Mo Allen, Jackson Stephen, Varma Kamini, Carpino Alan, Giardina Charles, Devers Thomas J, Rosenberg Daniel W
Center for Molecular Medicine, School of Medicine, UConn Health, Farmington, Connecticut. Colon Cancer Prevention Program, Neag Comprehensive Cancer Center, School of Medicine, UConn Health, Farmington, Connecticut.
Thermo Fisher Scientific, South San Francisco, California.
Mol Cancer Res. 2016 Sep;14(9):795-804. doi: 10.1158/1541-7786.MCR-16-0156. Epub 2016 Jun 27.
Although the progression of mutated colonic cells is dependent upon interactions between the initiated epithelium and surrounding stroma, the nature of these interactions is poorly understood. Here, the development of an ultrasensitive laser capture microdissection (LCM)/RNA-seq approach for studying the epithelial and stromal compartments of aberrant crypt foci (ACF) is described. ACF are the earliest identifiable preneoplastic lesion found within the human colon and are detected using high-definition endoscopy with contrast dye spray. The current analysis focused on the epithelium of ACF with somatic mutations to either KRAS, BRAF, or APC, and expression patterns compared with normal mucosa from each patient. By comparing gene expression patterns among groups, an increase in a number of proinflammatory NF-κB target genes was identified that was specific to ACF epithelium, including TIMP1, RELA, and RELB Distinct transcriptional changes associated with each somatic mutation were observed and a subset of ACF display BRAF(V600E)-mediated senescence-associated transcriptome characterized by increased expression of CDKN2A Finally, LCM-captured ACF-associated stroma was found to be transcriptionally distinct from normal-appearing stroma, with an upregulation of genes related to immune cell infiltration and fibroblast activation. Immunofluorescence confirmed increased CD3(+) T cells within the stromal microenvironment of ACF and an abundance of activated fibroblasts. Collectively, these results provide new insight into the cellular interplay that occurs at the earliest stages of colonic neoplasia, highlighting the important role of NF-κB, activated stromal fibroblasts, and lymphocyte infiltration.
Fibroblasts and immune cells in the stromal microenvironment play an important role during the earliest stages of colon carcinogenesis. Mol Cancer Res; 14(9); 795-804. ©2016 AACR.
尽管突变结肠细胞的进展依赖于起始上皮细胞与周围基质之间的相互作用，但这些相互作用的本质仍知之甚少。在此，描述了一种用于研究异常隐窝灶（ACF）上皮和基质成分的超灵敏激光捕获显微切割（LCM）/RNA测序方法的开发。ACF是在人类结肠中发现的最早可识别的癌前病变，通过使用高清内镜和对比染料喷雾进行检测。当前分析聚焦于具有KRAS、BRAF或APC体细胞突变的ACF上皮，并将其表达模式与每位患者的正常黏膜进行比较。通过比较各组之间的基因表达模式，发现了一些促炎NF-κB靶基因的增加，这些基因是ACF上皮特有的，包括TIMP1、RELA和RELB。观察到与每个体细胞突变相关的不同转录变化，并且一部分ACF表现出BRAF（V600E）介导的衰老相关转录组，其特征是CDKN2A表达增加。最后，发现LCM捕获的ACF相关基质在转录上与外观正常的基质不同，与免疫细胞浸润和成纤维细胞活化相关的基因上调。免疫荧光证实ACF基质微环境中CD3（+）T细胞增加以及活化成纤维细胞丰富。总体而言，这些结果为结肠肿瘤发生最早阶段发生的细胞相互作用提供了新见解，突出了NF-κB、活化的基质成纤维细胞和淋巴细胞浸润的重要作用。
基质微环境中的成纤维细胞和免疫细胞在结肠癌发生的最早阶段起重要作用。《分子癌症研究》；14（9）；795 - 804。©2016美国癌症研究协会。
