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

Comparison of the biological effects of various radiation therapy for keloid by single-cell RNA sequencing reveals IRF1 as a novel target in keloid therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparison of the biological effects of various radiation therapy for keloid by single-cell RNA sequencing reveals IRF1 as a novel target in keloid therapy Xiaoqian Li, Wei Li, Tao Yan, Linfen Guo, Yahui Feng, Yulan Liu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4780437/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Keloids are benign dermal tumors that arise from abnormal wound healing processes following skin lesions. Postoperative radiotherapy (PORT) is a clinically effective measure to reduce recurrence rates of keloid with various radiation modalities. Nevertheless, studies comparing the effectiveness and underlying mechanisms of distinct radiotherapy modalities for keloid remain absent in the literature. Methods We performed single-cell RNA sequencing (scRNA-Seq) analysis of primary keloid fibroblasts treated with photon irradiation, electron beam irradiation or β-radiation using a 90 Sr- 90 Y surface applicator to explore gene expression profiles. Comparative analyses were also performed to identify the dysregulated pathways, regulators and ligand receptor interactions in five groups. To validate our findings, molecular biological approaches were used to confirm the potential target. Results Unbiased clustering revealed a considerable degree of cellular heterogeneity within primary keloid fibroblasts, resulting in the identification of seven distinct clusters. Among of them, the cell proportions of Fib2- CCND1 high , Fib3- IGFBP7 high and Fib 4- APCDD1 high increased significantly in X-rays group, electron beam group and both 90 Sr groups, which 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 of 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. Conclusions This study illustrated the molecular alterations and driving transcription factors following various radiotherapy modalities at the single cell resolution. Notably, the identification of IRF1 as a prospective therapeutic target for keloid is of signification importance. keloid radiotherapy single-cell RNA sequencing (scRNA-Seq) interferon regulatory factor 1 (IRF1) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Keloid is a dermal fibroproliferative disorder resulting from abnormal healing of wounds, often extending beyond the edge of the injury. 1 – 3 Besides obvious cosmetic concerns, keloid leads to a serious physical and psychological burden for patients by causing pain, pruritus, tenderness, burning and contracture. 3 – 5 Surgical excision is the most radical therapeutic option, while the recurrence rates in patients who underwent surgery alone as high as 40–100%. 6–9 Postoperative radiotherapy (PORT) is a well-established adjuvant treatment to reduce the risk of recurrence to approximately 20%. 10–12 Current keloid radiotherapy modalities include electron beam therapy from linear accelerator, photon beam therapy from X-ray tubes and brachytherapy with in situ placement of a radioactive source. 13 , 14 Electron beam delivers accelerated high-energy electrons (MeV) to depths of 2–6 cm without significant damage to deeper structures. 15 Kilo-voltage X-rays are used for treating superficial lesions and increase the dose deposited in bone. 16 While brachytherapy, using uniformly adsorbing radionuclides, ensures the even and appropriate focus of the radiation onto the target site, thereby minimizing harm to the surrounding healthy tissue. 17 For strontium-90-yttrium-90 ( 90 Sr- 90 Y) applicator, both 90 Sr and its decay product 90 Y emit β-radiation. The radiation dose at a tissue depth of 2 mm is 20–30% of the surface dose, and this significantly drops to 1–3% at a depth of 5 mm. 18 A comprehensive meta-analysis, summarized the literature from 1942 to 2014, compared recurrence between radiotherapy modalities revealed that brachytherapy yielded the lowest recurrence rate (15%), with the two 90 Sr studies showing rates of 20.5% and 12.67%, while X-rays and electron beam were both 23%. 10,12 In addition, another systematic review reported that the acute complications (erythema, dehiscence and infection) and chronic complications (hyperpigmentation, hypopigmentation and telangiectasia) across all three modalities. Peeling was reported in X-rays and electron beam, while fibrosis was only observed in brachytherapy. It’s intriguing to note that the highest rate of hyperpigmentation was observed in brachytherapy at 25.6, which was lower than that of X-rays and electron beam (100%). However, a study based on 90 Sr treatment reported a 10.4% incidence of telangiectasia as a complication. 19 These findings, derived from retrospective clinical analysis, further molecular differences among the three radiotherapy modalities are not yet fully understood. Considering the heterogeneity of keloid conditions across various studies, it is challenging to compare the biological effects of radiation, even though numerous studies have investigated into the molecular mechanisms associated with individual radiotherapy modalities. X-rays radiation increases the expression of senescence-associated genes (p16, p21 and p27), induces apoptosis and LDH release through targeting forkhead box O1 (FOXO1). 20 , 21 While electron beam irradiation might hinder keloid formation through the interleukin 6 (IL-6) signaling pathway, decrease collagen production by modifying miR-21/smad7-mediated p38 activation, and inhibit autophagy by reducing miR-21-5p, which regulates migration and LC3B expression via PTEN/Akt signaling. 22 , 23 Single-cell RNA sequencing (scRNA-Seq) transcends the limitations of bulk RNA-seq by offering an unbiased and detailed view of cellular heterogeneity and regulatory networks at a single-cell level. To date, scRNA-Seq has been applied to explore the differences between keloid tissues and adjacent normal tissues, including the depiction of the cellular landscape, fibroblast heterogeneity, the transcriptional profile of Schwann cells, Schwann cell-macrophage cross-talk, lineage-specific regulatory changes, fibrovascular communication and mesenchymal activation of endothelial cells and immune profiles. 24 – 30 Furthermore, scRNA-Seq was utilized to investigate the changes of combined triamcinolone acetonide (TAC) and 5-fluorouracil (5-FU) treatments on keloids. 31 In our study, we isolated primary keloid fibroblasts from patients and performed scRNA-Seq to analyze gene expression profiles following various radiotherapy modalities. We further analyzed and characterized the transcription factors involved in keloid radiotherapy. These findings contributed to our understanding of keloid radiotherapy and provided potential targets for keloid therapies. Materials and methods Reagents and materials Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). IRF1 overexpression adenovirus and the control Ad-NC were obtained from HanBio (Shanghai, China). ATRA and 9-cis-RA were purchased from MedChemExpress (Monmouth Junction, NJ). Antimycin A was purchased from Maokang Biotechnology (Shanghai, China). Human keloid samples The human keloid and adjacent skin samples tissues were derived from patients who had received surgery at the Department of Plastic Surgery, Second Affiliated Hospital of Chengdu Medical College (Chengdu, China). The patients’ information is summarized in Table E1. Inclusion criterion: all patients in this study were treated for the first time. Hematoxylin and Eosin (H&E) staining 4% paraformaldehyde was used to fix keloid and adjacent skin tissues. And then the tissues were embedded in paraffin. Paraffin sections of 3-µm thickness were deparaffinized and treated in citrate buffer (pH 6.0) for 7 min. The epitope retrieval procedure was then performed. Finally, the sections of keloid and adjacent tissues were stained by Hematoxylin and eosin. Cell culture Keloid tissues were washed 3–5 times in phosphate-buffered saline (PBS) and cut into ~ 1 mm 3 . Then, they were adhered to 75 cm 2 tissue culture flasks. HaCaT (human immortalized keratinocyte) and HFF-1 (human foreskin fibroblasts) were used as reported previously. 32 DMEM medium supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel), 0.1 mg/mL streptomycin and 100 U/mL penicillin (Beyotime, Nantong, China) cultured in a 37°C incubator with 5% CO 2 . After primary keloids cells freed from the tissues, experiments were performed using 3rd -7th generation, with a doubling time of 50 h. Primary keloid cells irradiation When cells reached 50% confluence in 10.0 cm dishes, they were divided into five groups: Control, 5 Gy/f for continuous 4 days X-rays irradiation (X-rays 5 Gy×4) using X-ray irradiator (320 KV; KUB Technologies, Inc., Stratford, CT), 5 Gy/f for continuous 4 days electron beam irradiation (electron beam 5 Gy×4) using a linear accelerator (Varian Medical Systems, Inc., Palo Alto, GA), 5 Gy/f for continuous 2 days and 4 days β-radiation using a 90 Sr- 90 Y surface applicator (1361 MBq) ( 90 Sr 5 Gy×2 and 90 Sr 5 Gy×4). 11,33–35 Immunofluorescence assay Primary keloid fibroblasts were first fixed with 4% paraformaldehyde for 15 min, then permeabilized with 1% Triton X-100 for 15 min, and subsequently blocked with 5% BSA for 2 h at room temperature. Then anti-Vimentin antibody (arigo Biolaboratories Corp., Shanghai, China, #ARG66199, 1:200) and anti-IRF1 antibody (Cell Signaling Technology, Beverly, MA; #8478S, 1:200) were incubated with cells overnight at 4 ℃, followed by incubation of secondary antibody with Alexa Fluor Plus 647- labeled goat anti-rabbit antibody and Alexa Fluor Plus 488- labeled goat anti-rabbit antibody (Thermo Fisher Scientific Inc., Shanghai, China) at a dilution of 1:300 for 2 hours at room temperature, respectively. Cell nuclei were stained with antifade mounting medium containing DAPI (Vector Laboratories, Inc., Burlingame, CA) for 15 min, and a scanning laser confocal microscopy (Olympus, Tokyo, Japan) was used to capture images. Clonogenic assay Six-h after radiation, primary keloid cells (5×10 3 ) were seeded into 6.0 cm dishes. After culturing for 10 days, cells were fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime Biotechnology, Nantong, China). Cell cycle assay Primary keloid cells were collected 48 hours after irradiation and fixed overnight in pre-chilled 70% ethanol at -20°C. Next, cells were treated with 1 mL propidium iodide solution (10 mg/mL) at 37°C for 30 min in darkness. Samples were detected by FACS Celesta flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and analyzed with ModFit LT™ (Version 5). Cell apoptosis assay Primary keloid cells were harvested 48 h post-irradiation and infection, then the degree of apoptosis was detected by Annexin V/PI kit (Yeasen Biotechnology, Shanghai, China). Then, samples were detected by FACS Celesta flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and analyzed with FlowJoTM (Version 10.7). Lactate dehydrogenase (LDH) assay LDH activity was measured by an LDH Cytotoxicity Assay Kit (Beyotime, Nantong, China) at 48 h post-radiation and 24, 48 and 72 h after the treatment of ATRA and 9-cis-RA. Lastly, the absorbance was measured at 490 nm with a microplate reader (BioTek, Synergy HTX, Winooski, VT). Senescence-associated β-galactosidase (SA-β-gal) staining Primary keloids fibroblasts were seeded into 24 wells plates at 5000 cells/ well for 6 h after radiation. Then, after 48 h of exposure to radiation, a Senescence SA-β-gal Staining Kit (Beyotime Biotechnology, Shanghai, China) was used. Briefly, cells were fixed with 500 µl of fixative solution for 30 min at room temperature, followed by preparation of SA-β-gal staining solution incubated overnight at 37°C protected from light. Finally, a light microscopy (Olympus, Tokyo, Japan) was used to capture the images, and the percentage of SA-β-gal + cells in five random fields in each of the four wells were counted. Western blotting analysis Proteins of primary keloid fibroblasts were harvested and lysed in RIPA lysis buffer containing protease inhibitor cocktail and PMSF (Beijing Solarbio, Beijing, China) for 30 min at 4 ℃. BCA Protein Assay kit (Beyotime, Nantong, China) was used to measure protein concentration. Protein (40 g/lane) from each lysate was fractionated by 10% SDS-PAGE and transferred to 0.22 µm PVDF (polyvinylidene difluoride) membranes (Millipore, Billerica, MA). After blocking with 5% non-fat milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBST) for 2 hours at room temperature, the membranes were blotted with primary antibodies: IRF1 (Cell Signaling Technology, Beverly, MA; #8478S), H3 (Cell Signaling Technology, Beverly, MA; #4499S), GAPDH (1:5000, Abcam Plc., Cambridge, UK; #ab181602), PARP (Cell Signaling Technology, Beverly, MA; #9542S), Cleaved PARP (Cell Signaling Technology, Beverly, MA; #5625S), Bax (Cell Signaling Technology, Beverly, MA; #2772S), Bcl-2 (Cell Signaling Technology, Beverly, MA; #3498S) and SSBP1 (Proteintech Group, Inc, Wuhan, China; #12212-1-AP) overnight at 4 ℃. Before detecting by a Super ECL Dectection Reagent (Yeasen Biotech, Shanghai, China) and visualizing by a FluroChem MI imaging system (Shenhua Science Technology Co., Ltd., Hangzhou, China), horseradish peroxidase-conjugated goat anti- rabbit IgG (Abbkine Scientific, Wuhan, China) secondary antibodies were applied to incubate for 2 h. Single-cell RNA sequencing (scRNA-Seq) After 48 h of exposure to radiation, scRNA-Seq was performed by Singleron (Nanjing, China). The samples were stained with trypan blue (Sigma, Shanghai, China) and microscopically evaluated for cell viability. Single-cell suspensions at 1 × 10 5 cells/mL in concentration in PBS (HyClone, Shanghai, China) were prepared and loaded onto microfluidic devices and scRNA-Seq libraries were constructed according to Singleron GEXSCOPE® protocol by GEXSCOPE® Single-Cell RNA Library Kit (Singleron Biotechnologies) and Singleron Matrix® Automated single-cell processing system (Singleron Biotechnologies). Individual libraries were diluted to 4 ng/µL and pooled for sequencing. Pools were sequenced on Illumina novaseq6000 with 150 bp paired end reads. The GEO dataset (GSE) number is GSE261116. Other methods were detailed in the Supplementary Materials . Results Ionizing radiation inhibits the proliferation and induces cell death of primary keloid fibroblasts The keloids and adjacent skin tissues were obtained from surgical excisions of patients. H&E staining revealed that the keloid showed an increased number of dermal cells and large number of parallel and staggered