Establishment of syngeneic melanoma model

All animal studies were conducted under the guidelines approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology. Female C57BL/6 and BALB/c-nu mice (6–8 weeks) were obtained from Beijing HFK Bioscience. The C57BL/6 mice were numbered and divided into experimental group and control group by random number method. B16-F10 murine melanoma cells were purchased from a cell bank (Shanghai, China) and cultured in RPMI 1640. B16-F10 cells (1×10⁶) were prepared in a 1:1 (v:v) ratio in Matrigel (Corning) and injected subcutaneously into the right shoulder or bilateral shoulders of the mice to establish unilateral and bilateral tumor models.