Tumor Xenograft modeling and in vivo experiments

BALB/c nude mice of 4 weeks old (weighing approximately 15–17 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangdong Province, China). All mice were housed and bred in a specific-pathogen-free (SPF) grade animal facility, with 22–25 °C temperature, 40–60% humidity, and 12 h/12 h light/dark cycle. To generate tumor xenograft, 20 mice were used. The skin of the left forelimb near the armpit was disinfected and 0.1 mL SiHa cells suspended in serum-free medium (containing approximately 5 × 10⁶ cells) were injected. After inoculation of the cervical cancer cells, the nude mice were continuously housed under the same conditions. Once the subcutaneous nodules grown to a rice grain size (required approximately a week), the subcutaneous xenograft model of cervical cancer in nude mice was successfully constructed. The subcutaneous tumor size in each nude mouse was measured using a digital vernier caliper. Once the tumor diameter reached approximately 0.3–0.5 cm, the nude mice were numbered, randomly divided into four groups (with five mice per group), namely, control, model, 50 mg/kg/d metformin, and 250 mg/kg/d metformin groups. Metformin was given by gavage. All nude mice were closely monitored for tumor growth, skin condition, and behavior daily and any tumor ulceration or irritation was noted. The longest (A) and the shortest (B) diameters of the subcutaneous tumors were measured with a digital vernier caliper before each metformin administration to calculate the tumor volume (V) using the following formula: V = 0.5 × A × B². In addition, all nude mice were weighed daily, and their daily food intake was also measured. After the completion of the 23-day metformin administration, all nude mice were sacrificed and placed on ice, their skin was immediately cut open, and the subcutaneous tumor xenografts were collected. After weighing each tumor xenograft on a digital scale, one part of the tumor tissue was dissected and frozen in liquid nitrogen for western blotting. All experimental procedures were approved by the Institutional Animal Care and Use Committee of South Medical University.