bundles of collagen fibers compared to the adjacent-keloid (Fig. 1 A). Afterwards, primary keloid cells were isolated and characterized using phase-contrast optical microscopy and immunofluorescence methods. The former showed that these cells exhibited typical fibroblasts features, such as elongated, spindle-shaped cells with distinctive ridges and filamentous projections (Fig. 1 B). In addition, the immunofluorescence staining demonstrated that primary keloid cells were positive for vimentin expression (Fig. 1 C). Subsequently, we examined the effects of different types and different doses of rays on primary keloid fibroblasts (Fig. 1 D). As shown in Fig. 1 E, the results of clonogenic experiments showed that primary keloid fibroblasts lose the ability to form clones after X-rays and electron beam irradiation, while the two 90 Sr groups only inhibited the clone formation of cells in a dose-dependent manner. Similar results can be seen in the SA-β-gal assay for detecting cellular senescence. At a radiation dose of 5 Gy×4, the percentage of SA-β-gal + cells of X-rays, electron beam and 90 Sr groups were all about 45%, but the percentage of senescent cells was lower, at about 35%, when the dose of 90 Sr was reduced to 5 Gy×2 (Fig. 1 F). We also found that X-rays and electron beams induced more severe damage than that of equivalent 90 Sr exposure (Fig. 1 G-I). Overall, these results demonstrated that keloids exhibit distinct biological responses to different radiotherapy modalities. Single-cell transcriptomic map in primary keloid fibroblasts with different radiotherapy modalities To dissect cellular heterogeneity in human primary keloid fibroblasts treated with different radiotherapy modalities, we performed scRNA-Seq (Fig. 2 A). After standard data processing and quality filtering, a total of 43,867 single cells from the five groups (control: 14,351; X-rays 5 Gy×4: 3,488; electron beam 5 Gy×4: 8,870; 90 Sr 5 Gy×2: 7,458; 90 Sr 5 Gy×4: 9,700) were obtained. Using unbiased clustering segmented the cells into seven distinct clusters, all marked by elevated expression of fibroblast-specific markers ( Col1A1 , Col1A2 , DCN and LUM ); yet, each subcluster displayed distinct transcriptomic signatures (Fig. 2 B, C). Then, according to the top five differential genes of each cluster in Fig. 2 D, these subpopulations could be characterized by specific markers: Fib 1- CDK1 high , Fib 2- CCDN1 high , Fib 3- IGFBP7 high , Fib 4- APCDD1 high , Fib 5- PTTG1 high , Fib 6- SQSTM1 high and Fib 7- MALAT1 high , respectively (Fig. 2 E). As shown in Fig. E1A-G, functional enrichment analysis revealed that the signature genes of Fib 1- CDK1 high and Fib 5- PTTG1 high were both enriched with nuclear division, while Fib 2- CCDN1 high were enriched with terms related to microtubule. Both Fib 3- IGFBP7 high and Fib 4- APCDD1 high showed enrichment with ECM-related terms, thus representing two myofibroblast subpopulations. Additionally, Fib 6- SQSTM1 high and Fib 7- MALAT1 high were found to be involved in stress responses and RNA splicing, respectively. Taken together, these findings collectively highlighted the intricate heterogeneity within keloid fibroblasts and their varied responses to radiotherapy. Next, to define the cell-cell communication landscape between fibroblast subpopulations in five groups, we performed analysis using CellPhoneDB 2.0. 36 As detailed in Circos plots and bar plots, we found a denser interaction network and a more abundant number of intercellular interactions in X-rays 5 Gy×4 compared to that in the other groups (Fig. 2 F, G). As shown in Fig. 2 H, We observed disparities in immune-associated interactions between control and irradiated groups, such as PVR-TNFSF9, TNFSF9-IL13RA2 and ACKR3-CXCL12. These data suggested that radiation may induce immune-related responses in primary keloid fibroblasts. Differential proportion analysis reveals significant expansion of fibroblast subpopulations in keloids with different radiotherapy modalities In our subsequent analysis, we aimed to pinpoint the irradiation-associated cell clusters that expanded under 5 Gy×4 conditions in primary keloid fibroblasts. The visualization of cellular density and distribution revealed changes in the relative proportions of seven subpopulations among four groups (Fig. 3 A, B). Notably, the radio of Fib 2- CCDN1 high , Fib 3- IGFBP7 high and Fib 4- APCDD1 high increased following X-rays 5 Gy×4, electron beam and 90 Sr irradiation, respectively. This expansion suggested a crucial role for these subpopulations in the keloid's response to various radiotherapy modalities. Figure 3 C-E illustrated our findings that under X-rays irradiation at 5 Gy×4, the Fib 2- CCDN1 high exhibited a significant upregulation of genes involved in co-translational protein targeting to membrane and protein localization to endoplasmic reticulum, such as ribosomal protein L41 ( RPL41 ), ribosomal protein S28 ( RPS28 ), ribosomal protein L22 ( RPL22 ), ribosomal protein S10 ( RPS10 ) and ribosomal protein L39 ( RPL39 ). Extracellular matrix-associated genes, including collagen type VIII alpha 1 chain ( COL8A1 ), fibulin 2 ( FBLN2 ), sulfatase 1 ( SULF1 ), dermatopontin ( DPT ) and cartilage oligomeric matrix protein ( COMP ), were significantly increased in electron beam 5 Gy×4 Fib 3- IGFBP7 high . In 90 Sr 5 Gy×4 Fib 4- APCDD1 high , there was a notable increase in genes related to reproductive development, including JunB proto-oncogene, AP-1 transcription factor subunit ( JUNB ), odd-skipped related transcription factor 1 ( OSR1 ), aldo-keto reductase family 1 member C3 ( AKR1C3 ) and pleckstrin homology like domain family A member 2 ( PHLDA2 ). Gene ontology (GO) analysis confirmed these trends, indicating enrichment of “signal recognition particle (SRP)-dependent co-translational protein targeting to membrane”, “protein targeting to ER and co-translational protein targeting to membrane” in X-rays 5 Gy×4 Fib 2- CCDN1 high , “extracellular matrix organization”, “extracellular structure organization” and “external encapsulating structure organization” in electron beam 5 Gy×4 Fib 3- IGFBP7 high and “reproductive structure development” and “reproductive system development” in 90 Sr 5 Gy×4 Fib 4- APCDD1 high (Fig. 3 F-H). These results implied that different irradiation not only alters the proportion of fibroblast subpopulations but also modulates their functional characteristics in primary keloid fibroblasts. Differential proportion analysis reveals differences of fibroblast subpopulations in primary keloid fibroblasts with different doses of 90 Sr Brachytherapy offers precise targeting of keloid tissue with less toxicity to adjacent tissue. 16 Thus, we sought to determine the molecular changes of primary keloid fibroblasts among 0, 5 Gy×2 and 5 Gy×4 90 Sr irradiation. By calculating the proportion and numbers of differentially expressed genes (DEGs) of each cluster in the three groups, the percentage of Fib 4- APCDD1 high increased in both 90 Sr irradiation groups and had relatively large numbers of DEGs (Fig. 4 A, B). Notably, genes associated with oxidative phosphorylation and ATP metabolic process, including cytochrome c oxidase subunit 7A1 ( COX7A1 ), coiled-coil-helix-coiled-coil-helix domain containing 10 ( CHCHD10 ), nuclear protein 1, transcriptional regulator ( NUPR1 ), NADH: ubiquinone oxidoreductase subunit A13 ( NDUFA13 ) and NADH: ubiquinone oxidoreductase subunit B8 ( NDUFB8 ), were upregulated in Fib 4- APCDD1 high of both 90 Sr irradiation groups (Fig. 4 C). Gene ontology (GO) analysis confirmed enrichment of “oxidative phosphorylation” and “ATP metabolic process and electron transport chain” pathways were enriched in both 90 Sr irradiation groups, which indicating a significant metabolic shift (Fig. 4 D). While higher percentage of Fib 6- SQSTM1 high and Fib 7- MALAT1 high , as well as the largest numbers of DEGs of Fib 7- MALAT1 high were observed in 90 Sr 5 Gy×2, suggesting a significant role in primary keloid fibroblasts response to this dosage (Fig. 4 A, B). Comparative analysis of these subpopulations between the 5 Gy×2 and other groups revealed that pathways related to "oxidative phosphorylation" "reactive oxygen species" and "P53" were upregulated in the Fib 6- SQSTM1 high group following 90 Sr 5 Gy×2 irradiation (Fig. 4 E). In Fib 7- MALAT1 high , DEGs such as sulfatase 1 ( SULF1 ), splicing factor 3b subunit 1 ( SF3B1 ), integrin subunit alpha 11 ( ITGA11 ), adrenomedullin ( ADM ), eukaryotic translation initiation factor 4A2 ( EIF4A2 ) were commonly upregulated when comparing 90 Sr 5 Gy×2 to other two groups (Fig. 4 F). GO enrichment analysis highlighted the enrichment of pathways like “transmembrane receptor protein serine/threonine kinase signaling pathway”, “RNA splicing” and “mRNA splicing” in 90 Sr 5 Gy×2 Fib 7- MALAT1 high (Fig. 4 G). Overall, these results demonstrated that the subpopulations changes between two 90 Sr irradiation doses were not dose-dependent. Pseudotemporal analysis reveals a branched trajectory with a significant shift toward the myofibroblast phenotype in primary keloid fibroblasts treated with different rays To account for the significant changes in primary keloid fibroblasts during the radiotherapy process, we utilized Monocle2 for performed pseudotemporal ordering. 37 This analysis revealed a branched trajectory with two cell fates (Fig. 5 A). Fib 5- PTTG1 high predominantly occupied the initial state before branching (Fig. 5 B). Both Fib 3- IGFBP7 high and Fib 4- APCDD1 high were elevated in the cell fate 1 branch, which represents the myofibroblast phenotype (Fig. 5 B). Fib 7- MALAT1 high constituted the most of the cell fate 2 branch associated with RNA splicing. In the control group, the proportion of cells in states pre-branch, 1 and 2 was 95.42%, 2.63% and 1.95%, respectively (Fig. 5 C). Strikingly, irradiated groups showed a pronounced shift toward the myofibroblast phenotype in cell fate 1 (X-rays 5 Gy×4: 71%; electron beam 5 Gy×4: 95.5%; 90 Sr 5 Gy×2: 65%; 90 Sr 5 Gy×4: 61.17%; Fig. 5 C). Unexpected, the proportion of cells in state pre-branch was 19.52% in 90 Sr 5 Gy×4. Branched expression analysis identified 7357 genes with expression dynamics that corresponded to transitions between cellular states, clustering into seven distinct modules (Fig. 5 D). Module 1 genes, highly expressed in the myofibroblast branch (cell fate 1), were linked to GO terms such as "extracellular matrix organization" and "neutrophil-mediated immunity." Module 3 genes, enriched in the RNA splicing branch (cell fate 2), were associated with "covalent chromatin modification" and "histone modification." While module 7 genes, showing pre-branch enrichment, were involved in processes like "chromosome segregation" and "DNA replication" (Fig. 5 E). These data suggested that radiation induce primary keloid cells significant shift toward the myofibroblast phenotype. Activation of transcription factor interferon regulatory factor 1 (IRF1) serves as a novel approach for keloids treatment Due to the importance of transcription factors (TFs) in RNA profiles, we applied a single-cell regulatory network inference and clustering (SCENIC) method to score the activity of regulons by an AUCell algorithm (AUC score). 38 Our analysis revealed significant enrichment of IRF1 across all irradiation groups compared with the control (Fig. 6 A). Then, immunofluorescence staining revealed nuclear translocation of IRF1 in primary keloid fibroblasts post-irradiation (Fig. 6 B). To substantiate the therapeutic potential of IRF1 in keloids, we overexpressed IRF1 using an adenovirus vector (Ad-IRF1). This led to an increase in apoptosis and upregulation of related proteins (Cleaved-PARP and Bax) at 48 hours post-infection, indicating a role for IRF1 in keloid cell death (Fig. 6 C, D). Next, to elucidate the activation mechanism of IRF1 by radiation, we focused on single-strand DNA-binding protein 1 (SSBP1), which have recently reported the interacting proteins of IRF1. 39 In addition, recent studies showed that the interaction between SSBP1 and IFI6 protects against radiation-induced skin injury. 40 Then, confirming their interaction in primary keloid fibroblasts through immunoprecipitation (IP) (Fig. 6 E). Moreover, we found that SSBP1 knockdown resulted in elevated IRF1 levels and enhanced its nuclear translocation (Fig. 6 F-H). These findings reinforce the notion of SSBP1 as an inhibitory chaperone of IRF1 in response to radiation. Furthermore, we investigated the potential of pharmacological IRF1 activation to enhance keloid treatment efficacy. Utilizing the Comparative Toxicogenomics Database | (CTD) ( www.ctdbase.org ), we identified 213 candidate chemicals modulate IRF1 expression (Fig. 6 I). Retinoic acid (ATRA), 9-cis-retinoic acid (9-cis-RA) and antimycin A caught our attention due to their positive correlation with IRF1 expression, although their specific impact on keloids remained to be determined. Our experiments demonstrated that both ATRA and 9-cis-RA significantly elevated IRF1 mRNA and protein levels in primary keloid fibroblasts and facilitated its nuclear translocation, whereas antimycin A had a minor effect on IRF1 mRNA expression only (Fig. 6 J-L). Further analysis of the chemicals' impact on cell proliferation revealed that ATRA and 9-cis-RA were more lethal to primary keloid fibroblasts than two of immortalized normal skin cells HaCaT and HFF-1 (Fig. 6 M and Fig. E2A-C). Although, all three chemicals increased the release of LDH, indicating cytotoxicity (Fig. 6 M). Thus, these data implicated IRF1 as a potential target of keloid therapy. Discussion Radiotherapy has been an adjuvant treatment in keloid management for over a century. 13 Among these three radiotherapy modalities, both electron beams and 90 Sr emit beta particles. Simultaneously, both beta particles and X-rays are classified as low LET (linear energy transfer) radiation, with an RBE (relative biological effectiveness) of 1. 41 Despite theirs recognized effectiveness, there were significant differences in the clinical therapeutic effects. 