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Keywords: medulloblastoma; laminin; vitronectin; subtypes; hydrogel; ECM; metastasis; chemoresistance; 3D model Abstract Medulloblastoma (MB) is the most common malignant brain tumour in children and is subdivided into four subgroups: WNT, SHH, Group 3, and Group 4. These molecular subgroups differ in their metastasis patterns and related prognosis rates. Conventional 2D cell culture methods fail to recapitulate these clinical differences. Realistic 3D models of the cerebellum are therefore necessary to investigate subgroup-specific functional differences and their role in metastasis and chemoresistance. A major component of the brain extracellular matrix (ECM) is the glycosaminoglycan hyaluronan. MB cell lines encapsulated in hyaluronan hydrogels grew as tumour nodules, with Group 3 and Group 4 cell lines displaying clinically characteristic laminar metastatic patterns and levels of chemoresistance. The glycoproteins, laminin and vitronectin, were identified as subgroup-specific, tumour-secreted ECM factors. Gels of higher complexity, formed by incorporation of laminin or vitronectin, revealed subgroup-specific adhesion and growth patterns closely mimicking clinical phenotypes. ECM subtypes, defined by relative levels of laminin and vitronectin expression in patient tissue microarrays and gene expression data sets, were able to identify novel high-risk MB patient subgroups and predict overall survival. Our hyaluronan model system has therefore allowed us to functionally characterize the interaction between different MB subtypes and their environment. It highlights the prognostic and pathological role of specific ECM factors and enables preclinical development of subgroup-specific therapies. [c] 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland. Article Note: No conflicts of interest were declared. CAPTION(S): Supplementary materials and methods Figure S1. Global gene expression of medulloblastoma cell lines correlates with clinical subgroup data Figure S2. Comparison of MB cell line growth in 2D compared with long-term 3D culture Figure S3. Etoposide (ETO) treatment kills the SHH MB cell line ONS76 effectively, while Group 3 nodules display strong resistance comparable to clinical observations Figure S4. Differential gene expression analysis of the data set from Cavalli et al [6] reveals SHH and Group 3 specific ECM markers Figure S5. MB patients express subgroup-specific levels of laminin (LAM) and vitronectin (VTN) genes Figure S6. Protein expression of laminin and vitronectin in TMAs Figure S7. Analysis of the data set from Cavalli et al [6] reveals subgroup-specific differences in ECM protein expression patterns Figure S8. Cell growth on pure HA hydrogels reveals remarkable differences between SHH cell lines and Group 3 and Group 4 cell lines Figure S9. Cell growth on laminin-supplemented HA hydrogels revealed remarkable differences between SHH cell lines and Group 3 and Group 4 cell lines Figure S10. Cell growth on vitronectin-supplemented HA hydrogels revealed remarkable differences between SHH cell lines and Group 3 and Group 4 cell lines Figure S11. ECM subtypes are composed of different MB subgroup proportions Table S1. Clinicopathological characteristics of MB patients included in the TMAs Byline: Franziska Linke, Macha Aldighieri, Anbarasu Lourdusamy, Anna M Grabowska, Snow Stolnik, Ian D Kerr, Catherine LR Merry, Beth Coyle