Recommendations for reproducibility of cerebrospinal fluid extracellular vesicle studies.

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.

Extracellular Vesicles Potentiate Medulloblastoma Metastasis in an EMMPRIN and MMP-2 Dependent Manner.

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.

Drug Resistance in Medulloblastoma Is Driven by YB-1, ABCB1 and a Seven-Gene Drug Signature.

Therapy resistance represents an unmet challenge in the treatment of medulloblastoma. Accordingly, the identification of targets that mark drug-resistant cell populations, or drive the proliferation of resistant cells, may improve treatment strategies. To address this, we undertook a targeted approach focused on the multi-functional transcription factor YB-1. Genetic knockdown of YB-1 in Group 3 medulloblastoma cell lines diminished cell invasion in 3D in vitro assays and increased sensitivity to standard-of-care chemotherapeutic vincristine and anti-cancer agents panobinostat and JQ1. For vincristine, this occurred in part by YB-1-mediated transcriptional regulation of multi-drug resistance gene ABCB1, as determined by chromatin immunoprecipitation. Whole transcriptome sequencing of YB-1 knockdown cells identified a role for YB-1 in the regulation of tumourigenic processes, including lipid metabolism, cell death and survival and MYC and mTOR pathways. Stable cisplatin- and vincristine-tolerant Group 3 and SHH cell lines were generated to identify additional mechanisms driving resistance to standard-of-care medulloblastoma therapy. Next-generation sequencing revealed a vastly different transcriptomic landscape following chronic drug exposure, including a drug-tolerant seven-gene expression signature, common to all sequenced drug-tolerant cell lines, representing therapeutically targetable genes implicated in the acquisition of drug tolerance. Our findings provide significant insight into mechanisms and genes underlying therapy resistance in medulloblastoma.

Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging.

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.

Establishing an In Vitro 3D Spheroid Model to Study Medulloblastoma Drug Response and Tumor Dissemination.

Medulloblastoma is the most common malignant pediatric brain tumor. Current treatment involves surgery, chemotherapy, and craniospinal radiotherapy, and these are associated with a significant reduction in quality of life. Metastatic dissemination at diagnosis is found in up to 30% of medulloblastoma cases and, alongside therapy resistance, is a significant feature in determining poor outcome. Development of new therapeutic approaches requires models where drug resistance and migration can be readily quantified and that are representative of patient disease. 3D medulloblastoma (3D-MB) spheroids are a simple yet effective means of bridging the gap between 2D culture and in vivo methods, providing users with highly reproducible in vitro models that more accurately recapitulate tumor morphology, drug response, and migration from a tumor mass. Unlike other cancer types, medulloblastoma spheroids fail to grow in their different standard cell culture media; instead, each cell line requires the same stem cell-enriching conditions. This requirement, however, has the advantage that it allows direct comparison of growth and response between cell lines in the absence of any potential media bias. In addition, spheroids can be used to model the initial stages of metastatic dissemination, something that cannot be achieved in 2D culture, providing insight into key changes occurring in migratory cells. Here, we provide protocols that detail the initial generation and maintenance of 3D-MB spheroids from sonic-hedgehog, Group 3, and Group 4 medulloblastoma subgroups, as well as describing functional assays to study drug response and cell migration across hyaluronan matrices, which represent the extracellular matrix backbone of the brain parenchyma. Through application of these simple yet highly representative models, it will be possible to test novel therapeutics targeting metastasis and drug resistance, as well as to develop insights into the mechanistic processes driving relapse in this malignant pediatric brain tumor. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation and maintenance of 3D medulloblastoma (3D-MB) spheroids Support Protocol 1: Measuring spheroid size for coefficient-of-variation analysis Basic Protocol 2: Assessing drug response in 3D-MB spheroids Support Protocol 2: 384-well 3D-MB spheroid generation Basic Protocol 3: Immunohistochemical staining of 3D-MB spheroids Basic Protocol 4: Modeling metastatic dissemination using 3D-MB migration models Support Protocol 3: RNA extraction from 3D-MB spheroids.

Chemosensitization of Temozolomide-Resistant Pediatric Diffuse Midline Glioma Using Potent Nanoencapsulated Forms of a N(3)-Propargyl Analogue.

