A mouse gallbladder cancer cell line mGBC1-ZH and a construction method and application thereof

Abstract

The application discloses a mouse gallbladder cancer cell line mGBC1-ZH and a construction method and application thereof, and belongs to the technical field of tumor cell lines and tumor model preparation. The mouse gallbladder cancer cell line mGBC1-ZH is preserved in the China Center for Type Culture Collection on March 6, 2025, and the preservation number is CCTCC NO: C202585. The cell line has a high proliferation capacity, can be stably passed in vitro, can form subcutaneous tumors and orthotopic tumors in a mouse in vivo with complete immune function, has similar pathological characteristics to human gallbladder cancer, is sensitive to standard chemotherapy drugs for gallbladder cancer, and can be used for mechanism research and drug evaluation of gallbladder cancer, and provides a valuable experimental tool for basic and clinical research of gallbladder cancer.

Description

Technical Field

[0001] This invention belongs to the field of tumor cell line and tumor model preparation technology, specifically relating to a mouse gallbladder cancer cell line mGBC1-ZH and its construction method and application. Background Technology

[0002] Gallbladder cancer (GBC) is a rare but highly malignant cancer originating in the mucosal layer of the gallbladder. Despite its rarity, GBC accounts for 80-95% of all biliary system cancers, ranking sixth among digestive system malignancies worldwide, and has a poor prognosis. The current state of GBC treatment is particularly concerning. While surgical resection is the only potentially long-term survival method, less than 20% of patients are eligible for this intervention. Even with the latest chemotherapy and immunotherapy approaches, the median survival for GBC patients remains as low as 12.8 months, highlighting the urgent need to develop more effective treatment strategies.

[0003] Limited progress in gallbladder cancer treatment stems primarily from insufficient understanding of its biological complexity, largely attributed to the lack of well-developed animal models. Currently, human gallbladder cancer cell lines remain the most widely used research models in both in vitro and in vivo studies. Patient-derived xenografts (PDXs) and tumor organoid cultures retain the genetic heterogeneity of the original tumor, playing a crucial role in drug response assessment. However, these models still have significant limitations: firstly, they can only be studied in immunodeficient mice, preventing the investigation of the tumor immune microenvironment and immunotherapy response; secondly, these models originate from advanced malignant tumors with complex genetic backgrounds, providing limited information for studying the continuous process of tumorigenesis and progression. Mouse-derived gallbladder cancer cell lines can be grown in immunocompetent mice, offering significant advantages in tumor-immune interaction studies. Currently, only one mouse gallbladder cancer cell line, A2, has been reported domestically and internationally (Cancer Res (2008) 68 (9_Supplement): 2949.). A2 is derived from a transgenic mouse that overexpresses ErbB2 in the biliary epithelium under the control of the bovine keratin 5 promoter, resulting in tumors in both the gallbladder and bile ducts (Cancer Res. 2001 Oct 1;61(19):6971-6.). However, further research using this cell line has been very limited.

[0004] Recent genomic analyses have revealed a complex array of molecular alterations in gallbladder cancer, including repetitive alterations in key tumor suppressor genes (TP53, SMAD4, KMT2C, ARID1A) and oncogenes (KRAS, PIK3CA, ERBB2, CTNNB1). Of particular note are the early occurrences of TP53 and KRAS mutations, suggesting their fundamental role in malignant transformation. However, the exact mechanisms by which these genetic alterations drive gallbladder cancer development remain unclear due to the lack of suitable experimental models capable of reproducing the early stages of the disease.

[0005] Therefore, constructing a domestically developed gallbladder cancer cell line with a clear genetic background, stable long-term passage capability, and the ability to grow in immune-intact mice can provide a more effective platform for the development of gallbladder cancer-related immunotherapies. Summary of the Invention

[0006] In view of this, the present invention provides a mouse gallbladder cancer cell line mGBC1-ZH, wherein the mouse gallbladder cancer cell line mGBC1-ZH is generated by using a carrier… Kras Activation and Trp53 A cell line was established from a mouse gallbladder organoid culture containing a deletion mutation, through in vitro culture and in vivo passage. This cell line exhibits high proliferative capacity and can form subcutaneous tumors and in situ tumors in immunocompetent mice, with pathological features similar to human gallbladder cancer. Transcriptomic analysis revealed that this cell line activates the PI3K-Akt and MAPK signaling pathways, and genomic analysis showed copy number variations on multiple chromosomes. This cell line can be stably passaged in vitro and is sensitive to standard chemotherapeutic agents for gallbladder cancer, making it a valuable experimental tool for studying the mechanisms and evaluating drugs in gallbladder cancer, and providing a valuable experimental tool for basic and clinical research on gallbladder cancer.

