Human RT- and RT+ skin sample collection and analysis
The collection and usage of human skin samples were approved by the Stockholm Regional Ethics Committee (Stockholm, Sweden). All participants gave their written consent, and the study was conducted according to the Declaration of Helsinki’s principles.
Paired tissue biopsies were collected from surplus skin from breast cancer patients undergoing autologous-tissue breast reconstruction (n=43 donors, Table S1) at the Karolinska University Hospital (Stockholm, Sweden). These patients underwent a mastectomy followed by external beam RT with a total dose of 40-60 Gy high-energy X-rays. The length of the period from the end of the RT to the breast reconstruction surgery varied from one to 12 years among the patients. During the reconstruction surgery, previously irradiated skin was collected from one side of the breast (RT+) and non-irradiated skin from the other side of the breast or abdomen (RT-) from each patient (Table S1). In dermal fibroblasts isolated from these RT- and RT+ skin biopsies, in vitro experiments were conducted to explore DNA accessibility, transcription factor binding, histone modification, gene expression, and cellular functions. Human ex vivo wound models were also set up using the RT- and RT+ skin to study THBS1 expression and evaluate the therapeutic effects of THBS1 antibodies (Cat. MS5-13377, Invitrogen, Waltham, MA).
Moreover, to monitor the in vivo gene expression changes of human skin wound healing, we developed a human wound healing model at the Karolinska University Hospital by creating three full-depth wounds (3 mm in diameter) on the skin of each healthy donors (n=18 donors, Table S2), then collecting the wound-edge tissue with a 6 mm biopsy punch from the same donor one (D1), seven (D7), and thirty days (D30) after the injury.
Murine irradiated skin and excisional wound model
The protocols of murine experiments were approved by the Comité de Protection des Animaux de l’Université Laval (CPAUL), Cégep de Sainte-Foy Animal Protection Committees (Québec, Canada), and the North Stockholm Ethical Committee for Care and Use of Laboratory Animals (Stockholm, Sweden).
The protocol of murine irradiated skin and excisional wound model is detailed in 44. Unique doses of 6MV photons were delivered to the back skin of CD-1 mice (45, 60, or 80 Gy). Skin toxicity recovered over four weeks, at which time eight mm-biopsy punches were used to create full-thickness excisional wounds. Non-irradiated (0 Gy) control animals were also included in the wound healing study. The wounds were splinted with silicone rings and allowed to heal under moist conditions 44. After 33 days, the wounded tissues were harvested with wound edges for histological analyses. Formalin-fixed samples embedded in paraffin were used for FISH analysis.
The murine skin wound healing model was performed as previously described 22. Four mm-biopsy punches were used to create full-thickness excisional wounds on the back skin of C57BL/6 mice. On day-3 and day-6 after the injury, mice were euthanized, and skin biopsies at the wound site and intact area were collected for single-cell RNA sequencing analysis or FISH analysis.
Human ex vivo wound model
Human ex vivo wound model was performed as previously described 22, 23. Briefly, partial-thickness wounds were created on the human RT- and RT+ skin collected from surgeries using a 2 mm biopsy punch and then excised from the skin using a 6 mm biopsy punch. After removing subcutaneous fat, we placed the tissues in a 12-well cell culture plate. The Dulbecco’s Modified Eagle Medium high glucose, DMEM (Cat. 11965092, Gibco, Waltham, MA) supplemented with 10% fetal bovine serum (Cat. 2567819RP, Gibco), and antibiotics (1x penicillin and streptomycin, Cat. 15140122, Gibco) was added (800 µl per well) around the tissue, so the epidermal surface was exposed to the air to create a liquid-air interface and cultured at 37 °C in a humidified atmosphere of 5% CO2. THBS1 antibody (Cat. MS5-13377, Invitrogen) was diluted mix with PBS in a final concentration of 0.2 ng/µl. The THBS1 antibody mixture were then mixed with 30% pluronic F-127 gel in a 1:2 ratio (volumes) (Cat. P2443, Sigma-Aldrich, St Louis, MO). 5 µl of this mixture was topically applied on wounds immediately after injury, as well as two and four days later. Wound samples were collected six days after injury for histological analysis. Furthermore, we monitored ex vivo wound closure with CellTracker™ Green CMFDA Dye (Cat. C2925, Invitrogen, Waltham, MA) as previously described 67. Briefly, 4 µl dye (50 µM) was added to each wound tissue and incubated at 37°C with 5% CO2 for 30 minutes. The tissue was washed with PBS and imaged with a Nikon eclipse Ni-E fluorescence microscope. The wound areas were quantified by using Image J. Wound contraction was measured by assessing the changes of regions within the initial wound-edge (IWtime point) over time, i.e., wound contraction (%) = ΔIWtime point/IWD0×100%.
