Xenograft Experiment

Six-week-old BALB/c nude mice were obtained from the Shanghai Experimental Animal Center. There were two groups, gemcitabine group and gemcitabine + metformin group. Five mice were assigned per group. A total of 1 × 10⁷ MIAPaCa-2 cells were injected subcutaneously into the neck area of each mouse. After cell injection, the mice were treated with drugs, and the scheme was as following, gemcitabine only group (100 mg/kg of gemcitabine injected intraperitoneally twice weekly), combination gemcitabine with metformin group (gemcitabine 100 mg/kg injected intraperitoneally twice weekly and 800 mg/L metformin in drinking water each day). The tumor width, length, and height were measured by external caliper every 5 days and calculated as follows: Volume (mm³) = width × length × height/2. The mice were sacrificed by cervical dislocation following 25 days of treatment and the tumors were collected, fixed, and embedded. The animal experiments were approved by the Animal Care and Use Committee of The Second Affiliated Hospital of Nanjing Medical University, approval number 2018-KY-027, and in accordance with the Guide for the Care and Use of Laboratory Animals (eighth edition, 2011, National Institutes of Health, USA).