2. Animal model construction

After 7 days of feeding, 5 × 10⁵ B16 cells in 1,250 μL phosphate-buffered saline (PBS) were subcutaneously injected into the right thigh of mice to simulate a primary tumor (irradiated tumor), and 3 × 10⁵ B16 cells in 750 μL PBS were injected under the skin of the left thigh to simulate a secondary tumor (abscopal tumor), this timepoint was considered day 0. The B16-F10 mouse melanoma cell line were purchased from BeNa Culture Collection (Suzhou, China).

When the tumor volume reached 150 mm³, the mice were randomly divided into four groups: control group, immunotherapy group, radiotherapy group, and radiotherapy combined with immunotherapy group. The immunotherapy scheme was to intraperitoneally inject anti-mouse programmed cell death protein 1 (PD-1) (10 mg/kg) five times [19], specifically on the 10th, 12th, 14th, 16th, and 18th days of the experiment. Purified anti-mouse PD-1 was purchased from Shanghai Junshi Biological Science Co., Ltd. For the radiotherapy group, on the 14th day, the right thigh of each mouse was irradiated with a single dose of 20 Gy of 6 MeV electron rays from the Elekta Infinity linear accelerator. The irradiation field was 20 cm × 5 cm and the SSD was 100 cm. Irradiate five mice side by side each time. The control group did not receive any treatment.