Cell isolation and culture
The human skin samples were transported in phosphate buffered saline (PBS) on ice. The skin was wiped with 70% ethanol and PBS, 6 mm punch biopsies were taken, and the underlying adipose tissue was removed. Biopsies were rinsed in PBS and placed in 5 U/mL dispase II solution (Cat. 17105041, Gibco, Waltham, MA) overnight at 4°C. Dermis was separated from the epidermis, cut into smaller pieces, and moved onto a cell culture dish. Fibroblast growth medium (DMEM supplemented with 10% fetal bovine serum (Gibco), and antibiotics (1x penicillin and streptomycin)) were added to overlay the dermis pieces, and fresh growth medium was changed every three days during the fibroblast outgrowth. When 70% confluent, cells were digested with 0.025% trypsin EDTA (Cat. R001100, Gibco) for 5 min at 37°C and resuspended with fibroblast growth medium for passaging. Only cells propagated for ≤ 5 passages were used for the experiments.
In vitro irradiation of fibroblasts
Human dermal fibroblasts were irradiated at room temperature for 8 Gy of gamma radiation at a dose rate of 0.76 Gy/minute. The Scandritronix radiator (Scanditronix, Vislanda, Sweden) was equipped with a 137Cs source (activity 33.3 TBq as of June 1985). Six days after the irradiation, cells were treated with 5 ng/mL recombinant human TGFB1 (Cat. 11343161, ImmunoTools, Friesoythe, Germany) for 24 hours.
Ex vivo explant migration assay
The ex vivo explant migration assay was performed as previously described 68. Briefly, 3 mm punch biopsies were collected from the paired human RT- and RT+ skin, and the dermis was separated from the epidermis as described above. Dermis pieces were placed in cell culture plates and overlaid with fibroblast growth medium. Fibroblast outgrowth was analyzed ten days later by measuring the length from the migrated edge to the tissue (average of three measurements per sample).
Cell migration assays
Cells were plated on ImageLock 96-well plates (Cat. 4379, Essen Bioscience, Ann Arbor, MI) and adhered overnight. To inhibit cell proliferation, we treated cells with 5 µg/mL mitomycin C (Cat. J63193.MA, Thermo Scientific Chemicals, Waltham, MA) for 2 hours, and the cell monolayer was scratched using the IncuCyte wound maker (Essen BioScience). Cells were imaged every 2 hours using the IncuCyte ZOOM imaging system, and cell migration was quantitated with IncuCyte ZOOM 2018A software (Essen BioScience) or Image J software (Bethesda, Maryland).
siRNA transfection
To study the biological function of THBS1, RT+ fibroblasts at 70% confluence were transfected with a 60 nM predesigned siRNA targeting THBS1 (siTHBS1, Cat. s14100, Invitrogen) or negative control siRNAs (siCtr, Cat. AM4611, Invitrogen) for 24 hours with Lipofectamine™ RNAiMAX Transfection Reagent (Cat. 13778075, Invitrogen).
