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

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.

Keywords: CAFs; oesophageal cancer; periostin; tumour microenvironment.

© 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Figures

Figure 1

CAFs in the microenvironment are important in EAC. (A) Kaplan–Meier curve of overall survival after resection in patients with α-SMA-positive versus α-SMA-negative tumours; representative IHC images of negative/low and positive/high are in the right-hand panel. (B) Western blot for pan-cytokeratin and vimentin in FLO-1, OE33 and primary oesophageal NOFs and CAFs. (C) Immunocytochemistry for α-SMA in primary oesophageal NOFs and CAFs. (D) Photograph of a representative gel contraction assay at 72 h and gel weight with the incorporation of CAFs compared to NOFs (p < 0.01; n = 3). (E) Western blot and immunocytochemistry for α-SMA in NOFs and NOFs + TGFβ1, and a representative gel contraction assay

Figure 2

CAFs promote EAC cell invasion in vitro and tumour growth in vivo. (A) Transwell invasion assays comparing the effect of NOFs and CAFs conditioned medium on invasion of the EAC cell lines FLO-1 and OE33. (B) Organotypic models of EAC comparing the effect of NOFs and CAFs to promote invasion of FLO-1 and OE33. (C) Kaplan–Meier curves of overall survival of mice injected with OE33 alone or in combination with NOFs or CAFs. Mice were culled when the tumour volume reached 500 mm3. (D) Immunohistochemistry for Pan-CK and α-SMA in tumour xenografts containing OE33 and CAFs

Figure 3

Periostin is secreted by CAFs and promotes EAC cell invasion. (A) Unsupervised hierarchical clustering of the ECM module genes differentiated between normal oesophageal epithelium and EAC. (B) Exported network weights in BioLayout Express3D, graphically representing periostin as a central gene within the ECM module, with many connections to other ECM module genes and genes associated with the cell-cycle process. (C) Western blot of conditioned medium and immunocytochemistry, comparing the expression and secretion of periostin in NOFs and CAFs. (D) Transwell invasion assays, demonstrating the invasion of FLO-1 in response to CAF-conditioned medium ± periostin silencing, as indicated (*p < 0.05; **p < 0.001), with western blot of conditioned medium normalized to cell number confirming periostin knockdown. (E) Organotypic cultures of FLO-1 and CAFs, with and without periostin silencing. (F) Transwell invasion assays of FLO-1 towards CAF-conditioned medium, with or without periostin immunodepletion and rescue with recombinant periostin, as indicated (*p < 0.05). (G) Transwell invasion assay of FLO-1 towards conditioned medium from NOFs or NOFs + TGFβ1, with and without periostin silencing, and western blot analysis of periostin expression under the same conditions (**p < 0.001). (H) Transwell invasion assay of FLO-1 towards conditioned medium from NOFs ± recombinant periostin or CAFs, as indicated (**p < 0.001)

Figure 4

Periostin-dependent invasion of EAC cells is suppressed through inhibition of αvβ3 and αvβ5 integrins and PI3K. (A) Western blot for total and phospho-Akt in FLO-1 up to 120 min after stimulation with recombinant periostin. (B) Western blot for total and phospho-Akt in FLO-1, 30 min after periostin stimulation, in the presence or absence of blocking antibodies against integrins αvβ3 or αvβ5, as indicated. (C) Western blot for total and phospho-Akt in FLO-1, 30 min after periostin stimulation, in the presence or absence of the PI3K inhibitor LY294002. (D) Transwell invasion assays of FLO-1 towards NOFs or CAFs conditioned medium under varying conditions. Invasion towards NOF-conditioned medium is used as the internal control. Periostin was added to the NOF-conditioned medium and either LY294002 or blocking antibodies against integrins αvβ3 or αvβ5 were added to the FLO-1 cells, as indicated. (E) Organotypic models of FLO-1 + CAFs in the presence or absence of LY294002, including staining by H&E, pan-cytokeratin and Ki67. (F) Immunohistochemistry for p-Akt in representative human EAC tumours with strongest staining adjacent to CAFs. (G) Immunohistochemistry for periostin and p-Akt in tumour xenografts containing OE33 and CAFs

Figure 5

Stromal periostin expression correlates with poor survival in EAC. (A) Kaplan–Meier curves of overall survival after resection in patients with periostin-positive versus periostin-negative tumours. (B) Kaplan–Meier curves of disease-free survival after resection in patients with periostin-positive versus periostin-negative tumours. (C) Immunohistochemistry for periostin expression in human normal oesophagus (limited to the walls of blood vessels) and EAC. (D) Immunohistochemistry to compare expression patterns of periostin and α-SMA in EAC