that, The preparation method further includes the following step: immortalizing the gastric adenocarcinoma cells.

5. A type of gastric adenocarcinoma cell, characterized in that, The gastric adenocarcinoma cells are named ST-YC19 and classified as a mouse gastric adenocarcinoma cell line. They were deposited on April 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46342. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

6. A method for constructing a mouse model of gastric adenocarcinoma, comprising inoculating mice with the gastric adenocarcinoma cells described in claim 5.

7. The construction method according to claim 6, characterized in that, The method of inoculation is injection.

8. The application of the gastric adenocarcinoma cells according to claim 5 in any one of b1)-b6): b1) constructing a mouse model of gastric adenocarcinoma; b2) screening drugs for the prevention and / or treatment of gastric adenocarcinoma; b3) studying the pathogenesis, development and / or metastasis mechanism of gastric adenocarcinoma; b4) constructing a gastric adenocarcinoma cell model; b5) screening gastric adenocarcinoma-related biomarkers, or diagnostic and / or therapeutic targets; b6) preparing a product, wherein the product is used in any one of b1)-b5); the applications in b2), b3), and b5) do not involve the diagnosis or treatment of diseases.

9. The application of the gastric adenocarcinoma mouse model obtained by the construction method according to any one of claims 1-2, and / or the gastric adenocarcinoma mouse model obtained by the construction method according to any one of claims 6-7, in any one of b2)-b6): b2) screening drugs for the prevention and / or treatment of gastric adenocarcinoma; b3) studying the pathogenesis, development and / or metastasis mechanism of gastric adenocarcinoma; b4) constructing a gastric adenocarcinoma cell model; b5) screening gastric adenocarcinoma-related biomarkers, or diagnostic and / or therapeutic targets; b6) preparing a product, said product being used in any one of b2)-b5); the applications described in b2), b3), and b5) do not involve the diagnosis or treatment of the disease.

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