Studying medulloblastoma, the most common malignant paediatric brain tumour, requires simple yet realistic in vitro models. In this study, we optimised a robust, reliable, three-dimensional (3D) culture method for medulloblastoma able to recapitulate the spatial conformation, cell–cell and cell–matrix interactions that exist in vivo and in patient tumours. We show that, when grown under the same stem cell enriching conditions, SHH subgroup medulloblastoma cell lines established tight, highly reproducible 3D spheroids that could be maintained for weeks in culture and formed pathophysiological oxygen gradients. 3D spheroid culture also increased resistance to standard-of-care chemotherapeutic drugs compared to 2D monolayer culture. We exemplify how this model can enhance in vitro therapeutic screening approaches through dual-inhibitor studies and continual monitoring of drug response. Next, we investigated the initial stages of metastatic dissemination using brain-specific hyaluronan hydrogel matrices. RNA sequencing revealed downregulation of cell cycle genes and upregulation of cell movement genes and key fibronectin interactions in migrating cells. Analyses of these upregulated genes in patients showed that their expression correlated with early relapse and overall poor prognosis. Our 3D spheroid model is a significant improvement over current in vitro techniques, providing the medulloblastoma research community with a well-characterised and functionally relevant culture method.

Macrophages (Mφ) are abundantly present in the tumor microenvironment and may predict outcome in solid tumors and defined lymphoma subtypes. Mφ heterogeneity, the mechanisms of their recruitment, and their differentiation into lymphoma‐promoting, alternatively activated M2‐like phenotypes are still not fully understood. Therefore, further functional studies are required to understand biological mechanisms associated with human tumor‐associated Mφ (TAM). Here, we show that the global mRNA expression and protein abundance of human Mφ differentiated in Hodgkin lymphoma (HL)‐conditioned medium (CM) differ from those of Mφ educated by conditioned media from diffuse large B‐cell lymphoma (DLBCL) cells or, classically, by macrophage colony‐stimulating factor (M‐CSF). Conditioned media from HL cells support TAM differentiation through upregulation of surface antigens such as CD40, CD163, CD206, and PD‐L1. In particular, RNA and cell surface protein expression of mannose receptor 1 (MRC1)/CD206 significantly exceed the levels induced by classical M‐CSF stimulation in M2‐like Mφ; this is regulated by interleukin 13 to a large extent. Functionally, high CD206 enhances mannose‐dependent endocytosis and uptake of type I collagen. Together with high matrix metalloprotease9 secretion, HL‐TAMs appear to be active modulators of the tumor matrix. Preclinical in ovo models show that co‐cultures of HL cells with monocytes or Mφ support dissemination of lymphoma cells via lymphatic vessels, while tumor size and vessel destruction are decreased in comparison with lymphoma‐only tumors. Immunohistology of human HL tissues reveals a fraction of cases feature large numbers of CD206‐positive cells, with high MRC1 expression being characteristic of HL‐stage IV. In summary, the lymphoma‐TAM interaction contributes to matrix‐remodeling and lymphoma cell dissemination.

Extracellular vesicles (EVs) have emerged as pivotal mediators of communication in the tumour microenvironment. More specifically, nanosized extracellular vesicles termed exosomes have been shown to contribute to the establishment of a premetastatic niche. Here, we sought to determine what role exosomes play in medulloblastoma (MB) progression and elucidate the underlying mechanisms. Metastatic MB cells (D458 and CHLA-01R) were found to secrete markedly more exosomes compared to their nonmetastatic, primary counterparts (D425 and CHLA-01). In addition, metastatic cell-derived exosomes significantly enhanced the migration and invasiveness of primary MB cells in transwell migration assays. Protease microarray analysis identified that matrix metalloproteinase-2 (MMP-2) was enriched in metastatic cells, and zymography and flow cytometry assays of metastatic exosomes demonstrated higher levels of functionally active MMP-2 on their external surface. Stable genetic knockdown of MMP-2 or extracellular matrix metalloproteinase inducer (EMMPRIN) in metastatic MB cells resulted in the loss of this promigratory effect. Analysis of serial patient cerebrospinal fluid (CSF) samples showed an increase in MMP-2 activity in three out of four patients as the tumour progressed. This study demonstrates the importance of EMMPRIN and MMP-2-associated exosomes in creating a favourable environment to drive medulloblastoma metastasis via extracellular matrix signalling.

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. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.