Hyperthermia enhances the efficacy of anti-PD-L1 immunotherapy by promoting PD-L1 upregulation through PI3K/AKT signaling in non-small cell lung cancer

Although immune checkpoint blockade has significantly improved the treatment of non-small cell lung cancer (NSCLC), a substantial proportion of patients still experience limited therapeutic responses. Hyperthermia, as an adjuvant therapeutic modality, has shown antitumor effects in various malignancies by modulating tumor biological behavior and the tumor immune microenvironment, suggesting its potential to enhance immunotherapy. However, whether hyperthermia can enhance the efficacy of anti-PD-L1 immunotherapy in NSCLC and the underlying molecular mechanisms remain to be fully elucidated. Human NSCLC cell lines were used for in vitro assays of proliferation, migration, invasion, and apoptosis. A murine LLC subcutaneous tumor model was established to evaluate the effects of hyperthermia alone or in combination with anti-PD-L1 therapy. PD-L1 expression and changes in the tumor immune microenvironment were assessed by flow cytometry. RNA sequencing combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed to identify relevant signaling pathways, followed by Western blotting to assess PI3K/AKT signaling and PD-L1 expression. The regulatory role of the PI3K/AKT pathway in PD-L1 expression was further investigated using pharmacological inhibition, PIK3CG knockdown, and PIK3CG overexpression experiments. Hyperthermia significantly inhibited the proliferation, migration, and invasion of NSCLC cells and promoted apoptosis in vitro, while also suppressing tumor growth in vivo. Hyperthermia also upregulated PD-L1 expression in both tumor cells and tumor tissues. In vivo, hyperthermia enhanced the antitumor efficacy of anti-PD-L1 therapy and increased CD8⁺ T-cell effector activity. Transcriptomic analysis revealed significant enrichment of the PI3K/AKT signaling pathway, which was further supported by Western blotting. Knockdown and overexpression experiments, together with pharmacological inhibition of PI3K, further supported the involvement of PI3K/AKT signaling in hyperthermia-induced PD-L1 upregulation. Hyperthermia induces PD-L1 expression through activation of the PI3K/AKT signaling pathway and remodels the tumor immune microenvironment, thereby enhancing the therapeutic efficacy of PD-L1 blockade. These findings provide mechanistic support for combining hyperthermia with immunotherapy in NSCLC. Proposed mechanism of hyperthermia-enhanced immunotherapy in NSCLC: Hyperthermia activates the PI3K/AKT signaling pathway, leading to upregulation of PD-L1 expression in tumor cells, while simultaneously enhancing T cell effector function. This dual effect renders tumors more susceptible to PD-L1 blockade. Upon PD-L1 blockade, CD8⁺ T cells are relieved from immune suppression and secrete higher levels of granzyme B (GzmB) and IFN-γ, thereby driving tumor cell death.