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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.

Tumour diversity Ependymoma is a type of neural tumour that arises throughout the central nervous system. Using comparative transcriptomics in mouse and human tumours, Johnson et al . home in on mutations that are specific to individual tumour subgroups. In the course of their study, the authors generate the first mouse model of ependymoma and demonstrate the power of interspecific genomic comparisons to interrogate cancer subgroups. Ependymoma is a type of neural tumour that arises throughout the central nervous system. Using comparative transcriptomics in mouse and human tumours, these authors home in on mutations that are specific to individual tumour subgroups. In doing so, they generate the first mouse model of ependymoma and demonstrate the power of interspecific genomic comparisons to interrogate cancer subgroups. Understanding the biology that underlies histologically similar but molecularly distinct subgroups of cancer has proven difficult because their defining genetic alterations are often numerous, and the cellular origins of most cancers remain unknown 1 , 2 , 3 . We sought to decipher this heterogeneity by integrating matched genetic alterations and candidate cells of origin to generate accurate disease models. First, we identified subgroups of human ependymoma, a form of neural tumour that arises throughout the central nervous system (CNS). Subgroup-specific alterations included amplifications and homozygous deletions of genes not yet implicated in ependymoma. To select cellular compartments most likely to give rise to subgroups of ependymoma, we matched the transcriptomes of human tumours to those of mouse neural stem cells (NSCs), isolated from different regions of the CNS at different developmental stages, with an intact or deleted Ink4a/Arf locus (that encodes Cdkn2a and b). The transcriptome of human supratentorial ependymomas with amplified EPHB2 and deleted INK4A/ARF matched only that of embryonic cerebral Ink4a/Arf −/− NSCs. Notably, activation of Ephb2 signalling in these, but not other, NSCs generated the first mouse model of ependymoma, which is highly penetrant and accurately models the histology and transcriptome of one subgroup of human supratentorial tumour. Further, comparative analysis of matched mouse and human tumours revealed selective deregulation in the expression and copy number of genes that control synaptogenesis, pinpointing disruption of this pathway as a critical event in the production of this ependymoma subgroup. Our data demonstrate the power of cross-species genomics to meticulously match subgroup-specific driver mutations with cellular compartments to model and interrogate cancer subgroups.

Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world‐wide survey to ISEV members to assess methods considered ‘best practices’ for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.

Disruption of membrane potential (Vmem) can activate pathways associated with cancer proliferation. Manipulating ion channels may therefore present an effective strategy for treating cancers that fail to respond to conventional therapies. One approach to target these channels is to manipulate the membrane charge, which involves the use of wireless bipolar electrodes such as carbon nanotube porins (CNTPs) inserted into cell membranes to modulate membrane charge and ionic flux. By utilizing membrane dyes, we observed alterations in Vmem induced by CNTPs and externally applied voltages. Analyses of cellular behaviors and processes indicated that Vmem is more receptive to stimuli in invasive cancers, while it leads to increased metabolism in less invasive cancers, with notable changes in the cell cycle occurring at approximately 48 h post‐treatment in Glioblastoma (GB) cell lines. This work shows that CNTPs, in combination and with externally applied voltages, can modulate Vmem and alter cancer cell processes, supporting their potential as a therapeutic. A schematic illustrating glioblastoma cell membrane modulation using carbon nanotube porins (CNTPs) and an applied voltage. CNTPs enable ion flux leading to changes in membrane potential. This figure summarizes the concept of bioelectronic control of cell membrane voltage.

Three of the hallmarks of poor prognosis in paediatric ependymoma are drug resistance, local invasion and recurrence. We hypothesised that these hallmarks were due to the presence of a sub-population of cancer stem cells expressing the multi-drug efflux transporter ABCB1. ABCB1 gene expression was observed in 4 out of 5 paediatric ependymoma cell lines and increased in stem cell enriched neurospheres. Functional inhibition of ABCB1 using vardenafil or verapamil significantly (p ≤ 0.05–0.001) potentiated the response to three chemotherapeutic drugs (vincristine, etoposide and methotrexate). Both inhibitors were also able to significantly reduce migration (p ≤ 0.001) and invasion (p ≤ 0.001). We demonstrate that ABCB1 positive patients from an infant chemotherapy-led trial (CNS9204) had a shorter mean event free survival (EFS) (2.7 versus 8.6 years; p = 0.007 log-rank analysis) and overall survival (OS) (5.4 versus 12 years; p = 0.009 log-rank analysis). ABCB1 positivity also correlated with reduced event free survival in patients with incompletely resected tumours who received chemotherapy across CNS9204 and CNS9904 (a radiotherapy-led SIOP 1999-04 trial cohort; p = 0.03). ABCB1 is a predictive marker of chemotherapy response in ependymoma patients and vardenafil, currently used to treat paediatric pulmonary hypertension in children, could be repurposed to reduce chemoresistance, migration and invasion in paediatric ependymoma patients at non-toxic concentrations.

