Tumor xenograft mouse models

Eighteen female immunodeficient BALB/c nude mice (age, 4-6 weeks; weight, 16±2 g) were purchased from Vital River Laboratory Animal Technology Company Limited. All mice were raised under pathogen‑free conditions and maintained in a controlled environment (temperature, 25±2 °C; relative humidity, 70±5%; 12-h light/dark cycle) and fed standard laboratory food and water. The tumor xenografts were generated by a subcutaneous injection of GBC-SD cells (5×10⁷/ml × 125 μl) into the right flank of female nude mice. The mice were randomly divided into three groups of 6 mice: The control and the lenvatinib groups, administered with 15 or 30 mg/kg (intraperitoneal injection, daily) ^{9, 19}. Tumor size and weight were measured using calipers and electronic balance every 2 or 3 days. The tumor volume was determined according to the following formula: Tumor volume (mm³) = 0.5× (length × width²). The maximum tumor diameter in the present study was 15.74 mm, which was allowable by ethical guidelines. After 22 days, the animals were anesthetized with 1% pentobarbital (50 mg/kg) and sacrificed by breaking their neck. The tumor tissues were removed and measured. Xenograft tumors were collected and fixed in 10% formalin for 24 h at room temperature, following which they were paraffin-embedded and cut into 4-μm sections for immunohistochemical analysis.