Epigenetic memory of radiotherapy in dermal fibroblasts impairs wound repair capacity in cancer survivors (ATAC-Seq)
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ABSTRACT: Long-term toxicities caused by cancer treatments have recently gained increasing recognition due to a steadily growing population of cancer survivors. Radiotherapy (RT) is a common treatment known to unintentionally harm surrounding normal tissues including the skin, hindering wound healing even years after treatment. Our study aimed to elucidate the underlying mechanisms of these late-onset adverse effects caused by RT. By comparing paired skin biopsies from previously irradiated (RT+) and non-irradiated (RT-) sites in breast cancer survivors who underwent RT years ago, we discovered compromised wound healing capacity and impaired fibroblast functions in the RT+ skin. By employing ATAC-seq, we identified altered chromatin landscapes in RT+ fibroblasts, pinpointing THBS1 as a crucial epigenetically primed wound repair-related gene. Further confirmation of THBS1's significance during wound repair came from single-cell RNA-sequencing and spatial transcriptomic analysis of human wounds. Remarkably, heightened and sustained THBS1 expression was observed in RT+ fibroblasts in both mouse and human radiation wound models, leading to impaired fibroblast motility and contractility. Encouragingly, our study found that treatment with anti-THBS1 antibodies promoted ex vivo wound closure in RT+ skin from breast cancer survivors. These findings indicate that dermal fibroblasts retain a long-term radiation memory recorded in the form of epigenetic changes. Targeting this maladaptive epigenetic memory shows promise for mitigating the late-onset adverse effects caused by RT, offering potential solutions to improve the quality of life for cancer survivors.
Project description:Long-term toxicities caused by cancer treatments have recently gained increasing recognition due to a steadily growing population of cancer survivors. Radiotherapy (RT) is a common treatment known to unintentionally harm surrounding normal tissues including the skin, hindering wound healing even years after treatment. Our study aimed to elucidate the underlying mechanisms of these late-onset adverse effects caused by RT. By comparing paired skin biopsies from previously irradiated (RT+) and non-irradiated (RT-) sites in breast cancer survivors who underwent RT years ago, we discovered compromised wound healing capacity and impaired fibroblast functions in the RT+ skin. By employing ATAC-seq, we identified altered chromatin landscapes in RT+ fibroblasts, pinpointing THBS1 as a crucial epigenetically primed wound repair-related gene. Further confirmation of THBS1's significance during wound repair came from single-cell RNA-sequencing and spatial transcriptomic analysis of human wounds. Remarkably, heightened and sustained THBS1 expression was observed in RT+ fibroblasts in both mouse and human radiation wound models, leading to impaired fibroblast motility and contractility. Encouragingly, our study found that treatment with anti-THBS1 antibodies promoted ex vivo wound closure in RT+ skin from breast cancer survivors. These findings indicate that dermal fibroblasts retain a long-term radiation memory recorded in the form of epigenetic changes. Targeting this maladaptive epigenetic memory shows promise for mitigating the late-onset adverse effects caused by RT, offering potential solutions to improve the quality of life for cancer survivors.
Project description:Long-term toxicities caused by cancer treatments have recently gained increasing recognition due to a steadily growing population of cancer survivors. Radiotherapy (RT) is a common treatment known to unintentionally harm surrounding normal tissues including the skin, hindering wound healing even years after treatment. Our study aimed to elucidate the underlying mechanisms of these late-onset adverse effects caused by RT. By comparing paired skin biopsies from previously irradiated (RT+) and non-irradiated (RT-) sites in breast cancer survivors who underwent RT years ago, we discovered compromised wound healing capacity and impaired fibroblast functions in the RT+ skin. By employing ATAC-seq, we identified altered chromatin landscapes in RT+ fibroblasts, pinpointing THBS1 as a crucial epigenetically primed wound repair-related gene. Further confirmation of THBS1's significance during wound repair came from single-cell RNA-sequencing and spatial transcriptomic analysis of human wounds. Remarkably, heightened and sustained THBS1 expression was observed in RT+ fibroblasts in both mouse and human radiation wound models, leading to impaired fibroblast motility and contractility. Encouragingly, our study found that treatment with anti-THBS1 antibodies promoted ex vivo wound closure in RT+ skin from breast cancer survivors. These findings indicate that dermal fibroblasts retain a long-term radiation memory recorded in the form of epigenetic changes. Targeting this maladaptive epigenetic memory shows promise for mitigating the late-onset adverse effects caused by RT, offering potential solutions to improve the quality of life for cancer survivors.
Project description:Wound healing within the oral mucosa results in minimal scar formation compared to wounds within the skin. We have recently demonstrated distinct differences in the ageing profiles of cells (oral mucosal and patient-matched skin fibroblasts) isolated from these tissues. We hypothesize that the increased replicative potential of oral mucosal fibroblasts may confer upon them preferential wound healing capacities. Passage-matched early cultures of oral mucosal fibroblasts and skin fibroblasts demonstrated distinct gene expression profiles with a number of genes linked to wound healing/tissue repair. We analyzed the gene expression profiles of oral mucosal and patient-matched skin fibroblasts for multiple patients both prior to (0h) and (6h) following a wounding stimulus.
