Bortezomib enhances anti-tumor efficacy of PD-1 blockade in lung squamous cell carcinoma via innate immune activation

Background: Immune checkpoint inhibitors have improved survival outcomes for patients with lung squamous cell carcinoma compared to conventional chemotherapy. However, challenges such as low response rates and acquired resistance remain. Although proteasome inhibitors have shown antitumor potential owing to their unique mechanism of action, their ability to synergistically enhance immunotherapy is not well established. This study aimed to investigate a novel combinatory strategy of bortezomib and PD-1 blockade in lung squamous cell carcinoma. Methods: We established co-culture systems to assess lymphocyte cytotoxicity and cytokine production by flow cytometry. Syngeneic and humanized mouse models of lung squamous cell carcinoma were used to evaluate tumor growth and immune infiltration in response to bortezomib, alone or in combination with anti-PD-1. Immune infiltration in tumor tissues was analyzed by immunohistochemistry. Signaling pathway activation and chemokine expression were examined by Western blot and polymerase chain reaction. Key molecules of innate immune pathways were perturbed using genetic knockdown or pharmacological inhibition to delineate the underlying mechanism. Results: Pre-treatment of lung squamous cell carcinoma cells with bortezomib enhanced lymphocyte-mediated killing and increased secretion of IFN-gamma and granzyme B in co-culture. In syngeneic models, bortezomib promoted intratumoral lymphocyte infiltration and synergized with PD-1 blockade, leading to increased CD8⁺ T cell and granzyme B⁺ cell accumulation. These findings were corroborated in humanized mouse models. Mechanistically, bortezomib triggered chemokine expression (e.g., CCL5) and activated components of the innate immune pathway, including TBK, IRF3, IKK, and NF-κB. This effect was not abrogated by STING or RIG-I knockdown. In contrast, pharmacologic inhibition of MYD88 suppressed TBK and IKK phosphorylation and reversed CCL5 upregulation induced by bortezomib. Conclusion: Bortezomib augments the efficacy of PD-1 blockade in lung squamous cell carcinoma by activating a TLR/MYD88-dependent innate immune pathway, enhancing chemokine-mediated immune infiltration. These results support the further development of this combination as a promising therapeutic strategy.