Cancer-associated fibroblasts predict poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma.

Timothy J Underwood,Annette L Hayden,Abbie Mead,Gareth J Thomas,Michael J White,Nicholas Clemons,Fergus Noble,Jeremy P Blaydes,Massimiliano Mellone,Edwin Garcia,John N Primrose,Chudy Uzoho,Mathieu Derouet,Steve Thirdborough

doi:10.1002/path.4467

Abstract

Interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an important role in tumour development and progression. In this study we investigated the functional role of CAFs in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of 183 EAC patients for CAF markers related to disease mortality. We characterized CAFs and normal oesophageal fibroblasts (NOFs) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3D organotypic culture and xenograft models were used to examine the effects on EAC cell function and to dissect molecular mechanisms regulating invasion. Most EACs (93%) contained CAFs with a myofibroblastic (α-SMA-positive) phenotype, which correlated significantly with poor survival [p = 0.016; HR 7. 1 (1.7–29.4)]. Primary CAFs isolated from EACs have a contractile, myofibroblastic phenotype and promote EAC cell invasion in vitro (Transwell assays, p ≤ 0.05; organotypic culture, p < 0.001) and in vivo (p ≤ 0.05). In vitro, this pro-invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAFs and acts as a ligand for EAC cell integrins αvβ3 and αvβ5, promoting activation of the PI3kinase–Akt pathway. In patient samples, periostin expression at the tumour cell–stromal interface correlates with poor overall and disease-free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF-secreted periostin and EAC-expressed integrins results in PI3 kinase–Akt activation and increased tumour cell invasion. Most EACs contain a myofibroblastic CAF-rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patients.

Highlights

Oesophageal cancer is associated with extremely poor overall survival; 60–70% of patients present with late-stage disease, too advanced for treatment with curative intent, and in those patients who are suitable for multimodal therapy, 5 year survival is only 35% [1]
Having established the importance of periostin to cancer-associated fibroblast (CAF)-induced oesophageal adenocarcinoma (EAC) cell invasion in vitro, we examined our EAC patient cohort to confirm the presence of stromal periostin by IHC
We found that the vast majority of patients with EAC have tumours with high or moderate levels of stromal α-smooth muscle actin (α-SMA); this predicts poor survival and may account, in part, for the aggressive nature of the disease

Summary

Introduction

Oesophageal cancer is associated with extremely poor overall survival; 60–70% of patients present with late-stage disease, too advanced for treatment with curative intent, and in those patients who are suitable for multimodal therapy, 5 year survival is only 35% [1]. This is partly due to the mechanically compliant oesophageal anatomy, which allows symptomless tumour expansion [2]. Depth of invasion and nodal metastasis are both prognostic, and improving survival rates will require an increased understanding of the molecular mechanisms regulating local and regional tumour spread. Stromal features are prognostic in many tumour types [7,8,9,10,11], and expression of stromal genes has been shown to be associated with poor outcome in oesophageal cancer [12]

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Results

Conclusion