Abstract Nucleolin (NCL) is a highly conserved nucleocytoplasmic multifunctional protein predominantly localized at nucleolus. It shuttles to the cell surface in tumor, and cell-surface NCL has become a target for anti-cancer therapy. We have recently developed a several multivalent pseudopeptides family NUCANT for NCL and Nucleophosmin antagonist. Among these, N6L peptide, which strongly inhibits human tumor growth by inducing apoptosis of tumor cells (1), is currently in phase II clinical trial for cancer. Since NCL shuttles to the cell surface of endothelial cells under VEGF stimulation, and NUCANT inhibited developmental angiogenesis, we sought to investigate the effect of NUCANT on tumor vasculature and endothelial cell (EC) homeostasis. In human ECs, we showed that cell-surface NCL is glycosilated, N6L specifically induced the endocytosis of glycosilated NCL and its accumulation in the nucleus. NCL internalization by N6L blocked endothelial cell cycle progression and inhibits endothelial cell adhesion and migration. It is known that NCL belongs to intracrine pathways which form intracellular positive feed-back loops from the cell surface to the nucleus thereby maintaining a specific cellular state. Our findings suggest that N6L interferes with glycosilated NCL intracrine pathway. NCL is a marker of tumor vessels we investigated the effects of NUCANT on tumor angiogenesis in a mouse model of pancreatic ductal adenocarcinoma (PDAC) (2) and a model of pancreatic neuroendocrine tumors (RipTag2). We tested NUCANT in soluble and the clinically used poliplex form. As expected, NUCANT inhibited primary tumor growth and vessel area. Notably, NUCANT induced both vessel pruning and normalization of tumor vasculature. In addition, tumor vessel normalization correlated with stronger inhibition of tumor growth and metastasis achieved by the poliplex form. We conclude that NCL targeting acts both on tumor and endothelial cells and suggest that poliplex NUCANT blocks metastasis formation and normalizes tumor vasculature by interfering with NCL-mediated oncogenic and pro-angiogenic cellular states. 1. Destouches D, et al. (2011) A simple approach to cancer therapy afforded by multivalent pseudopeptides that target cell-surface nucleoproteins. Cancer Res 71(9):3296-3305. 2. Olson P, Chu GC, Perry SR, Nolan-Stevaux O, & Hanahan D (Imaging guided trials of the angiogenesis inhibitor sunitinib in mouse models predict efficacy in pancreatic neuroendocrine but not ductal carcinoma. Proc Natl Acad Sci U S A 108(49):E1275-1284. Citation Format: Maud-Emmanuelle Gilles, Damien Destouches, Gilles Carpentier, Enrico Giraudo, Federica Maione, José Courty, Ilaria Cascone. Nucleolin-targeting NUCANT normalizes tumor vasculature and inhibits tumor growth and metastasis formation in mouse models of cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 15. doi:10.1158/1538-7445.AM2014-15