Abstract
BACKGROUND: Glioblastoma (GBM) cell infiltration into the surrounding normal brain tissue where the blood brain barrier is intact, represents a major problem for clinical management and therapy. There is a vital need to understand the molecular mechanism that drives tumor cell invasion into the surrounding brain. We have previously developed a 3D coculture model where mature brain organoids are confronted with patient-derived glioblastoma stem-like cells (GSCs). In such a coculture system, single cell invasion into the normal brain tissue can be studied in detail. Here, we first describe in detail, by RNA-seq and proteomics, the differentiation of various neural cell lineages into mature brain organoids as well as their cellular organization. By real-time confocal microscopy and imaging analyses we also determine the speed of tumor cell invasion into the brain. Finally, we used this coculture system to delineate in detail the cellular heterogeneity within the invasive compartment and their gene expression. MATERIAL AND METHODS: Immunohistochemistry and immunofluorescence were used to determine the expression and distribution of mature neurons, astrocytes, oligodendrocytes, and microglia within the brain organoids. Proteomics and RNA-seq were used to determine brain development ex-vivo. To assess the clonal composition of the GBM-invasive compartment, we used cellular (RGB) barcoding technology. By advanced imaging, we tracked in real time the invasion of barcoded cells into the brain organoids. Finally, we isolated invasive cells and non-invasive cells from our coculture system and used single cell sequencing to analyze their gene expression profiles and molecular phenotypes. RESULTS: Immunohistochemistry and immunofluorescence showed that brain organoids, after 21 days of differentiation, display a highly cellular and structural organization. RNA-seq and proteomics, performed at different time points of organoid differentiation, revealed that the brain organoids develop into mature brain structures after 21 days as verified by a comparative analysis to normal rat brain development in vivo. Imaging analyses showed that multiple clones within the GBMs have the capacity to invade into the brain tissue with an average speed of ~ 20 μm/h. RNA-sec analysis of the invasive compartment revealed a strong up-regulation of genes and pathways associated with anaerobic respiration (glycolysis). CONCLUSION: We describe a highly standardized brain organoid coculture system that can be used to delineate GBM invasion ex-vivo. We demonstrate that this platform can be used to unravel the mechanisms that drive GBM invasion into the normal brain.