In vivo experiments

To study treatment-induced changes in tumors, 2.5 × 10⁵ B16.OVA melanoma cells were implanted subcutaneously on 4–6 week old female C57BL/6JOlaHsd mice (Envigo Labs, Huntingdon, UK). Eleven days after engraftment, animals were randomized into groups (n = 12-14/group). Then, they received systemic treatments of 0.1 mg of anti-PD-L1 (clone 10 F.9G2, BE0101, BioXCell, Lebanon, New Hampshire, USA) and intratumoral injections of 1 × 10⁸ vp (including equal amounts of Ad5-CMV-mIL2 and Ad5-CMV-mTNFa viruses, non-replicative in mice) on days 0, 1, 3, and 6. PBS was injected intratumorally for groups that did not receive virus. On day 7, 6 animals per group were sacrificed and tumors collected to investigate immune-cell phenotyping and cytokine signatures. The rest of the animals (n = 6–8/group) continued to a 90-d OS study, where the treatments continued once every 3 d, until maximum tumor size reached (18 mm) or complete tumor regression.