Xenograft mouse studies

To investigate whether metformin inhibited CoCl₂-induced EMT in vivo, the subcutaneous xenografted growth of OSCC cells was monitored. For the experiment, cells were cultured in DMEM with 20% FBS containing 300 µM CoCl₂, with or without 10 mM metformin, for 48 h at 37°C. There were 28 male BALB/C nude mice (Shanghai Vital River Laboratory Animal Technology Co., Ltd.; age, 4–6 weeks; weight, 15–20 g) were housed under specific pathogen-free conditions, with food and water provided ad libitum. After 1 week of acclimation, the mice were randomly divided into four groups (seven mice per group) and injected with 5×10⁶ indicated OSCC cells subcutaneously which was resuspended by PBS into the right flank. Xenograft tumor volume and weight were measured every other day. After 24 days, nude mice were euthanized by cervical dislocation and tumors were collected. Tumor volume (mm3) was calculated as follows: 1/2 × long diameter (mm) × short diameter (mm)². The present study was approved by the Animal Research Ethics Committee of Jilin University. All animal treatments were performed in accordance with the Regulations of the Administration of Affairs Concerning Experimental Animals.