The most common malignant brain tumour in children, medulloblastoma (MB), is subdivided into four clinically relevant molecular subgroups, although targeted therapy options informed by understanding of different cellular features are lacking. Here, by comparing the most aggressive subgroup (Group 3) with the intermediate (SHH) subgroup, we identify crucial differences in tumour heterogeneity, including unique metabolism-driven subpopulations in Group 3 and matrix-producing subpopulations in SHH. To analyse tumour heterogeneity, we profiled individual tumour nodules at the cellular level in 3D MB hydrogel models, which recapitulate subgroup specific phenotypes, by single cell RNA sequencing (scRNAseq) and 3D OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) imaging. In addition to identifying known metabolites characteristic of MB, we observed intra- and internodular heterogeneity and identified subgroup-specific tumour subpopulations. We showed that extracellular matrix factors and adhesion pathways defined unique SHH subpopulations, and made up a distinct shell-like structure of sulphur-containing species, comprising a combination of small leucine-rich proteoglycans (SLRPs) including the collagen organiser lumican. In contrast, the Group 3 tumour model was characterized by multiple subpopulations with greatly enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity. Extensive TCA cycle metabolite measurements revealed very high levels of succinate and fumarate with malate levels almost undetectable particularly in Group 3 tumour models. In patients, high fumarate levels (NMR spectroscopy) alongside activated stress response pathways and high Nuclear Factor Erythroid 2-Related Factor 2 (NRF2; gene expression analyses) were associated with poorer survival. Based on these findings we predicted and confirmed that NRF2 inhibition increased sensitivity to vincristine in a long-term 3D drug treatment assay of Group 3 MB. Thus, by combining scRNAseq and 3D OrbiSIMS in a relevant model system we were able to define MB subgroup heterogeneity at the single cell level and elucidate new druggable biomarkers for aggressive Group 3 and low-risk SHH MB.

The patterns of 5-methylcytosine (5mC) and its oxidized derivatives, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) are reportedly altered in a range of cancers. Likewise, Wilms' Tumor protein 1 (WT1), a transcription factor essential for urogenital, epicardium, and kidney development exhibits aberrant expression in multiple tumors. Interestingly, WT1 directly interacts with TET proteins that catalyze the enzymatic oxidation of 5mC and exhibits high affinity for 5caC-containing DNA substrates . Here we review recent developments in the fields of Tet-dependent 5mC oxidation and WT1 biology and explore potential perspectives for studying the interplay between TETs and WT1 in brain tumors.

Epigenetic alterations, including methylation, have been shown to be an important mechanism of gene silencing in cancer. Ependymoma has been well characterized at the DNA copy number and mRNA expression levels. However little is known about DNA methylation changes. To gain a more global view of the methylation profile of ependymoma we conducted an array-based analysis. Our data demonstrated tumors to segregate according to their location in the CNS, which was associated with a difference in the global level of methylation. Supratentorial and spinal tumors displayed significantly more hypermethylated genes than posterior fossa tumors, similar to the ‘CpG island methylator phenotype’ (CIMP) identified in glioma and colon carcinoma. This hypermethylated profile was associated with an increase in expression of genes encoding for proteins involved in methylating DNA, suggesting an underlying mechanism. An integrated analysis of methylation and mRNA expression array data allowed us to identify methylation-induced expression changes. Most notably genes involved in the control of cell growth and death and the immune system were identified, including members of the JNK pathway and PPARG. In conclusion, we have generated a global view of the methylation profile of ependymoma. The data suggests epigenetic silencing of tumor suppressor genes is an important mechanism in the pathogenesis of supratentorial and spinal, but not posterior fossa ependymomas. Hypermethylation correlated with a decrease in expression of a number of tumor suppressor genes and pathways that could be playing an important role in tumor pathogenesis.

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.

Background Alteration of DNA methylation (5-methylcytosine, 5mC) patterns represents one of the causes of tumorigenesis and cancer progression. Tet proteins can oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine (5caC). Although the roles of these oxidised forms of 5mC (oxi-mCs) in cancer pathogenesis are still largely unknown, there are indications that they may be involved in the mechanisms of malignant transformation. Thus, reduction of 5hmC content represents an epigenetic hallmark of human tumours, and according to our recent report, 5caC is enriched in a proportion of breast cancers and gliomas. Nevertheless, the distribution of oxi-mCs in paediatric brain tumours has not been assessed. Findings Here, we analyse the global levels and spatial distribution of 5hmC and 5caC in four brain tumour cell lines derived from paediatric sonic hedgehog (SHH) pathway-activated medulloblastomas (Daoy and UW228-3) and ependymomas (BXD-1425EPN and DKFZ-EP1NS). We show that, unlike HeLa cells, the paediatric tumour cell lines possess both 5hmC and 5caC at immunochemically detectable levels and demonstrate that both modifications display high degrees of spatial overlap in the nuclei of medulloblastomas and ependymomas. Moreover, although 5hmC levels are comparable in the four brain tumour cell lines, 5caC staining intensities differ dramatically between them with highest levels of this mark in a subpopulation of DKFZ-EP1NS cells. Remarkably, the 5caC enrichment does not correlate with 5hmC levels and is not associated with alterations in thymine DNA glycosylase ( TDG ) expression in SHH medulloblastoma and ependymoma cell lines but corresponds to elevated levels of TET1 transcript in UW228-3 and DKFZ-EP1NS cells. Conclusions We demonstrate that both 5caC enrichment and elevated TET1 expression are observed in SHH medulloblastomas and ependymomas. Our results suggest that increased Tet-dependent 5mC oxidation may represent one of the epigenetic signatures of cancers with neural stem cell origin and, thus, may contribute to development of novel approaches for diagnosis and therapy of the brain tumours.