Correlation of cancer-associated fibroblasts with tumour cell invasion and chemoresistance in oesophageal adenocarcinoma

Abstract

BackgroundThe incidence of oesophageal adenocarcinoma is rising rapidly; treatment options are associated with high morbidity and poor prognosis. The initiating events are believed to be related to chronic reflux in the lower oesophagus leading to mucosal and stromal inflammation. The transforming growth factor β (TGFβ) superfamily mediates the inflammatory response in a range of tissues, and TGFβ signalling is seen in the stroma of gastrointestinal cancers. Furthermore, TGFβ is the dominant driver in the activation of resident stromal fibroblasts into the secretory and contractile myofibroblast phenotype, characterised by expression of α smooth muscle actin (αSMA) and observed in most cancer-associated fibroblasts (CAF). We hypothesised that CAF are present in oesophageal adenocarcinoma and that they have a direct effect on tumour biology leading to increased invasion and chemoresistance. MethodsPrimary normal oesophageal fibroblasts (NOF), and CAF were cultured directly from oesophageal resection specimens, and their phenotype confirmed by western blotting and immunofluorescence for αSMA. We assessed functional ability of NOF and CAF to promote oesophageal adenocarcinoma cell invasion and chemoresistance using organotypic culture, transwell invasion assays, collagen-1 gel contraction assays, siRNA gene silencing, and colony forming assays. The multifunctional extracellular matrix protein, periostin, which has tumour promoting effects in other cancers, was examined as a potential CAF-secreted molecular target in oesophageal adenocarcinoma. t tests were done for all statistical analyses. Survival data were analysed by Kaplan-Meier plots (SPSS, version 19). FindingsCAF showed high αSMA expression and increased collagen contractility compared with NOF, suggesting a myofibroblast like phenotype (p<0·01). Compared with NOF, oesophageal adenocarcinoma cell invasion doubled in response to CAF-conditioned medium in transwell invasion assays and in organotypic models containing co-cultured fibroblasts and oesophageal adenocarcinoma cells (OE33 and FLO-1 cell lines, p<0·05). Reduction of CAF-secreted periostin by siRNA and immunodepletion resulted in a significant (p=0·02) reduction in cancer cell invasion in transwell invasion assays that could be rescued with recombinant periostin, and a complete loss of invasion in organotypic culture. Moreover, collagen gel contraction was abolished by periostin downregulation in CAF. NOF exposed to TGFβ for 72 h demonstrated myofibroblast transdifferentiation including high αSMA expression, secretion of periostin, and the ability to significantly promote oesophageal adenocarcinoma cell invasion to a similar degree as CAF. There was a significant correlation between periostin expression assessed in 183 oesophageal resections and disease-specific survival (high periostin=worse survival, p<0·05). CAF-conditioned medium supported more cancer cell colony formation in the presence of cisplatin and fluorouracil than did NOF-conditioned medium (p<0·05). When CAF-conditioned medium was depleted of periostin, this protective effect was lost, but could be rescued with recombinant periostin. InterpretationThis study highlights the role of the tumour microenvironment in cancer development and progression in the oesophagus. CAF are present in the oesophagus and promote oesophageal adenocarcinoma cell invasion and chemoresistance via periostin secretion in vitro. The CAF phenotype can be induced by exposing oesophageal NOF to TGFβ in vitro and this is likely to represent the molecular driving force for fibroblast transdifferentiation in vivo. Manipulation of CAF transdifferentiation and interruption of CAF-secreted periostin signalling in cancer cells might offer novel therapeutic targets in oesophageal adenocarcinoma. FundingNational Institute of Health Research, UK Medical Research Council.