In vivo tumor xenograft study

The protocols for the mouse experiments conformed to international regulations for animal care and maintenance and were approved by the Institutional Animal Ethical Committee, Experimental Animal Center of Southern Medical University. To evaluate tumor growth, a subcutaneous xenograft mouse model was established. Cells were resuspended in PBS and subcutaneously injected into the flanks of 4- to 5-week-old male BALB/c-nu mice (N = 5 per group). Tumors from HCC-bearing mice were excised, weighed, and subjected to further study at 15 days after cell inoculation. Tumor volumes were calculated as previously described.^45,46 A subcutaneous xenograft mouse model was also adopted to investigate the tumor formation ability as previously described.⁴³ In brief, a series of cells were inoculated into the animals (N = 6 per group), and tumor formation was measured after cell inoculation. In addition, a subcutaneous xenograft mouse model was also applied to determine HCC resistance to sorafenib. Male BALB/c-nu mice aged 4–5 weeks (N = 10 per group) were subcutaneously injected with 5 × 10⁶ cells in the flanks and then treated orally with the vehicle solution or sorafenib (30 mg/kg) once daily for 4 weeks.⁴⁷ Survival curves were generated by Kaplan–Meier analysis.

In vivo metastasis assays were performed using a pulmonary metastasis model and an orthotopic tumor model. Cells were intravenously inoculated into the tail veins of the animals (N = 5 per group) or inoculated under the liver capsule (N = 7 per group). The metastases were measured as previously described.⁴⁸ Optical and pathological images were obtained to evaluate the growth of primary tumors and the formation of metastatic lesions.