19 Furthermore, the underlying mechanisms and comparability of different irradiation modalities on keloids have not been fully elucidated. Our study aimed to provide a comprehensive cellular and molecular landscape of primary keloid fibroblasts under different irradiations, revealing both shared and unique changes that could inform new radiotherapeutic targets. Our findings indicated that equivalent X-rays, electron beam and 90 Sr radiation showed different inhibitory effect on primary keloid fibroblasts, accompanied by distinct gene expression profiles. Specifically, X-rays 5 Gy×4 increased the population of Fib 2- CCDN1 high enhancing the expression of genes related to microtubule-associated complexes and inflammation-associated receptor-ligand interactions, suggesting a role in cell proliferation and immune response. Electron beam 5 Gy×4 increased the Fib 3- IGFBP7 high cell population, driving a significant majority of primary keloid fibroblasts toward a pro-fibrosis cell fate, characterized by upregulated extracellular matrix organization genes. Although 90 Sr 5 Gy×2 and 90 Sr 5 Gy×4 both increased the population of Fib 4- APCDD1 high which enriched in oxidative phosphorylation, assessments of cellular damage, interactions, and pseudotemporal dynamics suggested that 90 Sr 5 Gy×2 was more effective. These insights contributed to understanding the radiotherapy's effects on keloid fibroblasts and may guide the optimization of treatment strategies. Previously, a study based on RNA-Seq and ChIP-Seq datasets revealed that IRF1 dominated the IFN response triggered by ionizing radiation in mouse bone marrow-derived macrophage. 42 In this study, we uncovered potential regulatory role of IRF1 across all keloid radiotherapy and affirmed the role of SSBP1 as an IRF1 chaperone that inhibits its nuclear translocation following radiation exposure. Previously, the downregulation of SSBP1 was reported to sensitize non-small cell lung cancer cells to ionizing radiation. 43 Furthermore, we used the database to predict potential IRF1 agonists. We focused on ATRA and 9-cis-RA, retinoid acid isomers derived from vitamin A. 44 ATRA is used for acute promyelocytic leukemia and being the first example of a cyto-differentiating agent to anti-tumor. It has been shown to activate IRF1 gene expression, thereby inhibiting cell growth in various cancer cell lines, including myeloid leukemia, cervical squamous carcinoma and lung cancer. 45 , 46 Recent studies have shown that ATRA upregulate expression of IRF1 by activating retinoic acid receptor (RAR)γ and interferon-β response pathway and resulted in cell death. 47 Furthermore, ATRA has been reported to increase nuclear IRF1 levels in human mammary epithelial cells. 48 Although there was no study about the relationship between 9-cis-RA and IRF1, 9-cis-RA was applied to treat skin diseases and involved in immune response, including chronic hand eczema, intractable prurigo and cutaneous T-cell lymphoma. 49 – 51 Taken together, we identified that ATRA and 9-cis-RA could be useful agents against keloids possibly via the activation of IRF1. However, there were several potential limitations of this study. Although abnormal fibroblast is the predominant cell type in keloid, other types of cells such as endothelia cells and immune cells are yet to be investigated. Stable animal models with keloid warrant further investigated. Conclusions Our study illustrated the mRNA profiles and driving transcription factors of primary keloid fibroblasts following various radiotherapy modalities by scRNA-Seq. Overexpression of IRF1 through adenovirus and IRF1 agonists (ATRA and 9-cis-RA) induced apoptosis and inhibited cell viability. Moreover, we affirmed the role of SSBP1 as an IRF1 chaperone following radiation exposure which may involve in keloid radioresistance. Thus, we provided reliable experimental support for the development of novel treatment strategies targeting IRF1 for keloids treatment. Declarations Ethics approval and consent to participate Before surgery, informed consents were obtained from all patients and approved by the ethics committee of Second Affiliated Hospital of Chengdu Medical College (Approval No. KJ20210035). All procedures comply with the guidelines and ethical principles. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work is supported by the National Natural Science Foundation of China (82073477 and 82373523), the Young Talent Project of China National Nuclear Corporation and Natural Science Foundation of Science and Technology of Sichuan Provincial (2023NSFSC0648 and 2024YFHZ0332). Authors' contributions Xiaoqian Li participated in the design and cellular experiments, conducted statistical analysis and drafted the manuscript. Wei Li, Tao Yan, Linfen Guo and Daojiang Yu collected the samples and conducted statistical analysis. Yahui Feng, Yulan Liu and Lu Ye participated in the design and cellular experiments. Yuehua Zhang and Hao Bai provided guidance in manuscript drafting. Nianyong Chen conducted the statistical analysis. Shuyu Zhang conceived the study, participated in the design and contributed to revised the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank all the participants of this study. References Stevenson AW, Deng Z, Allahham A, Prêle CM, Wood FM, Fear MW. The epigenetics of keloids. Exp Dermatol. 2021 Aug;30(8):1099-1114. Tsai CH, Ogawa R. Keloid research: current status and future directions. Scars Burn Heal. 2019 Aug 19; 5:2059513119868659. Tan S, Khumalo N, Bayat A. Understanding Keloid Pathobiology From a Quasi-Neoplastic Perspective: Less of a Scar and More of a Chronic Inflammatory Disease With Cancer-Like Tendencies. Front Immunol. 2019 Aug 7;10:1810. McGinty S, Siddiqui WJ. Keloid. 2022 Jul 19. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan–. Hawash AA, Ingrasci G, Nouri K, Yosipovitch G. Pruritus in Keloid Scars: Mechanisms and Treatments. Acta Derm Venereol. 2021 Oct 28;101(10): adv00582. Jfri A, Rajeh N, Karkashan E. A Case of Multiple Spontaneous Keloid Scars. Case Rep Dermatol. 2015 Jul 24;7(2):156-60. Arno AI, Gauglitz GG, Barret JP, Jeschke MG. Up-to-date approach to manage keloids and hypertrophic scars: a useful guide. Burns. 2014 Nov;40(7):1255-66. Furtado F, Hochman B, Ferreira LM. Evaluating keloid recurrence after surgical excision with prospective longitudinal scar assessment scales. J Plast Reconstr Aesthet Surg. 2012 Jul;65(7): e175-81. Kumar AS, Kamalasanan K. Drug delivery to optimize angiogenesis imbalance in keloid: A review. J Control Release. 2021 Jan 10;329:1066-1076. Mankowski P, Kanevsky J, Tomlinson J, Dyachenko A, Luc M. Optimizing Radiotherapy for Keloids: A Meta-Analysis Systematic Review Comparing Recurrence Rates Between Different Radiation Modalities. Ann Plast Surg. 2017 Apr;78(4):403-411. Kal HB, Veen RE. Biologically effective doses of postoperative radiotherapy in the prevention of keloids. Dose-effect relationship. Strahlenther Onkol. 2005 Nov;181(11):717-23. Wagner W, Alfrink M, Micke O, Schäfer U, Schüller P, Willich N. Results of prophylactic irradiation in patients with resected keloids--a retrospective analysis. Acta Oncol. 2000;39(2):217-20. Liu EK, Cohen RF, Chiu ES. Radiation therapy modalities for keloid management: A critical review. J Plast Reconstr Aesthet Surg. 2022 Aug;75(8):2455-2465. Cheraghi N, Cognetta A Jr, Goldberg D. RADIATION THERAPY for the Adjunctive Treatment of Surgically Excised Keloids: A Review. J Clin Aesthet Dermatol. 2017 Aug;10(8):12-15. Ogawa R, Tosa M, Dohi T, Akaishi S, Kuribayashi S. Surgical excision and postoperative radiotherapy for keloids. Scars Burn Heal. 2019 Dec 10; 5:2059513119891113. Goutos I, Ogawa R. Brachytherapy in the adjuvant management of keloid scars: literature review. Scars Burn Heal. 2017 Nov 10; 3:2059513117735483. Laskar S, Gurram L, Laskar SG, Chaudhari S, Khanna N, Upreti R. Superficial ocular malignancies treated with strontium-90 brachytherapy: long term outcomes. J Contemp Brachytherapy. 2015 Oct;7(5):369-73. Dong W, Qiu B, Fan F. Adjuvant Radiotherapy for Keloids. Aesthetic Plast Surg. 2022 Feb;46(1):489-499. Yan L, Wang LZ, Xiao R, Cao R, Pan B, Lv XY, Jiao H, Zhuang Q, Sun XJ, Liu YB. Inhibition of microRNA-21-5p reduces keloid fibroblast autophagy and migration by targeting PTEN after electron beam irradiation. Lab Invest. 2020 Mar;100(3):387-399. Hong M, Li X, Liu Y, Mo W, Shi B, Chen S, Yan T, Shi Y, Yu D, Zhang S. Molecular response of keloids to ionizing radiation: targeting FOXO1 radiosensitizes keloids. Int J Radiat Biol. 2022 Sep 28:1-10. Ji J, Tian Y, Zhu YQ, Zhang LY, Ji SJ, Huan J, Zhou XZ, Cao JP. Ionizing irradiation inhibits keloid fibroblast cell proliferation and induces premature cellular senescence. J Dermatol. 2015 Jan;42(1):56-63. Tosa M, Ghazizadeh M, Shimizu H, Hirai T, Hyakusoku H, Kawanami O. Global gene expression analysis of keloid fibroblasts in response to electron beam irradiation reveals the involvement of interleukin-6 pathway. J Invest Dermatol. 2005 Apr;124(4):704-13. Li S, Liu W, Lei Y, Long J. Regulatory effects of electronic beam irradiation on mir-21/smad7-mediated collagen I synthesis in keloid-derived fibroblasts. Biol Open. 2016 Nov 15;5(11):1567-1574. Liu X, Chen W, Zeng Q, Ma B, Li Z, Meng T, Chen J, Yu N, Zhou Z, Long X. Single-Cell RNA-Sequencing Reveals Lineage-Specific Regulatory Changes of Fibroblasts and Vascular Endothelial Cells in Keloids. J Invest Dermatol. 2022 Jan;142(1):124-135.e11. Direder M, Wielscher M, Weiss T, Laggner M, Copic D, Klas K, Bormann D, Vorstandlechner V, Tschachler E, Jan Ankersmit H, Mildner M. The transcriptional profile of keloidal Schwann cells. Exp Mol Med. 2022 Nov;54(11):1886-1900. Direder M, Weiss T, Copic D, Vorstandlechner V, Laggner M, Pfisterer K, Mildner CS, Klas K, Bormann D, Haslik W, Radtke C, Farlik M, Shaw L, Golabi B, Tschachler E, Hoetzenecker K, Ankersmit HJ, Mildner M. Schwann cells contribute to keloid formation. Matrix Biol. 2022 Apr;108:55-76. Gong T, Wang Y, Dong S, Ma X, Du D, Zou C, Zheng Q, Wen Z. Single-cell RNA-seq reveals the communications between extracellular matrix-related components and Schwann cells contributing to the earlobe keloid formation. Front Med (Lausanne). 2022 Oct 26; 9:1000324. Deng CC, Hu YF, Zhu DH, Cheng Q, Gu JJ, Feng QL, Zhang LX, Xu YP, Wang D, Rong Z, Yang B. Single-cell RNA-seq reveals fibroblast heterogeneity and increased mesenchymal fibroblasts in human fibrotic skin diseases. Nat Commun. 2021 Jun 17;12(1):3709. Shim J, Oh SJ, Yeo E, Park JH, Bae JH, Kim SH, Lee D, Lee JH. Integrated Analysis of Single-Cell and Spatial Transcriptomics in Keloids: Highlights on Fibrovascular Interactions in Keloid Pathogenesis. J Invest Dermatol. 2022 Aug;142(8):2128-2139.e11. Feng C, Shan M, Xia Y, Zheng Z, He K, Wei Y, Song K, Meng T, Liu H, Hao Y, Liang Z, Wang Y, Huang Y. Single-cell RNA sequencing reveals distinct immunology profiles in human keloid. Front Immunol. 2022 Aug 3;13:940645. Xia Y, Wang Y, Hao Y, Shan M, Liu H, Liang Z, Kuang X. Deciphering the single-cell transcriptome network in keloids with intra-lesional injection of triamcinolone acetonide combined with 5-fluorouracil. Front Immunol. 2023 May 19;14:1106289. Li X, Qian K, Zhang Y, Zhang Y, Liu Y, Sun C, Jiao Y, Yu D, Geng F, Cao J, Zhang S. Ubiquitin-specific peptidase 47 (USP47) regulates cutaneous oxidative injury through nicotinamide nucleotide transhydrogenase (NNT). Toxicol Appl Pharmacol. 2023 Dec 1;480:116734. Fraunholz IB, Gerstenhauer A, Böttcher HD. Results of postoperative (90) Sr radiotherapy of keloids in view of patients' subjective assessment. Strahlenther Onkol. 2005 Nov;181(11):724-9. Wang QG, Li XM, Zhang M, Li H, Wen B, Li HZ, Gao XS. [Effect of two dose fractionations on postoperative radiotherapy of keloid: an analysis of 107 patients]. Beijing Da Xue Xue Bao Yi Xue Ban. 2014 Feb 18;46(1):169-72. Chinese. Son Y, Phillips EON, Price KM, Rosenberg LZ, Stefanovic B, Wolfe CM, Shaath TS, Om A, Cohen GF, Gunjan A. Treatment of keloids with a single dose of low-energy superficial X-ray radiation to prevent recurrence after surgical excision: An in vitro and in vivo study. J Am Acad Dermatol. 2020 Nov;83(5):1304-1314. Efremova M, Vento-Tormo M, Teichmann SA, Vento-Tormo R. CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes. Nat Protoc. 2020 Apr;15(4):1484-1506. Qiu X, Hill A, Packer J, Lin D, Ma YA, Trapnell C. Single-cell mRNA quantification and differential analysis with Census. Nat Methods. 2017 Mar;14(3):309-315. Van de Sande B, Flerin C, Davie K, De Waegeneer M, Hulselmans G, Aibar S, Seurinck R, Saelens W, Cannoodt R, Rouchon Q, Verbeiren T, De Maeyer D, Reumers J, Saeys Y, Aerts S. A scalable SCENIC workflow for single-cell gene regulatory network analysis. Nat Protoc. 2020 Jul;15(7):2247-2276. Geng F, Chen J, Song B, Tang Z, Li X, Zhang S, Yang T, Liu Y, Mo W, Zhang Y, Sun C, Tan L, Tu W, Yu D, Cao J, Zhang S. Chaperone- and PTM-mediated activation of IRF1 tames radiation-induced cell death and the inflammatory response. Cell Mol Immunol. 2024 Jun 07; 2042-0226. Jia H, Mo W, Hong M, Jiang S, Zhang YY, He D, Yu D, Shi Y, Cao J, Xu X, Zhang S. Interferon-α inducible protein 6 (IFI6) confers protection against ionizing radiation in skin cells. J Dermatol Sci. 2020 Nov;100(2):139-147. Beheshti A, McDonald JT, Miller J, Grabham P, Costes SV. GeneLab Database Analyses Suggest Long-Term Impact of Space Radiation on the Cardiovascular System by the Activation of FYN Through Reactive Oxygen Species. Int J Mol Sci. 2019 Feb 3;20(3):661. Purbey PK, Scumpia PO, Kim PJ, Tong AJ, Iwamoto KS, McBride WH, Smale ST. Defined Sensing Mechanisms and Signaling Pathways Contribute to the Global Inflammatory Gene Expression Output Elicited by Ionizing Radiation. Immunity. 2017 Sep 19;47(3):421-434.e3. Wang Y, Hu L, Zhang X, Zhao H, Xu H, Wei Y, Jiang H, Xie C, Zhou Y, Zhou F. Downregulation of Mitochondrial Single Stranded DNA Binding Protein (SSBP1) Induces Mitochondrial Dysfunction and Increases the Radiosensitivity in Non-Small Cell Lung Cancer Cells. J Cancer. 2017 May 12;8(8):1400-1409. Zhu J, Hu L, Li L, Huang X, Shi H. Comparison of phenotypic and global gene expression changes in Xenopus tropicalis embryos induced by agonists of RAR and RXR. Toxicol Appl Pharmacol. 2017 Sep 1;330:40-47. Matikainen S, Ronni T, Hurme M, Pine R, Julkunen I. Retinoic acid activates interferon regulatory factor-1 gene expression in myeloid cells. Blood. 1996 Jul 1;88(1):114-23. Arany I, Whitehead WE, Grattendick KJ, Ember IA, Tyring SK. Suppression of growth by all-trans retinoic acid requires prolonged induction of interferon regulatory factor 1 in cervical squamous carcinoma (SiHa) cells. Clin Diagn Lab Immunol. 2002 Sep;9(5):1102-6. Wang Q, Lin Z, Wang Z, Ye L, Xian M, Xiao L, Su P, Bi E, Huang YH, Qian J, Liu L, Ma X, Yang M, Xiong W, Zu Y, Pingali SR, Xu B, Yi Q. RARγ activation sensitizes human myeloma cells to carfilzomib treatment through the OAS-RNase L innate immune pathway. Blood. 2022 Jan 6;139(1):59-72. Luo XM, Ross AC. Retinoic acid exerts dual regulatory actions on the expression and nuclear localization of interferon regulatory factor-1. Exp Biol Med (Maywood). 2006 May;231(5):619-31. Napolitano M, Potestio L, De Lucia M, Nocerino M, Fabbrocini G, Patruno C. Alitretinoin for the treatment of severe chronic eczema of the hands. Expert Opin Pharmacother. 