CRISPR-mediated transcriptional activation
To mediate efficient transcriptional activation at endogenous genomic THBS1 loci, we used a CRISPR/Cas9 Synergistic Activation Mediator (SAM) system, which uses a dCas9 devoid of endonuclease activity. The core component of this system is comprised of two plasmids, lentiMS2-P65-HSF1_Hygro (Plasmid #61426, Addgene, Cambridge, Massachusetts) and lentiSAM v2 (Puro) (Plasmid #92062, Addgene). Six single guide RNAs (sgRNAs) were designed in a previous study 45 or online tools UCSC (http://genome.ucsc.edu/index.html), typically sgRNA targeting a 300 nt long sequence upstream of the TSS and selecting the sgRNA with the highest on-target activity. Individual sgRNA expression plasmid was constructed by first annealing the oligonucleotides pair and then ligating them to a BsmBI-v2 (Cat. R0739S, New England Biolabs, Ipswich, MA) digested lentiMS2-P65-HSF1_Hygro backbone. Primers were designed by Addgene or online tools Benchling (https://benchling.com/), aiming for a 182 bp with the sgRNA binding site in the middle. The used sgRNAs and primer sequences are listed in Table S9. Primary human dermal fibroblasts (Cat. C0135C, Gibco) were transfected with Lipofectamine™ 3000 Transfection Reagent (Cat. L3000008, Invitrogen) and harvested 48 hours after transfection, THBS1 expression was analysed by qRT-PCR.
Masson’s trichrome staining and imaging
Tissue samples were fixed in 4% paraformaldehyde (Cat#HL96753.1000, HistoLab, Askim, Sweden) overnight at 4°C, placed in 70% ethanol, dehydrated and embedded in paraffin, and cut in 8 µm thickness. Tissue sections were stained by Trichrome Stain (Masson) Kit (Cat. HT15, Sigma-Aldrich) and imaged using a Nikon eclipse Ni-E bright field microscope (Tokyo, Japan).
Fluorescence In situ hybridization (FISH)
In situ hybridization probes for human and mouse THBS1 (Hs-THBS1, Cat No. 42658 and Mm-Thbs1, Cat No. 457891) or positive and negative control probes (Mm-Polr2a, Cat No. 312471 and DapB probe, Cat No. 310043) were designed and synthesized by Advanced Cell Diagnostics (ACD, Silicon Valley, CA). Tissues were prepared by following the manufacturer’s instructions. After paraffin removal, the slides were incubated in hydrogen peroxide, target retrieval reagent, and protease plus (ACD), following incubation with hybridization probes for two hours at 40 °C in HybEZ™ II Hybridization System using RNAscope® Multiplex Fluorescent Reagent Kit v2 (ACD). The hybridization signals were amplified via sequential hybridization of amplifiers and probes. Probe signals were visualized on Zeiss AxioScan.Z1 Slide Scanner (Oberkochen, Germany) and analyzed with Zen 3.4 software (Zeiss).
Immunofluorescence staining
Paraffin-embedded tissue sections were deparaffinized and rehydrated by passage through xylene and graded ethanol series. After antigen retrieval in citric acid buffer (10 mM, pH 6.0), sections were blocked with 5% bovine serum albumin (BSA, Cat. 9414, Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween-20 (TBST). Sections were incubated overnight at 4°C with primary antibody (1:100) targeting THBS1 protein (cat. sc-59887, Santa Cruz Biotechnology, Dallas, TX), followed by incubation with Alexa Fluor 555 Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed secondary antibody (cat. A-31570, Invitrogen) in 1:200 dilution in TBST. Sections were counter-stained with ProLong™ Diamond Antifade Mountant with DAPI (Cat. P36971, In Invitrogen) .Immunofluorescence staining was visualized using a Nikon eclipse Ni-E fluorescence microscope.
ATAC-seq library preparation, sequencing, and analysis
ATAC-seq was performed as previously described 69. Briefly, five pairs of RT- and RT+ fibroblasts (50,000 cells per sample) were subjected to nuclei extraction and library preparation, and the samples were sequenced by Illumina NovaSeq SP-100 (2x50bp) at the National Genomics Infrastructure at SciLifeLab Stockholm.
Raw data were processed using Trimmomatic v0.36 70 by removing reads of low quality and with a length of fewer than 30 nucleotides. The PCR duplicated reads were excluded using Picard (v2.20.4) tools after mapping to GRCh38 human reference genome using Bowtie2 (v2.3.5.1) 71. The uniquely mapped reads were shifted and converted into bigwig format with a 200-bp extension size for IGV visualization 72 using the deepTools 73. Peaks were called on each sample individually using MACS2 (v2.2.6) with the default parameter except for using the BAMPE option 74. Peaks overlapping with the repetitive regions in ENCODE blacklist downloaded from UCSC Table Browser were filtered out using the BEDTools suite 75.