The lack of clinical response to the alkylating agent temozolomide (TMZ) in pediatric diffuse midline/intrinsic pontine glioma (DIPG) has been associated with O6-methylguanine-DNA-methyltransferase (MGMT) expression and mismatch repair deficiency. Hence, a potent N(3)-propargyl analogue (N3P) was derived, which not only evades MGMT but also remains effective in mismatch repair deficient cells. Due to the poor pharmacokinetic profile of N3P (t1/2 < 1 h) and to bypass the blood-brain barrier, we proposed convection enhanced delivery (CED) as a method of administration to decrease dose and systemic toxicity. Moreover, to enhance N3P solubility, stability, and sustained distribution in vivo, either it was incorporated into an apoferritin (AFt) nanocage or its sulfobutyl ether ß-cyclodextrin complex was loaded into nanoliposomes (Lip). The resultant AFt-N3P and Lip-N3P nanoparticles (NPs) had hydrodynamic diameters of 14 vs 93 nm, icosahedral vs spherical morphology, negative surface charge (-17 vs -34 mV), and encapsulating ∼630 vs ∼21000 N3P molecules per NP, respectively. Both NPs showed a sustained release profile and instant uptake within 1 h incubation in vitro. In comparison to the naked drug, N3P NPs demonstrated stronger anticancer efficacy against 2D TMZ-resistant DIPG cell cultures [IC50 = 14.6 (Lip-N3P) vs 32.8 µM (N3P); DIPG-IV) and (IC50 = 101.8 (AFt-N3P) vs 111.9 µM (N3P); DIPG-VI)]. Likewise, both N3P-NPs significantly (P < 0.01) inhibited 3D spheroid growth compared to the native N3P in MGMT+ DIPG-VI (100 µM) and mismatch repair deficient DIPG-XIX (50 µM) cultures. Interestingly, the potency of TMZ was remarkably enhanced when encapsulated in AFt NPs against DIPG-IV, -VI, and -XIX spheroid cultures. Dynamic PET scans of CED-administered zirconium-89 (89Zr)-labeled AFt-NPs in rats also demonstrated substantial enhancement over free 89Zr radionuclide in terms of localized distribution kinetics and retention within the brain parenchyma. Overall, both NP formulations of N3P represent promising approaches for treatment of TMZ-resistant DIPG and merit the next phase of preclinical evaluation.

ABCB1 inhibition provides a novel therapeutic target to block TWIST1-induced migration in medulloblastoma.

BACKGROUND: Therapeutic intervention in metastatic medulloblastoma is dependent on elucidating the underlying metastatic mechanism. We investigated whether an epithelial-mesenchymal transition (EMT)-like pathway could drive medulloblastoma metastasis. METHODS: A 3D Basement Membrane Extract (3D-BME) model was used to investigate medulloblastoma cell migration. Cell line growth was quantified with AlamarBlue metabolic assays and the morphology assessed by time-lapse imaging. Gene expression was analyzed by qRT-PCR and protein expression by immunohistochemistry of patient tissue microarrays and mouse orthotopic xenografts. Chromatin immunoprecipitation was used to determine whether the EMT transcription factor TWIST1 bound to the promoter of the multidrug pump ABCB1. TWIST1 was overexpressed in MED6 cells by lentiviral transduction (MED6-TWIST1). Inhibition of ABCB1 was mediated by vardenafil, and TWIST1 expression was reduced by either Harmine or shRNA. RESULTS: Metastatic cells migrated to form large metabolically active aggregates, whereas non-tumorigenic/non-metastatic cells formed small aggregates with decreasing metabolic activity. TWIST1 expression was upregulated in the 3D-BME model. TWIST1 and ABCB1 were significantly associated with metastasis in patients (P = .041 and P = .04, respectively). High nuclear TWIST1 expression was observed in the invasive edge of the MED1 orthotopic model, and TWIST1 knockdown in cell lines was associated with reduced cell migration (P < .05). TWIST1 bound to the ABCB1 promoter (P = .03) and induced cell aggregation in metastatic and TWIST1-overexpressing, non-metastatic (MED6-TWIST1) cells, which was significantly attenuated by vardenafil (P < .05). CONCLUSIONS: In this study, we identified a TWIST1-ABCB1 signaling axis during medulloblastoma migration, which can be therapeutically targeted with the clinically approved ABCB1 inhibitor, vardenafil.

BLBP Is Both a Marker for Poor Prognosis and a Potential Therapeutic Target in Paediatric Ependymoma.

