While VEGF blockade has been validated as a therapy in human cancers, not all tumor types respond, suggesting that alternative pathways may contribute to tumor neoangiogenesis. Recent data support a critical role for the Notch pathway in some cancer models. Using the well-characterized NGP xenograft model of human neuroblastoma, we have previously shown that this system is relatively resistant to VEGF blockade. Further, inhibition of VEGF signaling in NGP tumors causes overexpression of Notch components in the vasculature, leading to an increase in notch signaling, as indicated by an increase in vascular HEY-2 staining. We hypothesized that Notch contributes to tumor neoangiogenesis in NGP tumors. METHODS: In order to study the role of Notch in our model, we used a novel construct based on the extracellular domain of Notch1 (N1ECDFc) and expressed it in the human NGP cell line. N1ECDFc was secreted from the cells, and blocked Notch signaling. 106 NGP-N1ECFc and NGP-LacZ cells were implanted intrarenally in NCR nude mice (N=13 and 11, respectively). Tumors were harvested after six weeks and collected for analysis. RESULTS: The expressed construct was detected through its human Fc tag via immunohistochemistry and Western blot in NGP-N1ECDFc tumors, but not in controls. NGP-N1ECDFc tumors did not differ significantly in weight from NGP-LacZ controls, but tumor viability as assessed by TUNEL quantitation was significantly decreased. Further, parenchymal erythrocyte content was significantly increased in NGP-N1ECDFc tumors, suggesting hemorrhage and physical disruption of vasculature. Consistent with this finding, specific staining for endothelial and vascular mural cells showed disorganization and detachment of vascular cells within an intact collagen IV sleeve. Recruitment of nascent pericytes was evidenced by a significant increase in the amount of NG2 expressed by the N1ECDFc tumors compared to controls by real time PCR. We also detected increases in VEGF and PlGF expression by real time PCR. CONCLUSIONS: We conclude that Notch signaling is critical for vascular assembly in the NGP neuroblastoma model. Notch inhibition using the N1ECDFc construct disrupts vasculature, causing hemorrhage, reducing tumor viability, and resulting in increased expression of the hypoxia-regulated growth factors VEGF and PlGF. These data lend support to the concept of Notch dependence of tumor vasculature, and suggest that this pathway may contribute to the relative VEGF-independence of some tumor systems.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA