Optimization and Evaluation of Conditions for Orthotopic Nude Mouse Models of Human Liver Tumor Cells

doi:10.12300/j.issn.1674-5817.2024.048

Abstract

Abstract:

Objective The study aims to optimize the conditions for constructing orthotopic nude mouse models of liver cancer by injecting human liver tumor cell lines and to explore appropriate timings for drug administration. Methods Human hepatocellular carcinoma Hep3B and hepatoblastoma HepG2 cell lines, which stably expressing the luciferase reporter gene (LUC), were selected. The linear correlation between the luciferase luminescence intensity and the number of liver tumor cells was analyzed using a Small Animal In Vivo Imaging system to verify the luminescent efficiency of the human liver tumor cells. Different concentrations (8×10⁶, 2.4×10⁷, 7.2×10⁷ cells/mL) and resuspension media (PBS, Matrigel) of human liver tumor cell suspensions HepG2-LUC and Hep3B-LUC were orthotopically inoculated into the liver lobes of 5-week-old female BALB/c nude mice (12 groups, 7 mice each) to construct human liver tumor nude mouse orthotopic cancer models. Every 7 days, the weights of mice were recorded, and the growth of orthotopic tumors was monitored using the Small Animal In Vivo Imaging system. On day 35 post-cell inoculation, mouse livers were dissected, and pathological slices were prepared for HE staining to observe histopathological changes in liver tissues. Results The luminescence intensity of human liver tumor cell lines was positively correlated with the number of cells (R²=0.983 1, R²=0.970 5), indicating their suitability for orthotopic model construction. Successful modeling was achieved in the high-concentration groups of HepG2-LUC, the low-, medium-, and high-concentration groups of HepG2-LUC+Matrigel, the medium- and high-concentration groups of Hep3B-LUC, and the low-, medium-, and high-concentration groups of Hep3B-LUC+Matrigel. For both HepG2-LUC+Matrigel and Hep3B-LUC+Matrigel groups, mice in the high-concentration groups exhibited significantly reduced body weight compared to the low- and medium-concentration groups (both with P<0.05). The luminescence intensity of successfully modeled mice increased exponentially over time (R²>0.950 0), and reached a minimum of 1.0×10⁷ p/(s·cm2·sr) by day 14 post-transplantation. Mice in the low- and medium-concentration groups of HepG2-LUC and the low-concentration group of Hep3B-LUC showed no significant pathological changes, while the other groups exhibited evident liver tumors and hepatocyte lesions. Conclusion For the HepG2-LUC cell line, the recommended injection volume is 50 μL with a cell density of 2.4×10⁷ cells/mL, resuspended with Matrigel, followed by drug administration or prognostic measures on day 7 post-modeling. For the Hep3B-LUC cell line, the recommended injection volume is 50 μL with a cell density of 7.2×10⁷ cells/mL, not resuspended with Matrigel, with administration or prognostic measures on day 14 post-modeling.

Key words: Liver cancer, Orthotopic transplantation, HepG2-LUC, Hep3B-LUC, BALB/c nude mice

CLC Number:

R73-3

MENG Yu,LIANG Dongli,ZHENG Linlin,et al. Optimization and Evaluation of Conditions for Orthotopic Nude Mouse Models of Human Liver Tumor Cells[J]. Laboratory Animal and Comparative Medicine, 2024, 44(5): 511-522. DOI: 10.12300/j.issn.1674-5817.2024.048.

Figures/Tables 4

Figure 1 Cell bioluminescence efficiency of human liver tumor cell linesNote: A, Bioluminescence efficiency of human hepatoblastoma HepG2 cells labeled with luciferase reporter gene (LUC) after incubating with D-luciferin potassium; B, Bioluminescence efficiency of human hepatocellular carcinoma Hep3B cells labeled with LUC after incubating with D-luciferin potassium. Simple linear regression and one-way ANOVA test showed that the number of each cell was positively correlated with the average bioluminescence intensity. In the 96-well plate, the number of cells in each well from left to right was 2×103，4×103， 8×103， 1.6×104， 3.2×104， 4.8×104, and 6.4×104, n=3.

Figure 2 Body weight changes of nude mouse liver tumor models with different cell concentrations and suspension mediaNote：A-D represent body weight over time in nude mouse orthotopic tumor models of HepG2-LUC [human hepatoblastoma HepG2 cells labeled with luciferase reporter gene (LUC) and diluted with PBS into different cell concentrations], HepG2-LUC+Matrigel (human hepatoblastoma HepG2 cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations), Hep3B-LUC (human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with PBS into different cell concentrations), and Hep3B-LUC+Matrigel (human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations) successively. Compared with the low-concentration (8×106 cells/mL) group of the same time, #P<0.05, ##P<0.01, ###P=0.000 1; Compared with the medium-concentration (2.4×107 cells/mL) group of the same time, #P<0.05, ##P<0.01, ###P=0.000 1; Compared among the three groups of the same time, nsP>0.05.

Figure 3 Bioluminescence monitoring of nude mouse liver tumor models with different cell concentrations and suspension mediaNote：A-D represent the weekly bioluminescence monitoring results of nude mouse orthotopic tumor models of HepG2-LUC [human hepatoblastoma HepG2 cells labeled with luciferase reporter gene (LUC) and diluted with PBS into different cell concentrations], HepG2-LUC+Matrigel (human hepatoblastoma HepG2 cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations), Hep3B-LUC (human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with PBS into different cell concentrations), and Hep3B-LUC+Matrigel (human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations) successively, which show the change trends of the luminescence intensity over time after in-situ modeling (dotted lines indicate exponential growth).

Figure 4 Gross images (A) and HE staining (B) of liver tissue in nude mouse liver tumor models with different cell concentrations and suspension mediaNote：HepG2-LUC, left liver lobe of nude mice 35 days after in-situ modeling with human hepatoblastoma HepG2 cells labeled with luciferase reporter gene (LUC) and diluted with PBS into different cell concentrations. HepG2-LUC+Matrigel, left liver lobe of nude mice 35 days after in-situ modeling with human hepatoblastoma HepG2 cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations. Hep3B-LUC, left liver lobe of nude mice 35 days after in-situ modeling with human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with PBS into different cell concentrations. Hep3B-LUC+Matrigel, left liver lobe of nude mice 35 days after in-situ modeling with human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations. The scale size is 5 mm in figure A, and the scale sizes of the left and right graphs in each cell concentration group are 50 μm and 20 μm respectively in figure B.

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