[0007] This invention includes the following technical solutions: In a first aspect, the present invention provides a mouse gallbladder cancer cell line, named mGBC1-ZH, which was deposited at the China Center for Type Culture Collection on March 6, 2025, with accession number CCTCCNO:C202585.

[0008] Furthermore, the mouse gallbladder cancer cell line provided by the present invention also includes daughter cells of the preserved cell line.

[0009] In a second aspect, the present invention provides a method for constructing the mouse gallbladder cancer cell line mGBC1-ZH, the method comprising the following steps: a) From genotype Kras LSL-G12D / Trp53 fl / flGallbladder epithelial tissue was isolated from C57BL / 6J mice and cultured to obtain organ-like cultures; b) Transfect the organ-like culture with a lentivirus expressing Cre recombinase and EGFP to activate it. Kras G12D Expression and absence Trp53 , obtained genotype Kras G12D / Trp53 - / - Organ-like cultures; c) The genotype is Kras G12D / Trp53 - / - Organoid-like cultures were passaged into a two-dimensional culture system and then injected subcutaneously into homologous wild-type mice. d) The isolated tumor tissue was digested into single cells and cultured in vitro to obtain the mouse gallbladder cancer cell line mGBC1-ZH.

[0010] In step a), the method for obtaining organ-like cultures from mouse gallbladder epithelial tissue includes: a.1) The tissue was cut into fragments and transferred to pre-cooled DMEM / F12 medium containing 1% BSA. The cells were digested with Dispase containing 10 μM Y27632, centrifuged, and the cell pellet was resuspended in mouse gallbladder organoid medium (mGOM) containing 10 μM Y27632. a.2) Mix the cell suspension with pre-cooled Matrigel containing growth factors on ice, plant it in the center of a low-adhesion 24-well plate, and incubate at 37°C and 5% CO2 until the Matrigel solidifies. a.3) Each droplet was then covered with mGOM medium containing 10 μM Y27632. After 24 hours, it was replaced with fresh mGOM medium without Y27632. The medium was replaced every 2-3 days. a.4) After the initial isolation, culture for 7-10 days, and then passage by mechanical separation every 5-7 days to obtain organ-like cultures.

[0011] In a specific embodiment of the present invention, the lentivirus expressing Cre recombinase and EGFP described in step b) is selected from VSVG-LENTAI-UbiC-Cre-EGFP-WPRE-pA (LTR) lentivirus. Those skilled in the art can transduce the lentivirus into organ-like cultures using conventional methods.

[0012] In one specific embodiment of the present invention, the transduction method includes the following steps: b.1) Digest the organoid culture from step a) into single cells using trypsin solution and resuspend them in mGOM medium; b.2) Containing 1×10 ^5 200 μl of culture medium per cell was incubated with VSVG-LENTAI-UbiC-Cre-EGFP-WPRE-pA (LTR) lentivirus at 37°C and 5% CO2. Gene modification was then performed on the organ-like culture to obtain the genotype […]. Kras G12D / Trp53 - / - Organ-like cultures; b.3) Following the methods described in steps a.2-a.4), the genotype is... Kras G12D / Trp53 - / - Organ-like cultures were cultured in Matrigel for passage.

[0013] Step c) of the present invention describes the process of transferring the genotype to... Kras G12D / Trp53 - / - Specific methods for two-dimensional culture of organ-like cultures include: culturing genotypes of... Kras G12D / Trp53 - / - The organoid cultures were passaged five times in Matrigel, digested into single cells, and then cultured in 12-well plates for two-dimensional culture. After 15 passages of two-dimensional culture, the cells were injected subcutaneously into mice to form tumors.

[0014] The two-dimensional culture comprises two stages: 1) passage three times in medium 1; 2) transferring cells to medium 2 and passage twelve times. Medium 1 is a 1:1 mixture of mGOM medium and DMEM / F12 containing 10% fetal bovine serum. Medium 2 is DMEM containing 10% fetal bovine serum.

[0015] In step d) of this invention, the enzyme used to digest the tumor tissue is selected from a combination of type I collagenase, type IV collagenase, and DNase I. Those skilled in the art can culture tumor cells in vitro using conventional methods. In one specific embodiment of this invention, the in vitro culture method includes culturing the cells for 20 generations using DMEM medium containing 10% fetal bovine serum to obtain the mouse gallbladder cancer cell line mGBC1-ZH.