2022 Feb;23(2):159-167. Chung BY, Um JY, Kang SY, Jung MJ, Kim JC, Kwak IS, Park CW, Kim HO. Oral Alitretinoin for Patients with Refractory Prurigo. Medicina (Kaunas). 2020 Nov 9;56(11):599. Kaemmerer T, Stadler PC, Helene Frommherz L, Guertler A, Einar French L, Reinholz M. Alitretinoin in the treatment of cutaneous T-cell lymphoma. Cancer Med. 2021 Oct;10(20):7071-7078. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4780437","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":332207119,"identity":"9a2bb7bd-7197-4dcd-a33e-11279438ecbb","order_by":0,"name":"Xiaoqian Li","email":"","orcid":"","institution":"Sichuan University West China School of Basic Medical Sciences and Forensic Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqian","middleName":"","lastName":"Li","suffix":""},{"id":332207120,"identity":"5f4b5024-63ff-4710-8267-ac48b14f3cb0","order_by":1,"name":"Wei Li","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Li","suffix":""},{"id":332207121,"identity":"df865039-f99b-49aa-8efa-5356b065e4ff","order_by":2,"name":"Tao Yan","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital: Chengdu Medical College Second Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Yan","suffix":""},{"id":332207122,"identity":"c7dc5190-5a64-45cf-921e-9ad1f4e68829","order_by":3,"name":"Linfen Guo","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Linfen","middleName":"","lastName":"Guo","suffix":""},{"id":332207123,"identity":"b08d43ad-1810-4916-9464-c240a3e1a153","order_by":4,"name":"Yahui Feng","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital: Chengdu Medical College Second Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yahui","middleName":"","lastName":"Feng","suffix":""},{"id":332207124,"identity":"69d3841d-961d-4736-86b0-8fe69d729902","order_by":5,"name":"Yulan Liu","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital: Chengdu Medical College Second Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yulan","middleName":"","lastName":"Liu","suffix":""},{"id":332207125,"identity":"f8f13b6d-2816-4096-a473-199f0bc8517a","order_by":6,"name":"Lu Ye","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital: Chengdu Medical College Second Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Ye","suffix":""},{"id":332207126,"identity":"8a316907-bfc9-4d9b-8220-345b2d26404b","order_by":7,"name":"Yuehua Zhang","email":"","orcid":"","institution":"Sichuan University West China Second University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuehua","middleName":"","lastName":"Zhang","suffix":""},{"id":332207127,"identity":"f6baf988-9171-46bd-9acd-edfd98dba653","order_by":8,"name":"Hao Bai","email":"","orcid":"","institution":"Sichuan University West China School of Basic Medical Sciences and Forensic Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Bai","suffix":""},{"id":332207128,"identity":"466eee93-8c7a-4ddf-9823-75e6ec0cc655","order_by":9,"name":"Nianyong Chen","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Nianyong","middleName":"","lastName":"Chen","suffix":""},{"id":332207129,"identity":"9731cb4f-b5d9-48bd-92f8-1105ca6e6da7","order_by":10,"name":"Daojiang Yu","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital: Chengdu Medical College Second Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Daojiang","middleName":"","lastName":"Yu","suffix":""},{"id":332207130,"identity":"9b16a27c-7570-49ad-95d4-a881a24e5e9f","order_by":11,"name":"Shuyu Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYDACCQiVAMSMj8FMZuYGorUwGzMwGAApRuK1sEmDtTAQ0CI/u/nZwy8MdXkGN3LMqgsq/kTztwO1/KjYhlML45xj5sYyDIeLQVpuzzhjkDvjMGMDY8+Z2zi1MEskmElLMBxI3ADSwttmkNsA1MLM2IZbC5tE+jegljqwlmKQlvmEtPBI5JhJfmBgBmthBmnZQEiLhEROmTSDweHEmWeeFUvznDHO3QjUchCfX+RnpG+T/FFRl9h3PHnjZ54Kudx55w8ffPCjArcWcBDwgGJDIAEhcgCveiBg/AEi+QmqGwWjYBSMgpEKABldVmzGsRW5AAAAAElFTkSuQmCC","orcid":"","institution":"Sichuan University West China School of Basic Medical Sciences and Forensic Medicine","correspondingAuthor":true,"prefix":"","firstName":"Shuyu","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-07-22 08:50:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4780437/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4780437/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63030613,"identity":"9fbdf0ba-0970-425c-9d92-f992bbd93e07","added_by":"auto","created_at":"2024-08-22 09:18:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4211121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of various radiotherapy modalities in the suppression of primary keloid fibroblasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e H\u0026amp;E staining of keloid and skin adjacent to keloid tissues from patients. Bar = 200 μm. \u003cstrong\u003e(B)\u003c/strong\u003e Phase-contrast optical microscopy imaging showed the morphology of primary keloid fibroblasts. Bar = 500 μm. \u003cstrong\u003e(C)\u003c/strong\u003e The presence of the Vimentin in primary keloid cells was confirmed by immunofluorescence staining. Bar = 20 μm. \u003cstrong\u003e(D)\u003c/strong\u003e Diagram of different radiation modalities and doses to primary keloid fibroblasts. \u003cstrong\u003e(E)\u003c/strong\u003e Clonogenic assay to detect the colony formation of primary keloid fibroblasts treated with different rays. \u003cstrong\u003e(F)\u003c/strong\u003e Cell senescence of primary keloid fibroblasts treated with different rays measured by SA-β-gal\u003csup\u003e+\u003c/sup\u003e staining. \u003cstrong\u003e(G)\u003c/strong\u003e Cell cycle of primary keloid fibroblasts treated with different rays. \u003cstrong\u003e(H)\u003c/strong\u003e Apoptosis rate of primary keloid fibroblasts treated with different rays.\u003cstrong\u003e (I)\u003c/strong\u003e LDH release of primary keloid fibroblasts treated with different rays. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared with the control group.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4780437/v1/e0098d10b6ce571c601e9fa8.png"},{"id":63029681,"identity":"2b38731d-8e82-42f3-822f-51cfa684212f","added_by":"auto","created_at":"2024-08-22 09:10:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4338132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell type identification by scRNA-Seq analysis of primary keloid fibroblasts with various\u003c/strong\u003e \u003cstrong\u003eradiotherapy modalities.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eFlowchart overview of scRNA-seq of primary keloid fibroblasts treated with different irradiation. \u003cstrong\u003e(B)\u003c/strong\u003e Stratification and cell-type identification of primary keloid fibroblasts from five groups. Clusters were distinguished by different colors. \u003cstrong\u003e(C)\u003c/strong\u003e Feature plots showed marker genes specific for each cluster. \u003cstrong\u003e(D)\u003c/strong\u003e The bubble plot of top five differentially expressed genes of seven clusters. \u003cstrong\u003e(E)\u003c/strong\u003e Molecular signatures for the seven fibroblast subpopulations. \u003cstrong\u003e(F) \u003c/strong\u003eCircos plots showed the interactions density between any two cell clusters in five groups. \u003cstrong\u003e(G)\u003c/strong\u003eBar plots showed the number of intercellular communication (up) and interaction strength (down) in five groups.\u003cstrong\u003e (H)\u003c/strong\u003e Bubble diagrams demonstrated the ligand-receptor pairs which significant changes in specificity between any two types of fibroblasts in irradiated groups versus control.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4780437/v1/061f777d394c1bcaa0024857.png"},{"id":63029683,"identity":"b1484db8-57b8-43fe-aa4b-0636afe4cdeb","added_by":"auto","created_at":"2024-08-22 09:10:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2424892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic profiles of primary keloid fibroblasts after different radiation using scRNA-Seq.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e UMAP plots of fibroblasts clusters in the groups of control, X-rays, electron beam, \u003csup\u003e90\u003c/sup\u003eSr radiation. \u003cstrong\u003e(B)\u003c/strong\u003e Percentage of each fibroblasts cluster in four groups. \u003cstrong\u003e(C-E) \u003c/strong\u003eStack\u003cstrong\u003e \u003c/strong\u003eviolin plots showed upregulated DEGs between X-rays 5 Gy×4 Fib 2-\u003cem\u003eCCDN1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and other 3 groups Fib 2-\u003cem\u003eCCDN1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, electron beam 5 Gy×4 Fib 3-\u003cem\u003eIGFBP7\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and other groups Fib 3-\u003cem\u003eIGFBP7\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, \u003csup\u003e90\u003c/sup\u003eSr 5 Gy×4 Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and other groups Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cstrong\u003e. (F-H)\u003c/strong\u003e Functional enrichment of upregulated DEGs in three fibroblast clusters with significance threshold set at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4780437/v1/fe69116d15c97613c6528b6b.png"},{"id":63029686,"identity":"35dfa9c8-bc3a-4fee-82e3-41ccd423c0db","added_by":"auto","created_at":"2024-08-22 09:10:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1796078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic profiles of primary keloid fibroblasts after different doses \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e90\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eSr irradiation using scRNA-Seq.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eProportion of each cluster in primary keloid fibroblasts treated with \u003csup\u003e90\u003c/sup\u003eSr 5 Gy×2 and\u003csup\u003e 90\u003c/sup\u003eSr 5 Gy×4. \u003cstrong\u003e(B)\u003c/strong\u003e DEGs number of each cell clusters were represented in a bar chart.\u003cstrong\u003e (C) \u003c/strong\u003eStack\u003cstrong\u003e \u003c/strong\u003eviolin plots showed representative common upregulated genes between \u003csup\u003e90\u003c/sup\u003eSr 5 Gy×2 Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e vs control Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh \u003c/sup\u003eand\u003csup\u003e 90\u003c/sup\u003eSr 5 Gy×4 Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e vs control Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e. \u003cstrong\u003e(D)\u003c/strong\u003e Functional enrichment of common upregulated DEGs between \u003csup\u003e90\u003c/sup\u003eSr 5 Gy×2 Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e vs control Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh \u003c/sup\u003eand\u003csup\u003e 90\u003c/sup\u003eSr 5 Gy×4 Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e vs control Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e with significance threshold set at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003e(E) \u003c/strong\u003eDifferences in pathway activity among Fib 6-\u003cem\u003eSQSTM1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e of three groups calculated based on GSVA scores. \u003cstrong\u003e(F) \u003c/strong\u003eStack\u003cstrong\u003e \u003c/strong\u003eviolin plots of the expression of top five common upregulated DEGs for the Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e from the \u003csup\u003e90\u003c/sup\u003eSr 5 Gy×2 vs control and \u003csup\u003e90\u003c/sup\u003eSr 5 Gy×2 vs\u003csup\u003e 90\u003c/sup\u003eSr 5 Gy×4. \u003cstrong\u003e(G) \u003c/strong\u003eFunctional enrichment of common upregulated DEGs between \u003csup\u003e90\u003c/sup\u003eSr 5 Gy×2 Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e vs control Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh \u003c/sup\u003eand \u003csup\u003e90\u003c/sup\u003eSr 5 Gy×2 Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e vs\u003csup\u003e 90\u003c/sup\u003eSr 5 Gy×4 Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e with significance threshold set at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4780437/v1/2c6db6824d5d786e3f90950b.png"},{"id":63030615,"identity":"69a7034e-4d9c-4456-b3d1-2f179f4cac4b","added_by":"auto","created_at":"2024-08-22 09:18:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2547870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePseudotemporal analysis reveals a significant shift toward the myofibroblast phenotype in primary keloid fibroblasts post-radiation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Pseudo-temporal ordering of primary keloid fibroblasts revealed a branched trajectory. (\u003cstrong\u003eB)\u003c/strong\u003eDistribution of the seven subpopulations on each of the branches. \u003cstrong\u003e(C)\u003c/strong\u003eCell fate transitions of fibroblasts in five groups.\u003cstrong\u003e (D)\u003c/strong\u003e Hierarchical clustering of the branch-dependent genes reveals seven gene modules. From the center to the left of the heatmap, the kinetic curve from the pre-branch along the trajectory to fate 1 branch. From the center to the right, the curve from pre-branch to fate 2 branch. \u003cstrong\u003e(E)\u003c/strong\u003e GO analysis of differently expressed genes associated with three gene clusters as in \u003cstrong\u003e(D)\u003c/strong\u003e identified unique response pathways for each branch.\u003cstrong\u003e \u003c/strong\u003eThe significance threshold was set to an adjusted \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4780437/v1/6cc8f76e4d952abc7f984b1c.png"},{"id":63030614,"identity":"e004a308-4716-4af2-b807-5e0c8e83e3b8","added_by":"auto","created_at":"2024-08-22 09:18:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2954712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of IRF1 plays an important role in the treatment of keloids.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eActivity of the major regulators identified by pySCENIC in fibroblast clusters. Heatmap showing the clustering of regulator AUC matrices in each cell cluster at the mean value. Horizontal coordinates represent five groups, vertical coordinates are regulon names, and numbers in parentheses represent the number of target genes of transcription factors. \u003cstrong\u003e(B) \u003c/strong\u003eImmunofluorescence analysis of primary keloid fibroblasts treated with X-rays 5 Gy×4 and Ad-IRF1.\u003cstrong\u003e (C)\u003c/strong\u003eWestern blotting analysis of IRF1, PARP, Cleaved-PARP, Bax and Bcl-2 protein levels after infection with Ad‑IRF1 and control in primary keloid fibroblasts.