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Effect of DLL4- and JAGGED1- Mediated NOTCH Blockade In Experimental Neuroblastoma

Saturday, October 20, 2012: 2:46 PM
Versailles Ballroom (Hilton Riverside)
Alejandro V. Garcia, MD1, Debarshi Banerjee, PhD2, Roderick M. Alfonso, BS1, Arul S. Thirumoorthi, MD1, Angela Kadenhe-Chiweshe, MD1, Darrell J. Yamashiro, MD, PhD2 and Jessica J. Kandel, MD1, (1)Surgery, Columbia University Medical Center, New York, NY, (2)Pediatrics, Columbia University Medical Center, New York, NY

Purpose:  Notch signaling critically regulates the vascular endothelial growth factor (VEGF) pathway, and  Notch inhibition has consequently been proposed as an anti-angiogenic therapy for cancer to complement VEGF blockade.  However, in experimental neuroblastoma combined inhibition of VEGF and Notch accelerates hepatic metastasis, despite having no effect on primary tumor growth.  We tested the hypothesis that a specific Notch ligand was responsible for the increase in neuroblastoma liver metastasis.

Methods:  10[6] NGP-luc neuroblastoma cells engineered to express a soluble Notch1-decoy construct (N1D) or Fc control were implanted intrarenally in immunodeficient mice, and spontaneous hepatic metastases evaluated, both during VEGF blockade (bevacizumab [BV] 10mg/kg intraperitoneally biweekly) and vehicle treatment (N=30 each). To study Notch ligand specificity, 10[6] NGP-luc neuroblastoma cells engineered to express a soluble Dll4-specific Notch decoy (N=22), Jagged1-specific Notch decoy (N=20), Notch1 decoy (Dll4+Jagged1) (N=22) or Fc control (N=19) were implanted intrarenally [Fig 1]. Mice were randomized to receive treatment with bevacizumab or vehicle, and liver metastasis was evaluated. Tumor growth was monitored by bioluminescence. Immunoblotting of conditioned media confirmed decoy secretion. BrdU cell proliferation and invasion through Matrigel assays were performed to measure proliferation and migration under normoxic and hypoxic conditions.  

Results:   Notch inhibition during VEGF blockade increased hepatic metastasis of experimental neuroblastoma (p=0.002) without affecting either primary tumor growth or circulating tumor cells. In-vitro ligand-specific inhibition did not affect proliferation or migration in either normoxia or hypoxia. Ligand-specific Notch inhibition led to delayed primary tumor growth in Dll4-specific Notch-inhibited mice treated with BV (p=0.059) but Jagged1-specific Notch inhibition + BV did not (p=0.658).  Increased liver metastasis was detected only in Notch1-decoy + BV treatment but not in either ligand-specific decoy + BV group (p=0.006) [Fig 2]. Circulating tumor cells did not change between groups (p=0.1415).   

Conclusion:  Our data indicates that concurrent inhibition of VEGF and Notch pathways, without selection for specific Notch ligands, leads to an increase in liver metastasis of experimental neuroblastoma. Intriguingly, selective Dll4-specific Notch and VEGF inhibition delayed primary tumor growth without promoting liver metastasis.  Our study suggests that defective Notch signaling can be permissive for liver metastasis, but that more selective targeting can restrict primary tumor growth without this deleterious effect.  Further study of Notch functions are required to better delineate the potential role of this pathway in cancer treatment.