BM-23 * Twist1 EXPRESSION CONFIRMS THE ROLE OF AN EMT-LIKE PROCESS IN MEDULLOBLASTOMA METASTASIS

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Medulloblastoma is an aggressive malignant embryonal tumour of the cerebellum that is frequently metastatic (one-third of primary and almost all recurrences) resulting in poor outcome. Epithelial-mesenchymal-transition (EMT) occurs during the early stages of metastasis where cells gain migratory and invasive abilities by altering phenotypically. By using a 3D in vitro culture system to better represent the in vivo microenvironment this study aims to investigate metastatic markers in medulloblastoma. Myc-immortalised cerebellar progenitor cells (C17.2), metastatic group 4 (MED1) and non-metastatic WNT subtype (MED6, C17.2 WNT) medulloblastoma cells were suspended in basement membrane extract to provide a 3D culture system. Expression of EMT genes was assessed by quantitative real time polymerase chain reaction on RNA extracted from 3D (on days 3, 6 and 8) and 2D monolayers. Twist1 expression was assessed in the MED1 primary tumour and a panel of 27 patient medulloblastomas by immunohistochemistry. Further markers were identified from published gene expression profiles of 62 medulloblastomas analysed according to metastatic status. 3D-culture of metastatic MED1 formed metabolically active branched interconnecting aggregates whilst MED6, C17.2WNT and non-tumourigenic C17.2 cells showed low metabolic activity and differentiation by day 3; supporting a non-metastatic phenotype. Expression of the Twist1 transcription factor doubled in MED1 cells grown in 3D compared to MED1 2D monolayers with no increase in expression observed in other cell types. The MED1 primary tumour showed focally high Twist1 positivity. Twist1 expression significantly correlated with metastasis in patient samples (p < 0.02). Twist1 was also expressed in spinal metastases in an orthotopic mouse model of MED1. Eight further markers differentially expressed in metastatic medulloblastoma have also been identified and are undergoing analysis. This data suggests that the EMT transcription factor Twist1 plays a role in an EMT-like process in metastatic medulloblastoma and supports the use of this 3D culture system to screen further metastatic markers.