Antitumor Effects of PD-1 Blockade Combined with Mild Hyperthermia in a Murine Osteosarcoma Model

<b>Background</b>: Osteosarcoma remains largely refractory to immune checkpoint inhibitor (ICI) monotherapy, and strategies to modulate the tumor immune microenvironment are being actively explored. Mild hyperthermia has been reported to influence antitumor immune responses; however, its impact in combination with PD-1 blockade in osteosarcoma has not been well characterized. <b>Methods</b>: Murine LM8 osteosarcoma cells were subjected to mild thermal stimulation, and changes in PD-L1 expression were evaluated. LM8-bearing mice were treated with mild hyperthermia, anti-PD-1 antibody, or their combination. Tumor growth, lung metastasis, and survival were assessed. Tumor-infiltrating immune cells were profiled using single-cell RNA sequencing to descriptively characterize immune-associated transcriptional features under each treatment condition. <b>Results</b>: Mild thermal stimulation (42 °C, 30 min) increased PD-L1 expression in LM8 cells in vitro. In vivo, combination therapy significantly suppressed primary tumor growth compared with control (χ² = 29.75, <i>p</i> = 1.6 × 10^-6) and reduced lung metastasis burden, with a significant decrease in metastatic nodules (<i>p</i> < 0.01). Kaplan-Meier analysis demonstrated a significant survival benefit in the combination group (log-rank <i>p</i> < 0.001). Single-cell RNA sequencing revealed an increased proportion of CD8⁺ T cells with reduced exhaustion-associated gene expression and a shift toward pro-inflammatory (M1-like) macrophage transcriptional profiles. <b>Conclusions</b>: PD-1 blockade combined with mild hyperthermia was associated with enhanced antitumor efficacy and immune-associated transcriptional remodeling in a murine osteosarcoma model, supporting further preclinical evaluation of this combination strategy.