In vivo tumorigenicity assay

BALB/c nude mice (SPF grade, 18–22 g, 8 weeks old, male) were purchased from Shanghai SLRC Laboratory Animal Co., Ltd. (Shanghai, China). CRC cell xenografts were performed on the BALB/C nude mice. A total of 5×10⁶ NC-OE or DAB2IP-OE HCT116 cells (NC, DAB2IP) were suspended in 100 µL of serum-free DMEM medium and inoculated subcutaneously in the flank of the nude mice (n=10, 5/group). Tumor volumes were measured at 7, 10, 14, 17, and 21 days after implantation and were calculated using the formula: volume = (length × width²)/2. At the end of the experiment, the nude mice were euthanized, and the tumors were excised, weighed, and subjected to other experiments. Experiments were performed under a project license (No. ISM-IACUC-0054-R) granted by Animal Ethics Committee of Suzhou Institute of Systems Medicine (Suzhou, China), in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.