[0016] In a third aspect, the present invention provides an application of the mouse gallbladder cancer cell line mGBC1-ZH in at least one of the following: 1) Application in the preparation of gallbladder cancer cell models; 2) Application in the preparation of animal models of gallbladder cancer; 3) Application in the preparation and / or screening of drugs for the prevention and treatment of gallbladder cancer; 4) Application in establishing a research platform for the mechanism of drug resistance in gallbladder cancer; 5) Application in establishing a research platform for early screening and grading methods for gallbladder cancer; 6) Application in establishing a research platform for gallbladder cancer treatment methods.

[0017] The cell and animal models described in this invention include cell or animal models of the occurrence, development, and metastasis stages of gallbladder cancer, which can be used to study the mechanisms of gallbladder cancer occurrence, development, and metastasis.

[0018] In a fourth aspect, the present invention provides a method for constructing a mouse model of gallbladder cancer, the method comprising implanting the mouse gallbladder cancer cell line mGBC1-ZH described in the first aspect of the present invention into mice and feeding them for 7-28 days to form a mouse model of gallbladder cancer.

[0019] The gallbladder cancer mouse models include gallbladder cancer subcutaneous tumor mouse models, gallbladder cancer in situ tumor mouse models, or gallbladder cancer metastatic tumor mouse models.

[0020] In this invention, the mice used to construct the mouse model are selected from immunodeficient mice or immunonormal mice. Immunonormal mice are preferred.

[0021] In a specific embodiment of the present invention, the mice used to construct the mouse model are homologous wild-type mice, namely immune-normal C57BL / 6J mice.

[0022] In one specific embodiment of the present invention, the mouse model is a subcutaneous gallbladder cancer mouse model. The method for constructing this model includes implanting the mouse gallbladder cancer cell line mGBC1-ZH described in the first aspect of the present invention subcutaneously into mice, feeding them for one week, resulting in the formation of a palpable tumor subcutaneously. Those skilled in the art can adjust the number of implanted cells according to actual circumstances.

[0023] In another specific embodiment of the present invention, the mouse model is a mouse model of gallbladder carcinoma in situ. The method for constructing the model includes: 1) fasting mice for 4 hours before surgery and then anesthetizing them; 2) suspending the mouse gallbladder cancer cell line mGBC1-ZH described in the first aspect of the present invention in DPBS and mixing it with Matrigel; 3) exposing the gallbladder, puncturing it, squeezing out the bile and cleaning it, injecting the cell suspension into the gallbladder using a syringe, and withdrawing the syringe after the suspension solidifies; 4) suturing the abdominal wall, feeding for 4 weeks, and forming a solid in situ tumor in the gallbladder.

[0024] Fifthly, the present invention provides a mouse model of gallbladder cancer constructed according to the method described in the fourth aspect of the present invention.

[0025] Sixthly, the present invention provides an application of the described gallbladder cancer mouse model in at least one of the following: 1) Application in the preparation and / or screening of drugs for the prevention and treatment of gallbladder cancer; 2) Used as an animal model for the preparation and / or screening of drugs for the prevention and treatment of gallbladder cancer; 3) Application in establishing a research platform for the mechanism of drug resistance in gallbladder cancer; 4) Application in establishing a research platform for early screening and grading methods for gallbladder cancer; 5) Application in establishing a research platform for gallbladder cancer treatment methods.

[0026] In a seventh aspect, the present invention provides a method for preparing or screening candidate drugs for the prevention and / or treatment of gallbladder cancer, said method being selected from at least one of the following: i) The candidate drug is applied to the gallbladder cancer cell model, and the candidate drug that inhibits cell proliferation or causes other programmed cell death is the effective drug, wherein the gallbladder cancer cell model is the mouse gallbladder cancer cell line mGBC1-ZH described in the first aspect of the present invention; ii) The candidate drug is administered to an animal model of gallbladder cancer, and the animal's body weight and tumor growth are monitored. The candidate drug that causes improvement or cure of gallbladder cancer symptoms in the animal model is an effective drug. The animal model of gallbladder cancer is constructed by the method described in the fourth aspect of the present invention.

[0027] Wherein, the method of administering the test substance described in method ii) is a drug administration method commonly used by those skilled in the art, including but not limited to one or a combination of two or more of the following: tail vein injection, intraperitoneal injection, gavage, subcutaneous injection, intramuscular injection, or local tumor administration.