Peaks were further extended to 500 bp windows centered on the summits, avoiding the bias of differential accessibility (DA) analysis due to the varying lengths of peaks. DA peaks were analyzed across different conditions with DESeq2 76. DA domains were defined as p-adjust value <0.05 (171 peaks). RT+ up domains were defined as log2(fold change) >0,p-adjust value <0.05 in RT+ fibroblasts. RT- up domains were defined as log2(fold change) <0,p-adjust value <0.05 in RT+ fibroblasts. The peaks were annotated using the ChIPseeker package with a promoter region ranging from -3K (upstream) to 3K (downstream) bp of the transcription starting site 77. Gene ontology (GO) analysis for peak-related genes was carried out using the Metascape 78. Significant GO terms with criteria p value less than 0.05 were shown. For motif analysis of ATAC peaks, HOMER known motif search was used to generate the list of enriched sequence motifs matching known transcription factor (TF) binding sites 27. To explore the TF footprint, we used TOBIAS software version 0.14.0 28 to predict the differential TF binding scores between the RT- and RT+ fibroblasts with bound-pvalue = 0.01. TF-TF gene regulation networks were created using TOBIAS’s built-in function ‘CreateNetwork’.
RNA extraction and qRT-PCR
Total RNA was extracted from fibroblasts using Trizol, followed by cDNA synthesis with RevertAid First Strand cDNA Synthesis Kit (Cat. K1621, Thermo Scientific, Waltham, MA). Specific premixed primers and probes were predesigned by Integrated DNA Technologies (IDT, Leuven, Belgium) for the detection of THBS1, ACTA2, FN1, ELN, CDKN1A, 18S, and GAPDH. Gene expression was determined by TaqMan expression assays (Cat. 4304437, ThermoScientific) and normalized based on the values of the housekeeping gene GAPDH or 18S. The comparative 2ΔΔCT method was used for the quantification of gene expression. All reactions were run by QuantStudio 6 or 7 (Applied Biosystems, Waltham, MA). Information for all the primers used in this study is listed in Table S9.
Chromatin immunoprecipitation (ChIP)
RT- and RT+ fibroblasts were treated with 5 ng/mL recombinant human TGFB1 (Cat. 11343161, ImmunoTools) in fibroblast culture medium for 24 hours. Cells were crosslinked with 1 % formaldehyde (Cat. 28908, Thermo Scientific) for 10 min and quenched with 0.125 M glycine (Cat. 50046, Sigma-Aldrich). MAGnify Chromatin Immunoprecipitation System kit (Cat. 492024, Applied Biosystems) was used for ChIP according to the manufacturer’s instructions. Briefly, 200,000 cells per sample were collected and lysed, following DNA sonication to achieve 200-500 bp fragments using Bioruptor UCD-200 (Diagenode, Seraing, Belgium). Protein A/G Dynabeads were mixed with RUNX1-targeting antibody (Cat. ab272456, Abcam, Cambridge, UK) or H3K4me1 (Cat. ab8895, Abcam). Sonicated cell lysates were incubated with the antibody-coated beads for two hours at 4°C, followed by washing, reverse crosslinking, and DNA purifying. Samples were analyzed by qPCR with primers designed to span the RUNX1-binding sites at the THBS1 promoter region (Table S9).
