Ivis imaging system

BALB/c nude mice were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and cultured in a sterile environment. After a week of acclimatization, MDA-MB-231-Luc cells were inoculated into nude mice at a density of 5 × 10⁶/mouse. The control group was treated with saline, while the experimental group received a daily intraperitoneal injection of SCA (0.3 mg/kg). One week after the tumor cell injection, the signal intensity of the tumor was detected using the IVIS Imaging System (Bruker, Madison, WI, United States), and treatment was initiated. Twenty-one days later, the tumor signal intensity of the nude mice was recorded again. The tumor, liver, spleen, lung, and kidney were removed, and the tumor weight was measured. Hematoxylin-eosin staining and TdT-mediated dUTP nick-end labeling were performed and the tissues were imaged.

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.