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INTRODUCTION: Micro-Ribonucleic acids (miRNAs) are short non-protein coding RNAs involved in post-transcriptional regulation of genes by targeting the 3’ region of mRNAs. They have been associated with dysregulation of oncogenes and tumour suppressors (i.e. Myc and PTEN) and signalling pathways (i.e. PDGF and EGF) in many human cancers including adult high grade glioma (HGG). However, limited work has been published about their role in paediatric astrocytoma. Here, we examine the miRNA profile of paediatric astrocytomas of different grades, their correlation to survival and micro-vascularity and their function in proliferation, invasion and migration. METHODS: The Nanostring nCounter miRNA array system analysed the expression levels of 665 miRNA sequences in 116 samples (101 astrocytoma tumours, 2 normal brain samples and 13 cell lines). Validation of selected miRNAs and their role in survival and micro-vascularity was studied by qPCR and immunohistochemistry. The functional effect of these miRNAs was evaluated by knock-down/knock-up of the sequences into the paediatric HGG cell lines SF-188 and KNS42 by transient transfection followed by Presto blue assay (assessing proliferation) and transwell assay in the presence (for invasion) or absence (for migration) of extra-cellular proteins. RESULTS: 551 miRNAs were significantly expressed between HGG, low grade glioma (LGG) and normal brain. 89 of them exhibited fold-differences higher than 3, including miR-451, miR-149 and miR-21. The latter has known oncogenic effects in adult HGG and we also observed that its overexpression in SF-188 and KNS42 resulted in significant increased proliferation of more than double after 5 days. The comparison between HGG and LGG displayed differences in the expression of 10 miRNAs, with miR-34a down-regulated in HGG and correlating to loss of its chromosomal locus in 12–13% of tumours. CONCLUSION: Specific miRNA profile signature of paediatric astrocytomas of different grades could potentially be used for prognosis and for identifying personalised therapeutic targets.

Aims: Recent studies have observed that cells from high-grade glial tumors are capable of assuming an endothelial phenotype and genotype, a process termed ‘vasculogenic mimicry’ (VM). Here we model and manipulate VM in dynamic 3-dimensional (3D) glioma cultures. Methods: The Rotary Cell Culture System (RCCS) was used to derive large macroscopic glioma aggregates, which were sectioned for immunohistochemistry and RNA extracted prior to angiogenic array-PCR. Results: A 3D cell culture induced microenvironment (containing only glial cells) is sufficient to promote expression of the endothelial markers CD105, CD31 and vWF in a proportion of glioma aggregates in vitro. Many pro-angiogenic genes were upregulated in glioma aggregates and in primary explants and glioma cells were capable of forming tubular- like 3D structures under endothelial-promoting conditions. Competitive inhibition of either vascular endothelial growth factor or fibroblast growth factor receptor was sufficient to impair VM and downregulate the tumor-derived angiogenic response, whilst impairing tumor cell derived tubule formation. Glioma xenografts using the same cells reveal tumor-derived vessel-like structures near necrotic areas, consistent with widespread tumor-derived endothelial expression in primary glioma tissue. Conclusions: Our findings support studies indicating that tumor-derived endothelial cells arise in gliomas and describe a dynamic 3D culture as a bona fide model to interrogate the molecular basis of this phenomenon in vitro. Resistance to current anti-angiogenic therapies and the contribution of tumor derived endothelial cells to such resistance are amenable to study using the RCCS.

Pediatric embryonal brain tumors can be difficult to classify. Atypical teratoid rhabdoid tumors (ATRT) contain rhabdoid cells, while primitive neuroectodermal tumors (PNETs) are composed of "small round blue cells." Loss of INI1 is a common event in ATRT; therefore, we investigated if the loss of INI1 protein expression was also observed in central nervous system (CNS) PNET and pineoblastoma. A histological review of 42 CNS PNETs and six pineoblastomas was performed. INI1 expression was assessed by immunohistochemistry. Sequencing was performed on the mutational hotspots of INI1. INI1-immunonegative tumors were further investigated using fluorescence in situ hybridization. Epithelial membrane antigen (EMA) protein expression was assessed in six CNS PNETs to further define the phenotype. Five CNS PNETs without rhabdoid cell morphology were immuno-negative for both INI1 and EMA. Of these primary CNS PNET patients, three died

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. Electronic supplementary material The online version of this article (doi:10.1007/s00401-011-0904-1) contains supplementary material, which is available to authorized users.

