Pathomorphological Study of Phototoxicity of Genetically-Encoded Photosensitizer KillerRed on Animal Tumors

The study of phototoxic effects of KillerRed in experimental tumors enables to understand the mechanisms of action of genetically-encoded photosensitizers and choose the directions for photodynamic therapy (PDT) development. The aim of the investigation was to study pathomorphological characteristics of the protein KillerRed effect on animal tumors in laser exposure modes close to those used in photodynamic therapy with chemically synthesized photosensitizers.Materials and Methods. The study was carried out on immunodeficient nude mice with subcutaneously implanted tumor HeLa (human cervical cancer). Tumor cells stably expressed genetically-encoded photosensitizer KillerRed in chromatin (KillerRed-Н2B). Control tumors were HeLa tumors without KillerRed. Tumor growth and fluorescence intensity change were observed in the process of radiation in vivo using fluorescence imaging on IVIS-Spectrum Sistem (Caliper Life Sciences, USA). Tumors were exposed to laser radiation at wavelength of 593 nm, with fluence of 180 J/cm2 three times every other day. 24 h after treatment the tumors were studied using confocal fluorescence microscopy and were analyzed histologically.Results. Fluorescence imaging techniques in vivo and ex vivo after laser treatment of the tumors revealed the bleaching of KillerRed that was the criterion for selection of treatment regimen, and indicated photodynamic reaction. In acute post-irradiationperiod the tumors expressing KillerRed were found to have signs of tissue destruction. Along with moderate, reversible dystrophic changes such as enlargement and reduction in cell size and cytoplasm vacuolization, irreversible damage signs were revealed in most cells — damage of cell and nuclear membrane integrity. The performed study showed for the first time the possibility in principle of tumor photodamage using phototoxic protein KillerRed in laser exposure in photodynamic therapy regimen.