Paediatric ependymomas are aggressive, treatment-resistant tumours with a tendency towards relapse, consistent with a sub-population of therapy-resistant cancer stem cells. These cells are believed to derive from brain lipid binding protein (BLBP)-expressing radial glia, hence we proposed that BLBP may be a marker for ependymoma therapy resistance. BLBP protein expression correlated with reduced overall survival (OS) in patients from two trials (CNS9204, a chemotherapy-led infant trial-5 y OS 45% vs. 80%, p = 0.011-and CNS9904, a radiotherapy-led trial-OS 38% vs. 85%, p = 0.002). All ependymoma cell lines examined by qRT-PCR expressed BLBP, with expression elevated in stem cell-enriched neurospheres. Modulation of BLBP function in 2D and 3D assays, using either peroxisome proliferator activated receptor (PPAR) antagonists or BLBP's fatty acid substrate docosahexaneoic acid (DHA), potentiated chemotherapy response and reduced cell migration and invasion in ependymoma cell lines. BLBP is therefore an independent predictor of poor survival in paediatric ependymoma, and treatment with PPAR antagonists or DHA may represent effective novel therapies, preventing chemotherapy resistance and invasion in paediatric ependymoma patients.

3D spheroid models of paediatric SHH medulloblastoma mimic tumour biology, drug response and metastatic dissemination.

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.

Corrigendum to: ABCB1 inhibition provides a novel therapeutic target to block TWIST1-induced migration in medulloblastoma.

[This corrects the article DOI: 10.1093/noajnl/vdab030.].

Y-Box Binding Protein-1: A Neglected Target in Pediatric Brain Tumors?

Brain and central nervous system tumors represent the most common childhood solid tumors. Comprising 21% of all pediatric cancers, they remain the leading cause of cancer-related mortality and morbidity in childhood. Due to advances in neurosurgical technique, radiotherapy and the use of combination therapy, survival rates have generally increased. However, by cause of the lesion itself, its surgical removal and subsequent treatment, survivors are at high risk of long-term neurocognitive sequelae and secondary cancer. Clearly, improvements in diagnosis and treatment are needed. Accordingly, current treatment is evolving away from conventional, uniform therapy and towards risk-stratified regimens and molecularly-targeted therapies, with the aim of diminishing adverse side effects while minimizing the risk of disease recurrence. The multifunctional oncoprotein Y-box binding protein 1 (YB-1) may serve as one such molecular target. Increased YB-1 levels have been reported in a number of pediatric brain tumors, where YB-1 appears to facilitate the advancement of malignant phenotypes. These include proliferation, invasion, and resistance to therapy, as well as the maintenance of brain tumor-initiating cells. Here we evaluate the current literature and show how YB-1 modulates signaling pathways driving each of these phenotypes. We also review the regulation of YB-1 at a transcriptional, translational, posttranslational and subcellular level and argue that there is strong and sufficient evidence to support the development of YB-1 as a biomarker and future therapeutic target in childhood brain tumors.

3D hydrogels reveal medulloblastoma subgroup differences and identify extracellular matrix subtypes that predict patient outcome.

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.

Leptomeningeal malignancy of childhood: sharing learning between childhood leukaemia and brain tumour trials.

Leptomeningeal malignancy complicates childhood cancers, including leukaemias, brain tumours, and solid tumours. In leukaemia, such malignancy is thought to invade leptomeninges via the vascular route. In brain tumours, dissemination from the primary tumour, before or after surgery, via CSF pathways is assumed; however, evidence exists to support the vascular route of dissemination. Success in treating leptomeningeal malignancy represents a rate-limiting step to cure, which has been successfully overcome in leukaemia with intensified systemic therapy combined with intra-CSF therapy, which replaced cranial radiotherapy for many patients. This de-escalated CNS-directed therapy is still associated with some neurotoxicity. The balanced benefit justifies exploration of ways to further de-escalate CNS-directed therapy. For primary brain tumours, standard therapy is craniospinal radiotherapy, but attendant risk of acute and delayed brain injury and endocrine deficiencies compounds post-radiation impairment of spinal growth. Alternative ways of treating leptomeninges by intensifying drug therapy delivered to CSF are being investigated-preliminary evidence suggests improved outcomes. This Review seeks to describe methods of intra-CSF drug delivery and drugs in use, and consider how the technique could be modified and additional drugs might be selected for this route of administration.

A role for ABCB1 in prognosis, invasion and drug resistance in ependymoma.

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.

Wilms' Tumor Protein 1 and Enzymatic Oxidation of 5-Methylcytosine in Brain Tumors: Potential Perspectives.

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 in vitro. 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.

A HIF-independent, CD133-mediated mechanism of cisplatin resistance in glioblastoma cells.