\u003cstrong\u003e (D)\u003c/strong\u003e Apoptosis rate of primary keloid fibroblasts after infection with Ad‑IRF1 and control. \u003cstrong\u003e(E) \u003c/strong\u003eCo-IP analysis of the interaction between IRF1 and SSBP1. \u003cstrong\u003e(F) \u003c/strong\u003eWestern blotting analysis of SSBP1 protein levels after transfection with siSSBP1 and controls in primary keloid fibroblasts. Influence of SSBP1 knockdown on the radiation-induced expression of IRF1 determined by western blotting \u003cstrong\u003e(G)\u003c/strong\u003e and nuclear translocation of IRF1 determined by immunofluorescence analysis \u003cstrong\u003e(H)\u003c/strong\u003e.\u003cstrong\u003e(I)\u003c/strong\u003e Schematic showed the workflow for identifying 3 chemicals interacted with IRF1. \u003cstrong\u003e(J)\u003c/strong\u003e IRF1 mRNA levels were analyzed using reverse transcription‑quantitative PCR after using three chemicals in primary keloid fibroblasts. Influence of three chemicals on the nuclear translocation of IRF1 determined by separating the nucleus and cytoplasm followed by western blotting\u003cstrong\u003e (K) \u003c/strong\u003eand performing immunofluorescence analysis\u003cstrong\u003e (L)\u003c/strong\u003e. \u003cstrong\u003e(M)\u003c/strong\u003e Primary keloid fibroblasts, HaCaT and HFF-1 cells were treated with ATRA and 9-cis-RA for 48 h and cell viability was measured by CCK-8 assays. \u003cstrong\u003e(N)\u003c/strong\u003e LDH release of primary keloid fibroblasts treated with ATRA and 9-cis-RA. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 and **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared with the control group.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4780437/v1/df8e56665b74b9fd04caabd5.png"},{"id":64187246,"identity":"e43f43b9-92f1-4655-9200-48f14df8f89e","added_by":"auto","created_at":"2024-09-09 16:36:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20137609,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4780437/v1/5cde1f9a-3891-48a7-a674-2d6462f1068d.pdf"},{"id":63029689,"identity":"2c8d3181-e224-4c37-9d38-b4e0e1364e7b","added_by":"auto","created_at":"2024-08-22 09:10:54","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":711188,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4780437/v1/588e86e0b08254d0522788c0.docx"}],"financialInterests":"","formattedTitle":"Comparison of the biological effects of various radiation therapy for keloid by single-cell RNA sequencing reveals IRF1 as a novel target in keloid therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eKeloid is a dermal fibroproliferative disorder resulting from abnormal healing of wounds, often extending beyond the edge of the injury.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Besides obvious cosmetic concerns, keloid leads to a serious physical and psychological burden for patients by causing pain, pruritus, tenderness, burning and contracture.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Surgical excision is the most radical therapeutic option, while the recurrence rates in patients who underwent surgery alone as high as 40\u0026ndash;100%.\u003csup\u003e6\u0026ndash;9\u003c/sup\u003e Postoperative radiotherapy (PORT) is a well-established adjuvant treatment to reduce the risk of recurrence to approximately 20%.\u003csup\u003e10\u0026ndash;12\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCurrent keloid radiotherapy modalities include electron beam therapy from linear accelerator, photon beam therapy from X-ray tubes and brachytherapy with \u003cem\u003ein situ\u003c/em\u003e placement of a radioactive source.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Electron beam delivers accelerated high-energy electrons (MeV) to depths of 2\u0026ndash;6 cm without significant damage to deeper structures.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Kilo-voltage X-rays are used for treating superficial lesions and increase the dose deposited in bone.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e While brachytherapy, using uniformly adsorbing radionuclides, ensures the even and appropriate focus of the radiation onto the target site, thereby minimizing harm to the surrounding healthy tissue.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e For strontium-90-yttrium-90 (\u003csup\u003e90\u003c/sup\u003eSr-\u003csup\u003e90\u003c/sup\u003eY) applicator, both \u003csup\u003e90\u003c/sup\u003eSr and its decay product \u003csup\u003e90\u003c/sup\u003eY emit β-radiation. The radiation dose at a tissue depth of 2 mm is 20\u0026ndash;30% of the surface dose, and this significantly drops to 1\u0026ndash;3% at a depth of 5 mm.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA comprehensive meta-analysis, summarized the literature from 1942 to 2014, compared recurrence between radiotherapy modalities revealed that brachytherapy yielded the lowest recurrence rate (15%), with the two \u003csup\u003e90\u003c/sup\u003eSr studies showing rates of 20.5% and 12.67%, while X-rays and electron beam were both 23%.\u003csup\u003e10,12\u003c/sup\u003e In addition, another systematic review reported that the acute complications (erythema, dehiscence and infection) and chronic complications (hyperpigmentation, hypopigmentation and telangiectasia) across all three modalities. Peeling was reported in X-rays and electron beam, while fibrosis was only observed in brachytherapy. It\u0026rsquo;s intriguing to note that the highest rate of hyperpigmentation was observed in brachytherapy at 25.6, which was lower than that of X-rays and electron beam (100%). However, a study based on \u003csup\u003e90\u003c/sup\u003eSr treatment reported a 10.4% incidence of telangiectasia as a complication.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e These findings, derived from retrospective clinical analysis, further molecular differences among the three radiotherapy modalities are not yet fully understood.\u003c/p\u003e \u003cp\u003eConsidering the heterogeneity of keloid conditions across various studies, it is challenging to compare the biological effects of radiation, even though numerous studies have investigated into the molecular mechanisms associated with individual radiotherapy modalities. X-rays radiation increases the expression of senescence-associated genes (p16, p21 and p27), induces apoptosis and LDH release through targeting forkhead box O1 (FOXO1).\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e While electron beam irradiation might hinder keloid formation through the interleukin 6 (IL-6) signaling pathway, decrease collagen production by modifying miR-21/smad7-mediated p38 activation, and inhibit autophagy by reducing miR-21-5p, which regulates migration and LC3B expression via PTEN/Akt signaling.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSingle-cell RNA sequencing (scRNA-Seq) transcends the limitations of bulk RNA-seq by offering an unbiased and detailed view of cellular heterogeneity and regulatory networks at a single-cell level. To date, scRNA-Seq has been applied to explore the differences between keloid tissues and adjacent normal tissues, including the depiction of the cellular landscape, fibroblast heterogeneity, the transcriptional profile of Schwann cells, Schwann cell-macrophage cross-talk, lineage-specific regulatory changes, fibrovascular communication and mesenchymal activation of endothelial cells and immune profiles.\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28 CR29\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Furthermore, scRNA-Seq was utilized to investigate the changes of combined triamcinolone acetonide (TAC) and 5-fluorouracil (5-FU) treatments on keloids.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn our study, we isolated primary keloid fibroblasts from patients and performed scRNA-Seq to analyze gene expression profiles following various radiotherapy modalities. We further analyzed and characterized the transcription factors involved in keloid radiotherapy. These findings contributed to our understanding of keloid radiotherapy and provided potential targets for keloid therapies.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and materials\u003c/h2\u003e \u003cp\u003eCell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). \u003cem\u003eIRF1\u003c/em\u003e overexpression adenovirus and the control Ad-NC were obtained from HanBio (Shanghai, China). ATRA and 9-cis-RA were purchased from MedChemExpress (Monmouth Junction, NJ). Antimycin A was purchased from Maokang Biotechnology (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHuman keloid samples\u003c/h2\u003e \u003cp\u003eThe human keloid and adjacent skin samples tissues were derived from patients who had received surgery at the Department of Plastic Surgery, Second Affiliated Hospital of Chengdu Medical College (Chengdu, China). The patients\u0026rsquo; information is summarized in Table E1. Inclusion criterion: all patients in this study were treated for the first time.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003eHematoxylin and Eosin (H\u0026amp;E) staining\u003c/b\u003e\u003c/h2\u003e \u003cp\u003e4% paraformaldehyde was used to fix keloid and adjacent skin tissues. And then the tissues were embedded in paraffin. Paraffin sections of 3-\u0026micro;m thickness were deparaffinized and treated in citrate buffer (pH 6.0) for 7 min. The epitope retrieval procedure was then performed. Finally, the sections of keloid and adjacent tissues were stained by Hematoxylin and eosin.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eKeloid tissues were washed 3\u0026ndash;5 times in phosphate-buffered saline (PBS) and cut into ~\u0026thinsp;1 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Then, they were adhered to 75 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e tissue culture flasks. HaCaT (human immortalized keratinocyte) and HFF-1 (human foreskin fibroblasts) were used as reported previously.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e DMEM medium supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel), 0.1 mg/mL streptomycin and 100 U/mL penicillin (Beyotime, Nantong, China) cultured in a 37\u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. After primary keloids cells freed from the tissues, experiments were performed using 3rd -7th generation, with a doubling time of 50 h.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003ePrimary keloid cells irradiation\u003c/h2\u003e \u003cp\u003eWhen cells reached 50% confluence in 10.0 cm dishes, they were divided into five groups: Control, 5 Gy/f for continuous 4 days X-rays irradiation (X-rays 5 Gy\u0026times;4) using X-ray irradiator (320 KV; KUB Technologies, Inc., Stratford, CT), 5 Gy/f for continuous 4 days electron beam irradiation (electron beam 5 Gy\u0026times;4) using a linear accelerator (Varian Medical Systems, Inc., Palo Alto, GA), 5 Gy/f for continuous 2 days and 4 days β-radiation using a \u003csup\u003e90\u003c/sup\u003eSr-\u003csup\u003e90\u003c/sup\u003eY surface applicator (1361 MBq) (\u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;2 and \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;4).\u003csup\u003e11,33\u0026ndash;35\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eImmunofluorescence assay\u003c/h2\u003e \u003cp\u003ePrimary keloid fibroblasts were first fixed with 4% paraformaldehyde for 15 min, then permeabilized with 1% Triton X-100 for 15 min, and subsequently blocked with 5% BSA for 2 h at room temperature. Then anti-Vimentin antibody (arigo Biolaboratories Corp., Shanghai, China, #ARG66199, 1:200) and anti-IRF1 antibody (Cell Signaling Technology, Beverly, MA; #8478S, 1:200) were incubated with cells overnight at 4 ℃, followed by incubation of secondary antibody with Alexa Fluor Plus 647- labeled goat anti-rabbit antibody and Alexa Fluor Plus 488- labeled goat anti-rabbit antibody (Thermo Fisher Scientific Inc., Shanghai, China) at a dilution of 1:300 for 2 hours at room temperature, respectively. Cell nuclei were stained with antifade mounting medium containing DAPI (Vector Laboratories, Inc., Burlingame, CA) for 15 min, and a scanning laser confocal microscopy (Olympus, Tokyo, Japan) was used to capture images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eClonogenic assay\u003c/h2\u003e \u003cp\u003eSix-h after radiation, primary keloid cells (5\u0026times;10\u003csup\u003e3\u003c/sup\u003e) were seeded into 6.0 cm dishes. After culturing for 10 days, cells were fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime Biotechnology, Nantong, China).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle assay\u003c/h2\u003e \u003cp\u003ePrimary keloid cells were collected 48 hours after irradiation and fixed overnight in pre-chilled 70% ethanol at -20\u0026deg;C. Next, cells were treated with 1 mL propidium iodide solution (10 mg/mL) at 37\u0026deg;C for 30 min in darkness. Samples were detected by FACS Celesta flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and analyzed with ModFit LT\u0026trade; (Version 5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell apoptosis assay\u003c/h2\u003e \u003cp\u003ePrimary keloid cells were harvested 48 h post-irradiation and infection, then the degree of apoptosis was detected by Annexin V/PI kit (Yeasen Biotechnology, Shanghai, China). Then, samples were detected by FACS Celesta flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and analyzed with FlowJoTM (Version 10.7).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLactate dehydrogenase (LDH) assay\u003c/h2\u003e \u003cp\u003eLDH activity was measured by an LDH Cytotoxicity Assay Kit (Beyotime, Nantong, China) at 48 h post-radiation and 24, 48 and 72 h after the treatment of ATRA and 9-cis-RA. Lastly, the absorbance was measured at 490 nm with a microplate reader (BioTek, Synergy HTX, Winooski, VT).