Deng T, DU B, Xi X
Department of Immunology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2023 Jun 20;43(6):943-951. doi: 10.12122/j.issn.1673-4254.2023.06.09.
To investigate the mechanism by which conditioned medium of colorectal cancer cells promotes the formation of cancer-associated fibroblasts (CAFs).
Normal human colorectal fibroblasts (CCD-18Co cells) in logarithmic growth phase were treated with the conditioned media of colorectal cancer HCT116 cells (HCT116-CM) or Caco-2 cells (Caco-2-CM) alone or in combination with 300 nmol/L ERK inhibitor SCH772984. The expression levels of CAFs-related molecular markers were detected in the treated cells with real-time quantitative PCR (RT- qPCR) and immunofluorescence assay, and the changes in cell proliferation, colony formation and migration were assessed with RTCA, colony formation and wound healing assays; Western blotting was performed to detect the activated signaling pathways in the fibroblasts and the changes in CAFs formation after blocking of the signaling pathway.
HCT116-CM and Caco-2-CM significantly upregulated mRNA expression levels of CAFs markers (including -SMA, FAP, FN and TGF-β) in CCD-18Co cells, and strongly promoted fibroblast transformation into CAFs ( < 0.05). The two conditioned media also promoted the proliferation, colony formation and migration of CCD-18Co cells ( < 0.05) and significantly increased the levels of -SMA protein and ERK phosphorylation in the cells ( < 0.05). The ERK inhibitor SCH772984 obviously inhibited the expression of -SMA and the transformation of CCD-18Co cells into CAFs induced by the conditioned medium of colorectal cancer cells ( < 0.05).
Colorectal cancer cells may induce the formation of colorectal CAFs by activating the ERK pathway in the fibroblasts.
探讨大肠癌细胞条件培养基促进癌相关成纤维细胞（CAFs）形成的机制。
将对数生长期的正常人结肠成纤维细胞（CCD-18Co细胞）分别用大肠癌细胞HCT116条件培养基（HCT116-CM）或Caco-2细胞条件培养基（Caco-2-CM）单独处理，或与300 nmol/L ERK抑制剂SCH772984联合处理。采用实时定量PCR（RT-qPCR）和免疫荧光法检测处理后细胞中CAFs相关分子标志物的表达水平，采用RTCA、集落形成和伤口愈合实验评估细胞增殖、集落形成和迁移的变化；采用蛋白质免疫印迹法检测成纤维细胞中激活的信号通路以及信号通路阻断后CAFs形成的变化。
HCT116-CM和Caco-2-CM显著上调CCD-18Co细胞中CAFs标志物（包括α-SMA、FAP、FN和TGF-β）的mRNA表达水平，并强烈促进成纤维细胞向CAFs转化（P<0.05）。这两种条件培养基还促进了CCD-18Co细胞的增殖、集落形成和迁移（P<0.05），并显著提高了细胞中α-SMA蛋白水平和ERK磷酸化水平（P<0.05）。ERK抑制剂SCH772984明显抑制了α-SMA的表达以及大肠癌细胞条件培养基诱导的CCD-18Co细胞向CAFs的转化（P<0.05）。
大肠癌细胞可能通过激活成纤维细胞中的ERK通路诱导大肠CAFs的形成。