PURPOSE: Glioblastoma (GBM) is the commonest brain tumour in adults. A sub-population of cells within these tumours, known as cancer stem cells (CSCs), is thought to mediate their chemo-/radiotherapy resistance. CD133 is a cell surface marker that is used to identify and isolate GBM CSCs. However, its functional significance, as well as the relevant microenvironment in which to study CD133, have so far remained unknown. Here, we examined the effect of hypoxia on the expression of CD133 and on that of the hypoxia-related factors HIF-1α and HIF-2α, and the potential functional significance of CD133 expression on the acquisition of chemo-resistance by GBM cells. METHODS: CD133, HIF-1α, HIF-2α, VEFG and (control) HPRT mRNA expression analyses were carried out on GBM cells (U251, U87 and SNB19; 2D or 3D cultures) under both normoxic and hypoxic conditions, using qRT-PCR. siRNA was used to downregulate CD133, HIF-1α and HIF-2α expression in the GBM cells, which was confirmed by flow cytometry and qRT-PCR, respectively. Drug sensitivity-related IC50 values were established using an Alamar Blue cell viability assay in conjunction with the Graphpad prism software tool. RESULTS: We found that the expression of CD133 was upregulated under hypoxic conditions in both the 2D and 3D GBM cell culture models. In addition, an increased resistance to cisplatin, temozolomide and etoposide was observed in the GBM cells cultured under hypoxic conditions compared to normoxic conditions. siRNA-mediated knockdown of either HIF-1α or HIF-2α resulted in a reduced CD133 expression, with HIF-2α having a more long-term effect. We also found that HIF-2α downregulation sensitized the GBM cells to cisplatin to a greater extent than HIF-1α, whereas CD133 knockdown had a more marked effect on cisplatin sensitisation than knockdown of either one of the HIFs, suggesting the existence of a HIF-independent cisplatin resistance mechanism mediated by CD133. This same mechanism does not seem to be involved in temozolomide resistance, since we found that HIF-1α downregulation, but not HIF-2α or CD133 downregulation, sensitized GBM cells to temozolomide. CONCLUSIONS: From our data we conclude that the mechanisms underlying hypoxia-induced CD133-mediated cisplatin resistance may be instrumental for the design of new GBM treatment strategies.

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Medulloblastoma and ependymoma cells display increased levels of 5-carboxylcytosine and elevated TET1 expression.

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.

In vitro models of medulloblastoma: Choosing the right tool for the job.

The recently-defined four molecular subgroups of medulloblastoma have required updating of our understanding of in vitro models to include molecular classification and risk stratification features from clinical practice. This review seeks to build a more comprehensive picture of the in vitro systems available for modelling medulloblastoma. The subtype classification and molecular characterisation for over 40 medulloblastoma cell-lines has been compiled, making it possible to identify the strengths and weaknesses in current model systems. Less than half (18/44) of established medulloblastoma cell-lines have been subgrouped. The majority of the subgrouped cell-lines (11/18) are Group 3 with MYC-amplification. SHH cell-lines are the next most common (4/18), half of which exhibit TP53 mutation. WNT and Group 4 subgroups, accounting for 50% of patients, remain underrepresented with 1 and 2 cell-lines respectively. In vitro modelling relies not only on incorporating appropriate tumour cells, but also on using systems with the relevant tissue architecture and phenotype as well as normal tissues. Novel ways of improving the clinical relevance of in vitro models are reviewed, focusing on 3D cell culture, extracellular matrix, co-cultures with normal cells and organotypic slices. This paper champions the establishment of a collaborative online-database and linked cell-bank to catalyse preclinical medulloblastoma research.

ABCB1 in children's brain tumours.

Tumours of the central nervous system are the most common solid tumour, accounting for a quarter of the 1500 cases of childhood cancer diagnosed each year in the U.K. They are the most common cause of cancer-related death in children. Treatment consists of surgery followed by adjuvant chemotherapy and/or radiotherapy. Survival rates have generally increased, but many survivors suffer from radiotherapy-related neurocognitive and endocrine side effects as well as an increased risk of secondary cancer. Adjuvant chemotherapy is normally given in combination to circumvent chemoresistance, but several studies have demonstrated it to be ineffective in the absence of radiotherapy. The identification of children with drug-resistant disease at the outset could allow stratification of those that are potentially curable by chemotherapy alone. Ultimately, however, what is required is a means to overcome this drug resistance and restore the effectiveness of chemotherapy. Medulloblastomas and ependymomas account for over 30% of paediatric brain tumours. Advances in neurosurgery, adjuvant radiotherapy and chemotherapy have led to improvements in 5-year overall survival rates. There remain, however, significant numbers of medulloblastoma patients that have intrinsically drug-resistant tumours and/or present with disseminated disease. Local relapse in ependymoma is also common and has an extremely poor prognosis with only 25% of children surviving first relapse. Each of these is consistent with the acquisition of drug and radiotherapy resistance. Since the majority of chemotherapy drugs currently used to treat these patients are transport substrates for ATP-binding cassette sub-family B member 1 (ABCB1) we will address the hypothesis that ABCB1 expression underlies this drug resistance.