Intratumor heterogeneity exists in many cancers, with clear evidence of differential gene expression temporo-spatially in glioblastoma multiforme (GBM). MicroRNA (miRNA) profiling studies have identified risk-stratifying signatures in GBM, but it has not been clarified whether microRNA profiles vary in a similar fashion to gene expression within individual GBM tumors. This study utilized primary GBM specimens resected from patients undergoing surgery in a regional UK Neurosurgery unit. Fresh frozen tissue underwent RNA extraction and miScript Human Brain Cancer miRNA PCR Array was used to assess 84 miRNA's expression levels between distinct spatial regions within primary GBMs. miRNA expression was analyzed with quantitative RT-PCR and gene targets for analysis were selected with miRtarget gene prediction software. Protein levels for the in silico predicted targets based on miRNA expression were validated through immunohistochemistry (IHC). A large degree of intratumor heterogeneity was apparent in the samples. Homology existed between individuals with 8 common miRNAs differentially expressed between central and edge regions of tumors. Protein levels of CDK6 and c-KIT nuclear accumulation were significantly elevated at tumor edges relative to core samples in concordance with the microRNA profiling. Intratumor heterogeneity in GBM produces significant miRNA profiling variation depending on spatial location of the sample within the tumor. Therefore, assessment of GBM miRNA signatures requires selective sampling from multiple distinct tumor regions and consideration of the overall patterns of miRNA expression across the entirety of the tumor.

INTRODUCTION: Local neurosurgically-applied drug-delivery systems offer an opportunity to target glioblastoma (GBM) residual disease, but consideration must be given to the uptake of delivered agents once diffused from a drug-delivery depot. We assess the glycosaminoglycan-binding enhanced transduction of nanoparticles coated with genetically-engineered peptides, as a direct corollary to biomaterial-based localised drug-delivery systems. METHOD: GBM cells were transduced with biocompatible iron oxide magnetic nanoparticles (dMNP) coated with membrane docking (P21) and cell penetrating (8R) peptides. Protein transduction efficiency was determined by cytochemical staining and metabolic assays. To test the efficacy of the cytotoxic peptide SHEPHERDIN (SHEP), which disrupts the interaction of Survivin with HSP 90, GBM cells were exposed to acute doses of dMNP-P21-8R-SHEP and effects on metabolism measured. RESULTS: Nuclear and cytoplasmic protein transduction of dMNP-8R-P21 was evident in 100% of adult and paediatric GBM cells after 3h exposure, whereas after 16h, only 25% of uncoated nanoparticles traverse the cellular membrane by direct translocation and/or endocytosis. Protein-mediated transduction of dMNP-P21-8R-SHEP results in dose-dependent proliferation impairment of adult and paediatric GBM cells (IC50 25 µM and 20 µM respectively). CONCLUSION: Protein-mediated cellular transduction via membrane docking and cell penetrating peptides has the potential to deliver anti-cancer cargos to brain tumours and diversifies nanoparticle functionality. Early findings characterising SHEP-mediated GBM viability loss and controlled release of dMNP-P21-8R-SHEP from a polymeric microparticle drug-delivery matrix will be discussed.

