Purpose: To evaluate the antitumor efficacy in local and distant tumors induced by local hyperthermia with CTLA-4 blockade Methods: A mouse breast cancer cell line was inoculated into both sides of the legs of mice. The mice were treated with three administrations of CTLA-4 blockade, a single application of local hyperthermia (42.5 C for 20 min) to the tumor on one side of the leg, or the combination of the two. Tumor growth in locally heated tumors (HT tumors) and unheated distant tumors (UnHT tumors) and overall survival were evaluated. Results: In the combination group, tumor volume significantly decreased for both HT and UnHT tumors compared with the tumors in the untreated and local hyperthermia monotherapy groups. Remarkable efficacy was only observed in the combination therapy group, in which 7 of 18 mice responded to HT and UnHT tumors, with significant prolonged overall survival. Conclusions: Combination therapy enhanced the antitumor response not only in HT tumors but also in UnHT tumors and prolonged overall survival.

Recent studies demonstrate that immune checkpoint blockade (ICB) increases the chances of the abscopal effect, an anti-tumor effect outside the radiation field in radiation therapy. However, the optimal sequence between radiation and ICB remains unclear. To investigate the impact of sequence of radiation in anti-PD-L1 antibody (P1) therapy on immune microenvironments and antitumor efficacies in local and abscopal tumors, metastatic LM8 osteosarcoma cells were inoculated into both legs of C3H mice. For irradiation, only one side leg was irradiated at 10 Gy. Then mice were divided into four groups: administrated anti-PD-L1 antibody three times (P1 monotherapy), receiving radiation 3 days prior to P1 therapy (P1+pre-Rad), and receiving concurrent radiation with P1 therapy (P1+conc-Rad). Thereafter, tumor immune microenvironment and tumor volume changes were analyzed in irradiated and unirradiated tumors. The P1+pre-Rad regimen increased the proportion of CD8+ programmed cell death 1 (PD-1)+ granzyme B (GzmB)+ reinvigorated T cells and decreased the proportion of CD8+ PD-1+ GzmB- exhausted T cells than P1+conc-Rad regimen in unirradiated tumors. Combination regimens suppressed tumor growth in irradiated tumors compared with that in P1 monotherapy. In both irradiated and unirradiated tumors, significant tumor growth suppression and prolonged overall survival were observed under both combination treatment regimens compared with P1 monotherapy. However, no distinct differences in unirradiated tumor volume and survival were observed between P1+pre-Rad and P1+conc-Rad groups. These results suggest that local irradiation is necessary to improve systemic treatment efficacy in P1 therapy regardless of sequence of local irradiation.