Construction of an orthotopic mouse model of intrahepatic cholangiocarcinoma resistant to BGJ398 using hydrodynamic injection
Objective:To establish a BGJ398-resistant orthotopic model of intrahepatic cholangiocarcinoma(ICC)in mice that simulates the fibroblast growth factor receptor 2(FGFR2)gene rearrangement/fusion mutations and FGFR2 kinase domain mutations in clinical patients,in order to provide a tool for relavant research.Methods:Modified FGFR2 gene encoding FGFR2 protein that's prone to phosphorylation activation was introduced into the mouse liver using hydrodynamic tail vein injection technology to establish a BGJ398-sensitive orthotopic model of ICC.Plasmids injected include pT3-myr-AKT-HA,pT3-EF1a-NICD1,pCMV(CAT)T7-SB100 and pSB[Exp]-myr-FGFR2-FLAG.Then,the mice were treated with BGJ398 for 4 weeks to induce resistance to BGJ398.Ultrasound imaging was used to monitor bile duct obstruction lesions in the model mice in order to access the extent of resistance to BGJ398.HE staining and oil red O staining were used to analyze liver tissue sections for the histological features of the BGJ398-resistant orthotopic ICC model.Immunofluorescence staining was used to analyze the origin of the ICC cells from BGJ398-resistant mice.Primary tumor cells were extracted from the model mice and CCK-8 assay was performed to analyze their sensitivity to BGJ398 in vitro as well as the median inhibition concentration(IC50)of BGJ398.The model mice were treated with BGJ398 and the survival curves were plotted to verify the resistance of the ICC mouse model to drug treatment in vivo.DNA was extracted from drug-resistant ICC tissues and genomic resequencing was used to verify mutations in the kinase domain of the FGFR2 sequence.Results:The successful rate of the BGJ398-resistant orthotopic ICC mouse model construction was 93.33%,with significant tumor burden observed in the liver.Histologically,ICC tumor tissue was arranged in glandular duct patterns in the liver.Large numbers of lipid droplet vacuoles appeared in HE staining and oil red O staining.Immunofluorescence staining of the liver tissue showed positive results for biliary epithelial cell marker cytokeratin 19(CK19)and negative results for hepatocyte marker albumin(ALB).Primary ICC tumor cells were extracted and cultured,and CCK-8 results showed that the IC50 value for BGJ398 was 95.22 nmol in BGJ398-sensitive cells while the number increased to 348.4 nmol in BGJ398-resistant cells.After treatment with BGJ398 through intraperitoneal injection,the survival rate for BGJ398-resistnat mice was significantly lower than that of BGJ398-sensitive mice(P=0.001 7).Subsequent genomic resequencing revealed significant mutations in the FGFR2 gene,verifying that the mutations in the orthotopic ICC mouse model was consistent with those found in clinical ICC patients.Conclusion:This study has successfully established a BGJ398-resistant orthotopic ICC mouse model,providing valuable tools for further exploration of the molecular mechanisms of resistance to FGFR inhibitors and the development of new treatment methods.
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