In vivo tumor growth in a xenograft model

The acquisition and housing of the nude mice used as xenograft models matched the conditions described for the in vivo metastasis assays. Animal studies were approved by the Fourth Military Medical University Animal Care Committee. Stable miR-302a-overexpressing Caco-2 cells labeled with luciferase were harvested by trypsinization, washed with PBS, and resuspended in PBS. A total of 5×10⁶ cells in 150 µl of PBS were subcutaneously injected into the flanks (the left flank was injected with miR ctrl cells, and the right flank was injected with miR-302a cells). When the tumor size reached approximately 100 mm³ (approximately 2 weeks after injection), the mice were randomly assigned to the control and CTX treatment groups. CTX was injected intraperitoneally at a dose of 0.3 mg/mouse twice weekly. The tumor size was measured every six days. One month after the CTX treatment, the mice were sacrificed according to institutional ethical guidelines, and the tumor size and weight were measured. Subsequently, the tumors were paraffin-embedded for H&E staining and IHC staining for NFIB or CD44. Additionally, Ki-67 and Cleaved Caspase-3 staining were performed to further evaluate tumor growth and the efficacy of CTX treatment.