Tumor xenograft and immunohistochemistry

The animal experiments were approved by the Ethics Committee of the Academy of Military Medical Sciences of the People's Liberation Army. Female BALB/c nude mice, aged five weeks, were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were raised in specific pathogen‐free conditions with a 12 hour light‐dark cycle. Next, 2 × 10⁶ H3255 cells were subcutaneously injected into the right flank of the mice. When the tumor volume reached approximately 100 mm³, the mice were randomly assigned to six groups (n = 6) and treated with dimethyl sulfoxide (DMSO), CM082 (80 mg/kg b.i.d.), CM082 (160 mg/kg b.i.d.), sunitinib (50 mg/kg q.d.), gefitinib (10 mg/kg q.d.), and gefitinib (10 mg/kg q.d.) combined with CM082 (80 mg/kg b.i.d.), respectively. Bodyweight and tumor volume of the mice were measured every three days. After 21 days, mice were killed, and the xenograft tumors were weighed and imaged. The tumors were used for western blotting and immunohistochemistry (IHC). Tumor volume was calculated as (length × width²)/2. Tumor growth inhibition (TGI) was calculated from the start of treatment by comparing changes in tumor volumes for control and treatment groups.

IHC was used to detect the expression of Ki‐67, CD31, and VEGF‐A in the tumor tissues according to the guidelines provided by Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). Meanwhile, apoptotic cells in the tumor tissues were detected using the TUNEL method.