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSenescence-associated β-galactosidase (SA-β-gal) staining\u003c/h2\u003e \u003cp\u003ePrimary keloids fibroblasts were seeded into 24 wells plates at 5000 cells/ well for 6 h after radiation. Then, after 48 h of exposure to radiation, a Senescence SA-β-gal Staining Kit (Beyotime Biotechnology, Shanghai, China) was used. Briefly, cells were fixed with 500 \u0026micro;l of fixative solution for 30 min at room temperature, followed by preparation of SA-β-gal staining solution incubated overnight at 37\u0026deg;C protected from light. Finally, a light microscopy (Olympus, Tokyo, Japan) was used to capture the images, and the percentage of SA-β-gal\u0026thinsp;+\u0026thinsp;cells in five random fields in each of the four wells were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting analysis\u003c/h2\u003e \u003cp\u003eProteins of primary keloid fibroblasts were harvested and lysed in RIPA lysis buffer containing protease inhibitor cocktail and PMSF (Beijing Solarbio, Beijing, China) for 30 min at 4 ℃. BCA Protein Assay kit (Beyotime, Nantong, China) was used to measure protein concentration. Protein (40 g/lane) from each lysate was fractionated by 10% SDS-PAGE and transferred to 0.22 \u0026micro;m PVDF (polyvinylidene difluoride) membranes (Millipore, Billerica, MA). After blocking with 5% non-fat milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBST) for 2 hours at room temperature, the membranes were blotted with primary antibodies: IRF1 (Cell Signaling Technology, Beverly, MA; #8478S), H3 (Cell Signaling Technology, Beverly, MA; #4499S), GAPDH (1:5000, Abcam Plc., Cambridge, UK; #ab181602), PARP (Cell Signaling Technology, Beverly, MA; #9542S), Cleaved PARP (Cell Signaling Technology, Beverly, MA; #5625S), Bax (Cell Signaling Technology, Beverly, MA; #2772S), Bcl-2 (Cell Signaling Technology, Beverly, MA; #3498S) and SSBP1 (Proteintech Group, Inc, Wuhan, China; #12212-1-AP) overnight at 4 ℃. Before detecting by a Super ECL Dectection Reagent (Yeasen Biotech, Shanghai, China) and visualizing by a FluroChem MI imaging system (Shenhua Science Technology Co., Ltd., Hangzhou, China), horseradish peroxidase-conjugated goat anti- rabbit IgG (Abbkine Scientific, Wuhan, China) secondary antibodies were applied to incubate for 2 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSingle-cell RNA sequencing (scRNA-Seq)\u003c/h2\u003e \u003cp\u003eAfter 48 h of exposure to radiation, scRNA-Seq was performed by Singleron (Nanjing, China). The samples were stained with trypan blue (Sigma, Shanghai, China) and microscopically evaluated for cell viability. Single-cell suspensions at 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL in concentration in PBS (HyClone, Shanghai, China) were prepared and loaded onto microfluidic devices and scRNA-Seq libraries were constructed according to Singleron GEXSCOPE\u0026reg; protocol by GEXSCOPE\u0026reg; Single-Cell RNA Library Kit (Singleron Biotechnologies) and Singleron Matrix\u0026reg; Automated single-cell processing system (Singleron Biotechnologies). Individual libraries were diluted to 4 ng/\u0026micro;L and pooled for sequencing. Pools were sequenced on Illumina novaseq6000 with 150 bp paired end reads. The GEO dataset (GSE) number is GSE261116.\u003c/p\u003e \u003cp\u003eOther methods were detailed in the \u003cb\u003eSupplementary Materials\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIonizing radiation inhibits the proliferation and induces cell death of primary keloid fibroblasts\u003c/h2\u003e \u003cp\u003eThe keloids and adjacent skin tissues were obtained from surgical excisions of patients. H\u0026amp;E staining revealed that the keloid showed an increased number of dermal cells and large number of parallel and staggered bundles of collagen fibers compared to the adjacent-keloid (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Afterwards, primary keloid cells were isolated and characterized using phase-contrast optical microscopy and immunofluorescence methods. The former showed that these cells exhibited typical fibroblasts features, such as elongated, spindle-shaped cells with distinctive ridges and filamentous projections (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, the immunofluorescence staining demonstrated that primary keloid cells were positive for vimentin expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we examined the effects of different types and different doses of rays on primary keloid fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, the results of clonogenic experiments showed that primary keloid fibroblasts lose the ability to form clones after X-rays and electron beam irradiation, while the two \u003csup\u003e90\u003c/sup\u003eSr groups only inhibited the clone formation of cells in a dose-dependent manner. Similar results can be seen in the SA-β-gal assay for detecting cellular senescence. At a radiation dose of 5 Gy\u0026times;4, the percentage of SA-β-gal\u0026thinsp;+\u0026thinsp;cells of X-rays, electron beam and \u003csup\u003e90\u003c/sup\u003eSr groups were all about 45%, but the percentage of senescent cells was lower, at about 35%, when the dose of \u003csup\u003e90\u003c/sup\u003eSr was reduced to 5 Gy\u0026times;2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). We also found that X-rays and electron beams induced more severe damage than that of equivalent \u003csup\u003e90\u003c/sup\u003eSr exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I). Overall, these results demonstrated that keloids exhibit distinct biological responses to different radiotherapy modalities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSingle-cell transcriptomic map in primary keloid fibroblasts with different radiotherapy modalities\u003c/h2\u003e \u003cp\u003eTo dissect cellular heterogeneity in human primary keloid fibroblasts treated with different radiotherapy modalities, we performed scRNA-Seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After standard data processing and quality filtering, a total of 43,867 single cells from the five groups (control: 14,351; X-rays 5 Gy\u0026times;4: 3,488; electron beam 5 Gy\u0026times;4: 8,870; \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;2: 7,458; \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;4: 9,700) were obtained. Using unbiased clustering segmented the cells into seven distinct clusters, all marked by elevated expression of fibroblast-specific markers (\u003cem\u003eCol1A1\u003c/em\u003e, \u003cem\u003eCol1A2\u003c/em\u003e, \u003cem\u003eDCN\u003c/em\u003e and \u003cem\u003eLUM\u003c/em\u003e); yet, each subcluster displayed distinct transcriptomic signatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Then, according to the top five differential genes of each cluster in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, these subpopulations could be characterized by specific markers: Fib 1-\u003cem\u003eCDK1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, Fib 2-\u003cem\u003eCCDN1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, Fib 3-\u003cem\u003eIGFBP7\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, Fib 5-\u003cem\u003ePTTG1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, Fib 6-\u003cem\u003eSQSTM1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). As shown in Fig. E1A-G, functional enrichment analysis revealed that the signature genes of Fib 1-\u003cem\u003eCDK1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and Fib 5-\u003cem\u003ePTTG1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e were both enriched with nuclear division, while Fib 2-\u003cem\u003eCCDN1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e were enriched with terms related to microtubule. Both Fib 3-\u003cem\u003eIGFBP7\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e showed enrichment with ECM-related terms, thus representing two myofibroblast subpopulations. Additionally, Fib 6-\u003cem\u003eSQSTM1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e were found to be involved in stress responses and RNA splicing, respectively. Taken together, these findings collectively highlighted the intricate heterogeneity within keloid fibroblasts and their varied responses to radiotherapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, to define the cell-cell communication landscape between fibroblast subpopulations in five groups, we performed analysis using CellPhoneDB 2.0.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e As detailed in Circos plots and bar plots, we found a denser interaction network and a more abundant number of intercellular interactions in X-rays 5 Gy\u0026times;4 compared to that in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, We observed disparities in immune-associated interactions between control and irradiated groups, such as PVR-TNFSF9, TNFSF9-IL13RA2 and ACKR3-CXCL12. These data suggested that radiation may induce immune-related responses in primary keloid fibroblasts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDifferential proportion analysis reveals significant expansion of fibroblast subpopulations in keloids with different radiotherapy modalities\u003c/h2\u003e \u003cp\u003eIn our subsequent analysis, we aimed to pinpoint the irradiation-associated cell clusters that expanded under 5 Gy\u0026times;4 conditions in primary keloid fibroblasts. The visualization of cellular density and distribution revealed changes in the relative proportions of seven subpopulations among four groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Notably, the radio of Fib 2-\u003cem\u003eCCDN1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, Fib 3-\u003cem\u003eIGFBP7\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e increased following X-rays 5 Gy\u0026times;4, electron beam and \u003csup\u003e90\u003c/sup\u003eSr irradiation, respectively. This expansion suggested a crucial role for these subpopulations in the keloid's response to various radiotherapy modalities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-E illustrated our findings that under X-rays irradiation at 5 Gy\u0026times;4, the Fib 2-\u003cem\u003eCCDN1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e exhibited a significant upregulation of genes involved in co-translational protein targeting to membrane and protein localization to endoplasmic reticulum, such as ribosomal protein L41 (\u003cem\u003eRPL41\u003c/em\u003e), ribosomal protein S28 (\u003cem\u003eRPS28\u003c/em\u003e), ribosomal protein L22 (\u003cem\u003eRPL22\u003c/em\u003e), ribosomal protein S10 (\u003cem\u003eRPS10\u003c/em\u003e) and ribosomal protein L39 (\u003cem\u003eRPL39\u003c/em\u003e). Extracellular matrix-associated genes, including collagen type VIII alpha 1 chain (\u003cem\u003eCOL8A1\u003c/em\u003e), fibulin 2 (\u003cem\u003eFBLN2\u003c/em\u003e), sulfatase 1 (\u003cem\u003eSULF1\u003c/em\u003e), dermatopontin (\u003cem\u003eDPT\u003c/em\u003e) \u003cem\u003eand\u003c/em\u003e cartilage oligomeric matrix protein (\u003cem\u003eCOMP\u003c/em\u003e), were significantly increased in electron beam 5 Gy\u0026times;4 Fib 3-\u003cem\u003eIGFBP7\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e. In \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;4 Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, there was a notable increase in genes related to reproductive development, including JunB proto-oncogene, AP-1 transcription factor subunit (\u003cem\u003eJUNB\u003c/em\u003e), odd-skipped related transcription factor 1 (\u003cem\u003eOSR1\u003c/em\u003e), aldo-keto reductase family 1 member C3 (\u003cem\u003eAKR1C3\u003c/em\u003e) and pleckstrin homology like domain family A member 2 (\u003cem\u003ePHLDA2\u003c/em\u003e). Gene ontology (GO) analysis confirmed these trends, indicating enrichment of \u0026ldquo;signal recognition particle (SRP)-dependent co-translational protein targeting to membrane\u0026rdquo;, \u0026ldquo;protein targeting to ER and co-translational protein targeting to membrane\u0026rdquo; in X-rays 5 Gy\u0026times;4 Fib 2-\u003cem\u003eCCDN1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, \u0026ldquo;extracellular matrix organization\u0026rdquo;, \u0026ldquo;extracellular structure organization\u0026rdquo; and \u0026ldquo;external encapsulating structure organization\u0026rdquo; in electron beam 5 Gy\u0026times;4 Fib 3-\u003cem\u003eIGFBP7\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and \u0026ldquo;reproductive structure development\u0026rdquo; and \u0026ldquo;reproductive system development\u0026rdquo; in \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;4 Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-H). These results implied that different irradiation not only alters the proportion of fibroblast subpopulations but also modulates their functional characteristics in primary keloid fibroblasts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferential proportion analysis reveals differences of fibroblast subpopulations in primary keloid fibroblasts with different doses of\u003c/b\u003e \u003csup\u003e\u003cb\u003e90\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eSr\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBrachytherapy offers precise targeting of keloid tissue with less toxicity to adjacent tissue.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Thus, we sought to determine the molecular changes of primary keloid fibroblasts among 0, 5 Gy\u0026times;2 and 5 Gy\u0026times;4 \u003csup\u003e90\u003c/sup\u003eSr irradiation. By calculating the proportion and numbers of differentially expressed genes (DEGs) of each cluster in the three groups, the percentage of Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e increased in both \u003csup\u003e90\u003c/sup\u003eSr irradiation groups and had relatively large numbers of DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Notably, genes associated with oxidative phosphorylation and ATP metabolic process, including cytochrome c oxidase subunit 7A1 (\u003cem\u003eCOX7A1\u003c/em\u003e), coiled-coil-helix-coiled-coil-helix domain containing 10 (\u003cem\u003eCHCHD10\u003c/em\u003e), nuclear protein 1, transcriptional regulator (\u003cem\u003eNUPR1\u003c/em\u003e), NADH: ubiquinone oxidoreductase subunit A13 (\u003cem\u003eNDUFA13\u003c/em\u003e) and NADH: ubiquinone oxidoreductase subunit B8 (\u003cem\u003eNDUFB8\u003c/em\u003e), were upregulated in Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e of both \u003csup\u003e90\u003c/sup\u003eSr irradiation groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Gene ontology (GO) analysis confirmed enrichment of \u0026ldquo;oxidative phosphorylation\u0026rdquo; and \u0026ldquo;ATP metabolic process and electron transport chain\u0026rdquo; pathways were enriched in both \u003csup\u003e90\u003c/sup\u003eSr irradiation groups, which indicating a significant metabolic shift (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile higher percentage of Fib 6-\u003cem\u003eSQSTM1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, as well as the largest numbers of DEGs of Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e were observed in \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;2, suggesting a significant role in primary keloid fibroblasts response to this dosage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Comparative analysis of these subpopulations between the 5 Gy\u0026times;2 and other groups revealed that pathways related to \"oxidative phosphorylation\" \"reactive oxygen species\" and \"P53\" were upregulated in the Fib 6-\u003cem\u003eSQSTM1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e group following \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;2 irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, DEGs such as sulfatase 1 (\u003cem\u003eSULF1\u003c/em\u003e), splicing factor 3b subunit 1 (\u003cem\u003eSF3B1\u003c/em\u003e), integrin subunit alpha 11 (\u003cem\u003eITGA11\u003c/em\u003e), adrenomedullin (\u003cem\u003eADM\u003c/em\u003e), eukaryotic translation initiation factor 4A2 (\u003cem\u003eEIF4A2\u003c/em\u003e) were commonly upregulated when comparing \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;2 to other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). GO enrichment analysis highlighted the enrichment of pathways like \u0026ldquo;transmembrane receptor protein serine/threonine kinase signaling pathway\u0026rdquo;, \u0026ldquo;RNA splicing\u0026rdquo; and \u0026ldquo;mRNA splicing\u0026rdquo; in \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;2 Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Overall, these results demonstrated that the subpopulations changes between two \u003csup\u003e90\u003c/sup\u003eSr irradiation doses were not dose-dependent.