In Vivo Tumor Growth Xenograft Model

22RV1 subcutaneous xenografts were performed, as previously described (29). BALB/c-nude mice (6 weeks old, male) were purchased from the Sino-British Sippr/BK Lab Animal Co., Ltd. (Shanghai, China). Mice were randomly divided into three groups (eight animals/group). The animal use protocol was approved by the Institutional Animal Care and Use Committee of East China Normal University. The 22RV1 xenograft tumor model was developed by injecting 3 × 10⁶ 22RV1 cell suspension into the right side of the dorsal area of a BALB/c-nude mouse using 100 μL PBS or 50% Matrigel. Tumor nodules were allowed to grow to a volume of approximately 100 mm³ before initiating the treatment. Tumor-bearing BALB/c-nude mice were randomly assigned to three groups and treated with the indicated compound or drug. The body weight of each mouse was recorded, and tumor volume was determined using a digital vernier caliper measurement every 3 days. At the end of the experiment, mice were sacrificed. Solid tumors were removed and processed for immunohistochemical analysis.