Cancer-associated fibroblasts induce epithelial-mesenchymal transition of bladder cancer cells through paracrine IL-6 signalling
Cancer-associated fibroblasts induce epithelial-mesenchymal transition of bladder cancer cells through paracrine IL-6 signalling
复制标题
DOI：
10.1186/s12885-019-5353-6
复制
发表时间：
2019-02-11
期刊：
影响因子：
3.8
通讯作者：
Bolduc, Stephane
中科院分区：
文献类型：
--
作者：
Goulet, Cassandra Ringuette;Champagne, Audrey;Bolduc, Stephane
癌症相关成纤维细胞（cancer -associated fibroblasts, CAFs）被肿瘤细胞激活，是肿瘤组织中主要的基质细胞类型，在与肿瘤细胞相互作用促进肿瘤进展中发挥重要作用。上皮间质转化（Epithelial-mesenchymal transition， EMT）是转移细胞的一个重要特征。然而，CAFs在膀胱癌细胞中诱导EMT程序的机制尚不清楚。方法利用膀胱癌源性外泌体将健康原代膀胱成纤维细胞（HFs）诱导为CAFs (iCAFs)，研究CAFs在膀胱癌进展中的作用。采用qPCR和Western blot检测iCAF条件培养基（CM (iCAF)）对非侵袭性RT4膀胱癌细胞株EMT标志物表达的影响。ELISA法和Western blot法检测iCAFs中IL6的表达。分别采用cyQUANT法、划痕法和transwell室法检测CM (iCAF) +/-抗il - 6中和抗体对RT4细胞增殖、迁移和侵袭的影响。我们通过查询来自TCGA和GEO基因组数据平台的人类膀胱癌标本的基因表达数据集，研究了IL6表达与膀胱癌进展的相关性。结果经癌外泌体处理的HFs具有高表达SMA和FAP的cas特征。我们发现，在非侵袭性RT4细胞中，CM （iCAF）诱导间充质标记物，如N-cadherin和vimentin的上调，同时抑制上皮标记物E-cadherin和p-ss-catenin的表达。此外，与对照组相比，EMT转录因子SNAIL1、TWIST1和ZEB1在CM （iCAF）培养的RT4细胞中上调。我们还发现，细胞因子IL-6在cas中高表达，其受体IL-6R在RT4膀胱癌细胞中被发现。CM （iCAF）培养RT4膀胱癌细胞可显著促进细胞生长、迁移和侵袭。重要的是，通过中和抗体抑制cafs分泌的IL-6可显著逆转IL-6诱导的EMT表型，这表明该细胞因子在人类膀胱癌进展中是cafs诱导的EMT所必需的。最后，我们观察到IL6在侵袭性膀胱癌中的表达上调，并与CAF标志物ACTA2相关。我们得出结论，CAFs通过分泌IL-6诱导的EMT促进非侵袭性膀胱癌细胞的侵袭性表型。
BackgroundCancer-associated fibroblasts (CAFs), activated by tumour cells, are the predominant type of stromal cells in cancer tissue and play an important role in interacting with neoplastic cells to promote cancer progression. Epithelial-mesenchymal transition (EMT) is a key feature of metastatic cells. However, the mechanism by which CAFs induce EMT program in bladder cancer cells remains unclear.MethodsTo investigate the role of CAFs in bladder cancer progression, healthy primary bladder fibroblasts (HFs) were induced into CAFs (iCAFs) by bladder cancer-derived exosomes. Effect of conditioned medium from iCAFs (CM (iCAF)) on EMT markers expression of non-invasive RT4 bladder cancer cell line was determined by qPCR and Western blot. IL6 expression in iCAFs was evaluated by ELISA and Western blot. RT4 cell proliferation, migration and invasion were assessed in CM (iCAF) +/- anti-IL6 neutralizing antibody using cyQUANT assay, scratch test and transwell chamber respectively. We investigated IL6 expression relevance for bladder cancer progression by querying gene expression datasets of human bladder cancer specimens from TCGA and GEO genomic data platforms.ResultsCancer exosome-treated HFs showed CAFs characteristics with high expression levels of SMA and FAP. We showed that the CM (iCAF) induces the upregulation of mesenchymal markers, such as N-cadherin and vimentin, while repressing epithelial markers E-cadherin and p-ss-catenin expression in non-invasive RT4 cells. Moreover, EMT transcription factors SNAIL1, TWIST1 and ZEB1 were upregulated in CM (iCAF)-cultured RT4 cells compared to control. We also showed that the IL-6 cytokine was highly expressed by CAFs, and its receptor IL-6R was found on RT4 bladder cancer cells. The culture of RT4 bladder cancer cells with CM (iCAF) resulted in markedly promoted cell growth, migration and invasion. Importantly, inhibition of CAFs-secreted IL-6 by neutralizing antibody significantly reversed the IL-6-induced EMT phenotype, suggesting that this cytokine is necessary for CAF-induced EMT in the progression of human bladder cancer. Finally, we observed that IL6 expression is up-regulated in aggressive bladder cancer and correlate with CAF marker ACTA2.ConclusionsWe conclude that CAFs promote aggressive phenotypes of non-invasive bladder cancer cells through an EMT induced by the secretion of IL-6.