Identification and Targeted Inhibition of a Fibroblast Lineage Responsible for Scarring and Cancer Stroma

Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined due to a lack of functional subclasses. Here we reveal the presence of multiple lineages of dermal fibroblasts within the dorsal back. Genetic lineage tracing and transplantation assays demonstrate that the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation is carried out by a single, somitic-derived fibroblast lineage. Reciprocal transplantation of distinct fibroblast lineages between the dorsal back and oral cavity induced ectopic dermal architectures that mimic their place–of-origin. These studies demonstrate that intra and inter-site diversity of dermal architectures are set embryonically and maintained postnatally by distinct lineages of fibroblasts. Lineage-specific cell ablation using transgenic-mediated expression of the simian diphtheria toxin receptor in conjunction with localized administration of diphtheria toxin led to diminished connective tissue deposition in wounds and significantly reduced melanoma growth in the dorsal skin of mice. Using flow cytometry and in silico approaches, we identify CD26/DPP4 as a surface marker that allows for the isolation of this fibrogenic, scar-forming lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. The identification and prospective isolation of these lineages holds promise for translational medicine aimed at in vivo modulation of their fibrogenic behavior. Dermal fibroblasts were harvested for FACS from the oral dermis and cranial dermis of Wnt1Cre; R26mTmG mice, and from ventral and dorsal dermis of En1Cre; R26mTmG mice as previously described. For all microarray analysis, EPFs and ENFs where harvested from backskin of mice at 30 days of age. Positivity for GFP or RFP allowed for the separation of EPFs from ENFs and WPFs from WNFs. RNA was precipitated via chloroform-phenol extraction. Samples were processed for cleanup and concentration using Rneasy MinElute cleanup kit (cat. 74204, QIAGEN). RNA yield was typically 0.5-1 µg RNA/sorted subpopulations. RNA samples from all sorted populations were converted to cDNA using SuperScript III first strand synthesis system for RT-PCR (cat. 18080-051, Invitrogen), and hybridized to Affymetrix Mouse Genome 430 2.0 arrays. Microarrays were normalized by robust multichip average (RMA) and quantile normalization in R. Cluster analysis was performed with AutoSOME (33), using the following settings for gene expression clustering: p-value threshold of 0.05, 100 ensemble iterations, unit variance normalization of arrays, median centering of genes, and sum of squares=1 normalization for both genes and arrays.

皮肤成纤维细胞是一类具有多样表型的异质性细胞群体，但因缺乏功能性亚类的精准界定，其诸多特征迄今仍未得到充分阐明。本研究首次揭示了小鼠背部真皮中存在多谱系皮肤成纤维细胞。通过遗传谱系示踪与移植实验证实，在胚胎发育、皮肤创伤愈合、放射性纤维化以及癌症基质形成过程中，大部分结缔组织的沉积均由单一的体节起源成纤维细胞谱系完成。将不同成纤维细胞谱系在背部与口腔黏膜间进行相互异位移植，可诱导出与供体起源部位特征一致的异位真皮结构。本研究证实，真皮结构的位点内与位间多样性在胚胎发育阶段即已确立，并在出生后由不同的成纤维细胞谱系维持。通过在特定谱系细胞中过表达猿猴白喉毒素受体，并结合局部注射白喉毒素的谱系特异性细胞清除策略，可减少小鼠伤口处的结缔组织沉积，并显著抑制其背部皮肤黑色素瘤的生长。本研究借助流式细胞术与生物信息学分析方法，鉴定出CD26/DPP4可作为分离该致纤维化、瘢痕形成谱系细胞的表面标志物。在创伤愈合过程中，通过小分子化合物抑制CD26/DPP4的酶活性，可减轻皮肤瘢痕形成。对这些成纤维细胞谱系的鉴定与前瞻性分离，为旨在在体调控其致纤维化行为的转化医学研究提供了新的思路。如前文所述，本研究从Wnt1Cre; R26mTmG小鼠的口腔真皮与颅部真皮，以及En1Cre; R26mTmG小鼠的腹部与背部真皮中分离皮肤成纤维细胞，用于荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）。所有芯片分析实验中，EPF与ENF均取自30日龄小鼠的背部皮肤。通过GFP或RFP的荧光表达可将EPF与ENF、WPF与WNF分离开来。通过氯仿-酚抽提法对RNA进行沉淀。使用RNeasy MinElute 纯化试剂盒（货号74204，QIAGEN公司）对样品进行纯化与浓缩处理。分选得到的各细胞亚群的RNA产量通常为0.5~1 µg。使用SuperScript III 第一链合成试剂盒（适用于RT-PCR，货号18080-051，Invitrogen公司）将所有分选群体的RNA样品反转录为cDNA，随后与Affymetrix小鼠基因组430 2.0芯片进行杂交。使用R语言中的稳健多芯片平均（Robust Multichip Average, RMA）算法与分位数归一化方法对芯片数据进行标准化处理。使用AutoSOME软件（参考文献33）进行基因表达聚类分析，聚类参数设置如下：p值阈值为0.05，100次集合迭代，芯片数据的单位方差归一化，基因的中位数中心化，以及基因与芯片样本均采用平方和为1的归一化处理。