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. 2019 Feb 11;19(1):137.
doi: 10.1186/s12885-019-5353-6.

Cancer-associated fibroblasts induce epithelial-mesenchymal transition of bladder cancer cells through paracrine IL-6 signalling

Cassandra Ringuette Goulet  1   2   3 , Audrey Champagne  2   3 , Geneviève Bernard  1   2 , Dominique Vandal  1 , Stéphane Chabaud  1   2 , Frédéric Pouliot  2   3 , Stéphane Bolduc  4   5   6
Affiliations

Cancer-associated fibroblasts induce epithelial-mesenchymal transition of bladder cancer cells through paracrine IL-6 signalling

Cassandra Ringuette Goulet et al. BMC Cancer. .

Abstract

Background: 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. 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.

Methods: To 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.

Results: Cancer 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.

Conclusions: We conclude that CAFs promote aggressive phenotypes of non-invasive bladder cancer cells through an EMT induced by the secretion of IL-6.

Keywords: Bladder cancer; CAFs; EMT; IL-6.

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Conflict of interest statement

Ethics approval and consent to participate

Bladder biopsies from paediatric patients undergoing non-oncologic urologic surgery were obtained at the CHU de Québec-Université Laval Research Center in accordance with the institutional review board. All legal guardians of donors provided their formal, informed and written consent, each agreeing to supply a biopsy for this study.

Consent for publication

Not applicable.

Competing interests

Frédéric Pouliot has received speaker’s bureau honoraria from Sanofi, Genzyme, Amgen, Astellas, and Janssen; he is a consultant/advisory board member of Sanofi, Abbvie, Astellas, Janssen, Genzyme and Roche. No potential conflicts of interest were disclosed by the other authors.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Healthy vesical primary fibroblasts (HFs) treated with bladder cancer-derived exosomes exhibit characteristics of cancer-associated fibroblasts (CAFs). HFs were treated with 1 mg/mL of bladder cancer cells-derived exosomes for 48 h to induce their activation in CAFs (iCAFs). HFs treated with TGFβ1 (4 ng/mL) served as a positive control. a. Cells were examined by immunocytochemistry for the expression of αSMA (α-smooth muscle actin, green). Nuclei were stained with DAPI (blue). Scale bar = 100 μm. b. The protein expression of αSMA, fibroblast-activating protein (FAP) was determined by western blot. The tubulin was used as loading control. The graph shows mean +/− SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ****P < 0.0001, n = 3
Fig. 2
Fig. 2
The conditioned medium (CM) from CAFs induces epithelial-mesenchymal transition (EMT) programming in RT4 bladder cancer cells. a. Experimental design. b. The expression of epithelial markers E-cadherin, phospho-ß-catenin (control: total ß-catenin) and phospho-GSK3ß (control: GSK3ß total) and of mesenchymal markers N-cadherin and vimentin was analyzed by Western blotting. The ß-actin was used as loading control. c. The expression of EMT-related transcription factors (EMT-TFs) was determined by qPCR or d. Western blotting. The graphs show mean +/− SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. = non-significant, n = 3–5
Fig. 3
Fig. 3
iCAFs express IL-6 and induce the activation of the STAT3 signaling pathway in RT4 bladder cancer cells. a. Interleukin-6 (IL-6) mRNA expression levels in HFs, HFs + TGFß and iCAFs cells were determined by qPCR. b. The expression of the IL-6 protein was measured in supernatants from HFs, HFs + TGFß and iCAFs cells using enzyme-linked immunosorbent assay (ELISA). c. The presence of the IL-6 receptor (IL-6R) in iCAFs and RT4 cells was detected by Western blotting. d. The activation of the signal transducer and activator of transcription 3 (STAT3) and AKT signaling pathway in RT4 cells cultured in conditionned medium (CM) was evaluated by Western blotting. The graphs show mean +/− SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ***P = 0.001, n.s. = non-significant, n = 3–5
Fig. 4
Fig. 4
CAFs-derived IL-6 enhances survival and promotes migration and invasion in RT4 bladder cancer cells. RT4 cells were cultured in the CM from HFs, HFs + TGFß or iCAFs with or without the anti-IL-6 antibody (1 μg/mL; x-IL-6). a. The proliferation of the RT4 cells was evaluated using the CyQUANT Kit. b. The migration of RT4 cells was determined by using Ibidi-silicone insert. Insert margins were marked by white vertical lines on optical micrographs. After 6 h and 12 h, the percentage of wound closure was evaluated by measuring migration distances (spaces between the white vertical lines). c. The invasion of RT4 cells was measured by transwell assays. Cells that had migrated through the membrane were fixed and nuclei were stained with DAPI. Representative photographs of migratory cells on the membrane are shown. The relative cell migration was determined by the number of migrated cells in 10 randomly selected fields. d. The sensitivity of RT4 cells to mitomycin C (0.5 μg/mL; Mito C) was evaluated using The CyQUANT Kit. RFU means relative fluorescence unit. All values are averages of three independent replicates. The graphs show mean +/− SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. = non-significant, n = 5–25
Fig. 5
Fig. 5
IL6 mRNA expression is up-regulated in aggressive bladder cancer patient tumor specimens, correlates with CAF marker ACTA2 expression, and is associated with poor prognosis. Boxplots of IL6 (a-c) and ATCA2 (d-f) mRNA expression with respect to tumor stage, grade or invasiveness for TCGA (a-b, d-e, g) and GSE13507 (c, f, h) bladder cancer data. Number of tissues in each group is shown in brackets. The difference between groups was analyzed by Mann-Whitney test. ****P < 0.0001. g. Spearman’s correlation between ACTA2 and IL6 gene expression (r = 0.4543; ****P < 0.0001). h. Kaplan-Meier cancer specific survival curves according to lower versus higher half of ACTA2/IL6 mRNA co-expression level in bladder cancer patients (HR = 2.738; *P = 0.0471). Non-muscle invasive bladder cancer (NMIBC); muscle invasive bladder cancer (MIBC)
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