PDF(Pubmed)
Abstract：
BACKGROUND: Radiation-induced fibrosis (RIF) is an important late complication of radiation therapy, and the resulting damaging effects of RIF can significantly impact reconstructive outcomes. There is currently a paucity of effective treatment options available, likely due to the continuing knowledge gap surrounding the cellular mechanisms involved. In this study, detailed analyses of irradiated and non-irradiated human skin samples were performed incorporating histological and single-cell transcriptional analysis to identify novel features guiding development of skin fibrosis following radiation injury.
METHODS: Paired irradiated and contralateral non-irradiated skin samples were obtained from six female patients undergoing post-oncologic breast reconstruction. Skin samples underwent histological evaluation, immunohistochemistry, and biomechanical testing. Single-cell RNA sequencing was performed using the 10X single cell platform. Cells were separated into clusters using Seurat in R. The SingleR classifier was applied to ascribe cell type identities to each cluster. Differentially expressed genes characteristic to each cluster were then determined using non-parametric testing.
RESULTS: Comparing irradiated and non-irradiated skin, epidermal atrophy, dermal thickening, and evidence of thick, disorganized collagen deposition within the extracellular matrix of irradiated skin were readily appreciated on histology. These histologic features were associated with stiffness that was higher in irradiated skin. Single-cell RNA sequencing revealed six predominant cell types. Focusing on fibroblasts/stromal lineage cells, five distinct transcriptional clusters (Clusters 0-4) were identified. Interestingly, while all clusters were noted to express Cav1, Cluster 2 was the only one to also express Cav2. Immunohistochemistry demonstrated increased expression of Cav2 in irradiated skin, whereas Cav1 was more readily identified in non-irradiated skin, suggesting Cav1 and Cav2 may act antagonistically to modulate fibrotic cellular responses.
CONCLUSIONS: In response to radiation therapy, specific changes to fibroblast subpopulations and enhanced Cav2 expression may contribute to fibrosis. Altogether, this study introduces a novel pathway of caveolin involvement which may contribute to fibrotic development following radiation injury.
METHODS: Paired irradiated and contralateral non-irradiated skin samples were obtained from six female patients undergoing post-oncologic breast reconstruction. Skin samples underwent histological evaluation, immunohistochemistry, and biomechanical testing. Single-cell RNA sequencing was performed using the 10X single cell platform. Cells were separated into clusters using Seurat in R. The SingleR classifier was applied to ascribe cell type identities to each cluster. Differentially expressed genes characteristic to each cluster were then determined using non-parametric testing.
RESULTS: Comparing irradiated and non-irradiated skin, epidermal atrophy, dermal thickening, and evidence of thick, disorganized collagen deposition within the extracellular matrix of irradiated skin were readily appreciated on histology. These histologic features were associated with stiffness that was higher in irradiated skin. Single-cell RNA sequencing revealed six predominant cell types. Focusing on fibroblasts/stromal lineage cells, five distinct transcriptional clusters (Clusters 0-4) were identified. Interestingly, while all clusters were noted to express Cav1, Cluster 2 was the only one to also express Cav2. Immunohistochemistry demonstrated increased expression of Cav2 in irradiated skin, whereas Cav1 was more readily identified in non-irradiated skin, suggesting Cav1 and Cav2 may act antagonistically to modulate fibrotic cellular responses.
CONCLUSIONS: In response to radiation therapy, specific changes to fibroblast subpopulations and enhanced Cav2 expression may contribute to fibrosis. Altogether, this study introduces a novel pathway of caveolin involvement which may contribute to fibrotic development following radiation injury.
摘要：
背景：放射性纤维化（RIF）是放射治疗的重要晚期并发症，RIF的破坏性影响会显著影响重建结果。目前缺乏有效的治疗方案,可能是由于围绕所涉及的细胞机制的持续知识差距。在这项研究中,我们对辐照和未辐照人类皮肤样本进行了详细分析,结合组织学和单细胞转录分析,以确定指导放射性损伤后皮肤纤维化发展的新特征.
方法：从6名接受肿瘤后乳房再造的女性患者中获得了配对的照射和对侧未照射的皮肤样本。皮肤样本进行组织学评估，免疫组织化学，和生物力学测试。使用10X单细胞平台进行单细胞RNA测序。使用R中的Seurat将细胞分成簇。应用SingleR分类器将细胞类型身份归因于每个簇。然后使用非参数测试确定每个簇的差异表达基因特征。
结果：比较辐照和未辐照的皮肤，表皮萎缩,真皮增厚，和厚的证据，在组织学上很容易理解辐照皮肤的细胞外基质内无序的胶原蛋白沉积。这些组织学特征与辐照皮肤中更高的硬度相关。单细胞RNA测序揭示了六种主要的细胞类型。专注于成纤维细胞/基质谱系细胞，鉴定了五个不同的转录簇(簇0-4)。有趣的是,尽管注意到所有簇都表达Cav1，但簇2是唯一也表达Cav2的簇。免疫组织化学显示照射的皮肤中Cav2的表达增加，而Cav1在未照射的皮肤中更容易识别，提示Cav1和Cav2可能拮抗调节纤维化细胞反应。
结论：对放射治疗的反应，成纤维细胞亚群的特异性变化和Cav2表达增强可能导致纤维化。总之,这项研究引入了一个新的小窝蛋白参与途径，该途径可能有助于放射损伤后的纤维化发展。
方法：从6名接受肿瘤后乳房再造的女性患者中获得了配对的照射和对侧未照射的皮肤样本。皮肤样本进行组织学评估，免疫组织化学，和生物力学测试。使用10X单细胞平台进行单细胞RNA测序。使用R中的Seurat将细胞分成簇。应用SingleR分类器将细胞类型身份归因于每个簇。然后使用非参数测试确定每个簇的差异表达基因特征。
结果：比较辐照和未辐照的皮肤，表皮萎缩,真皮增厚，和厚的证据，在组织学上很容易理解辐照皮肤的细胞外基质内无序的胶原蛋白沉积。这些组织学特征与辐照皮肤中更高的硬度相关。单细胞RNA测序揭示了六种主要的细胞类型。专注于成纤维细胞/基质谱系细胞，鉴定了五个不同的转录簇(簇0-4)。有趣的是,尽管注意到所有簇都表达Cav1，但簇2是唯一也表达Cav2的簇。免疫组织化学显示照射的皮肤中Cav2的表达增加，而Cav1在未照射的皮肤中更容易识别，提示Cav1和Cav2可能拮抗调节纤维化细胞反应。
结论：对放射治疗的反应，成纤维细胞亚群的特异性变化和Cav2表达增强可能导致纤维化。总之,这项研究引入了一个新的小窝蛋白参与途径，该途径可能有助于放射损伤后的纤维化发展。