[0028] The technical solution provided by this invention has the following beneficial technical effects: 1. The mouse gallbladder cancer cell line mGBC1-ZH constructed in this invention can grow in immune-intact homologous wild-type mice, and can better mimic the tumor microenvironment and immune response of gallbladder cancer.

[0029] 2. The mouse gallbladder cancer cell line mGBC1-ZH constructed in this invention is established through a clear gene driver, namely Kras activation and Trp53 deletion, which is consistent with the main driver genes of human gallbladder cancer and has good clinical relevance.

[0030] 3. The mouse gallbladder cancer cell line mGBC1-ZH constructed in this invention can be used to construct subcutaneous and in situ gallbladder cancer models, providing a reliable platform for mechanism research and drug evaluation of gallbladder cancer.

[0031] 4. Verification shows that the transcriptome and genomic characteristics of the mouse gallbladder cancer cell line mGBC1-ZH constructed in this invention are highly similar to those of human gallbladder cancer, better reflecting the molecular characteristics of the disease. Moreover, the cell line is stably passaged in vitro, which facilitates experimental operation and result reproducibility. Attached Figure Description

[0032] Figure 1 This represents EGFP expression after organoid transfection with virus.

[0033] Figure 2 The images show the morphology and immunohistochemical staining results of normal gallbladder organoids and transfected organoids.

[0034] Figure 3 Morphological image of the mGBC1-ZH cell line in vitro.

[0035] Figure 4 Immunohistochemical staining results for cell lines CK7, CK19, and EpCAM.

[0036] Figure 5 This refers to tumors formed subcutaneously by mGBC1-ZH cells in homologous wild-type mice.

[0037] Figure 6 Immunohistochemical staining results of CK19, CD31, CD3 and CD20 in subcutaneous tumor tissue.

[0038] Figure 7 This is an in situ tumor formed by mGBC1-ZH cells in the gallbladder of a homologous wild-type mouse.

[0039] Figure 8 The results of H&E and immunohistochemical staining of the in situ tumor tissue are shown.

[0040] Figure 9 The results of chromosome karyotype analysis for the mGBC1-ZH cell line.

[0041] Figure 10 The cell line exhibits proliferation curves, colony formation, and Transwell migration ability.

[0042] Figure 11 This study examines the expression of biliary system markers in transcriptomic analysis.

[0043] Figure 12 KEGG plot showing differential gene expression in the mGBC1-ZH cell line compared to normal gallbladder epithelial cells.

[0044] Figure 13 This is a somatic mutation of the mGBC1-ZH cell line relative to normal gallbladder epithelial cells.

[0045] Figure 14 This is a graph showing the chromosome copy number variation of the mGBC1-ZH cell line relative to normal gallbladder epithelial cells.

[0046] Figure 15 gemcitabine and cisplatin IC50 for mGBC1-ZH cell line 50 curve.

[0047] Figure 16 This study compares different drug treatments after subcutaneous tumor formation in the mGBC1-ZH cell line. Detailed Implementation

[0048] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Terminology Explanation Kras LSL-G12D This represents a conditional point mutation in the KRAS gene. In the label, "LSL" stands for "Lox-stop-lox," a gene expression regulatory system used to control the spatiotemporally specific expression of genes; while "G12D" refers to the mutation of glycine (Gly) to aspartic acid (Asp) at codon 12 of the KRAS gene, which is one of the most common oncogenic mutations in the KRAS gene.

[0050] Example 1: Establishment of the mouse gallbladder cancer cell line mGBC1-ZH (1) Establishment of gallbladder organoid culture from Kras LSL-G12D / Trp53 fl / flGallbladder tissue was isolated from male C57BL / 6J mice (gifted by He Rui's group from the School of Basic Medical Sciences, Fudan University) and washed with pre-chilled PBS. The tissue was cut into fragments of approximately 0.5-1 mm³ and transferred to ice-cold DMEM / F12 medium containing 1% BSA. The fragments were digested by shaking in a dispase solution containing 10 μM Y27632 at 37°C for 15-20 minutes. The cell suspension was centrifuged at 300g for 5 minutes at 4°C, and the pellet was washed with 10 ml of pre-chilled DMEM / F12 medium. After removing the supernatant, the cell pellet was resuspended in 100 μl of mouse gallbladder organoid culture medium (mGOM) containing 10 μM Y27632. Cell suspension was mixed with 100 μL of cold Matrigel containing growth factors on ice. 50 μL of the mixture (containing 2,000 cells) was seeded in the center of each well of a low-adhesion 24-well plate and incubated at 37°C and 5% CO2 for approximately 20 minutes until the Matrigel solidified. Each droplet was then covered with 500 μL of mGOM medium containing 10 μM Y27632. After 24 hours, the medium was replaced with fresh mGOM medium without Y27632. The medium was changed every 2–3 days. Organoid cultures were cultured for 7–10 days after initial isolation, followed by passage every 5–7 days using a P200 pipette tip via mechanical separation.

