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

doi:10.21203/rs.3.rs-4780437/v1

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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

https://doi.org/10.21203/rs.3.rs-4780437/v1

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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 ⁹⁰Sr-⁹⁰Y 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 ⁹⁰Sr 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)

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 (⁹⁰Sr-⁹⁰Y) applicator, both ⁹⁰Sr and its decay product ⁹⁰Y 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 ⁹⁰Sr 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 ⁹⁰Sr 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.

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 mm³. Then, they were adhered to 75 cm² 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% CO₂. 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 ⁹⁰Sr-⁹⁰Y surface applicator (1361 MBq) (⁹⁰Sr 5 Gy×2 and ⁹⁰Sr 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 (5×10³) 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 × 10⁵ 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.

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. 1A). 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. 1B). In addition, the immunofluorescence staining demonstrated that primary keloid cells were positive for vimentin expression (Fig. 1C).

Subsequently, we examined the effects of different types and different doses of rays on primary keloid fibroblasts (Fig. 1D). As shown in Fig. 1E, 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 ⁹⁰Sr 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 ⁹⁰Sr groups were all about 45%, but the percentage of senescent cells was lower, at about 35%, when the dose of ⁹⁰Sr was reduced to 5 Gy×2 (Fig. 1F). We also found that X-rays and electron beams induced more severe damage than that of equivalent ⁹⁰Sr exposure (Fig. 1G-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. 2A). 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; ⁹⁰Sr 5 Gy×2: 7,458; ⁹⁰Sr 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. 2B, C). Then, according to the top five differential genes of each cluster in Fig. 2D, 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. 2E). 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. 2F, G). As shown in Fig. 2H, 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. 3A, 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 ⁹⁰Sr irradiation, respectively. This expansion suggested a crucial role for these subpopulations in the keloid's response to various radiotherapy modalities.

Figure 3C-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 ⁹⁰Sr 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 ⁹⁰Sr 5 Gy×4 Fib 4-APCDD1^high (Fig. 3F-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 ⁹⁰Sr

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 ⁹⁰Sr 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 ⁹⁰Sr irradiation groups and had relatively large numbers of DEGs (Fig. 4A, 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 ⁹⁰Sr irradiation groups (Fig. 4C). Gene ontology (GO) analysis confirmed enrichment of “oxidative phosphorylation” and “ATP metabolic process and electron transport chain” pathways were enriched in both ⁹⁰Sr irradiation groups, which indicating a significant metabolic shift (Fig. 4D).

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 ⁹⁰Sr 5 Gy×2, suggesting a significant role in primary keloid fibroblasts response to this dosage (Fig. 4A, 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 ⁹⁰Sr 5 Gy×2 irradiation (Fig. 4E). 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 ⁹⁰Sr 5 Gy×2 to other two groups (Fig. 4F). GO enrichment analysis highlighted the enrichment of pathways like “transmembrane receptor protein serine/threonine kinase signaling pathway”, “RNA splicing” and “mRNA splicing” in ⁹⁰Sr 5 Gy×2 Fib 7-MALAT1^high (Fig. 4G). Overall, these results demonstrated that the subpopulations changes between two ⁹⁰Sr 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. 5A). Fib 5-PTTG1^high predominantly occupied the initial state before branching (Fig. 5B). Both Fib 3-IGFBP7^high and Fib 4-APCDD1^high were elevated in the cell fate 1 branch, which represents the myofibroblast phenotype (Fig. 5B). 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. 5C). 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%; ⁹⁰Sr 5 Gy×2: 65%; ⁹⁰Sr 5 Gy×4: 61.17%; Fig. 5C). Unexpected, the proportion of cells in state pre-branch was 19.52% in ⁹⁰Sr 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. 5D). 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. 5E). 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. 6A). Then, immunofluorescence staining revealed nuclear translocation of IRF1 in primary keloid fibroblasts post-irradiation (Fig. 6B). 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. 6C, 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. 6E). Moreover, we found that SSBP1 knockdown resulted in elevated IRF1 levels and enhanced its nuclear translocation (Fig. 6F-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. 6I). 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. 6J-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. 6M and Fig. E2A-C). Although, all three chemicals increased the release of LDH, indicating cytotoxicity (Fig. 6M). Thus, these data implicated IRF1 as a potential target of keloid therapy.

Radiotherapy has been an adjuvant treatment in keloid management for over a century.¹³ Among these three radiotherapy modalities, both electron beams and ⁹⁰Sr 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 ⁹⁰Sr 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 ⁹⁰Sr 5 Gy×2 and ⁹⁰Sr 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 ⁹⁰Sr 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.

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.

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.

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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

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Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials and methods

Reagents and materials

Human keloid samples

Hematoxylin and Eosin (H&E) staining

Cell culture

Primary keloid cells irradiation

Immunofluorescence assay

Clonogenic assay

Cell cycle assay

Cell apoptosis assay

Lactate dehydrogenase (LDH) assay

Senescence-associated β-galactosidase (SA-β-gal) staining

Western blotting analysis

Single-cell RNA sequencing (scRNA-Seq)

Results

Ionizing radiation inhibits the proliferation and induces cell death of primary keloid fibroblasts

Single-cell transcriptomic map in primary keloid fibroblasts with different radiotherapy modalities

Differential proportion analysis reveals significant expansion of fibroblast subpopulations in keloids with different radiotherapy modalities

Discussion

Conclusions

Declarations

References

Supplementary Files

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