Neuroblastoma (NB) is a highly heterogenous paediatric malignancy of the developing sympathetic nervous system. Approximately half of patients have metastatic disease at the time of diagnosis, coinciding with a survival rate of less than 50%. Therefore, elucidating the molecular basis of the metastatic process may guide novel strategies for drug discovery and therapy. With this in mind, we aimed to explore the invasion of NB in an ex vivo culture system. To this end, we isolated NB organoids from patient-derived xenografts (PDXs) and embedded them in 3D extracellular matrix (ECM) hydrogels. We also cultured clonal clusters of NB cell lines as well as organoids isolated from cell line xenografts to compare their behaviour to that of PDX organoids. We used 3D culture assays to directly observe local invasion under different microenvironmental conditions in high-resolution and real-time. We found that the invasion of NB organoids was dependent on the composition of the matrix, where Matrigel was the preferred substratum. Based on organoid morphologies, we have identified and characterised four distinct invasive phenotypes used by NB cells to overcome environmental challenges. Culturing organoids in medium supplemented with FBS and bFGF induced the most aggressive cellular behaviour and the widest range of phenotypes. Unlike organoids isolated from PDXs, we found that NB cell lines were phenotypically confined during ECM invasion and while organoids isolated from cell line xenografts displayed a broader range of phenotypes compared to clonal cell line clusters, they could not recapitulate the diversity seen in PDX organoids. We also used 3D invasion assays to show that repression of the prognostic marker MYCN, resulted in less aggressive cellular behaviour in a NB cell line. Overall, we propose the pairing of PDX organoid culture and 3D invasion assays as a valuable tool to discover the molecular mechanisms controlling local invasion in NB.
