IN VITRO MODELLING OF CROSSTALK BETWEEN PAEDIATRIC CEREBELLAR TUMOURS AND HEALTHY BRAIN TISSUE IN THE POST-SURGICAL MICROENVIRONMENT

Abstract

Abstract AIMS Atypical Teratoid Rhabdoid Tumours (ATRT) and Medulloblastoma are embryonal brain tumours arising in the cerebellum. Many in vitro models of paediatric brain tumours fail to recapitulate key pathophysiological characteristics. We aim to address this gap by developing a bespoke model incorporating cellular and acellular aspects of the TME. METHOD Cerebellar tissue from a healthy human autopsy donor brain was decellularized to form cerebellar ECM and incorporated into a “blank slate” hydrogel. Spheroid-forming multi-well plates were coated with the hydrogel and used to culture patient-derived Medulloblastoma and ATRT cells in co-culture with primary human cerebellar astrocytes. RESULTS Tissue decellularization and retention of ECM components was demonstrated using colorimetric assays and Orbitrap Secondary Ion Mass Spectrometry. Biocompatibility of the ECM hydrogel was shown in 2D and 3D culture conditions using the CellTiter-Glo® 3D cell viability assay. Furthermore, the size and morphology of spheroids grown on the hydrogel was characterised using a high-throughput imaging and analysis methodology. Cell lines were labelled to facilitate discrimination between tumour cells and astrocytes. Optical sections of co-culture spheroids generated using confocal microscopy showed cell-cell contact between tumour cells and astrocytes. Finally, co-culture spheroids were separated by fluorescence activated cell sorting to facilitate downstream analyses on individual cell types. CONCLUSIONS This work presents a highly characterised model of medulloblastoma and ATRT interactions with healthy brain tissue, mimicking the immediate post-surgical microenvironment. Future work will utilise transcriptomics to assess the tumour-astrocyte crosstalk modelled using this system.