[0051] (2) Gene modification of gallbladder organoid cultures The established gallbladder organoid culture was digested into single cells using trypsin solution; 1×10 ^5 200 μl of mGOM medium per cell was incubated with VSVG-LENTAI-UbiC-Cre-EGFP-WPRE-pA (LTR) lentivirus at 37°C and 5% CO2 for 12 hours to genetically modify gallbladder organoid cultures and introduce... Kras Activation and Trp53 Missing. Transduced cells were re-seeded in Matrigel cells using the organoid culture method described above. Successfully transduced organoid cultures expressed EGFP fluorescence (e.g., within 48 hours). Figure 1 (As shown).

[0052] The transduced organoid cultures formed irregular cystic structures with thicker walls than normal organoid cultures. Immunohistochemical analysis showed that the modified organoid cultures maintained CK7 and CK19 expression, but Ki-67 staining was enhanced compared to normal organoid cultures, indicating enhanced proliferative capacity (e.g., ...). Figure 2 (As shown).

[0053] (3) The transduced gallbladder organoid culture was cultured in two dimensions and then subcutaneously implanted into mice. After transduction, the organoids were passaged five times. The modified gallbladder organoid cultures were digested into single cells and seeded in 12-well plates for two-dimensional culture in a 1:1 mixture of mGOM and DMEM / F12 supplemented with 10% fetal bovine serum. After three passages, the cells were transferred to DMEM medium supplemented with 10% fetal bovine serum for further culture. After 15 passages of two-dimensional culture, the cells were injected subcutaneously into mice.

[0054] (4) Isolate tumor tissue and obtain cell lines through in vitro culture. Two weeks after injection, subcutaneous tumors formed. The cells were digested with 1 mg / ml type I collagenase, 1 mg / ml type IV collagenase, and 0.5 mg / ml DNase I. The isolated cells were then cultured for 20 generations in DMEM supplemented with 10% fetal bovine serum. Mycoplasma contamination was detected weekly during the culture process.

[0055] Initially separated epithelial cells are polygonal and tightly connected, containing many multinucleated cells. During passage, the cells gradually transform into elongated spindle shapes, indicating the occurrence of epithelial-mesenchymal transition (EMT). Figure 3 (As shown). Immunofluorescence showed that the cells expressed CK7, CK19, and EpCAM, confirming their epithelial origin. Strong Ki-67 expression indicated that the cells had a high proliferation rate (e.g., ...). Figure 4 As shown in the figure, this newly established cell line was named mGBC1-ZH.

[0056] Example 2: Tumorigenicity of mGBC1-ZH cell line in homologous wild-type mice To evaluate the tumorigenic capacity of the mGBC1-ZH cell line under an intact immune system, this invention involved subcutaneous injection of the cells into homologous wild-type mice. In all 10 test mice, these cells formed palpable tumors within one week, demonstrating their strong tumorigenic potential (e.g., Figure 5 (As shown). Histochemical analysis of the subcutaneous tumor showed CK19 expression, confirming its origin in gallbladder epithelium. Abundant CD31-positive vascular structures were observed in the tumor, indicating extensive angiogenesis, which may support the high metabolic demands of these rapidly proliferating cells. Tumor immune microenvironment analysis showed that CD3+ T cells and CD20+ B cells aggregated at the tumor periphery (e.g., ...). Figure 6 (As shown). Notably, these immune cells are sparsely distributed in the tumor core region, indicating a "cold" tumor microenvironment, a common feature of human gallbladder cancer.