INTRODUCTION: Ependymoma is the second commonest central nervous system malignancy in children. 50% of sufferers relapse and 80% of these subsequently die. Few prognostic factors have been identified but improved outcomes are seen in children over 3 years old and those with more complete surgical resection. Intratumour heterogeneity has not been previously described in ependymoma and refers to the diversity within a tumour. It has implications because, if present, single biopsies may ‘miss’ important areas whilst conventional treatments may ‘select’ for more resistant clonal populations. Overexpression of RelA protein has been described in ependymomas with the C11Orf95-RELA fusion gene; previously identified in 70% of supratentorial ependymomas. We aimed to interrogate a cohort of ependymomas for evidence of heterogeneity using RelA protein as a marker. METHODS: Tissue microarrays (TMAs) and whole sections from a group of children with ependymoma underwent immunohistochemistry for RelA protein. Each TMA core was scored as negative, positive or strongly positive (+/− nuclear staining) for RelA protein expression. RESULTS: 746 cores from 283 tumours (Mean = 2.63 cores/tumour) were scored. RelA protein was expressed in posterior fossa (34.2%), supratentorial (46.2%) and spinal (50.0%) tumours. 42 tumours had only 1 core available so were excluded. Of the remaining 241 tumours, 63 (26.1%) had multiple heterogeneous cores. Tumours with more cores were more likely to demonstrate heterogeneity. Heterogeneity was also seen across 23 tumour sections. CONCLUSIONS: Knowledge of ependymoma heterogeneity will facilitate approaches to diagnosis and development of targeted therapies. RelA protein is expressed heterogeneously in ependymoma and work based on protein expression in TMAs should therefore consider the risk of sampling bias. The extent of heterogeneity in ependymoma merits further study; including investigation of the relationship between RelA protein expression and the C11Orf95-RELA fusion genes and assessment of the association between RelA expression and clinical features.

In the preparation of biomimetic materials it is often required that efficient methods of polymerization be used, often methods that can lead to biomimetic polymers with relatively narrow molecular weight distribution. Living radical polymerization techniques have successfully been used to create low polydispersity linear polymers by free-radical polymerizations. Although this technique slows down the polymerization of multifunctional monomers, there is little effect on the network structure due to the high concentration of pendent double bonds. There are applications of the living radical polymerization in the synthesis of block copolymers. Essentially, the technique involves polymerizing a single type of monomer first to create a macromonomer that is capable of acting as an initiator because of the reversible bond between the polymer end group and the terminating group. This terminating group may be a thiol or a halogen and, under the right conditions, will dissociate to form radicals. A second monomer is then added to the system and the polymerization proceeds with the second monomer chemically attached to the polymer of the first monomer. We review methods of creating biomimetic block copolymers using the iniferter radical polymerization technique. The block copolymers would be used in the synthesis of micropatterned polymer films for use in biomaterials and other biomedical applications.

The design of novel biomaterials for applications in biological recognition, drug delivery, or diagnostics requires a judicious choice of preparation conditions and methods for the production of well-characterized 3-dimensional structures, preferably by benign processes. In this work, the polymerization of poly(ethylene glycol) (PEG) methacrylates was examined by kinetic gelation modeling and kinetic analysis in order to ascertain the factors affecting the resulting structure. The kinetics of the polymerization and structure of the final polymer network are strongly affected by the length of the PEG graft chain. The propagation of the polymer chains becomes increasingly diffusion limited with the incorporation of longer PEG grafts. In addition, a more heterogeneous network consisting of numerous microgel regions is produced as the length of the PEG graft is increased. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 3506–3519, 2003

The polymerizations of multifunctional monomers with an iniferter, p-xylylene bis-(N,N-diethyldithiocarbamate) (XDT), were studied with differential photocalorimetry. The iniferter was used to simulate a ‘living’ radical polymerization. The normal autoacceleration behavior that is often observed in the polymerization of multifunctional monomers was not evident in the reactions with the iniferter. The reversible reaction between the propagating polymer chain and the sulfur radical from the XDT molecule dominated the reaction and drastically decreased the rate of polymerization. Various parameters of the reaction (length between functional groups in the monomer, iniferter concentration, UV light intensity, and temperature) were investigated to determine the effect on the polymerization. The rate of polymerization and the final conversion of the polymer were increased significantly by increasing the temperature, initiator concentration and UV light intensity, and decreasing the length between functional groups. Mechanical properties of the resulting polymers were also examined. It was concluded that the presence of the iniferter during the polymerization of the multifunctional monomers had no effect on the heterogeneity of the polymer network. This was in agreement with previous modeling results.

