5-week-old immunodeficient nude mice (male, weight, 16-20 g, n = 40 mice for each group) were purchased from Cyagen bio. Co. (Beijing, China). Before experiments, the mice were adapted to the breeding environment for two weeks. All mice were maintained at a 12 h/12 h light/dark cycle with free access to water and food. A total of 5 x 10⁶ HT-29 or HCT-116 cells were suspended in 100 uL PBS and injected subcutaneously into the right posterior flanks of nude mice. After three weeks, the mice were killed and the tumor xenografts were dissected, weighed and fixed in 10% buffered formaldehyde for further IHC analysis.

BALB/c nude mice (18-20 g, 4-week-old, male) (n = 6) were purchased and applied for the tumor growth analysis of FTC133 cellsin vivo. About 1 x 10⁶ FTC133 cells were transfected with control shRNA or circ_0067934 shRNA and were subcutaneously injected into the left fat pad of mice. After 5 days of injection, we measured tumor growth every 5 days. We sacrificed the mice after 30 days of injection. The tumor volume was calculated by (length x width2)/2. And the tumor weight was calculated at day 30.