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

Photodynamic therapy (PDT) is effective for early epithelial pre-cancers, yet its efficacy in fully-developed cutaneous squamous cell carcinoma (SCC) is limited by immunosuppression in the tumor microenvironment. Vitamin D (VitD) has emerged as a potential neoadjuvant to enhance photodynamic priming in several cancers. Here, we investigated how VitD pretreatment modulates local and systemic immune responses to aminolevulinic acid-based PDT in two immunocompetent murine SCC models (chronic UV-induced SCC, and subcutaneously implanted PDVC57B cells). VitD combined with PDT significantly amplified hallmarks of immunogenic cell death, including calreticulin and HMGB1 expression, and increased recruitment of neutrophils, macrophages, dendritic cells, and CD8 T cells into tumors. Importantly, VitD+PDT produced a higher M1/M2 macrophage ratio, and reduced the number of exhausted (PD-1 expressing) T cells compared to PDT alone. Immune profiling of blood demonstrated enhanced T-cell activation (CD69) and reduced TIM-3 expression on cytotoxic T cells. Transcriptomic analysis revealed pathway enrichment for interferon-/{gamma} signaling and suppression of pro-tumorigenic epithelial-mesenchymal transition and of angiogenesis after VitD+PDT. Collectively, these findings demonstrate that VitD reprograms the immune response to PDT by enhancing cytotoxic immunity while limiting immunosuppressive features. This suggests an immune-priming strategy to consider, possibly alongside immune checkpoint blockade, for treating cutaneous SCC.