Methylene blue

GBM orthotopic model: SB mGSC (5 × 10³/mouse) or GL261 (10⁵/mouse) were stereotactically injected into the right striatum of six-to-eight-week-old male C57BL/6N mice (Charles River). Melanoma subcutaneous model: B16F10 (2 × 10⁵) was subcutaneously injected into the right axilla of six-to-eight-week-old male C57BL/6N mice (Charles River). Following the tumor injection, mice were randomly divided into four groups: control, anti-mouse PD-L1 mAb (Bio X Cell, BE0101) only, Methylene Blue or Belnacasan (MedChemExpress, HY-13205) only, and the combined treatment group. Methylene Blue (3 mg/kg body weight) was intraperitoneally injected once every two days for two weeks beginning after 3^rd day of tumor injection. Belnacasan (50 mg/kg body weight) was intraperitoneally injected every day for two weeks beginning after 3^rd day of tumor injection. Anti-mouse PD-L1 mAb (200 µg/mice) was intraperitoneally injected once every three days for three times beginning after 5^th day or 7^th day of tumor injection in GBM model or melanoma model, respectively. For the melanoma model, tumor volume was measured every five days, and tumor volume was calculated according to the formula: length × (width)² × 1/2. 20 days after the tumor injection, tumor-bearing mice were sacrificed, and tumor masses were resected for further analysis.