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePseudotemporal analysis reveals a branched trajectory with a significant shift toward the myofibroblast phenotype in primary keloid fibroblasts treated with different rays\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo account for the significant changes in primary keloid fibroblasts during the radiotherapy process, we utilized Monocle2 for performed pseudotemporal ordering.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e This analysis revealed a branched trajectory with two cell fates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Fib 5-\u003cem\u003ePTTG1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e predominantly occupied the initial state before branching (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Both Fib 3-\u003cem\u003eIGFBP7\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e were elevated in the cell fate 1 branch, which represents the myofibroblast phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Fib 7-\u003cem\u003eMALAT1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e constituted the most of the cell fate 2 branch associated with RNA splicing. In the control group, the proportion of cells in states pre-branch, 1 and 2 was 95.42%, 2.63% and 1.95%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Strikingly, irradiated groups showed a pronounced shift toward the myofibroblast phenotype in cell fate 1 (X-rays 5 Gy\u0026times;4: 71%; electron beam 5 Gy\u0026times;4: 95.5%; \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;2: 65%; \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;4: 61.17%; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Unexpected, the proportion of cells in state pre-branch was 19.52% in \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBranched expression analysis identified 7357 genes with expression dynamics that corresponded to transitions between cellular states, clustering into seven distinct modules (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Module 1 genes, highly expressed in the myofibroblast branch (cell fate 1), were linked to GO terms such as \"extracellular matrix organization\" and \"neutrophil-mediated immunity.\" Module 3 genes, enriched in the RNA splicing branch (cell fate 2), were associated with \"covalent chromatin modification\" and \"histone modification.\" While module 7 genes, showing pre-branch enrichment, were involved in processes like \"chromosome segregation\" and \"DNA replication\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These data suggested that radiation induce primary keloid cells significant shift toward the myofibroblast phenotype.\u003c/p\u003e \u003cp\u003e \u003cb\u003eActivation of transcription factor interferon regulatory factor 1 (IRF1) serves as a novel approach for keloids treatment\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDue to the importance of transcription factors (TFs) in RNA profiles, we applied a single-cell regulatory network inference and clustering (SCENIC) method to score the activity of regulons by an AUCell algorithm (AUC score).\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Our analysis revealed significant enrichment of IRF1 across all irradiation groups compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Then, immunofluorescence staining revealed nuclear translocation of IRF1 in primary keloid fibroblasts post-irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). To substantiate the therapeutic potential of IRF1 in keloids, we overexpressed IRF1 using an adenovirus vector (Ad-IRF1). This led to an increase in apoptosis and upregulation of related proteins (Cleaved-PARP and Bax) at 48 hours post-infection, indicating a role for IRF1 in keloid cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, to elucidate the activation mechanism of IRF1 by radiation, we focused on single-strand DNA-binding protein 1 (SSBP1), which have recently reported the interacting proteins of IRF1.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e In addition, recent studies showed that the interaction between SSBP1 and IFI6 protects against radiation-induced skin injury.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Then, confirming their interaction in primary keloid fibroblasts through immunoprecipitation (IP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Moreover, we found that SSBP1 knockdown resulted in elevated IRF1 levels and enhanced its nuclear translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-H). These findings reinforce the notion of SSBP1 as an inhibitory chaperone of IRF1 in response to radiation.\u003c/p\u003e \u003cp\u003eFurthermore, we investigated the potential of pharmacological IRF1 activation to enhance keloid treatment efficacy. Utilizing the Comparative Toxicogenomics Database | (CTD) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.ctdbase.org\" target=\"_blank\"\u003ewww.ctdbase.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.ctdbase.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), we identified 213 candidate chemicals modulate IRF1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Retinoic acid (ATRA), 9-cis-retinoic acid (9-cis-RA) and antimycin A caught our attention due to their positive correlation with IRF1 expression, although their specific impact on keloids remained to be determined. Our experiments demonstrated that both ATRA and 9-cis-RA significantly elevated IRF1 mRNA and protein levels in primary keloid fibroblasts and facilitated its nuclear translocation, whereas antimycin A had a minor effect on IRF1 mRNA expression only (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ-L). Further analysis of the chemicals' impact on cell proliferation revealed that ATRA and 9-cis-RA were more lethal to primary keloid fibroblasts than two of immortalized normal skin cells HaCaT and HFF-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM and Fig. E2A-C). Although, all three chemicals increased the release of LDH, indicating cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). Thus, these data implicated IRF1 as a potential target of keloid therapy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRadiotherapy has been an adjuvant treatment in keloid management for over a century.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Among these three radiotherapy modalities, both electron beams and \u003csup\u003e90\u003c/sup\u003eSr emit beta particles. Simultaneously, both beta particles and X-rays are classified as low LET (linear energy transfer) radiation, with an RBE (relative biological effectiveness) of 1.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Despite theirs recognized effectiveness, there were significant differences in the clinical therapeutic effects.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Furthermore, the underlying mechanisms and comparability of different irradiation modalities on keloids have not been fully elucidated. Our study aimed to provide a comprehensive cellular and molecular landscape of primary keloid fibroblasts under different irradiations, revealing both shared and unique changes that could inform new radiotherapeutic targets. Our findings indicated that equivalent X-rays, electron beam and \u003csup\u003e90\u003c/sup\u003eSr radiation showed different inhibitory effect on primary keloid fibroblasts, accompanied by distinct gene expression profiles. Specifically, X-rays 5 Gy\u0026times;4 increased the population of Fib 2-\u003cem\u003eCCDN1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e enhancing the expression of genes related to microtubule-associated complexes and inflammation-associated receptor-ligand interactions, suggesting a role in cell proliferation and immune response. Electron beam 5 Gy\u0026times;4 increased the Fib 3-\u003cem\u003eIGFBP7\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e cell population, driving a significant majority of primary keloid fibroblasts toward a pro-fibrosis cell fate, characterized by upregulated extracellular matrix organization genes. Although \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;2 and \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;4 both increased the population of Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e which enriched in oxidative phosphorylation, assessments of cellular damage, interactions, and pseudotemporal dynamics suggested that \u003csup\u003e90\u003c/sup\u003eSr 5 Gy\u0026times;2 was more effective. These insights contributed to understanding the radiotherapy's effects on keloid fibroblasts and may guide the optimization of treatment strategies.\u003c/p\u003e \u003cp\u003ePreviously, a study based on RNA-Seq and ChIP-Seq datasets revealed that IRF1 dominated the IFN response triggered by ionizing radiation in mouse bone marrow-derived macrophage.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e In this study, we uncovered potential regulatory role of IRF1 across all keloid radiotherapy and affirmed the role of SSBP1 as an IRF1 chaperone that inhibits its nuclear translocation following radiation exposure. Previously, the downregulation of SSBP1 was reported to sensitize non-small cell lung cancer cells to ionizing radiation.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFurthermore, we used the database to predict potential IRF1 agonists. We focused on ATRA and 9-cis-RA, retinoid acid isomers derived from vitamin A.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e ATRA is used for acute promyelocytic leukemia and being the first example of a cyto-differentiating agent to anti-tumor. It has been shown to activate IRF1 gene expression, thereby inhibiting cell growth in various cancer cell lines, including myeloid leukemia, cervical squamous carcinoma and lung cancer.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Recent studies have shown that ATRA upregulate expression of IRF1 by activating retinoic acid receptor (RAR)γ and interferon-β response pathway and resulted in cell death.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e Furthermore, ATRA has been reported to increase nuclear IRF1 levels in human mammary epithelial cells.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e Although there was no study about the relationship between 9-cis-RA and IRF1, 9-cis-RA was applied to treat skin diseases and involved in immune response, including chronic hand eczema, intractable prurigo and cutaneous T-cell lymphoma.\u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e Taken together, we identified that ATRA and 9-cis-RA could be useful agents against keloids possibly via the activation of IRF1. However, there were several potential limitations of this study. Although abnormal fibroblast is the predominant cell type in keloid, other types of cells such as endothelia cells and immune cells are yet to be investigated. Stable animal models with keloid warrant further investigated.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study illustrated the mRNA profiles and driving transcription factors of primary keloid fibroblasts following various radiotherapy modalities by scRNA-Seq.\u0026nbsp;Overexpression of IRF1 through adenovirus and IRF1 agonists (ATRA and 9-cis-RA) induced apoptosis and inhibited cell viability. Moreover, we affirmed the role of SSBP1 as an IRF1 chaperone following radiation exposure which may involve in keloid radioresistance. Thus, we provided reliable experimental support for the development of novel treatment strategies targeting IRF1 for keloids treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore surgery, informed consents were obtained from all patients and approved by the ethics committee of Second Affiliated Hospital of Chengdu Medical College (Approval No. KJ20210035). All procedures comply with the guidelines and ethical principles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by the National Natural Science Foundation of China (82073477 and 82373523), the Young Talent Project of China National Nuclear Corporation and Natural Science Foundation of Science and Technology of Sichuan Provincial (2023NSFSC0648 and 2024YFHZ0332).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaoqian Li participated in the design and cellular experiments, conducted statistical analysis and drafted the manuscript. Wei Li, Tao Yan, Linfen Guo and Daojiang Yu collected the samples and conducted statistical analysis. Yahui Feng, Yulan Liu and Lu Ye participated in the design and cellular experiments. Yuehua Zhang and Hao Bai provided guidance in manuscript drafting. Nianyong Chen conducted the statistical analysis. Shuyu Zhang conceived the study, participated in the design and contributed to revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the participants of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eStevenson AW, Deng Z, Allahham A, Pr\u0026ecirc;le CM, Wood FM, Fear MW. The epigenetics of keloids. Exp Dermatol. 2021 Aug;30(8):1099-1114.\u003c/li\u003e\n \u003cli\u003eTsai CH, Ogawa R. Keloid research: current status and future directions. Scars Burn Heal. 2019 Aug 19; 5:2059513119868659.\u003c/li\u003e\n \u003cli\u003eTan S, Khumalo N, Bayat A. Understanding Keloid Pathobiology From a Quasi-Neoplastic Perspective: Less of a Scar and More of a Chronic Inflammatory Disease With Cancer-Like Tendencies. Front Immunol. 2019 Aug 7;10:1810.\u003c/li\u003e\n \u003cli\u003eMcGinty S, Siddiqui WJ. Keloid. 2022 Jul 19. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan\u0026ndash;.