Single-cell RNA library preparation, sequencing, and analysis
After separating epidermis and dermis of the skin or wound samples using 5 U/mL dispase II solution (Cat. 17105041, Gibco), we digested the epidermis in 0.025% trypsin-EDTA (Gibco) for 15 minutes at 37°C, which was quenched with defined trypsin inhibitor (Cat. R007100, Gibco), and strained through a 70 µm filter. Red blood cells and dead cells were removed by red blood cell lysis solution kit (Cat. 130-094-183, Miltenyi Biotec, Bergisch Gladbach, Germany) and a dead cell removal kit (Cat. 130-090-101, Miltenyi Biotec), respectively. Dermis was cut into small pieces and further dissociated into single-cell suspension using a human enzyme mixture from a whole skin dissociation kit (Cat. 130-101-540, Miltenyi Biotec). Epidermal and dermal cells were combined in a 1:1 ratio, and libraries were constructed using a 10x chromium system with chemistry v3. Libraries were then sequenced with the Illumina NovaSeq 6000 sequencer to generate 150-base pair paired-end reads. Raw single-cell sequencing data were processed using the standard 10X Cell Ranger (v5.0.1) analysis workflow, including demultiplexing, aligning to the GRCh38 human reference genome, barcode counting, and unique molecular identifier (UMI) quantification. The doublets of cells predicted by Scrublet 79 and DoubletFinder 80 were excluded. The clean filtered feature barcode matrices were used as input into a Seurat pipeline 81. Within the Seurat, we removed mitochondrial genes, hemoglobin genes, ribosomal genes, genes expressed in less than ten cells, and cells with less than 500 detected genes, less than 1000 UMIs, and with more than 20% mitochondrial gene expression. Finally, 11,800 cells from the skin and 16,098 cells from day one post-wounding were retained for all the subsequent analyses. The data were first normalized using the SCTransform 82 function. Uniform manifold approximation and projection (UMAP) plots were generated using the 'RunUMAP' function with the first 40 harmonies. The clusters were obtained using the FindNeighbors and FindClusters functions with a resolution of 0.8. The cluster marker was identified using the function ‘FindAllMarker’. The cell types were annotated according to the overlaps between the cluster markers and well-known signature genes of each cell type from previous studies. The ligand-receptor (L-R) analysis was performed by the CellChat package 43 to access the potential cell-cell crosstalk among different cell types.
Magnetic activation cell sorting
Fibroblasts were isolated from human skin and acute wound tissues with magnetic activation cell sorting (MACS). Fresh tissue samples were washed 2–3 times in PBS and incubated in 5 U/mL dispase II solution (Cat. 17105041, Gibco) supplemented with antibiotics (1x penicillin and streptomycin, Cat. 15140122, Gibco) overnight at 4 °C. The epidermis was separated from the dermis. The dermis was incubated in the enzyme mix from the whole skin dissociation kit (Cat. 130-101-540, Miltenyi Biotec) for 3 hours according to the manufacturer’s instructions and further processed by Medicon tissue disruptor (BD Biosciences, Stockholm, Sweden). The dermal cell suspension was incubated with CD90 microbeads (Cat. 130-096-253, Miltenyi Biotec), and CD90+ fibroblasts were isolated with MACS MS magnetic columns according to the manufacturer’s instructions (Miltenyi Biotec). The isolated fibroblasts were used for qRT-PCR analysis directly without cell culture.
Tissue preparation and sequencing for spatial transcriptomics (ST)
Human skin and wound tissues were gently washed with cold PBS and embedded in optimal cutting temperature compound (OCT, Cat. 4583, Sakura Finetek USA, Torrance, CA) and snap-frozen on dry ice. The samples were then processed for the ST experiment by the Visium Spatial platform of 10x Genomics as per the manufacturer’s instructions. Cryosections were cut and mounted onto the ST arrays and stored at a −80 °C freezer. The tissue was dehydrated and stained with haematoxylin and eosin staining to assess the morphology and quality. After permeabilization, reverse transcription and second-strand synthesis were performed on the slides. cDNA Library preparation, clean up, and indexing were conducted following standard procedures. The pooled libraries are sequenced on NovaSeq6000 S4-200 (Illumina), generating ∼300 M reads per section. The raw ST data were processed using the standard Space Ranger pipeline (version 1.2) with the GRCh38 human reference genome and GENCODE v38 gene annotations and visualized by BBrowser (BioTuring).
Schematics
Schematic cartoons in Fig. 1A-B, 2H, 3C, 3G, 3J, 4G, 5A and 7 were created with BioRender.com.
Statistical information
The number of biological replicates used in each experiment is indicated in the respective method sections and figure legends. Comparison between groups was performed using paired or unpaired student’s t-test or two-way analysis of variance (ANOVA). Differences were considered statistically significant when P < 0.05. Statistical analysis was performed using GraphPad Prism software version 9 (San Diego, CA).