Tumor Modeling and Immunotherapy Protocols

MC38 cell line was generously provided by Z. Guo (University of Pittsburgh, School of Medicine) and cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P.S). Generation of B16–IL-33 and B16-vec tumor cell lines was previously reported (10 (link)). All cell lines were tested for mycoplasma routinely and with negative results. B16 and B16–IL-33 cells were cultured in RPMI 1640 medium with 10% FBS and 1% P.S. One million MC38 cells were injected intradermally into the right flank of the mice. For B16 models, 0.1 million cells were injected intradermally. IL-33 protein (10 μg), anti–PD-1 (200 μg per mouse; Bio X Cell, clone: J43), or anti-Areg (50 μg per mouse; R&D Systems, clone: 206220) was injected intraperitoneally on the fifth day after tumor inoculation, for a total of four times with 4-day intervals.