P06.09 Sequential photodynamic therapy and alphavirus-mediated interferon-γ delivery remodel the tumour microenvironment in breast cancer

Background Breast tumours with an immunosuppressive microenvironment respond poorly to immunotherapy, highlighting the need for strategies that simultaneously induce tumour cell death and reprogram tumour-associated immune cells. Photodynamic therapy (PDT) induces local oxidative stress and immunogenic cell death, while virus-based cytokine gene delivery offers a means to modulate immune function directly within the tumour microenvironment. However, the therapeutic interplay between PDT and virus-mediated cytokine delivery remains largely unexplored. This study investigates the combined potential of chlorin e6–based PDT and replication-deficient Semliki Forest virus–mediated interferon-γ (IFNγ) delivery for immune remodelling in breast cancer.Methods Murine 4T1 breast cancer cells were analysed in 2D cultures and 3D spheroid models to assess treatment-induced cytotoxicity, immunogenic cell death markers, viral transgene expression, and macrophage phagocytosis. Treatment sequencing was systematically evaluated to define optimal conditions for combination therapy. Therapeutic efficacy and immune modulation were further assessed in an orthotopic, immunocompetent 4T1 mouse model using sequential PDT and intratumoral viral administration. Tumour burden was quantified, and immune cell composition was analysed using high-dimensional spectral flow cytometry.Results PDT induced reactive oxygen species production and immunogenic tumour cell death, enhancing macrophage-mediated phagocytosis but not directly driving macrophage polarisation. In 3D tumour spheroids, treatment order was critical: PDT impaired viral gene expression when applied first, whereas viral priming followed by PDT resulted in pronounced synergistic tumour cell killing. In vivo, sequential PDT followed by IFNγ gene delivery significantly reduced tumour burden compared with either monotherapy. Immune profiling revealed that PDT primarily activated innate immunity, increasing dendritic cells, natural killer cells, and iNOS-positive myeloid populations, while IFNγ delivery reduced immunosuppressive cell subsets, including regulatory T cells, CD206-positive macrophages, and myeloid-derived suppressor cells.Conclusions This study demonstrates that rationally sequenced PDT and virus-mediated IFNγ delivery synergistically remodel the breast tumour microenvironment toward an immunostimulatory state. The findings underscore the importance of treatment timing and highlight innate immune activation as a key mechanism driving therapeutic efficacy, supporting further development of combined photo-immunogene strategies for resistant breast cancers.