Anti-PD-1 Therapy in Murine Colorectal Cancer

BALB/C mice were obtained from CLEA Japan (Tokyo, Japan) and maintained at a constant temperature (23 ± 1 °C) with a 12 h light/dark cycle under specific pathogen-free conditions. The experimental protocols were approved by the Institutional Animal Care and Use Committee of the Showa University, and all experiments were conducted according to relevant guidelines and regulations. This study is a research report following the ARRIVE guidelines. Age-matched 8–12-week-old male mice were subcutaneously injected with 2 × 10⁵ CT26 cells in the right flank. Mice received intraperitoneal injections of 150 μg of anti-PD-1 (clone RMP1-14; Lebanon, NH, USA) per injection or its isotype control (IgG2b; Lebanon, NH, USA) on days 4, 11, 18, and 25. Mice received isobutyric acid (100 mM) in the drinking water or pH-match water (control group) for 2 weeks prior to tumour inoculation, and the water solution was changed weekly. Tumour size was measured using a calliper twice a week until it reached the size that should be sacrificed, and tumour volume was determined as length × width² × 0.5.