Single-cell suspensions of 1 × 106 HCT116 cells in 50 μl of diluted Matrigel (1:1; BD Biosciences, #356234) were injected subcutaneously into the dorsal flank of male nude mice at 6 to 8 weeks of age. Mice were randomized into three groups until their tumors reached a size of approximately 100 mm3. Mice were then treated with vehicle [formulated in ethanol/cremophor/water at 10:10:80 (v/v/v)], CYD19 (30 mg/kg), or CYD19 (50 mg/kg) via intraperitoneal injection for two consecutive weeks. Tumor volumes were measured every 1 day using the formula π × length × width2/6. At the end point of treatment, mice were euthanized, and tumors and key organs were dissected, photographed, and weighed. Tissues were either fixed in 4% paraformaldehyde (PFA) for immunohistochemical and histological analyses or snap-frozen in liquid N2 and stored at −80°C for immunoblot analysis. In some experiments, 1 × 106 control-shRNA–expressing cells and 2 × 106 (or 1 × 106) Snail-shRNA2–expressing cells were used to form tumor xenografts in comparable sizes. For liver metastasis assay, a left subcostal surgical incision was created, and 1 × 106 GFP-labeled HCT116 cells were intrasplenically injected into the spleen of male nude mice (6 to 8 weeks of age). Mice were then treated intraperitoneally with vehicle or CYD19 (30 mg/kg) for three consecutive weeks starting from the third day after surgery, and livers were then harvested for analysis.

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