4.11. Tumor Xenograft Model and Experiment

All animal experiments were performed according to the Japanese Welfare and Management of Animals Act and conducted in accordance with institutional guidelines at Shigei Medical Research Institute, Okayama, Japan. All animal experiments were approved by the Ethics Review Committee for Animal Experimentation of Shigei Medical Research Institute (#160401-1), Okayama, Japan. Female BALB/c (nu/nu) mice were purchased from CLEA Japan (Tokyo, Japan). Female BALB/c (nu/nu) mice were purchased from CLEA Japan. The experiment started when the mice were 9 weeks of age. HSC-2 cells in culture were harvested and resuspended in a 1:1 ratio of PBS and Matrigel (BD Biosciences). HSC-2 cells (3.0 × 10⁶) were injected subcutaneously into the right flank. When the tumor size reached 150~200 mm³, the mice were randomly divided into four groups (control group, CDDP group, DFX group, combination group, n = 6 per group). Each group was treated with oral gavage of saline or DFX (160 mg/kg) three times per week for 3 weeks and by intraperitoneal injection of saline or CDDP (6 mg/kg) once per week for 3 weeks. Tumor size and body weight were measured every 3 days. The tumor volume (mm³) was calculated with the formula d2 × D/2 where d and D are the shortest and longest diameters in mm, respectively. At the end of the experiment, the mice were sacrificed, and the tumors were excised, weighed, and processed for histological analysis.