Introduction The median survival of patients with glioblastoma multiforme (astrocytoma grade 4) remains less than 18 months despite radical surgery, radiotherapy and systemic chemotherapy. Surgical implantation of chemotherapy eluting wafers into the resection cavity has been shown to improve length of survival but the current licensed therapy has several drawbacks. This paper investigates in vivo efficacy of a novel drug eluting paste in glioblastoma. Methods Poly(lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) self-sintering paste was loaded with the chemotherapeutic agent etoposide and delivered surgically into partially resected tumours in a flank murine glioblastoma xenograft model. Results Surgical delivery of the paste was successful and practical, with no toxicity or surgical morbidity to the animals. The paste was retained in the tumour cavity, and preliminary results suggest a useful antitumour and antiangiogenic effect, particularly at higher doses. Bioluminescent imaging was not affected significantly by the presence of the paste in the tumour. Conclusions Chemotherapy loaded PLGA/PEG paste seems to be a promising technology capable of delivering active drugs into partially resected tumours. The preliminary results of this study suggest efficacy with no toxicity and will lead to larger scale efficacy studies in orthotopic glioblastoma models.

Background Telomeric 3’ overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4), a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure. Methods We exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels. Results Although all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo. Conclusion This study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.

The "living" radical polymerization with an ini- ferter was used to create micropatterned biomedical sur- faces. Novel, photosensitive biomedical polymers were cre- ated by the incorporation of dithiocarbamate groups from iniferters. A second monomer layer was then irradiated onto the photosensitive polymer substrate created with the ini- ferter to form a copolymer. Patterns were created on the films by application of modified microfabrication-based photolithographic techniques. The technique was used to create patterns with depths from 5 to 80 mm. In addition, various polymers were incorporated, including polyethyl- ene glycol methacrylates, styrene, and methacrylic acid, to synthesize regions with different physico-chemical proper- ties. Applications include novel surfaces for biosensors and biomaterials for the selective adhesion of cells and proteins. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 351-360, 2001

A robust H∞ controller was developed to deliver insulin via a mechanical pump in Type I diabetic patients. A fundamental nonlinear diabetic patient model was linearized and then reduced to a third-order linear form for controller synthesis. Uncertainty in the nonlinear model was characterized by up to ± 40% variation in eight physiological parameters. A sensitivity analysis identified the three-parameter set having the most significant effect on glucose and insulin dynamics over the frequency range of interest ω = [0.002, 0.2] (rad/min). This uncertainty was represented in the frequency domain and incorporated in the controller design. Controller performance was assessed in terms of its ability to track a normoglycemic set point (81.1 mg/dL) in response to a 50 g meal disturbance. In the nominal continuous-time case, the controller maintained glucose concentrations within ± 3.3 mg/dL of set point. A controller tuned to accommodate uncertainty yielded a maximum deviation of 17.6 mg/dL for the worst-case parameter variation.

A model was developed to describe the overall dissolution kinetics of semicrystalline PVA gels prepared by freezing and thawing techniques. The dissolution process was described as a three-step mechanism: detachment-, diffusion-, and disentanglement-controlled dissolution. The unfolding of crystalline regions due to detachment and diffusion was calculated. The lamellar thickness of a PVA crystal was found to significantly change the rate of unfolding and thus, the overall dissolution kinetics. The model predicted the effect of the lamellar thickness, time of swelling, and sample thickness on the crystal chain unfolding and potential dissolution of freeze/thawed PVA hydrogels. q 2000 Elsevier Science Ltd. All rights reserved.

