Irradiated Fibroblast Sub-populations Alter Gene Expression To Heal Murine Skin
Darren B. Abbas, M.D., Christopher V. Lavin, MS, Evan J. Fahy, MB Bch BAO, MCh, Michelle Griffin, MBChB, Ph.D, Shamik Mascharak, BS, Nicholas Guardino, BS, Hendrik Lintel, BS, Michael Januszyk, MD, PhD, Michael T. Longaker, MD, MBA, Derrick C. Wan, MD.
Stanford University, Stanford, CA, USA.
Introduction: Radiation-induced skin fibrosis is a well-documented and burdensome sequela of radiation therapy, however the exact mechanism behind this effect is not well understood. We aim to better delineate the cellular mechanisms of acute and chronic radiation-induced skin fibrosis. Methods and Materials: Thirty adult B6 mice, with equal proportion of males and females underwent 5 Gy of radiation every other day for 12 days, equaling 30 Gy total, of the dorsal skin. At weeks 0, 1, 2, 4, and 8 after radiation, the skin was harvested and analyzed using histology and immunofluorescence. Dorsal skin also underwent single-cell RNA sequencing, data was demultiplexed and underwent dimension reduction to identify transcriptionally distinct subpopulations, cross-referenced with Enrichr to identify relevant genetic pathways. Results: Dermal thickness was noted to progressively, significantly increase from week 0 to 8. Dermal collagen deposition and disorganization also significantly increased throughout the experiment (A). CD31+ expression increased in the acute phase, but decreased below baseline levels by the chronic phase (B). Four distinct fibroblast subpopulations were identified, and the genetic expression of these individual subpopulations demonstrated dynamic changes throughout the experiment (C). Sca, Dlk-1, and CD26+ immunofluorescence staining further confirmed this dynamicity as their expression fluctuated throughout the 8-week course of the experiment (D). Conclusion: Identification of this dynamic process amongst the various fibroblast subpopulations during the healing process of irradiation wounds in wild-type mice is a novel finding and paramount to better understanding and treatment of radiation-induced skin fibrosis.
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