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. 2016 Apr 12;7(15):20440-54.
doi: 10.18632/oncotarget.7812.

Glutamate dependent NMDA receptor 2D is a novel angiogenic tumour endothelial marker in colorectal cancer

Henry J M Ferguson  1   2 , Joseph W Wragg  1 , Stephen Ward  2 , Victoria L Heath  1 , Tariq Ismail  2 , Roy Bicknell  1
Affiliations

Glutamate dependent NMDA receptor 2D is a novel angiogenic tumour endothelial marker in colorectal cancer

Henry J M Ferguson et al. Oncotarget. .

Abstract

Current vascular-targeted therapies in colorectal cancer (CRC) have shown limited benefit. The lack of novel, specific treatment in CRC has been hampered by a dearth of specific endothelial markers. Microarray comparison of endothelial gene expression in patient-matched CRC and normal colon identified a panel of putative colorectal tumour endothelial markers. Of these the glutamate dependent NMDA receptor GRIN2D emerged as the most interesting target. GRIN2D expression was shown to be specific to colorectal cancer vessels by RTqPCR and IHC analysis. Its expression was additionally shown be predictive of improved survival in CRC. Targeted knockdown studies in vitro demonstrated a role for GRIN2D in endothelial function and angiogenesis. This effect was also shown in vivo as vaccination against the extracellular region of GRIN2D resulted in reduced vascularisation in the subcutaneous sponge angiogenesis assay. The utility of immunologically targeting GRIN2D in CRC was demonstrated by the vaccination approach inhibiting murine CRC tumour growth and vascularisation. GRIN2D represents a promising target for the future treatment of CRC.

Keywords: GRIN2D; active immunotherapy; colorectal cancer; tumour endothelial marker; vaccination.

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Conflict of interest statement

CONFLICTS OF INTEREST

The Authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

Figures

Figure 1
Figure 1. GRIN2D is a specific marker of tumour vasculature in colorectal cancer
A. quantitative real-time analysis of relative candidate target gene levels in endothelium isolated from malignant and healthy tissue. Gene expression levels were normalised to flotillin-2. Average gene expression ± SEM (n=8, Mann-Whitney U, ***p<0.0001, **p<0.001). B. representative images of PECAM-1 and GRIN2D staining in healthy colon and colorectal cancer (scale bar = 50 μm). C. multi-organ tissue array analysis of GRIN2D expression, showing the frequency of positive GRIN2D vascular staining. D. representative images of positive and negative GRIN2D staining in CRC by IHC (scale bar = 50 μm) and Kaplan-Meier analysis of survival in CRC with (blue) or without (green) GRIN2D vascular expression. Statistical analysis = Log-ranks test, P and N-numbers shown.
Figure 2
Figure 2. Loss of GRIN2D impairs endothelial function in in vitro angiogenesis assays
GRIN2D was knocked down by transfection of two siRNA duplexes into 3 separate HUVEC isolates. A–C, the cells were plated on matrigel and endothelial tube formation and integrity observed over 16 hours. A, representative images of tube formation in each condition. B, the average number of nodes per field of view ± SEM. C, the average number of sprouts per node ± SEM (n=6 per condition [3] and isolate [3]). D–E. HUVEC were plated and allowed to grow to confluence. The monolayer was scratched and wound closure observed at nine scratch intersections over time. D, representative images of wound closure from initial scratch to end-stage. E, quantification of percentage wound closure over the time course of the experiment ± SEM (n=9 per condition [3] and isolate [3]). F–G. HUVEC were subjected to the transfilter (modified Boyden chamber) assay, the set up of which is shown in F. G, quantification of the average percentage of endothelial cells that have migrated through the filter, after 16 hours, per field of view ± SEM (n=6 per condition [3] and isolate [3]). Statistical analysis for all, Mann-Whitney U, ***p<0.0001.
Figure 3
Figure 3. The physiological effects of GRIN2D-Fc vaccination
A-B. quantitation of immune response to vaccination with GRIN2D-Fc fusion protein by ELISA, showing A, overall response and B, IgG specific response. Vaccinated mice had sponges introduced into their flank and angiogenesis was stimulated into the sponge with FGF infusions. C. representative images of macroscopic vascular invasion into the sponge in the treated and untreated groups. A mask was generated in image J [54] for each sponge and the percentage sponge invasion quantified, D, E. representative H&E images of subcutaneous sponge morphology. F. quantitation from the H&E images of sponge vessel density. Statistical analysis for all, Mann-Whitney U, ***P<0.0001, **P<0.001. 6 sponges in each group, 5 microscopic fields per sponge.
Figure 4
Figure 4. GRIN2D-Fc vaccination decreases subcutaneous CT26 tumour growth and vascularity
CT26 tumour cells were inoculated into vaccinated and non-vaccinated Balb/c mice. A. tumour growth was monitored by caliper measurement. Tumours were excised at day 21, imaged, B. and weighed, C, D. representative H&E images of subcutaneous tumour morphology. E. quantitation from the H&E images of tumour vessel density. Statistical analysis for all, Mann-Whitney U, ***P<0.0001, **P<0.001.5 mice per group; 5 microscopic fields per tumour.
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