SPHEROIDS AND PERFUSION BIOREACTORS AS INNOVATIVE 3D CELL CULTURE SYSTEMS TO INVESTIGATE THE ROLE OF EXTRACELLULAR VESICLES IN NEUROBLASTOMA PROGRESSION

Neuroblastoma (NB) is the most prevalent extracranial solid tumour in childhood, with an overall 5 year-survival rate of 50%. Extracellular vesicles (EVs) are known mediators of intercellular communication in the tumour microenvironment (TME), but their role in NB needs to be further investigated. The aim of this study is to shed light on the involvement of EVs on two crucial aspects of NB progression: drug resistance and the conversion of Bone Marrow-Mesenchymal Stem Cells (BM-MSCs) to cancer-associated fibroblasts (CAFs). Our approach was based on the development of NB cell lines-derived 3D models. Spheroids were chosen for their relative simplicity but yet complex 3D structure capable of resembling the cellular heterogeneity of in vivo solid tumours and the establishment of oxygen gradients. After developing a protocol for the separation of the hypoxic core and the proliferating periphery in NB spheroids, we thoroughly characterized the two cell subpopulations highlighting differences in terms of oxygen concentration and cell-cycle phases distribution. We then purified their EVs and studied their effects on Doxorubicin (Doxo) response, one of the main chemotherapy drugs used for NB treatment. Compared to Doxo treatment alone, the combination of hypoxic core-derived EVs provided an increase in cells viability, indicative of increased drug resistance. To understand the role of EVs in BM-MSCs to CAFs conversion, we used perfusion bioreactors (BRs). These devices guarantee continuous perfusion of the medium across a collagen-based scaffold, where cells attach and grow in a 3D organization, similar to in vivo physiological tissues. NB cell lines were cultured in normoxic and hypoxic conditions, in both BRs and 2D flasks. The EVs isolated from these different culture systems were used to treat primary BM-MSCs, BM-272. Gene expression analysis revealed that only BRs-derived EVs could induce the conversion of BM-272 to CAFs. Overall, this study provided new insights into the role of EVs in NB progression, highlighting their effects in the modulation of drug response and in the conversion of BM-MSCs to CAFs, via the use of optimized and complex 3D models.