2.9. Xenograft model

The xenograft model was established as previously described [³⁹]. SW620 and SW620/Ad300 cells were harvested and washed twice with PBS. Then, both cell lines (1 × 10⁷ cells in 0.1 mL PBS) were injected subcutaneously under armpits of the nude mice. Once the tumor volume reached 500 mm³, the mice were euthanized, and the tumor tissues were dissected. Each tumor tissue, except the necrotic part, was cut into 2 × 2 mm in size. The small tumor pieces were implanted subcutaneously under armpits of the nude mice. When the tumor reached approximately 100 mm³, the mice were randomly assigned to the following four groups (n = 6 each treatment group). Sitravatinib was prepared in a final concentration of 0.5% hydroxypropyl methylcellulose (HPMC) and 0.1% Tween‐80 solution (pH 1.4) (vehicle A) [²⁷], while vincristine was prepared in autoclaved water (vehicle B) [⁴⁰]. Group 1 mice received vehicle A (administered orally) 1 h prior to vehicle B (administered intraperitoneally). Group 2 mice received sitravatinib (prepared in vehicle A, 2 mg/kg, administered orally) 1 h prior to vehicle B (administered intraperitoneally). Group 3 mice received vehicle A (administered orally) 1 h prior to vincristine (prepared in vehicle B, 0.4 mg/kg, administered intraperitoneally). Group 4 mice received sitravatinib 1 h prior to vincristine at indicated dosages. All treatments were given every other day for 14 days.

The dosage of vincristine was determined by Zhang et al. [⁴⁰], and the dosage of sitravatinib was determined through preliminary experiments and previous pharmacokinetic studies [²⁸]. During the treatment, the animal behavior was monitored daily. The body weights as well as the length and width of tumor tissues were recorded using vernier caliper every other day. At the end of treatment period, all mice were euthanized, and the tumor tissues were excised, weighed, and measured. The tumor volume (V) and the growth inhibition rates (IR) for tumor volume (IRV) and tumor weight (IRW) were calculated using followed formulae [²⁶, ⁴¹‐⁴³]: