C. Berry1, M. Griffin1, D. Abbas1, H. Lintel1, L. Kameni1, C. Kendig1, T. Le1, A. Fazilat1, M. T. Longaker1, D. Wan1 1Stanford University, Plastic And Reconstructive Surgery, Palo Alto, CA, USA
Introduction: The rise in radiation-induced fibrosis (RIF) has paralleled advancements in oncological outcomes through contemporary chemoradiation treatments. Research has explored the utilization of topical deferoxamine (DFO) patch delivery to counteract the fibrotic changes in irradiated tissue. Our objective was to evaluate transcriptomic shifts in fibroblast phenotypes underlying the efficacy of this treatment to alleviate RIF in chronically irradiated excisional wounds.
Methods: Adult C57BL/6J (8-12 weeks) mice underwent dorsal irradiation and were separated into 3 conditions: 1) non-irradiated skin, 2) chronically irradiated skin, 3) chronically irradiated skin treated with topical DFO patch, and 4) chronically irradiated skin treated with a topical patch not containing DFO. All groups underwent excisional wounding of the dorsal skin four weeks following irradiation. Wounds were collected at closure and analyzed histologically, biomechanically, and with single-cell RNA sequencing 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: Histological stains with H+E, Masson’s Trichrome, and picrosirius red stains recapitulated evidence of wound healing closer to that of normal skin in irradiated wounds treated with DFO. Biomechanical testing demonstrated increased strength of wounds treated with DFO. Single-cell analysis identified seven transcriptionally distinct fibroblast subpopulations, with irradiation enhancing mechanosensitive and immunoregulatory fibroblast phenotypes (Figure 1A). These same groups decreased in proportional representation following DFO treatment, driven by decreased expression of pro-fibrotic signalling pathways associated with TIMP1 (Figure 1B).
Conclusion: This study characterizes the cellular contributions to the therapeutic effect of topical DFO treatment in the context of chronically irradiated excisional wounds and demonstrates that the downregulation of pro-fibrotic pathways drives a shift in proportional fibroblast cluster representation away from proinflammatory and mechanosensitive phenotypes related to shifts in TIMP1 expression.