[0057] This invention further evaluated the in situ tumorigenicity of the mGBC1-ZH cell line. Mice were anesthetized with tribromoethanol after fasting for 4 hours before surgery. 2×10 ^5Cells were suspended in 10 μl of DPBS and mixed with 10 μl of Matrigel. The gallbladder was exposed through a 0.8–1.0 cm midline abdominal incision and punctured with a 31G insulin syringe. After squeezing out the bile and cleaning, 20 μl of the cell suspension was injected into the gallbladder using a 29G insulin syringe. The syringe was withdrawn after the suspension solidified. The abdominal wall was sutured, and the mice were placed on a 37°C heating pad for recovery. The mice were monitored daily during the first week, and then every three days thereafter. After four weeks, the animals were sacrificed, and dissection revealed the formation of a solid in situ tumor in the gallbladder (e.g., ...). Figure 7 (As shown). Pathological histological analysis confirmed it to be moderately differentiated adenocarcinoma with liver invasion. Consistent with subcutaneous tumors, the in situ tumor maintained CK19 expression, and Ki-67 levels were higher compared to normal gallbladder tissue (e.g., ...). Figure 8 (As shown).

[0058] Example 3: Characterization of the biological characteristics of the mGBC1-ZH cell line After confirming its tumorigenic capacity in vivo, the mGBC1-ZH cell line was comprehensively characterized in this invention. Eighteen short tandem repeat (STR) sites were amplified using multiplex PCR. An additional marker (Human TH01) was added to detect the presence of the human species (as shown in Table 1).

[0059] Table 1. STR analysis results of the mGBC1-ZH cell line The results confirmed that the cell line was of mouse origin. Karyotype analysis revealed significant chromosomal instability, manifested as numerical and structural abnormalities. Chromosomal analysis showed counts ranging from 53 to 80, with most cells containing 60 to 70 chromosomes. This aneuploidy is characteristic of cancer cells and is commonly seen in TP53-mutant tumors exhibiting similar copy number alterations (e.g., Figure 9 (As shown).

[0060] The CCK8 assay showed that the average doubling time of mGBC1-ZH cells was 25.2 ± 4.03 hours. Cell colony formation assays showed that significant colony formation was observed within 10-12 days. Transwell assays demonstrated the migration potential of mGBC1-ZH cells (e.g., ...). Figure 10 (As shown). Overall, these results indicate that mGBC1-ZH is a highly proliferating, colony-forming, and migratory cell line, and a valuable tool for studying gallbladder cancer biology.

[0061] Example 4: Transcriptome analysis of the mGBC1-ZH cell line To elucidate the transcriptomic landscape of mGBC1-ZH, this invention performed transcriptomic analysis on initial normal gallbladder organoid cultures and established gallbladder cancer cell lines. In mGBC1-ZH, gallbladder and biliary tract markers ( Aldoa , Krt19 (CK19) Krt7 (CK7) Epcam and S100a6 The expression level of liver cancer markers ( ) is high. Afp and Gpc3 ) and hepatocyte markers ( Alb , Ttr , Apoa1 and Apoe ) showed a lower expression level, demonstrating the maintenance of its gallbladder lineage identity (e.g. Figure 11 (As shown).

[0062] Differential expression analysis of the transcriptomes of the two cell groups revealed 5,588 differentially expressed genes, including 2,312 upregulated and 3,276 downregulated genes. Pathway analysis showed that the PI3K-Akt and MAPK signaling pathways were significantly upregulated in mGBC1-ZH cells, consistent with the characteristic signaling patterns of KRAS-mutant cancers. Furthermore, extracellular matrix receptor interaction and local adhesion pathways, which regulate cell-matrix interactions, migration, and invasion, were upregulated. Conversely, gene pathways involved in gallbladder-specific functions, including glutathione metabolism and cytochrome P450 foreign body metabolism, were significantly downregulated. The p53 signaling pathway was significantly downregulated, which is consistent with the presence of... Trp53 The mutations are consistent (e.g.) Figure 12 (As shown).

[0063] Example 5: Genomic alterations in the mGBC1-ZH cell line To elucidate the genomic landscape of mGBC1-ZH, whole-exome sequencing (WES) was performed on the cell line and its corresponding normal gallbladder organoid cultures. Analysis revealed 193 somatic mutations and variants in mGBC1-ZH, including 179 missense mutations. Single nucleotide polymorphisms (SNPs) were the predominant variant type. The distribution of single base substitutions showed a G>A / C>T and T>C / A>G switching predominance, consistent with the mutation spectrum previously described in human gallbladder cancer samples (e.g., ...). Figure 13 (As shown). Copy number analysis revealed deletions on chromosomes 4, 7, 9, and 16, and amplifications on chromosomes 3, 15, and 17. On chromosome 11... Trp53 Deletion of the locus was detected. Copy number variations are also found in other known gallbladder cancer driver genes, such as those on chromosome 4. Cdkn2a Deletion of loci, and on chromosomes 3 and 17 Pik3ca and Vegfa Amplification of loci (e.g.) Figure 14 (As shown).

