Understanding The Tumour Microenvironment Of Neuroblastoma By Modelling Stromal Schwann Cell Interaction And 3D In Vitro Culturing.

Neuroblastoma (NB) is an aggressive paediatric cancer that affects the sympathetic nervous system. Current therapies are ineffective long term for 80% of patients with clinically aggressive, high-risk disease. The major barriers in developing effective treatments against high-risk NB include the lack of preclinical models and insufficient knowledge of cell communication within the tumour microenvironment (TME) of NB. With this in mind, the aims of this work include:

1) Generate 3D in vitro neuroblastoma spheroids for therapeutic screening of platinum-based chemotherapeutics.

2) Investigate the Schwann cell-neuroblastoma interaction.

In chapter one, Spheroicalplate5D (SP5D) was used to generated spheroids to study the phenotypes of NB cell lines in three dimensional (3D) culture. All tested seeding densities formed large, uniform clusters after 4-5 days in culture. Spheroids generated using MYCN amplified Kelly cells did not display a more aggressive phenotype compared to non-MYCN amplified SH-SY5Y spheroids. In contrast, spheroids generated using cisplatin resistant KellyCis83 cells displayed an increased metabolism and aggressive phenotype compared to the cisplatin sensitive Kelly cells. The novel fluorophore conjugated compound (Pt(tet)(DMSO)(Cl)2) demonstrated comparable IC50 values in two dimensional (2D) culture to standard chemotherapies. 3D NB spheroids demonstrated reduced sensitivity to the novel platinum-based chemotherapy compared to 2D culture.

In chapter two, the direct and paracrine cell communication between neuroblasts and stromal Schwann cells (SCs) was investigated using co-culture and Schwann cell- derived exosomes (SC-EVs). It was found that direct contact between SCs and neuroblasts led to increased single-cell dispersion of neuroblasts. Whereas SC-EVs reduced the proliferation, migration, and invasion of NB by inducing a differentiated phenotype in NB cells. Ultimately, uniform and reproducible NB spheroids were generated and optimised for therapeutic screening. While the data is preliminary, an important interaction between neuroblast and SCs in the TME of NB was highlighted.