Abstract
The aim of the study is to investigate the potential of using three-dimensional (3D) in vitro neuroblastoma models to mimic the neuroblastoma microenvironment by testing a potential therapeutic compound-the natural extract epigallocatechin-3-gallate (EGCG), and to further elucidate the roles of DYRK1A in the growth and differentiation of neuroblastoma tissue. In vitro models based on a classic neuroblastoma cell line SH-SY5Y were employed, including 3D models with extracellular matrix and co-cultured with vascular endothelial cells. Cell viability was tested using AlamarBlue and Resazurin assay. The growth and differentiation of in vitro models of SH-SY5Y were analysed based on microscopy images obtained from immunofluorescence or real-time imaging. Protein expression level was investigated using immunoblotting analysis. The two-dimensional (2D) in vitro model implies the cytotoxicity and DYRK1A inhibition effect of EGCG and shows the induction of neuronal differentiation marker TuJ1. 3D in vitro models suggest that EGCG treatment compromised the growth of SH-SY5Y multicellular 3D spheroids and the viability of SH-SY5Y cultured in 3D Matrigel matrix. In addition, co-culture of SH-SY5Y with human vascular umbilical vein endothelial cells implied the inhibitory effects by EGCG in a vascularised microenvironment. In this study, novel 3D in vitro models of neuroblastoma were established in the application of testing a potential anti-cancer candidate compound EGCG. In pursuit of the goals of the 3Rs (replacement, reduction and refinement), the usage of these 3D in vitro models has the potential to reduce and eventually replace current animal models used in neuroblastoma research. The DYRK1A inhibiting nature of EGCG, together with the facts that EGCG inhibits the growth and induces the differentiation of neuroblastoma in vitro models, suggests an oncogene role of DRYK1A.