[0064] Example 6: Evaluation of treatment response in a homologous gallbladder cancer model To evaluate the therapeutic potential of the homologous mouse gallbladder cancer model of the present invention, its response to standard chemotherapy drugs used in the treatment of human gallbladder cancer was evaluated through comprehensive in vitro and in vivo studies. In vitro dose-response analysis of gemcitabine (GEM) and cisplatin (CIS) showed a half-maximal inhibitory concentration (IC50). 50 The concentrations were 80.17 nM and 23.75 μM, respectively (e.g., ...). Figure 15 (As shown).

[0065] Subsequently, the therapeutic effects of GEM, CIS, and their combination (GC) were evaluated in a subcutaneous mouse model. Twenty mice carrying established tumors were randomly assigned to four treatment groups (n=5 per group) 10 days after tumor growth, ensuring comparable initial tumor volumes. After three cycles of treatment, all treatment groups showed a significant reduction in tumor volume and weight compared to the control group (e.g., ...). Figure 16 (As shown). These findings validate the model's potential for preclinical research, providing a valuable platform for evaluating novel treatment strategies and understanding drug resistance mechanisms in gallbladder cancer.

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mouse gallbladder cancer cell line, named mGBC1-ZH, was deposited at the China Center for Type Culture Collection on March 6, 2025, with accession number CCTCC NO:C202585.

2. A method for constructing the mouse gallbladder cancer cell line mGBC1-ZH as described in claim 1, characterized in that, The method includes the following steps: a) From genotype Kras LSL-G12D / Trp53 fl / fl Gallbladder epithelial tissue was isolated from C57BL / 6J mice and cultured to obtain organ-like cultures; b) Transfect the organ-like culture with a lentivirus expressing Cre recombinase and EGFP to activate it. Kras G12D Expression and absence Trp53 , obtained genotype Kras G12D / Trp53 - / - Organ-like cultures; c) The genotype is Kras G12D / Trp53 - / - Organoid-like cultures were passaged into a two-dimensional culture system and then injected subcutaneously into homologous wild-type mice. d) The isolated tumor tissue was digested into single cells and cultured in vitro to obtain the mouse gallbladder cancer cell line mGBC1-ZH.

3. The construction method according to claim 2, characterized in that, In step a), the method for obtaining organ-like cultures from mouse gallbladder epithelial tissue includes: a.1) The tissue was cut into fragments and transferred to pre-cooled DMEM / F12 medium containing 1% BSA. The cells were digested with Dispase containing 10 μM Y27632, centrifuged, and the cell pellet was resuspended in mouse gallbladder organoid medium (mGOM) containing 10 μM Y27632. a.2) Mix the cell suspension with pre-cooled Matrigel containing growth factors on ice, plant it in the center of a low-adhesion 24-well plate, and incubate at 37°C and 5% CO2 until the Matrigel solidifies. a.3) Each droplet was then covered with mGOM medium containing 10 μM Y27632. After 24 hours, it was replaced with fresh mGOM medium without Y27632. The medium was replaced every 2-3 days. a.4) After the initial isolation, culture for 7-10 days, and then passage by mechanical separation every 5-7 days to obtain organ-like cultures.

4. The construction method according to claim 2, characterized in that, The lentivirus expressing Cre recombinase and EGFP mentioned in step b) is selected from VSVG-LENTAI-UbiC-Cre-EGFP-WPRE-pA(LTR) lentivirus; the transduction method includes the following steps: b.1) Digest the organoid culture from step a) into single cells using trypsin solution and resuspend them in mGOM medium; b.2) Containing 1×10 ^5 200 μl of culture medium per cell was incubated with VSVG-LENTAI-UbiC-Cre-EGFP-WPRE-pA (LTR) lentivirus at 37°C and 5% CO2. Gene modification was then performed on the organ-like culture to obtain the genotype […]. Kras G12D / Trp53 - / - Organ-like cultures; b.3) Following the methods described in steps a.2-a.4), the genotype is... Kras G12D / Trp53 - / - Organ-like cultures were cultured in Matrigel for passage.

5. The construction method according to claim 2, characterized in that, Step c) describes transferring the genotype to Kras G12D / Trp53 - / - Specific methods for two-dimensional culture of organ-like cultures include: culturing genotypes of... Kras G12D / Trp53 - / - The organoid cultures were passaged five times in Matrigel, digested into single cells, and then cultured in 12-well plates for two-dimensional culture. After 15 passages of two-dimensional culture, the cells were injected subcutaneously into mice to form tumors.

