Abstract
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 αSMA 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.
Highlights
Cancer-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
Healthy bladder primary fibroblasts treated with bladder cancer-derived exosomes exhibit characteristics of CAFs To induce the activation of healthy bladder primary fibroblasts (HFs) into CAFs, we cultured Healthy fibroblast (HF) with 1 mg/mL of bladder cancer-derived exosomes for 48 h [20]
HFs + TGFß was used as positive control for activated fibroblasts [8]. Induced cancer-associated fibroblast (iCAF) were positive for αSMA staining and exhibited a spindle-shape morphology, similar to CAFs (Fig. 1a)
Summary
Cancer-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. Previous studies suggest that CAFs play a pivotal role in establishing a metastatic niche and promoting tumor cell proliferation, invasion and metastasis by secretion of chemokines and cytokines in the microenvironment [9, 11, 12]. It is still unclear by which mechanisms CAFs affect the metastatic potential of bladder cancer cells
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