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Isabella Orienti,1 Ferro Nguyen,2 Peng Guan,2 Venkatadri Kolla,2 Natalia Calonghi,1 Giovanna Farruggia,1 Michael Chorny,2 Garrett M Brodeur2 1Department of Pharmacy and Biotechnology, University of Bologna, Bologna 40127, Italy; 2Divisions of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USACorrespondence: Isabella Orienti Email isabella.orienti@unibo.itPurpose: Currently >50% of high-risk neuroblastoma (NB) patients, despite intensive therapy and initial partial or complete response, develop recurrent NB due to the persistence of minimal residual disease (MRD) that is resistant to conventional antitumor drugs. Indeed, their low therapeutic index prevents drug-dose escalation and protracted administration schedules, as would be required for MRD treatment. Thus, more effective and less toxic therapies are urgently needed for the management of MRD. To address this aim, we evaluated a new combination of fenretinide and lenalidomide, both endowed with antitumor activity and low-toxicity profiles. New nanomicelles were prepared as carriers for this combination to maximize bioavailability and accumulation at the tumor site because of the enhanced permeability and retention (EPR) effect.Experimental design: New nanomicelles containing the fenretinide–lenalidomide combination (FLnMs) were prepared by a one-step method, providing high drug encapsulation and micelle dimensions suitable for tumor accumulation. Their administration to mice bearing human NB xenografts allowed us to evaluate their efficacy in comparison with the nanomicelles containing fenretinide alone (FnMs).Results: Treatment by FLnMs significantly decreased the tumor growth of NB xenografts. FLnMs were more active than FnMs despite comparable fenretinide concentrations in tumors, and lenalidomide alone did not show cytotoxic activity in vitro against NB cells. The tumor mass at the end of treatment with FLnMs was predominantly necrotic, with a decreased Ki-67 proliferation index.Conclusion: FLnMs provided superior antitumor efficacy in NB xenografts compared to FnMs. The enhanced efficacy of the combination was likely due to the antiangiogenic effect of lenalidomide added to the cytotoxic effect of fenretinide. This new nanomicellar combination is characterized by a low-toxicity profile and offers a novel therapeutic option for the treatment of high-risk tumors where the persistence of MRD requires repeated administrations of therapeutic agents over long periods of time to avoid recurrent disease.Keywords: neuroblastoma, drugs combination, nanomicelles, fenretinide, lenalidomide

Purpose: In a previous study, we demonstrated that the combination of fenretinide with lenalidomide, administered by a novel nanomicellar formulation (FLM), provided a strong antitumor effect in a neuroblastoma TrkB-expressing tumor. In this study, we tested the nanomicellar combination in an MYCN amplified neuroblastoma xenograft to assess its efficacy in different tumor genotypes and evaluate the interactions of the nanomicelles with the tumor cells. Experimental Design: FLM was administered to mice bearing human NLF xenografts to evaluate its efficacy in comparison with the nanomicelles containing fenretinide alone (FM). Confocal laser-scanning fluorescence microscopy images of the NLF cells treated with FLM and FM allowed us to estimate the nanomicelle ability to transport the encapsulated drugs inside the tumor cells. Flow cytometric analysis of the cells from treated tumors was performed to assess the effect of treatment on GD2 expression and NK cell infiltration. Results: FLM and FM decreased the growth of NLF xenografts at comparable extents during the treatment period. Afterwards, FLM induced a progressive tumor regression without regrowth, while FM treatment was followed by regrowth within 15-20 days after the end of treatment. Both FLM and FM were able to penetrate the tumor cells transporting the encapsulated drugs. FLM transported higher amount of fenretinide inside the cells. Also, FLM treatment strongly increased GD2 expression in treated tumors and slightly decreased the NK infiltration compared to FM. Conclusion: FLM treatment induced a superior antitumor response than FM in NLF xenografts, presumably due to the combined effects of fenretinide cytotoxicity and lenalidomide antiangiogenic activity. The ability of FLM to penetrate tumor cells, transporting the encapsulated drugs, substantially improved the therapeutic efficiency of this system. Moreover, the enhancement of GD2 expression in FLM treated tumors offers the possibility to further increase the antitumor effect by the use of anti-GD2 CAR-T cells and anti-GD2 antibodies in combination with FLM in multimodal therapies.

Angiogenesis is essential for tumor growth and metastasis and depends on the production of angiogenic factors. Mechanisms regulating the expression of angiogenic factors in tumor cells are largely unknown. High expression of the neurotrophin receptor TrkA in neuroblastomas (NBs) is associated with a favorable prognosis, whereas TrkB is mainly expressed on aggressive, MYCN-amplified NBs. To investigate the biological effects of TrkA and TrkB expression on angiogenesis in NB, we examined the expression of angiogenic factors in the human NB cell line SY5Y and its TrkA and TrkB transfectants. In comparison with parental SY5Y cells, mRNA and protein levels of the examined angiogenic factors were significantly reduced in SY5Y-TrkA cells, whereas SY5Y-TrkB cells did not demonstrate a significant change. Conditioned medium of TrkB transfectants and parental SY5Y cells induced endothelial cell proliferation and migration, but this effect was completely absent in SY5Y-TrkA cells. TrkA expression also resulted in severely impaired tumorigenicity in a mouse xenograft model and was associated with reduced angiogenic factor expression and vascularization of tumors, as determined by immunohistochemistry and an in vivo Matrigel assay. TrkA expression inhibits angiogenesis and tumor growth in SY5Y NB cells by down-regulation of angiogenic factors, whereas expression of TrkB does not down-regulate the production of these angiogenic factors. The biologically different behavior of TrkA- and TrkB-expressing NBs may be explained in part by their effects on angiogenesis.

We describe a child with dysmorphic features, as well as developmental and growth delay, who developed neuroblastoma at 5 mo of age. Cytogenetic analysis of blood lymphocytes revealed an interstitial deletion of 1p36.1-->1p36.2, which was apparent only with high-resolution banding. Molecular analysis with a collection of polymorphic DNA probes for 1p confirmed an interstitial deletion involving subbands of 1p36. Deletions of this region are a common finding in neuroblastoma cells from patients with advanced stages of disease. Therefore, these results (a) suggest that constitutional deletion of this region predisposed the patient to the development of neuroblastoma and (b) support the localization of a neuroblastoma tumor-suppressor locus to 1p36.