Gastric cancer xenograft model and therapeutic method

MGC803 cells were digested with 0.25% trypsin (cat. no. 59427C; Sigma-Aldrich; Merck KGaA), then washed using sterile PBS for three times, and injected into nude mice subcutaneously in the right flank of nude mice (1×10⁶/100 µl). Day 0 was marked when subcutaneous tumor nodules grew up to ~0.1 mm³ (~10 days). The nude mice were randomly divided into three experimental groups, with each group containing 8 mice: The untreated T-cells treatment group (PBS-treated CD3⁺ T-cells), the treatment group using galectin-9-stimulated T-cells (CD3⁺ T-cells that were cultured for 72 h with 5 µg/ml final concentration of galectin-9) and the PBS control group. Then, the intratumor injection treatment was performed. All experiments were approved by the Institutional Animal Care and Utilization Committee of Medical Ethics committee of Provincial Hospital Affiliated to Shandong University and Weihai Municipal Hospital. The treatments were performed on day 5 after MGC803 cells injection, and there were 4 treatments, with the injection being repeated every 3 days. The untreated CD3⁺ T-cells (1×10⁶/50 µl per mice; total of 8 mice) were injected into mice in the conventional T-cells treatment group; Tim-3-stimulated CD3⁺ T-cells (1×10⁶/50 µl, 8 mice) were injected into the mice in the treatment group of the Tim-3-stimulated CD3⁺ T-cells; the same volume of PBS was injected into the mice in the PBS control group (8 mice) instead of the cell suspension. At the same time, 5,000 U/kg interleukin-2 (Biolegend, Inc.) was injected into abdominal cavity or each mouse. The general state of the mice and the volume of the tumor nodules were observed. The duration of the experiments was 1 month, and following that all mice were anesthetized/euthanized using cervical dislocation. The computational formula for tumor volume used was: Tumor volume (mm³)=axb²x0.5, where ‘a’ denotes the long diameter of tumor (mm) and ‘b’ denotes the short diameter of tumor (mm).