A kinetic gelation model has been developed that incorporates the kinetics of a living/controlled radical polymerization. Specifically, the kinetics of an iniferter polymerization were implemented in the model to determine the effect of the kinetics on the polymer structure. In particular, the replacement of the termination step with a reversible termination was examined. The model predicts the formation of more monodisperse linear polymers in the presence of an iniferter and a reversible termination reaction. When termination by combination is allowed to occur, the polydispersity increases. For cross-linking reactions, it was concluded that the iniferter only had an effect on reactions with small amounts of cross-linking agent, resulting in a delay in the gel point and a more heterogeneous structure. When the amount of cross-linking agent is increased, there is no difference in the network structure obtained by a cross-linking reaction with an iniferter versus a cross-linking reaction with the classical ...

Background: Central nervous system primitive neuroectodermal tumours (CNS PNETs) are embryonal tumours occurring predominantly in children. Current lack of knowledge regarding their underlying biology hinders development of more effective treatments. We previously identified WNT/β-catenin pathway activation in one-third of CNS PNETs, which was potentially linked to a better prognosis. In this study, we have extended our cohort, achieving a statistically significant correlation with prognosis. We additionally investigated the biological effects of WNT/β-catenin pathway activation in tumour pathogenesis. Methods: A total of 42 primary and 8 recurrent CNS PNETs were analysed for WNT/β-catenin pathway status using β-catenin immunohistochemistry. Genomic copy number and mRNA expression data were analysed to identify a molecular profile linked to WNT/β-catenin pathway activation. results: Pathway activation was seen in 26% of CNS PNETs and was significantly associated with longer overall survival. Genes displaying a significant difference in expression levels, between tumours with and without WNT/β-catenin pathway activation, included several involved in normal CNS development suggesting aberrant pathway activation may be disrupting this process. conclusion: We have identified WNT/β-catenin pathway status as a marker, which could potentially be used to stratify disease risk for patients with CNS PNET. Gene expression data suggest pathway activation is disrupting normal differentiation in the CNS.

Purpose: The high incidence of recurrence and unpredictable clinical outcome for pediatric ependymoma reflect the imprecision of current therapeutic staging and need for novel risk stratification markers. We therefore evaluated 1q25 gain across three age- and treatment-defined European clinical trial cohorts of pediatric intracranial ependymoma. Experimental Design: Frequency of 1q gain was assessed across 48 ependymomas (42 primary, 6 recurrent) using Affymetrix 500K single-nucleotide polymorphism arrays. Gain of 1q25 was then evaluated by interphase FISH across 189 tumors treated on the Children's Cancer Leukaemia Group/International Society for Pediatric Oncology (SIOP) CNS9204 (n = 60) and BBSFOP (n = 65) adjuvant chemotherapy trials, or with primary postoperative radiotherapy (SIOP CNS9904/RT, n = 64). Results were correlated with clinical, histologic, and survival data. Results: Gain of 1q was the most frequent imbalance in primary (7/42, 17%) and recurrent ependymomas (2/6, 33%). Gain of 1q25 was an independent predictor of tumor progression across the pooled trial cohort [HR = 2.55; 95% confidence interval (CI): 1.56–4.16; P = 0.0002] and both CNS9204 (HR = 4.03; 95% CI: 1.88–8.63) and BBSFOP (HR = 3.10; 95% CI: 1.22–7.86) groups. The only clinical variable associated with adverse outcome was incomplete tumor resection. Integrating tumor resectability with 1q25 status enabled stratification of cases into disease progression risk groups for all three trial cohorts. Conclusions: This is the first study to validate a prognostic genomic marker for childhood ependymoma across independent trial groups. 1q25 gain predicts disease progression and can contribute to patient risk stratification. We advocate the prospective evaluation of 1q25 gain as an adverse marker in future international clinical trials. Clin Cancer Res; 18(7); 2001–11. ©2012 AACR.

