Comparison of the biological effects of various radiation therapies for keloids by single-cell RNA sequencing reveals IRF1 as a novel target in keloid therapy

Keloids are benign dermal tumors that arise from abnormal wound healing processes following skin lesions. Keloids are present in all human populations but occur most frequently in Africans. A significantly higher incidence is observed in female populations. Consequently, keloid patients not only endure physical burdens caused by symptoms such as pain, pruritus, and burning, but also suffer from psychological distress due to altered appearance, significantly compromising their quality of life. Surgical excision, as one of the various treatment options, demonstrates rapid efficacy but carries an unacceptably high recurrence rate of 40%–100% when used as a standalone therapy. Postoperative radiotherapy (PORT) is a clinically effective measure to reduce recurrence rates of keloid. Nevertheless, studies comparing the effectiveness and underlying mechanisms of distinct radiotherapy modalities for keloid remain absent in the literature. In our study, we performed single-cell RNA sequencing (scRNA-Seq) of primary keloid fibroblasts treated with three kinds of radiotherapy modalities, and unbiased clustering revealed the identification of seven distinct clusters. Among them, the cell proportions of Fib2-CCND1^high, Fib3-IGFBP7^high and Fib 4-APCDD1^high increased significantly in the X-ray group, electron beam group and both ⁹⁰Sr groups, which are involved in microtubule-associated complex, extracellular matrix organization and oxidative phosphorylation, respectively. In addition, the bioinformatics analysis highlighted the alteration of immune-associated interactions, such as PVR-TNFSF9 and TNFSF9-IL13RA2, and provided a basis for the activation of interferon regulatory factor 1 (IRF1) in all radiotherapy modalities. Then, functional analysis revealed that Ad-IRF1 and pharmacological activation of IRF1 (ATRA and 9-cis-RA) significantly induced apoptosis and suppressed cell viability. Mechanistically, reciprocal regulation between the single-stranded DNA sensors SSBP1 and IRF1 was revealed. Thus, this study illustrates the molecular alterations and driving transcription factors following various radiotherapy modalities at the single-cell resolution. Notably, IRF1 is regulated by SSBP1 and identified as a novel therapeutic target for keloid.