Mouse models

HT29 shNT cells or HT29 shSREBP2 cells (1 × 10⁶) were subcutaneously implanted into both flanks of nude mice (5 mice per group) to establish subcutaneous tumor models. Tumor volumes were assessed on indicated days by a caliper and calculated using the formula: volume = (Length × Width²) / 2.

When the volume of subcutaneous HT29 xenograft tumors reached to 0.5 cm³, the tumors were removed and dissected into 1-mm³ sections. These tumor sections were incubated into cecum of nude mice to establish cecum orthotopic xenograft models.

To establish intra-splenic injection models, HT29 cells (1 × 10⁶/200 μL PBS) were injected into spleen and observed for 6 weeks.

Different HT29 stable cell lines (1 × 10⁶) were injected under the liver capsule (Glisson's capsule) of mice to establish liver orthotropic injection model. For drug treatment experiment, after 3 days, mice were randomly separated into four groups (five per group) were treated by intragastric administration with vehicle control, simvastatin (30 mg/kg/day), or betulin (15 or 30 mg/kg/day) daily. At the end of each procedure (about 6 weeks), mice were sacrificed, and tumors were dissected, weighed, taken photos, and used for different experiments.

All experiments were performed in 6 weeks old male nude mice (BALB/c nu/nu) obtained from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China) and maintained on a 12 h light dark cycle at 22 ºC. All mice were treated in strict accordance with animal care procedures and methods approved by the institutional review board of Department of Laboratory Animal Science, Fudan University, and conformed to the guidelines of National Institutes of Health on the ethical use of animals.