Pediatric embryonal brain tumors can be difficult to classify. Atypical teratoid rhabdoid tumors (ATRT) contain rhabdoid cells, while primitive neuroectodermal tumors (PNETs) are composed of “small round blue cells.” Loss of INI1 is a common event in ATRT; therefore, we investigated if the loss of INI1 protein expression was also observed in central nervous system (CNS) PNET and pineoblastoma. A histological review of 42 CNS PNETs and six pineoblastomas was performed. INI1 expression was assessed by immunohistochemistry. Sequencing was performed on the mutational hotspots of INI1. INI1‐immunonegative tumors were further investigated using fluorescence in situ hybridization. Epithelial membrane antigen (EMA) protein expression was assessed in six CNS PNETs to further define the phenotype. Five CNS PNETs without rhabdoid cell morphology were immuno‐negative for both INI1 and EMA. Of these primary CNS PNET patients, three died

BACKGROUND: Molecularly targeted approaches informing next-generation glioblastoma (GBM) chemotherapy must account for the intra-heterogeneous genome landscape and aim to eradicate invasive residual sub-populations. We outline an analytical strategy that forms a functional genomic corollary to existing genome-wide mutation and gene expression studies. METHODS: Biopsies from spatially distinct intra-GBM regions were surgically resected via Stealth and 5-ALA fluorescence guidance, including core, enhancing and invasive margin regions. Cellular intra-tumour heterogeneity (ITH) was confirmed by immunohistochemistry and qPCR arrays, whilst molecular analytics were conducted using liquid chromatography-mass spectrometry (metabolomic/lipidomic) magnetic resonance spectroscopy (metabolic) and SDS PAGE phosphoprotein enrichment (proteomic). RESULTS: Cellular ITH was confirmed via distinct neural stem cell expression in medial and posterior rims relative to the central enhancement region. Metabolites present in the invasive margin (5-ALA-determined), central enhancement and peri-necrotic core were distinct within each GBM. Controlling for the high proportion of normal brain cells surrounding the invasive margin, high tumour-specific glutamine levels were consistently present. Relative abundance of cytosolic phosphoproteins distinguished active protein pathways within intra-GBM regions, including invasive margin. Metabolomic network analyses confirmed that GBM residual disease is distinct from core ITH regions with respect to metabolic dependence. CONCLUSIONS: This innovative concept is the first to address ITH with multi-region biopsies using advanced analytical bioscience techniques. Identification of metabolites, lipids and phospho-proteins that are functional determinants of GBM infiltration reveal putative molecular targets on residual disease cells and represent a route to expedite new chemotherapy agents to clinic within a precision medicine framework.

INTRODUCTION: It is now clear that intratumour heterogeneity exists in many cancers, with clear evidence of differential gene expression temporo-spatially in glioblastoma multiforme (GBM). MicroRNA (miRNA) profiling studies have identified risk-stratifying signatures in GBM, but it has not been clarified whether microRNA profiles vary in a similar fashion to gene expression within individual GBM tumours. METHODS: miScript Human Brain Cancer miRNA PCR Array was used to assess 84 miRNA's expression levels between distinct regions within primary GBMs. Primary tumour specimens (n = 2) were sampled in 4 distinct regions including enhancing and necrotic core, invasive margin and medial rim. miRNA expression was analysed with quantitative RT-PCR. Gene targets for analysis were selected with miRtarget gene prediction software, and protein levels were validated through immunohistochemistry (IHC). RESULTS: A large degree of intratumour heterogeneity was apparent in the samples. Among the most differentially expressed miRNAs were miR-137, miR-182-5p, miR-183-5p and miR-218-5p. Conversely, miR-129-5p, miR-15b-5p and miR-185-5p were the most consistently expressed miRNAs across regions. Homology existed between individuals with 8 common miRNAs differentially expressed between both central and edge regions. Protein levels of CDK6 and c-KIT nuclear accumulation were significantly elevated at tumour edges relative to core samples as predicted by microRNA profiling. CONCLUSION: The miRNA intratumour heterogeneity in GBM could produce significant profiling variation depending on spatial location of the sample within the tumour. Therefore, assessment of GBM miRNA signatures requires selective sampling from multiple distinct tumour regions.

