Optimization of a Neuroblastoma 3D model to decipher the role of hypoxic extracellular vesicles in drug resistance

Neuroblastoma (NB) is the most common extra-cranial embryonal solid tumor in childhood. Despite multimodal treatment including Doxorubicin (Doxo) as one of the most common chemotherapy drugs, High-Risk (HR) NB patients show very poor long-term survival. The aim of this study is to decipher the role of extracellular vesicles (EVs) in NB drug resistance and progression, focusing on the contribution of hypoxia and the tumor microenvironment (TME). For this purpose, we exploited an optimized NB three-dimensional (3D) spheroid model to define EVs’ role in drug resistance. We first used our 3D model for Doxo treatments, and carried out cytotoxicity/viability assays, both on two-dimensional (2D) and 3D cultures of IMR-32 (N-myc-amplified) and SK-N-AS (non-N-myc-amplified) NB cell lines observing that 3D model mimicked better in vivo tumor behavior compared with 2D cultures and provided a better prediction of drug efficacy. Moreover, to take into account the heterogeneity of the tumor mass and the contribution of the tumor milieu, we separated and characterized two sub-populations from the NB spheroids: “migrating” and “non-migrating cells”. We isolated and characterized EVs from the two populations and used them in combination with Doxo treatments on the SK-N-AS cell line. We noticed that EVs significantly increased drug resistance, and that the effect was higher for combined treatments with EVs derived from migrating compared non-migrating cells. EVs from hypoxic cells induced an even greater contribution to drug resistance. In conclusion, the study provides novel findings on NB drug resistance exploring the role of hypoxia and the TME in tumor dissemination, and highlights the complexity of the tumor mass. Although additional studies are mandatory to better define the role of EVs derived from the two spheroids cell sub-populations, our findings are an important starting point for the development of novel alternative therapeutic strategies for NB that take in account the important role of the TME.