Background: Photodynamic therapy (PDT) leading to immunogenic cell death (ICD) may serve as a promising basis for the development of antitumor therapeutic strategies. However, the mechanisms of action of photoinduced ICD in primary tumor cultures, including human glioma, remain unexplored. Methods: In the present study, the features of regulated cell death induced by photodynamic therapy using a previously described ICD inducer, porphyrazine III (pz III), were investigated. Cell death was studied in 7 primary cultures of high-grade human gliomas (astrocytomas, oligodendrogliomas, and glioblastomas). Results: Accumulation of porphyrazine III was observed in the endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and mitochondria; however, the distribution of the photosensitizer varied across different cultures. A narrow concentration window of porphyrazine III was established to effectively reach IC85, primarily inducing ferroptosis with contributions from apoptosis and necroptosis accompanied by superoxide anion generation and mitochondrial dysfunction. Conclusions: Given the immunogenic potential of ferroptosis, apoptosis and necroptosis we hypothesize that the induction of PDT using porphyrazine III in glioma will trigger immunogenic cell death.

Fluorescence lifetime imaging microscopy (FLIM) has developed significantly over the past two decades and is now a powerful tool in biomedical research. Recent advances in fluorescent probes have greatly expanded the range of its potential applications. As fluorescence lifetime is highly sensitive to microenvironmental and molecular changes, FLIM is a promising technique for detecting pathological conditions, including cancer, and monitoring the efficacy of antineoplastic therapies. This technology allows for the observation of tumor structure and the real-time monitoring of dynamic processes, enabling researchers to probe living cancer cells and their microenvironment with remarkable precision. FLIM is especially valuable for developing and evaluating immunotherapeutic strategies. In solid tumor therapy; in particular, it is crucial to assess how treatment affects tumor metabolism and heterogeneity, cell death mechanisms, and immune response dynamics. This review provides a comprehensive analysis of current research supporting the feasibility of FLIM as a key research technique to advance cancer immunotherapy.