Animal tumor xenograft model

Twelve male nude mice (athymic, Balb/c nu/nu, aged 5–6 weeks, weighting 18–20 g) were purchased from the Slac Laboratory Animal Center (Shanghai, China) and kept under specific pathogen-free conditions. 1 × 10⁶ PC3 cells stably transfected with sh-circDPP4 or sh-NC were resuspended in 200 μl of PBS and inoculated subcutaneously into a single side of the posterior flank of nude mice. Six mice were included in each group. Tumors were measured using a caliper every week and tumor volume was calculated following the formula: V = 0.5 × D × d² (V, volume; D, longitudinal diameter; d, latitudinal diameter). Five weeks after cell inoculation, mice were sacrificed by CO₂ asphyxiation, and the xenograft tumors were excised, weighted and subjected to H&E staining and immunohistochemistry (IHC) analysis of Ki-67 protein.

All experimental procedures involving the use of animals were approved by the Ethics Committee of Zhongshan Hospital, and all efforts were made to minimize animal suffering, according to the NIH Guide for the Care and Use of Laboratory Animals.