Tumor Xenograft Formation in Nude Mice

Ten nude mice [Shanghai Branch of Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China; SYXK (Shanghai) 2017–0014] were randomly allocated into the shBMP2 group (injected with HepG2-CSCs transfected with shBMP2) and the scramble group (injected with HepG2-CSCs transfected with scramble), with 5 mice in each group. Briefly, HepG2-CSCs in logarithmic growth phase stably transfected with shBMP2 or scramble were collected, detached with 0.25% trypsin and dispersed into single-cell suspension in PBS. Next, the suspension (2 × 10⁶ cells) was injected subcutaneously into the right armpit of mice. The health status of nude mice was monitored every day after injection. The tumor size was measured every 4 days, and calculated by the following formula: V = W² × L × 0.52.²³ The nude mice were euthanized by intraperitoneal injection of pentobarbital sodium (≥100 mg/kg) on the 28th day after injection, and the tumors were removed under sterile conditions. After PBS washing, the tumor weights were recorded.