Photodynamic priming with Vitamin D and ALA-based PDT induces intratumoral immune cell recruitment and signaling pathway activation in cutaneous squamous cell carcinoma.

Photodynamic therapy (PDT) is an effective non-invasive treatment for epithelial pre-cancers, yet its efficacy in cutaneous squamous cell carcinoma (SCC) is limited by the immunosuppressive microenvironment of SCC. Vitamin D (VitD) has emerged as a potential neoadjuvant to enhance photodynamic priming in several cancers, but its immunologic effects in SCC remain poorly defined. Here, we investigated how VitD pretreatment modulates local and systemic immune responses to aminolevulinic acid-based PDT in two immunocompetent murine SCC models. VitD combined with PDT significantly amplified hallmarks of immunogenic cell death, including calreticulin and HMGB1 expression. Combination therapy increased intratumoral infiltration of neutrophils, macrophages, dendritic cells, and CD8⁺ T cells, while preserving a favorable M1/M2 macrophage ratio and reducing PD-1 expression on cytotoxic T cells. Peripheral immune profiling demonstrated enhanced T-cell activation (CD69) and reduced TIM-3 expression on cytotoxic T cells. Transcriptomic analysis revealed robust enrichment of interferon-α/γ signaling and suppression of pro-tumorigenic epithelial-mesenchymal transition and angiogenesis pathways following VitD + PDT. Collectively, these findings demonstrate that VitD reprograms the immune response to PDT in SCC, enhancing cytotoxic immunity while limiting immunosuppressive features. This combination suggests an immune-priming strategy that might be considered, together with immune checkpoint blockade, for SCC and other immunosuppressive cancers.