Division of Gastroenterology, Department of Internal Medicine, Oklahoma City, OK, USA.
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA.
Oncogene. 2014 May 15;33(20):2639-54. doi: 10.1038/onc.2013.210. Epub 2013 Jun 10.
DCLK1 and Lgr5 have recently been identified as markers of quiescent and cycling stem cells in the small intestinal crypts, respectively. Epithelial-mesenchymal transition (EMT) is a key development program that is often activated during cancer invasion and metastasis, and also imparts a self-renewal capability to disseminating cancer cells. Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we observed a relative decrease in DCLK1 expression in the colonic crypts, with significant shift towards stromal staining at peak (12 days post infection) hyperplasia, whereas staining for Lgr5 and Msi-1 increased several fold. When hyperplasia was regressing (days 20-34), an expansion of DCLK1+ve cells in the CR-infected crypts compared with that seen in uninfected control was recorded. Purified colonic crypt cells exhibiting epigenetic modulation of the transforming growth factor-β (TGFβ), Wnt and Notch pathways on 12 or 34 days post infection formed monolayers in vitro, and underwent trans-differentiation into fibroblast-like cells that stained positive for vimentin, fibronectin and DCLK1. These cells when trypsinized and regrown in soft agar, formed colonospheres/organoids that developed into crypt-like structures (colonoids) in Matrigel and stained positive for DCLK1. Mice exhibiting 12 or 34 days of TMCH were given azoxymethane once for 8 h (Gp1) or weekly for 3 weeks (Gp2), and subjected to crypt isolation. Crypt cells from Gp1 animals formed monolayers as well as colonospheres in soft agar and nodules/tumors in nude mice. Crypt cells isolated from Gp2 animals failed to form the monolayers, but developed into colonospheres in soft agar and nodules/tumors in nude mice. Thus, both hyperplasia and increased presence of DCLK1+ve cells promote cellular transformation in response to a second hit. The TMCH model, therefore, provides an excellent template to study how alterations in intestinal stem cells promote trans-differentiation, crypt regeneration or colon carcinogenesis following bacterial infection.
DCLK1 和 Lgr5 最近分别被鉴定为小肠隐窝中静止和循环干细胞的标志物。上皮-间充质转化 (EMT) 是一种关键的发育程序，在癌症侵袭和转移过程中经常被激活，并且赋予扩散癌细胞的自我更新能力。利用柠檬酸杆菌 (CR) 诱导的可传播的小鼠结肠增生 (TMCH) 模型，我们观察到结肠隐窝中 DCLK1 表达相对减少，在增生高峰期（感染后 12 天）明显向基质染色转移，而 Lgr5 和 Msi-1 的染色增加了几倍。当增生消退（第 20-34 天）时，与未感染对照相比，CR 感染隐窝中 DCLK1+ve 细胞的扩张被记录下来。在感染后 12 或 34 天，经纯化的结肠隐窝细胞表现出转化生长因子-β (TGFβ)、Wnt 和 Notch 途径的表观遗传调节，在体外形成单层，并转分化为成纤维细胞样细胞，这些细胞对波形蛋白、纤连蛋白和 DCLK1 染色呈阳性。这些细胞在用胰蛋白酶处理并在软琼脂中重新生长时，形成结肠球体/类器官，在 Matrigel 中发育成类似隐窝的结构（类器官），并对 DCLK1 染色呈阳性。在 TMCH 中表现出 12 或 34 天的小鼠，单次给予 8 小时的氧化偶氮甲烷（Gp1）或每周给予 3 周（Gp2），并进行隐窝分离。来自 Gp1 动物的隐窝细胞在软琼脂中形成单层和结肠球体以及裸鼠中的结节/肿瘤。来自 Gp2 动物的隐窝细胞无法形成单层，但在软琼脂中发育成结肠球体，并在裸鼠中形成结节/肿瘤。因此，增生和 DCLK1+ve 细胞的增加都促进了对第二次打击的细胞转化。因此，TMCH 模型为研究肠道干细胞的改变如何促进细菌感染后细胞转分化、隐窝再生或结肠癌发生提供了一个极好的模板。