6. The construction method according to claim 2, characterized in that, The specific method for obtaining the mouse gallbladder cancer cell line mGBC1-ZH by digesting the isolated tumor tissue into single cells and culturing them in vitro in step d) includes: after separating the subcutaneous tumor formed in step c), digesting it with collagenase, and then culturing the separated cells in DMEM with 10% fetal bovine serum for 20 generations to obtain the mouse gallbladder cancer cell line mGBC1-ZH.

7. The use of the mouse gallbladder cancer cell line mGBC1-ZH according to claim 1 in at least one of the following: 1) Application in the preparation of gallbladder cancer cell models; 2) Application in the preparation of animal models of gallbladder cancer; 3) Application in the preparation and / or screening of drugs for the prevention and treatment of gallbladder cancer; 4) Application in establishing a research platform for the mechanism of drug resistance in gallbladder cancer; 5) Application in establishing a research platform for early screening and grading methods for gallbladder cancer; 6) Application in establishing a research platform for gallbladder cancer treatment methods.

8. A method for constructing a mouse model of gallbladder cancer, the method comprising implanting the mouse gallbladder cancer cell line mGBC1-ZH of claim 1 into mice and feeding them for 7-28 days to form a mouse model of gallbladder cancer.

9. The construction method according to claim 8, characterized in that, The gallbladder cancer mouse models include gallbladder cancer subcutaneous tumor mouse models, gallbladder cancer in situ tumor mouse models, or gallbladder cancer metastatic tumor mouse models.

10. The construction method according to claim 8, characterized in that, The mice used to construct the mouse model were selected from immunodeficient mice or immunonormal mice, preferably immunonormal mice.

11. The construction method according to claim 8, characterized in that, The mouse model is a subcutaneous gallbladder cancer mouse model. The method for constructing this model includes implanting the mouse gallbladder cancer cell line mGBC1-ZH as described in claim 1 into the subcutaneous tissue of mice, feeding them for 1 week, and forming a palpable tumor in the subcutaneous tissue.

12. The construction method according to claim 8, characterized in that, The mouse model is a mouse model of gallbladder carcinoma in situ. The construction method of the model includes: 1) fasting mice for 4 hours before surgery and then anesthetizing them; 2) suspending the mouse gallbladder cancer cell line mGBC1-ZH as described in claim 1 in DPBS and mixing it with Matrigel; 3) exposing the gallbladder, puncturing it, squeezing out the bile and cleaning it, injecting the cell suspension into the gallbladder with a syringe, and withdrawing the syringe after the suspension solidifies; 4) suturing the abdominal wall, feeding for 4 weeks, and forming a solid in situ tumor in the gallbladder.

13. A mouse model of gallbladder cancer constructed according to any one of claims 8-12.

14. The use of the gallbladder cancer mouse model of claim 13 in at least one of the following: 1) Application in the preparation and / or screening of drugs for the prevention and treatment of gallbladder cancer; 2) Used as an animal model for the preparation and / or screening of drugs for the prevention and treatment of gallbladder cancer; 3) Application in establishing a research platform for the mechanism of drug resistance in gallbladder cancer; 4) Application in establishing a research platform for early screening and grading methods for gallbladder cancer; 5) Application in establishing a research platform for gallbladder cancer treatment methods.

15. A method for preparing or screening candidate drugs for the prevention and / or treatment of gallbladder cancer, said method being selected from at least one of the following: i) The candidate drug is applied to the gallbladder cancer cell model, and the candidate drug that inhibits cell proliferation or causes other programmed cell death is the effective drug, wherein the gallbladder cancer cell model is the mouse gallbladder cancer cell line mGBC1-ZH as described in claim 1; ii) The candidate drug is administered to an animal model of gallbladder cancer, and the animal's body weight and tumor growth are monitored. The candidate drug that causes improvement or cure of gallbladder cancer symptoms in the animal model is an effective drug. The animal model of gallbladder cancer is constructed by the method described in any one of claims 8-12.

16. The method according to claim 15, characterized in that, The method of administering the test substance described in method ii) is a drug administration method commonly used by those skilled in the art, including one or a combination of two or more of the following: tail vein injection, intraperitoneal injection, gavage, subcutaneous injection, intramuscular injection, or local tumor administration.

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