B16bl6 melanoma cell line

The B16BL6 melanoma cell line (hereafter termed ‘B16 melanoma’) was purchased from the Korean Cell Line Bank (Seoul, Korea). The Lewis lung carcinoma (LLC) cell line was obtained from the American Type Culture Collection (Manassas, Virginia, USA). The MC38 colon adenocarcinoma cell line was kindly provided by Dr Tai-Gyu Kim (The Catholic University of Korea, Seoul, Korea). All cell lines were cultured in Dulbecco’s Modified Eagle Medium (Welgene, Gyeongsan, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL Fungizone (Gibco; Thermo Fisher Scientific, Waltham, Massachusetts, USA). All cell lines used in this study were negative for Mycoplasma, when tested using an e-Myco Mycoplasma PCR Detection Kit (iNtRON Biotec, Seongnam, Korea). After being resuspended in phosphate buffered saline (PBS), 5×10⁵ B16 melanoma cells, 1×10⁵ MC38 cells or 2.5×10⁵ LLC cells were injected subcutaneously into the right flank of mice aged 8–12 weeks. Tumor volumes were measured every 2–3 days with a caliper and calculated according to the following formula: V(mm³)=D×d²×0.52, where D (mm) and d (mm) are the largest and smallest perpendicular tumor diameters, respectively.