Balance of pro- versus anti-angiogenic splice isoforms of vascular endothelial growth factor as a regulator of neuroblastoma growth

Neuroblastoma (NB) is the second most common extracranial tumour of childhood. Angiogenesis plays a crucial role in the growth and development of NB and vascular endothelial growth factor (VEGF), one of the most potent stimuli of angiogenesis, has been studied extensively in vitro. VEGF₁₆₅ has been shown to be the predominant angiogenic isoform expressed in NB cell lines and tumours. In this study, we investigated the anti-angiogenic isoform of VEGF-A, generated from distal splice site selection in the terminal exon of VEGF (VEGF₁₆₅b) and shown to be down-regulated in epithelial malignancies. The expression of both the pro- (VEGFxxx) and the anti-angiogenic (VEGFxxxb) isoforms was compared in a range of NB and ganglioneuroma (GN) tumours. Whereas VEGFxxxb and VEGFxxx were both expressed in GN, specific up-regulation of the VEGFxxx isoforms was seen in NB at RNA and protein levels. Highly tumourigenic NB cell lines also showed up-regulation of the angiogenic isoforms relative to VEGFxxxb compared to less tumourigenic cell lines, and the isoforms were differentially secreted. These results indicate that VEGF₁₆₅ is up-regulated in NB and that there is a difference in the balance of isoform expression from anti-angiogenic VEGF₁₆₅b to angiogenic VEGF₁₆₅. Treatment with recombinant human VEGF₁₆₅b significantly reduced the growth rate of established xenografts of SK-N-BE(2)-C cells (4.24 ± 1.01 fold increase in volume) compared with those treated with saline (9.76 ± 3.58, p < 0.01). Microvascular density (MVD) was significantly decreased in rhVEGF₁₆₅b-treated tumours (19.4 ± 1.9 vessels/mm³) in contrast to the saline-treated tumours (45.5 ± 8.6 vessels/mm³). VEGF₁₆₅b had no significant effect on the proliferative or apoptotic activity, viability or cytotoxicity of SK-N-BE(2)-C cells after 48 h. In conclusion, VEGF₁₆₅b is an effective inhibitor of NB growth. These findings provide the rationale for further investigation of VEGF₁₆₅b in NB and other paediatric malignancies. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.