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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

Background: Anti-cancer drugs-loaded targeted nanocarriers can reduce side-effects and improve therapeutic efficacy in preclinical studies. However, poorly perfused and dysfunctional tumour vessels limit the transport of the payload into the parenchyma of solid tumours. The use of tumour-penetrating nanocarriers might enhance tumour penetration and anti-tumour effects. Methods: A consensus motif, mediator of tissue penetration (TP) was added to a previously characterized neuroblastoma (NB)-targeting peptide (pep). In vitro NB cell association and internalization of TP-pep, either free or coupled to Stealth Liposomes (SL), were tested by FACS and confocal microscopy. In vitro cytotoxic potential of a novel doxorubicin (DXR)-loaded liposomal (TP-pep-SL[DXR]) was evaluated by MTS assay. Three mouse xenograft models mimicking the growth and spread of NB in humans (injection routes: subcutaneous; adrenal gland; tail vein) were enrolled to examine in vivo penetration, vascular permeability, tumour glucose consumption and sensitivity in response to TP-pep-SL[DXR]. Results: Compared to pep, TP-pep increases its cellular association in vitro on cell lines and cells derived from NB patients. When coupled to SL, TP-pep enhances liposomes penetration and cytotoxic effects in vitro and increases binding and penetration in a mouse model of NB. Moreover, in vivo accumulation of Evans Blue dye within the tumour mass reveals that TP-pep-SL[DXR] increases the tumour vascular permeability into NB tumour mass, but not in non-tumour tissues. Compared to pep-targeted liposomes, TP-pep-SL[DXR] leads to an increased anti-NB effect towards all the animal models tested. Conclusion: Our findings demonstrate that the enhancement of tumour penetration by drug-loaded nanoparticles might represent an innovative targeted strategy for NB. Citation Format: Mirco Ponzoni, Chiara Brignole, Laura Emionite, Silvia Bruno, Daniela Guarnieri, Leopoldo Sitia, Matteo Bauckneht, Ambra Buschiazzo, Andrea Rossi, Daniela Di Paolo, Patrizia Perri, Flavio Curnis, Alessandro Gori, Angela Rita Sementa, Michele Cilli, Pier Paolo Pompa, Gianmario Sambuceti, Angelo Corti, Fabio Pastorino. Enhancement of tumor penetration by drug-loaded nanoparticles: An innovative targeted strategy for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3879.

Anticancer drugs loaded into tumor- and vasculature-targeted nanocarriers (NC) can reduce side-effects and improve therapeutic efficacy in pre-clinical studies. However, poorly perfused and dysfunctional tumor vessels and lymphatics limit the transport of the payload into the parenchyma of solid tumors. The use of NC decorated with tumor-penetrating peptides (TPPs) might enhance tumor penetration and antitumor effects. A previously characterized neuroblastoma (NB)-targeting peptide ligand was here modified (now referred as TPP-NB) by adding a consensus motif as a mediator of cell, vascular and tissue penetration via neuropilin-1 (NRP-1) receptor recognition. NPR-1 expression was validated by FACS analysis in NB cell lines and by IHC staining in tumor cells and tumor stroma from NB-bearing mice. Recombinant NRP-1 was used to validate TPP-NB specificity. In vitro and in vivo cell association and internalization of TPP-NB, either free or coupled to Liposomes (L) were tested by FACS and confocal microscopy. Vascular permeability assay after treatment with TPP-NB-targeted, doxorubicin-loaded Liposomes (TPP-NB-L[DXR]) was performed evaluating the in vivo accumulation of Evans Blue dye within the tumor mass. Therapeutic experiments with TPP-NB-L[DXR] were performed in mice orthotopically injected with human NB cells. NRP-1 expression is validated in a panel of NB cells and in tumors from NB-bearing mice. Differently from the original peptide and some control ones, TPP-NB is able to recognize recombinant NRP-1. The addition of the NRP-1-recognizing sequence to the original peptide significantly increases its NB cellular association in vitro. Interestingly, the results seem to indicate that the enhanced capability by TPP-NB in binding NB cells is related to the combination of the NRP-1-recognizing and the original sequence. Importantly, TPP-NB coupled at the external surfaces of L[DXR] significantly increases their cellular association on NB cells in vitro. Competitive binding assay reveals that binding of TPP-NB is specific and can be inhibited by an excess of the unlabeled free peptide. The localization and the cellular distribution of L evaluated by confocal microscopy in vitro and in mouse models of NB, confirm the binding specificity, showing an increased selective internalization of TPP-NB-L-FITC compared to that obtained with either untargeted L or L decorated with the scrambled peptide. Moreover, TPP-NB-L[DXR] further increases the vascular permeability into the NB tumor mass, but not in non-tumor tissues. The therapeutic efficacy of TPP-NB-L[DXR] has been investigating in terms of overall survival. On running results indicate that the novel NC exerts an increased anti-NB effect compared to DXR-loaded L decorated with the original peptide. Our findings demonstrate that the achieved penetrating features by a NB-targeting peptide might increase liposomal drug binding, homing and antitumor efficacy. Citation Format: Fabio Pastorino, Chiara Brignole, Laura Emionite, Silvia Bruno, Flavio Curnis, Daniela Di Paolo, Patrizia Perri, Alessandro Gori, Renato Longhi, Michele Cilli, Angelo Corti, Mirco Ponzoni. Tumor-penetrating peptide-coated nanoparticles as a novel strategy for the targeted therapy of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5130. doi:10.1158/1538-7445.AM2017-5130