\u003c/li\u003e\n \u003cli\u003eHawash AA, Ingrasci G, Nouri K, Yosipovitch G. Pruritus in Keloid Scars: Mechanisms and Treatments. Acta Derm Venereol. 2021 Oct 28;101(10): adv00582.\u003c/li\u003e\n \u003cli\u003eJfri A, Rajeh N, Karkashan E. A Case of Multiple Spontaneous Keloid Scars. Case Rep Dermatol. 2015 Jul 24;7(2):156-60.\u003c/li\u003e\n \u003cli\u003eArno AI, Gauglitz GG, Barret JP, Jeschke MG. Up-to-date approach to manage keloids and hypertrophic scars: a useful guide. Burns. 2014 Nov;40(7):1255-66.\u003c/li\u003e\n \u003cli\u003eFurtado F, Hochman B, Ferreira LM. Evaluating keloid recurrence after surgical excision with prospective longitudinal scar assessment scales. J Plast Reconstr Aesthet Surg. 2012 Jul;65(7): e175-81.\u003c/li\u003e\n \u003cli\u003eKumar AS, Kamalasanan K. Drug delivery to optimize angiogenesis imbalance in keloid: A review. J Control Release. 2021 Jan 10;329:1066-1076.\u003c/li\u003e\n \u003cli\u003eMankowski P, Kanevsky J, Tomlinson J, Dyachenko A, Luc M. Optimizing Radiotherapy for Keloids: A Meta-Analysis Systematic Review Comparing Recurrence Rates Between Different Radiation Modalities. Ann Plast Surg. 2017 Apr;78(4):403-411.\u003c/li\u003e\n \u003cli\u003eKal HB, Veen RE. Biologically effective doses of postoperative radiotherapy in the prevention of keloids. Dose-effect relationship. Strahlenther Onkol. 2005 Nov;181(11):717-23.\u003c/li\u003e\n \u003cli\u003eWagner W, Alfrink M, Micke O, Sch\u0026auml;fer U, Sch\u0026uuml;ller P, Willich N. Results of prophylactic irradiation in patients with resected keloids--a retrospective analysis. Acta Oncol. 2000;39(2):217-20.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLiu EK, Cohen RF, Chiu ES. Radiation therapy modalities for keloid management: A critical review. J Plast Reconstr Aesthet Surg. 2022 Aug;75(8):2455-2465.\u003c/li\u003e\n \u003cli\u003eCheraghi N, Cognetta A Jr, Goldberg D. RADIATION THERAPY for the Adjunctive Treatment of Surgically Excised Keloids: A Review. J Clin Aesthet Dermatol. 2017 Aug;10(8):12-15.\u003c/li\u003e\n \u003cli\u003eOgawa R, Tosa M, Dohi T, Akaishi S, Kuribayashi S. Surgical excision and postoperative radiotherapy for keloids. Scars Burn Heal. 2019 Dec 10; 5:2059513119891113.\u003c/li\u003e\n \u003cli\u003eGoutos I, Ogawa R. Brachytherapy in the adjuvant management of keloid scars: literature review. Scars Burn Heal. 2017 Nov 10; 3:2059513117735483.\u003c/li\u003e\n \u003cli\u003eLaskar S, Gurram L, Laskar SG, Chaudhari S, Khanna N, Upreti R. Superficial ocular malignancies treated with strontium-90 brachytherapy: long term outcomes. J Contemp Brachytherapy. 2015 Oct;7(5):369-73.\u003c/li\u003e\n \u003cli\u003eDong W, Qiu B, Fan F. Adjuvant Radiotherapy for Keloids. Aesthetic Plast Surg. 2022 Feb;46(1):489-499.\u003c/li\u003e\n \u003cli\u003eYan L, Wang LZ, Xiao R, Cao R, Pan B, Lv XY, Jiao H, Zhuang Q, Sun XJ, Liu YB. Inhibition of microRNA-21-5p reduces keloid fibroblast autophagy and migration by targeting PTEN after electron beam irradiation. Lab Invest. 2020 Mar;100(3):387-399.\u003c/li\u003e\n \u003cli\u003eHong M, Li X, Liu Y, Mo W, Shi B, Chen S, Yan T, Shi Y, Yu D, Zhang S. Molecular response of keloids to ionizing radiation: targeting FOXO1 radiosensitizes keloids. Int J Radiat Biol. 2022 Sep 28:1-10.\u003c/li\u003e\n \u003cli\u003eJi J, Tian Y, Zhu YQ, Zhang LY, Ji SJ, Huan J, Zhou XZ, Cao JP. Ionizing irradiation inhibits keloid fibroblast cell proliferation and induces premature cellular senescence. J Dermatol. 2015 Jan;42(1):56-63.\u003c/li\u003e\n \u003cli\u003eTosa M, Ghazizadeh M, Shimizu H, Hirai T, Hyakusoku H, Kawanami O. Global gene expression analysis of keloid fibroblasts in response to electron beam irradiation reveals the involvement of interleukin-6 pathway. J Invest Dermatol. 2005 Apr;124(4):704-13.\u003c/li\u003e\n \u003cli\u003eLi S, Liu W, Lei Y, Long J. Regulatory effects of electronic beam irradiation on mir-21/smad7-mediated collagen I synthesis in keloid-derived fibroblasts. Biol Open. 2016 Nov 15;5(11):1567-1574.\u003c/li\u003e\n \u003cli\u003eLiu X, Chen W, Zeng Q, Ma B, Li Z, Meng T, Chen J, Yu N, Zhou Z, Long X. Single-Cell RNA-Sequencing Reveals Lineage-Specific Regulatory Changes of Fibroblasts and Vascular Endothelial Cells in Keloids. J Invest Dermatol. 2022 Jan;142(1):124-135.e11.\u003c/li\u003e\n \u003cli\u003eDireder M, Wielscher M, Weiss T, Laggner M, Copic D, Klas K, Bormann D, Vorstandlechner V, Tschachler E, Jan Ankersmit H, Mildner M. The transcriptional profile of keloidal Schwann cells. Exp Mol Med. 2022 Nov;54(11):1886-1900.\u003c/li\u003e\n \u003cli\u003eDireder M, Weiss T, Copic D, Vorstandlechner V, Laggner M, Pfisterer K, Mildner CS, Klas K, Bormann D, Haslik W, Radtke C, Farlik M, Shaw L, Golabi B, Tschachler E, Hoetzenecker K, Ankersmit HJ, Mildner M. Schwann cells contribute to keloid formation. Matrix Biol. 2022 Apr;108:55-76.\u003c/li\u003e\n \u003cli\u003eGong T, Wang Y, Dong S, Ma X, Du D, Zou C, Zheng Q, Wen Z. Single-cell RNA-seq reveals the communications between extracellular matrix-related components and Schwann cells contributing to the earlobe keloid formation. Front Med (Lausanne). 2022 Oct 26; 9:1000324.\u003c/li\u003e\n \u003cli\u003eDeng CC, Hu YF, Zhu DH, Cheng Q, Gu JJ, Feng QL, Zhang LX, Xu YP, Wang D, Rong Z, Yang B. Single-cell RNA-seq reveals fibroblast heterogeneity and increased mesenchymal fibroblasts in human fibrotic skin diseases. Nat Commun. 2021 Jun 17;12(1):3709.\u003c/li\u003e\n \u003cli\u003eShim J, Oh SJ, Yeo E, Park JH, Bae JH, Kim SH, Lee D, Lee JH. Integrated Analysis of Single-Cell and Spatial Transcriptomics in Keloids: Highlights on Fibrovascular Interactions in Keloid Pathogenesis. J Invest Dermatol. 2022 Aug;142(8):2128-2139.e11.\u003c/li\u003e\n \u003cli\u003eFeng C, Shan M, Xia Y, Zheng Z, He K, Wei Y, Song K, Meng T, Liu H, Hao Y, Liang Z, Wang Y, Huang Y. Single-cell RNA sequencing reveals distinct immunology profiles in human keloid. Front Immunol. 2022 Aug 3;13:940645.\u003c/li\u003e\n \u003cli\u003eXia Y, Wang Y, Hao Y, Shan M, Liu H, Liang Z, Kuang X. Deciphering the single-cell transcriptome network in keloids with intra-lesional injection of triamcinolone acetonide combined with 5-fluorouracil. Front Immunol. 2023 May 19;14:1106289.\u003c/li\u003e\n \u003cli\u003eLi X, Qian K, Zhang Y, Zhang Y, Liu Y, Sun C, Jiao Y, Yu D, Geng F, Cao J, Zhang S. Ubiquitin-specific peptidase 47 (USP47) regulates cutaneous oxidative injury through nicotinamide nucleotide transhydrogenase (NNT). Toxicol Appl Pharmacol. 2023 Dec 1;480:116734.\u003c/li\u003e\n \u003cli\u003eFraunholz IB, Gerstenhauer A, B\u0026ouml;ttcher HD. Results of postoperative (90) Sr radiotherapy of keloids in view of patients\u0026apos; subjective assessment. Strahlenther Onkol. 2005 Nov;181(11):724-9.\u003c/li\u003e\n \u003cli\u003eWang QG, Li XM, Zhang M, Li H, Wen B, Li HZ, Gao XS. [Effect of two dose fractionations on postoperative radiotherapy of keloid: an analysis of 107 patients]. Beijing Da Xue Xue Bao Yi Xue Ban. 2014 Feb 18;46(1):169-72. Chinese.\u003c/li\u003e\n \u003cli\u003eSon Y, Phillips EON, Price KM, Rosenberg LZ, Stefanovic B, Wolfe CM, Shaath TS, Om A, Cohen GF, Gunjan A. Treatment of keloids with a single dose of low-energy superficial X-ray radiation to prevent recurrence after surgical excision: An in vitro and in vivo study. J Am Acad Dermatol. 2020 Nov;83(5):1304-1314.\u003c/li\u003e\n \u003cli\u003eEfremova M, Vento-Tormo M, Teichmann SA, Vento-Tormo R. CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes. Nat Protoc. 2020 Apr;15(4):1484-1506.\u003c/li\u003e\n \u003cli\u003eQiu X, Hill A, Packer J, Lin D, Ma YA, Trapnell C. Single-cell mRNA quantification and differential analysis with Census. Nat Methods. 2017 Mar;14(3):309-315.\u003c/li\u003e\n \u003cli\u003eVan de Sande B, Flerin C, Davie K, De Waegeneer M, Hulselmans G, Aibar S, Seurinck R, Saelens W, Cannoodt R, Rouchon Q, Verbeiren T, De Maeyer D, Reumers J, Saeys Y, Aerts S. A scalable SCENIC workflow for single-cell gene regulatory network analysis. Nat Protoc. 2020 Jul;15(7):2247-2276.\u003c/li\u003e\n \u003cli\u003eGeng F, Chen J, Song B, Tang Z, Li X, Zhang S, Yang T, Liu Y, Mo W, Zhang Y, Sun C, Tan L, Tu W, Yu D, Cao J, Zhang S. Chaperone- and PTM-mediated activation of IRF1 tames radiation-induced cell death and the inflammatory response. Cell Mol Immunol. 2024\u0026nbsp;Jun 07;\u0026nbsp;2042-0226.\u003c/li\u003e\n \u003cli\u003eJia H, Mo W, Hong M, Jiang S, Zhang YY, He D, Yu D, Shi Y, Cao J, Xu X, Zhang S. Interferon-\u0026alpha; inducible protein 6 (IFI6) confers protection against ionizing radiation in skin cells. J Dermatol Sci. 2020 Nov;100(2):139-147.\u003c/li\u003e\n \u003cli\u003eBeheshti A, McDonald JT, Miller J, Grabham P, Costes SV. GeneLab Database Analyses Suggest Long-Term Impact of Space Radiation on the Cardiovascular System by the Activation of \u003cem\u003eFYN\u003c/em\u003e Through Reactive Oxygen Species. Int J Mol Sci. 2019 Feb 3;20(3):661.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePurbey PK, Scumpia PO, Kim PJ, Tong AJ, Iwamoto KS, McBride WH, Smale ST. Defined Sensing Mechanisms and Signaling Pathways Contribute to the Global Inflammatory Gene Expression Output Elicited by Ionizing Radiation. Immunity. 2017 Sep 19;47(3):421-434.e3.\u003c/li\u003e\n \u003cli\u003eWang Y, Hu L, Zhang X, Zhao H, Xu H, Wei Y, Jiang H, Xie C, Zhou Y, Zhou F. Downregulation of Mitochondrial Single Stranded DNA Binding Protein (SSBP1) Induces Mitochondrial Dysfunction and Increases the Radiosensitivity in Non-Small Cell Lung Cancer Cells. J Cancer. 2017 May 12;8(8):1400-1409.\u003c/li\u003e\n \u003cli\u003eZhu J, Hu L, Li L, Huang X, Shi H. Comparison of phenotypic and global gene expression changes in Xenopus tropicalis embryos induced by agonists of RAR and RXR. Toxicol Appl Pharmacol. 2017 Sep 1;330:40-47.\u003c/li\u003e\n \u003cli\u003eMatikainen S, Ronni T, Hurme M, Pine R, Julkunen I. Retinoic acid activates interferon regulatory factor-1 gene expression in myeloid cells. Blood. 1996 Jul 1;88(1):114-23.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eArany I, Whitehead WE, Grattendick KJ, Ember IA, Tyring SK. Suppression of growth by all-trans retinoic acid requires prolonged induction of interferon regulatory factor 1 in cervical squamous carcinoma (SiHa) cells. Clin Diagn Lab Immunol. 2002 Sep;9(5):1102-6.\u003c/li\u003e\n \u003cli\u003eWang Q, Lin Z, Wang Z, Ye L, Xian M, Xiao L, Su P, Bi E, Huang YH, Qian J, Liu L, Ma X, Yang M, Xiong W, Zu Y, Pingali SR, Xu B, Yi Q. RAR\u0026gamma; activation sensitizes human myeloma cells to carfilzomib treatment through the OAS-RNase L innate immune pathway. Blood. 2022 Jan 6;139(1):59-72.\u003c/li\u003e\n \u003cli\u003eLuo XM, Ross AC. Retinoic acid exerts dual regulatory actions on the expression and nuclear localization of interferon regulatory factor-1. Exp Biol Med (Maywood). 2006 May;231(5):619-31.\u003c/li\u003e\n \u003cli\u003eNapolitano M, Potestio L, De Lucia M, Nocerino M, Fabbrocini G, Patruno C. Alitretinoin for the treatment of severe chronic eczema of the hands. Expert Opin Pharmacother. 2022 Feb;23(2):159-167.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChung BY, Um JY, Kang SY, Jung MJ, Kim JC, Kwak IS, Park CW, Kim HO. Oral Alitretinoin for Patients with Refractory Prurigo. Medicina (Kaunas). 2020 Nov 9;56(11):599.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKaemmerer T, Stadler PC, Helene Frommherz L, Guertler A, Einar French L, Reinholz M. Alitretinoin in the treatment of cutaneous T-cell lymphoma. Cancer Med. 2021 Oct;10(20):7071-7078. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"keloid, radiotherapy, single-cell RNA sequencing (scRNA-Seq), interferon regulatory factor 1 (IRF1)","lastPublishedDoi":"10.21203/rs.3.rs-4780437/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4780437/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eKeloids are benign dermal tumors that arise from abnormal wound healing processes following skin lesions. Postoperative radiotherapy (PORT) is a clinically effective measure to reduce recurrence rates of keloid with various radiation modalities. Nevertheless, studies comparing the effectiveness and underlying mechanisms of distinct radiotherapy modalities for keloid remain absent in the literature.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe performed single-cell RNA sequencing (scRNA-Seq) analysis of primary keloid fibroblasts treated with photon irradiation, electron beam irradiation or β-radiation using a \u003csup\u003e90\u003c/sup\u003eSr-\u003csup\u003e90\u003c/sup\u003eY surface applicator to explore gene expression profiles. Comparative analyses were also performed to identify the dysregulated pathways, regulators and ligand receptor interactions in five groups. To validate our findings, molecular biological approaches were used to confirm the potential target.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUnbiased clustering revealed a considerable degree of cellular heterogeneity within primary keloid fibroblasts, resulting in the identification of seven distinct clusters. Among of them, the cell proportions of Fib2-\u003cem\u003eCCND1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, Fib3-\u003cem\u003eIGFBP7\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e and Fib 4-\u003cem\u003eAPCDD1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e increased significantly in X-rays group, electron beam group and both \u003csup\u003e90\u003c/sup\u003eSr groups, which 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 of 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.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study illustrated the molecular alterations and driving transcription factors following various radiotherapy modalities at the single cell resolution. Notably, the identification of IRF1 as a prospective therapeutic target for keloid is of signification importance.\u003c/p\u003e","manuscriptTitle":"Comparison of the biological effects of various radiation therapy for keloid by single-cell RNA sequencing reveals IRF1 as a novel target in keloid therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 09:10:48","doi":"10.21203/rs.3.rs-4780437/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c3fa5b8f-3fda-42eb-abc9-a6394e32a3d7","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-09T16:28:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-22 09:10:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4780437","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4780437","identity":"rs-4780437","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}