Abstract Background: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Since the use of targeted therapies is severely limited by the lack of specific receptors, chemotherapy and radiotherapy remain the main treatment choices, which, nevertheless expose high-risk NB patients to unacceptable toxicity and to the development of drug resistance. Nucleolin (NCL), a multifunctional protein involved in both physiological and pathological conditions, is overexpressed and partially localized on the cell surface of tumor cells of adult cancers. Little is reported about NCL in pediatric tumors, including NB. Methods: To test the expression of NCL as a potential and targetable cell surface marker in NB, human NB cell lines were evaluated in vitro and after injection in mice, by Imaging Flow Cytometry (IFC) and immunohistochemistry (IHC) analyses, respectively. Cell binding and internalization of rhodamine-labeled nanocarriers, decorated with the NCL-recognizing F3 peptide (Rhod-F3-SL) were evaluated in vitro by FACS and confocal microscopy. The cytotoxic potential of a doxorubicin (DXR)-loaded, pH-sensitive nanocarrier and decorated with the F3 peptide was evaluated in vitro, in terms of inhibition of NB cells proliferation and induction of cell death by CSFE and MTS assays, respectively. Orthotopic and pseudometastatic animal models of human NB, mimicking the growth and spread of NB in patients, were finally enrolled to examine in vivo the anti-tumor activity of DXR-loaded, pH-sensitive nanocarriers decorated with F3 peptide. Results: IFC analyses demonstrated that NCL is expressed at different extent on the surface of all NB cells analyzed. Notably, IHC evaluations displayed NCL staining on both tumor and endothelial tumor cells in well-established NB xenografts. Moreover, in vitro binding studies showed that Rhod-F3-SL strongly associated to NB cells, exhibiting a specific co-localization with cell surface NCL. The cellular distribution of Rhod-F3-SL also confirms the binding specificity, showing an increased selective internalization of NCL-targeted nanoparticles, compared to that obtained with untargeted formulations. Finally, F3-targeted, DXR-loaded, pH-sensitive nanocarriers resulted significantly more effective, in terms of inhibition of cell proliferation and reduction of cell viability in vitro, and in terms of delay of tumor growth in each NB animal model tested, when compared to DXR-loaded, pH-sensitive untargeted liposomes. Conclusion: Our findings demonstrate that NCL represents a novel cell surface protein to be targeted by “drug”-loaded nanoparticles as an innovative therapeutic strategy for high-risk NB. Citation Format: Brignole Chiara, Veronica Bensa, Genny Del Zotto, Silvia Bruno, Nuno A Fonseca, Ana F Cruz, Daniela Di Paolo, Patrizia Perri, Vera Moura, Laura Emionite, Michele Cilli, Mirco Ponzoni, Joao N Moreira, Fabio Pastorino. Nucleolin: A novel cell surface protein for neuroblastoma targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A101. doi:10.1158/1535-7163.TARG-19-A101