4.9. Tumor Xenograft and Immunohistochemistry

Female BALB/C nude mice (age, 4 weeks) were randomly distributed into four groups, the NC group, the NC + RSL3 group, the oeFTO group, and the oeFTO + RSL3 group (n = 6 per group). To establish the cell line-derived subcutaneous transplanted model, approximately 1.2 × 10⁷ SCC25 cells per mouse, diluted in 150 μL of phosphate-buffered saline, were injected subcutaneously into the right posterior flanks. Two weeks after tumor transplantation, RSL3 was injected in a manner of 10 mg/kg intratumorally every other day. The transplanted tumors were measured every 3 days to observe tumor growth before the intratumoral RSL3 injection and every 2 days after the RSL3 injection. The tumor volume (V) was calculated using the formula V = 0.5 × (length × width²). After treatment for 10 days, the tumor xenografts were harvested and fixed for immunohistochemistry. This animal experiment was approved by the Sun Yat-Sen University’s Animal Experiment Ethics Committee. Immunohistochemistry on the tumor xenograft tissue was performed according to our previously described protocol [69]. The 4-HNE immunohistochemistry staining scores were calculated according to the stain intensity and staining sections. The staining intensity was scored as 0 (negative staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The staining sections were scored as 1 (cells with <10% staining), 2 (cells with 10–49% staining), 3 (cells with 50–74% staining), and 4 (cells with 75–100% staining). The final score was defined as the staining intensity score multiplied by the staining section score.