Living image software

All experiments were performed according to the institutional guidelines for the care and use of animals and were approved by the Animal Care and Use Committee of the Second Xaingya Hospital, Central South University. To establish LLC mouse models with subcutaneous tumors and metastatic tumors, LLC-sgNTC and LLC-sgAtrx cells were suspended in 150 μl of PBS at a density of 1 × 10⁶/ml. Then, these cell suspensions were subcutaneously injected or intravenously injected via the tail vein into C57BL/6 mice with a 1-ml syringe. The anti-CTLA4 and anti–PD-1 were given at a dose of 200 ug/mice at 9, 12, and 15 days after the establishment of models. The sizes of the subcutaneous tumors were measured by Vernier calipers every 3 days [tumor volume = 1/2 × (L × W)²]. For the metastasis model, tumor volume was monitored in vivo by bioluminescence detected by the IVIS imaging system (Bruker, USA) once every month. A D-luciferin potassium salt solution (Goldbio. St. Louis, MO, USA) was injected intraperitoneally (150 mg/kg), and 10–15 min after injection, the mice were imaged for in vivo tumor growth using an IVIS machine (PerkinElmer). Living Image Software (Bruker MI, USA) was used to measure the total flux of the metastatic lung tumor.