24. Prevention Of Radiation-induced Ferroptosis In The Skin Alleviates Formation Of Chronic Fibrosis

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Purpose: Radiation-induced fibrosis (RIF) is a debilitating sequelae of radiation therapy that has been shown to improve with topical treatment with the iron chelator deferoxamine (DFO). We investigated whether ferroptosis, a recently described type of iron-dependent pathway of cell death contributes to the pathogenesis of RIF and is attenuated with DFO treatment. Methods: Adult C57BL/6J (8-12 weeks) mice were split into 5 treatment groups: 1) Untreated non-irradiated, 2) Untreated irradiated, 3) Irradiated with prophylactic DFO treatment, 4) Irradiated with continuous ferrostatin treatment, and 5) Irradiated with subsequent DFO treatment. Irradiated groups received 30 Gys of irradiation to the dorsal skin resulting in the development of chronic RIF. Perfusion testing with laser doppler was performed throughout the healing interval. Four weeks following radiation, dorsal skin was harvested and analyzed histologically and biomechanically. Immunofluorescent staining with 4 Hydroxynonenal (4-HNE) antibody was carried out to assess ferroptosis activity and imaged using confocal microscopy. Tissue was collected for single cell RNA sequencing and analyzed using the 10x platform. Cell annotations were ascribed using SingleR to identify fibroblasts and cell-type marker lists were generated using Seurat’s native FindMarkers function. Results: IHC staining showed decreased 4-HNE staining in non-irradiated skin, DFO-treated skin, and ferrostatin-treated skin compared to irradiated untreated skin. Prophylactic treatment with DFO decreased 4-HNE staining and produced histological measures closer to normal skin more so than subsequent DFO treatment. While perfusion levels increased with DFO treatment, they were not affected by ferrostatin treatment. Histological measures of RIF (dermal thickness via Hemotoxylin and Eosin and collagen deposition via Massons Trichrome) were closer to normal skin with both DFO and ferrostatin treatment. Single cell RNA sequencing analysis demonstrated that an expression module of genes associated with ferroptosis decreased in groups treated with DFO compared to irradiated untreated skin. Conclusion: Ferroptosis occurs more frequently in the dermis of murine skin following irradiation and is decreased by DFO topical treatment. Inhibition of ferroptosis with DFO and Ferrostatin alleviates RIF in murine skin. Single cell analysis indicated DFO treatment decreased transcription of drivers of ferroptosis in irradiated murine skin.

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