Project description:Impaired skin wound healing is a significant global health issue, especially among the elderly. Wound healing is a well-orchestrated process involving the sequential phases of inflammation, proliferation, and tissue remodeling. Although wound healing is a highly dynamic and energy-requiring process, the role of metabolism remains largely unexplored. By combining transcriptomics and metabolomics of human skin biopsy samples, we mapped the core bioenergetic and metabolic changes in normal acute as well as chronic wounds in elderly subjects. We found upregulation of glycolysis, the tricarboxylic acid cycle, glutaminolysis, and β-oxidation in the later stages of acute wound healing and in chronic wounds. To ascertain the role of these metabolic pathways on wound healing, we targeted each pathway in a wound healing assay as well as in a human skin explant model using metabolic inhibitors and stimulants. Enhancement or inhibition of glycolysis and, to a lesser extent, glutaminolysis had a far greater impact on wound healing than similar manipulations of oxidative phosphorylation and fatty acid β-oxidation. These findings increase the understanding of wound metabolism and identify glycolysis and glutaminolysis as potential targets for therapeutic intervention.
Project description:Wound healing within the oral mucosa results in minimal scar formation compared to wounds within the skin. We have recently demonstrated distinct differences in the ageing profiles of cells (oral mucosal and patient-matched skin fibroblasts) isolated from these tissues. We hypothesize that the increased replicative potential of oral mucosal fibroblasts may confer upon them preferential wound healing capacities. Passage-matched early cultures of oral mucosal fibroblasts and skin fibroblasts demonstrated distinct gene expression profiles with a number of genes linked to wound healing/tissue repair. We analyzed the gene expression profiles of oral mucosal and patient-matched skin fibroblasts for multiple patients both prior to (0h) and (6h) following a wounding stimulus. Differences in the gene expression profiles of oral mucosal and patient-matched skin fibroblasts were anlazyed for multiple patients both prior to (0h) and (6h) following a wounding stimulus. Serum starvation and subsequent stimulation provides a model for wounding and RNA extracted at 0h and 6h following this stimulus was hybridized to Affymetrix microarrays for analysis. We sought to compare the expression profiles both between oral and normal fibroblasts, in both serum depleted and stimulated conditions and also compare differences between patients.
Project description:Proteinases play a pivotal role in wound healing by degrading molecular barriers, regulating cell-matrix interactions and availability of bioactive molecules. Matrix metalloproteinase-13 (MMP-13, collagenase-3) is a wide spectrum proteinase. Its expression and function is linked to the growth and invasion of many epithelial cancers such as squamous cell carcinoma. Moreover, the physiologic expression of MMP-13 is associated e.g. to scarless healing of human fetal skin and adult gingival wounds. While MMP-13 is not found in the normally healing skin wounds in human adults, it is expressed in mouse skin during wound healing. Thus, mouse wound healing models can be utilized for studying the role of MMP-13 in the events of wound healing. As the processes such as the migration and proliferation of keratinocytes, angiogenesis, inflammation and activation of fibroblasts are components of wound repair as well as of cancer, many results received from wound healing studies are also adaptable to cancer research. Classically, the process of wound healing can be devided into three phases which are histologically and functionally separate but temporally overlapping: 1) hemostasis and inflammation, 2) re-epithelialization and granulation tissue formation, and 3) matrix remodeling. Granulation tissue is formed into the wound via fibroplasia, angiogenesis and extracellular matrix (ECM) deposition by fibroblasts. Granulation tissue is rich in inflammatory cells, fibroblasts, myofibroblasts and blood vessels. After epidermal recovery, the granulation tissue is resolved via matrix remodeling and cell apoptosis. A sterile viscose cellulose sponge (VCS) characterized by defined size and structure can be used to experimentally induce formation of subcutaneous granulation tissue. Compared to normal granulation tissue, this model allows easy examination of the granulation tissue in its entirety but leaving out epidermal keratinocytes in the sample preparation. In this study, we studied the role of MMP-13 in the formation of mouse VCS-induced granulation tissue. We performed gene expression profiling of the granulation tissue samples of Mmp13-/- (KO) and wild type (WT) mice harvested at day 7, day 14 and day 21 after VCS implantation. Mmp13-/- (KO) mice were generated as described (Inada et al. 2004, PNAS, 101: 17192-17197) and used in these experiments after backcrossing at least seven generations into C57BL6 mice. The WT mice were generated from the backcrossed heterozygote Mmp13-/- (KO) mice. Granulation tissues were harvested at three time points (7d, 14d, 21d) from Mmp13-/- (KO) and WT mice. One sample of each mouse was analyzed (n=3, 7d; n=4, 14d; n=4, 21d; for each genotype). The samples were processed for RNA extraction and Affymetrix 3'IVT DNA microarray gene expression analysis.
