Abstract A72: VAP-1 expression is significantly reduced in colorectal cancer compared to normal colon tissue despite elevated sVAP-1 levels.
Authors: Ward Stephen T. , Weston Christopher J. , Shepherd Emma L. , Hepburn Elizabeth A. , Li Ka-kit , Hejmadi Rahul K. , Ismail Tariq , Adams David H. 
Published: August 2014

Introduction: Lymphocyte infiltration in human colorectal cancer is associated with favorable outcome, both in terms of overall survival and recurrence-free survival (1). Lymphocytes are recruited to tissues after binding to receptors on endothelium. These include classical adhesion molecules belong to the immunoglobulin superfamily but also other molecules including the ectoenzyme vascular adhesion protein-1 (VAP-1) which is implicated in lymphocyte trafficking to the gut and liver.

The role of VAP-1 in lymphocyte recruitment to colorectal cancer is unknown and tissue VAP-1 expression in colorectal cancer has not been documented. The aim of this study was to determine if increased VAP-1 expression in CRC supports lymphocyte recruitment to the tumor. The infiltration of specific lymphocyte subsets in colorectal cancer has a profound effect on prognosis and VAP may provide a means of cancer-specific lymphocyte recruitment.

Methods: Normal colon and colorectal cancer tissue was obtained from patients undergoing resection for colorectal cancer having given informed consent. The study was approved by the Local Research and Ethics Committee. Paraffin-embedded sections were stained for VAP-1 and CD31 and visualised using chromogenic methods or by dual-color immunofluorescence.

VAP-1 gene expression in whole tumor and colon tissue was analyzed by real-time PCR. Tissue lysates were used to estimate VAP-1 protein expression by Western blotting. CD31 positive cells were isolated from fresh tissue by a preliminary immunomagnetic depletion of contaminating EpCAM+ epithelial cells followed by a positive immunomagnetic selection of CD31+ cells. Cultured CD31+ cells were then stained for VAP-1 and analyzed by flow cytometry.

Results: VAP-1 gene expression is reduced in colorectal cancer tissue compared to matched colon. VAP-1 protein was detected in the colonic mucosa to a far greater extent than in matched colorectal cancer for 8 matched samples. Densitometric analysis of protein bands showed that this difference was highly statistically significant.

Ten matched colon and colorectal cancer sections demonstrated a striking paucity of VAP-1 staining in cancer tissue. Positive VAP-1 staining was demonstrated in colonic tissue, specifically the muscularis muscosae and surrounding lamina proprial and submucosal vessels. Dual-colour immunofluorescence demonstrated co-localization of VAP-1 with alpha-SMA rather than CD31.

Cultured endothelial cells from colon and tumor expressed CD31 but no VAP-1 by flow cytometry.

Conclusion: sVAP levels have been shown to be elevated in the serum of colorectal cancer compared to healthy controls. We have demonstrated by gene expression analysis, protein detection and immunohistochemistry that little VAP-1 is expressed in colorectal cancer tissue or tumor endothelial cells. This suggests that downregulation of VAP-1 may be a mechanism of tumor escape from immune surveillance by reducing the recruitment of effector lymphocytes to the tumor. Previously reported elevated sVAP levels could be explained by increased turnover and cleaving of tissue VAP-1 in CRC by matrix-metalloproteinases -2 and -9 which are elevated in colorectal cancer.

Citation Format: Stephen T. Ward, Christopher J. Weston, Emma L. Shepherd, Elizabeth A. Hepburn, Ka-kit Li, Rahul K. Hejmadi, Tariq Ismail, David H. Adams. VAP-1 expression is significantly reduced in colorectal cancer compared to normal colon tissue despite elevated sVAP-1 levels. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A72.