INTRODUCTION: Intratumour heterogeneity (ITH) may account for therapy failure and tumour recurrence in adult glioblastoma multiforme (GBM). Low availability of fresh frozen tissue has hindered characterisation of ITH in paediatric GBM. Here, we propose the application of desorption electrospray ionisation mass spectrometry (DESI-MS) to assess spatial lipidomic profiles of formalin-fixed paraffin-embedded (FFPE) tissue of paediatric GBM. METHOD: Optimisation of DESI-MS has been conducted on FFPE tissue of normal adult brain prior to transfer of the technology to paediatric GBM tissue. Immunohistochemical analyses for proteins associated with lipid biosynthesis delineated distinct intratumour regions. Spatial characterisation of lipids synthesised by these metabolic proteins will permit optimisation of DESI-MS, prior to unbiased lipidomic analyses. RESULTS: Paediatric GBM cell lines grown under lipoprotein-deficient conditions demonstrated greater resilience compared to an adult cell line, indicating distinct lipid metabolism that will be revealed through lipidomic analyses. Proteins associated with lipid biosynthesis demonstrated evidence of ITH in paediatric GBM, with 69% of patients displaying heterogeneous expression of fatty acid synthase that correlated with areas of high cellularity. CONCLUSION: DESI-MS has the potential to characterise early indications of paediatric GBM ITH through characterisation of lipid markers associated with distinct intratumour regions. Proteins associated with the synthesis of identified lipid markers may represent potential therapeutic targets. We anticipate the establishment of an optimised DESI-MS method that will facilitate lipidomic studies of other paediatric brain tumours.

▪ Abstract Hydrogels are cross-linked hydrophilic polymers that can imbibe water or biological fluids. Their biomedical and pharmaceutical applications include a very wide range of systems and processes that utilize several molecular design characteristics. This review discusses the molecular structure, dynamic behavior, and structural modifications of hydrogels as well as the various applications of these biohydrogels. Recent advances in the preparation of three-dimensional structures with exact chain conformations, as well as tethering of functional groups, allow for the preparation of promising new hydrogels. Meanwhile, intelligent biohydrogels with pH- or temperature-sensitivity continue to be important materials in medical applications.

UV free-radical polymerization techniques are often used to synthesize hydrogels for controlled release applications. Numerous techniques exist for immobilizing drugs or solutes in the gel. This work focuses on the entrapment of solute in a hydrogel by conducting a photopolymerization in the presence of the monomer and the solute. A kinetic gelation model has been developed to examine the effect of the solute material on the polymerization process and the ensuing network structure. Kinetic experiments have also been conducted of the polymerization of poly(ethylene glycol) methacrylate in the presence of theophylline. It was found that the presence of the solute led to a more heterogeneous network with numerous microgel regions present. The effect of the size of the solute on the polymerization was also investigated.

We have developed novel techniques for the preparation of micropatterned structures from thin films prepared by the block copolymerization of monomers using UV free-radial polymerizations. The process involves polymerizing the first monomer layer in the presence of an iniferter (initator-transfer agent-terminator) with a dithiocarbamate group to make a photosensitive polymer. Upon application of the second monomer layer on the first polymer layer and irradiation, a copolymer is formed between the two layers. Patterns are created on the films by applying a mask and selectively irradiating the surface. We have successfully polymerized poy (ethylene glycol) (PEG) onto a highly crosslinked material of poly(ethylene glycol) dimethacrylate. Various patterns have been created to determine the precision that can be achieved with this method. Preliminary results show that the patterns in the second monomer layer can be from 5 micrometers to 100 micrometers thick, with feature size as small as 5 micrometers , allowing the use of this material to high aspect ratio structures for micro-fluidics. In addition, applications of this type of material are also in bioMEMS, biomaterials, and biosensors for the selective adhesion of cells and proteins.© (2000) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.