Project description:Overexpression of activin A by keratinocytes accelerates excisional wound healing in mice. Activin-promoted wound healing is mediated via the stroma, specifically by the wound immune cells and fibroblasts. To determine if activin A alters the gene expression profile of wound fibroblasts, we performed RNA-sequencing of fibroblasts FACS-sorted from excisional skin wounds of activin overexpressing mice. We found that activin induces a pro-fibrotic gene expression profile of these fibroblasts, leading to the upregulation of genes involved in collagen biosynthesis and remodeling.
Project description:Proteinases play a pivotal role in wound healing by degrading molecular barriers, regulating cell-matrix interactions and availability of bioactive molecules. Matrix metalloproteinase-13 (MMP-13, collagenase-3) is a wide spectrum proteinase. Its expression and function is linked to the growth and invasion of many epithelial cancers such as squamous cell carcinoma. Moreover, the physiologic expression of MMP-13 is associated e.g. to scarless healing of human fetal skin and adult gingival wounds. While MMP-13 is not found in the normally healing skin wounds in human adults, it is expressed in mouse skin during wound healing. Thus, mouse wound healing models can be utilized for studying the role of MMP-13 in the events of wound healing. As the processes such as the migration and proliferation of keratinocytes, angiogenesis, inflammation and activation of fibroblasts are components of wound repair as well as of cancer, many results received from wound healing studies are also adaptable to cancer research. Classically, the process of wound healing can be devided into three phases which are histologically and functionally separate but temporally overlapping: 1) hemostasis and inflammation, 2) re-epithelialization and granulation tissue formation, and 3) matrix remodeling. Granulation tissue is formed into the wound via fibroplasia, angiogenesis and extracellular matrix (ECM) deposition by fibroblasts. Granulation tissue is rich in inflammatory cells, fibroblasts, myofibroblasts and blood vessels. After epidermal recovery, the granulation tissue is resolved via matrix remodeling and cell apoptosis. A sterile viscose cellulose sponge (VCS) characterized by defined size and structure can be used to experimentally induce formation of subcutaneous granulation tissue. Compared to normal granulation tissue, this model allows easy examination of the granulation tissue in its entirety but leaving out epidermal keratinocytes in the sample preparation. In this study, we studied the role of MMP-13 in the formation of mouse VCS-induced granulation tissue. We performed gene expression profiling of the granulation tissue samples of Mmp13-/- (KO) and wild type (WT) mice harvested at day 7, day 14 and day 21 after VCS implantation.
Project description:Mammalian skin wounds heal by forming fibrotic scars. We report that reindeer antler velvet exhibits regenerative wound healing, whereas identical injury to back skin forms scar. This regenerative capacity was retained following ectopic transplantation of velvet to scar-forming sites. Single-cell mRNA/ATAC-Sequencing revealed that while uninjured velvet fibroblasts resembled human fetal fibroblasts, back skin fibroblasts were enriched in pro-inflammatory features resembling adult human fibroblasts. Injury elicited site-specific immune polarization; back skin fibroblasts amplified the immune response, whereas velvet fibroblasts adopted an immunosuppressive state leading to restrained myeloid maturation and hastened immune resolution ultimately enabling myofibroblast reversion to a regeneration-competent state. Finally, regeneration was blunted following application of back skin associated immunostimulatory signals or inhibition of pro-regenerative factors secreted exclusive to velvet fibroblasts. This study highlights a unique model to interrogate mechanisms underlying divergent healing outcomes and nominates both decoupling of stromal-immune crosstalk and reinforcement of pro-regenerative fibroblast programs to mitigate scar.
Project description:Ionizing radiation (IR) therapy for malignant tumors can damage adjacent tissues, leading to severe wound complications. Plasma-derived exosome treatment has recently emerged as a safe and impactful cell-free therapy. Herein, we aimed to determine whether plasma-derived exosomes could improve the healing of post-radiation wound. Rat plasma-derived exosomes (RP-Exos) were locally injected on cutaneous wounds created on the backs of irradiated rats and boosted the healing process as well as the deposition and remodeling of the extracellular matrix with collagen formation. Subsequently, the effects of RP-Exos were further evaluated on irradiated fibroblasts in vitro. The results suggested that exosomes promoted fibroblast proliferation, migration, cell cycle progression, and cell survival. Moreover, transcriptome sequencing, analysis, and quantitative polymerase chain reaction validation were performed to identify the underlying molecular mechanisms. RP-Exos enhanced the expression of cell proliferation and radioresistance-related genes, and yet downregulated ferroptosis pathway in irradiated fibroblasts. Inhibition of ferroptosis by RP-Exos was further confirmed through colorimetric assay, fluorescence probe and flow cytometry in ferroptosis-induced fibroblasts. Our results suggest that RP-Exos regulate cell proliferation and ferroptosis in radiated fibroblasts, thereby boosting the healing of radiated wounds. These findings support plasma-derived exosomes as a potential therapeutic method for post-radiation wound complications.