Single-cell RNA Sequencing Revealed Fibro-adipogenic Progenitors As The Major Contributor To Radiation-induced Skeletal Muscle Fibrosis

Radiation therapy causes skeletal muscle atrophy, and fibrosis in cancer survivors due to alterations in skeletal muscle mononuclear cells. However, a lack of mechanistic understanding hinders effective therapies on skeletal muscle side effects of radiation. PURPOSE: To identify transcriptional changes at the single-cell level induced by radiation and how these changes may influence cell-cell communication within skeletal muscle in a murine model of therapeutic radiation. METHODS: A fractionated radiation dose (8.2 Gray/dose) was administered for three alternating days to one hindlimb of C57Bl/6 J male mice age five weeks (IR). The rest of the body was lead-shielded, and the contralateral limb was used as a non-IR control (CON). Single-cell RNA sequencing was performed at 24 hours and 56-days post-IR. RESULTS: Transcriptomic profiling revealed 13 distinct cell populations, including muscle satellite cells (MuSCs), fibro-adipogenic progenitors (FAPs), and macrophages. A deep quiescent MuSC population expressing CD34+ was significantly reduced (p-adjusted<0.05, from 55% CON to 25% IR limb), and a subpopulation of macrophages type 2 expressing Ccr2+ was substantially increased at 56-days post-IR compared to CON (p-adjusted<0.05, from 11.19% CON to 43.06% IR limb). Genes related to fibroblast proliferation were up-regulated in FAPs at 24-hours post-IR, and a fibrotic phenotype persisted in FAPs at 56-days post-IR (Differentially expressed genes (DEGs), p-adjusted<0.05). A conserved Cdkn1a signature was observed in all cells post-IR (DEGs, p-adjusted<0.05) and inflammatory signalling mediators such as SPP1, TNF-α, and PARs were upregulated in the IR-limb at 56-days (DEGs, p-adjusted p < 0.05). Intercellular communication analysis revealed FAPs as the primary contributor of extracellular matrix and target of TGF-β signalling at both 24-hours and 56-days post-IR (DEGs, p-adjusted<0.05). CONCLUSIONS: Radiation depletes deep quiescent MuSCs, induces a conserved senescent transcriptomic profile across all muscle mononuclear cells, and increases pro-fibrotic signaling, particularly in FAPs. This work provides novel targets for reducing the long-term late effects of radiation in skeletal muscle. Natural Sciences and Engineering Research Council of Canada (RGPIN-2017-04320)