NON-CELL AUTONOMOUS INDUCTION OF A CANCER STEM CELL MOLECULAR PHENOTYPE IN AN IN VITRO MODEL OF SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract

Abstract AIMS • Design a novel in vitro model of sonic hedgehog (SHH) medulloblastoma (SHH-MB) by co-culture of two distinct human SHH-MB cell lines with wild-type cerebellar organoids derived from human induced pluripotent stem cells. • Investigate how the phenotype of SHH-MB cells grown in co-culture differs from conventional monolayer or tumour spheroid cultures. METHOD DAOY and ONS76 cell lines were cultured as monolayers, tumour spheroids or co-cultured with wild type cerebellar organoids for twenty-five days. Cells were then processed for single cell RNA-seq yielding thousands of sequenced transcriptomes, which were analysed using the R package, Seurat and other software. RESULTS Tumour cells in co-culture could be distinguished from non-malignant cells by their distinct transcriptional profiles. Subpopulations of both cancer cell lines in co-culture upregulated neuronal differentiation genes. In co-culture only, a distinct subset of DAOY cells expressed multiple cancer stem cell markers including SOX2, OTX2, GLI2 and NES. The upregulation of SOX2 by these cells correlated with the co-expression of ERBB4, which encodes a tyrosine kinase receptor. CONCLUSIONS SHH-MB cells exhibited striking differences in their phenotype when grown under physiological conditions in co-culture. First, subpopulations of both types of cancer cells expressed a neuronal differentiation programme. Second, a cancer stem cell phenotype is revealed in TP53 mutant tumour cells that is centred on SOX2. Signalling by ERBB4 is a candidate in the upregulation of SOX2 in these cells and has previously been implicated in the pathogenesis of Group 4 medulloblastoma. Future experiments will test whether SOX2 and ERBB4 are co-expressed in patient tumour cells.