A common tumour in a rare location: a single centre case series of cerebellar glioblastoma.

Although glioblastoma is the commonest primary brain tumour in adults, its location in the cerebellum is extremely rare. We present thirteen cases (3 female, 10 male; median age at presentation 56 [age range 21-77]) of surgically managed, histologically confirmed, primary cerebellar glioblastoma (cGB) over a 17 year period (2005-2022). Pre-operative radiological diagnosis was challenging given cGB rarity, although MRI demonstrated ring enhancement in all cases. Surgical management included posterior fossa craniectomy and debulking in 11 cases and burr hole biopsy in two. CSF diversion was necessary in four cases. No evidence of IDH or ATRX gene mutations was found when tested. Survival ranged from 1 to 22 months after diagnosis (mean 10.9 months). We also seek to understand why glioblastoma is rare in this location and discuss potential reasons for this. We hypothesise that increasing anatomical distance from germinal regions and decreased local endogenous neural stem cell activity (which has been associated with glioblastoma) may explain why glioblastoma is rare in the cerebellum. We hereby seek to add to the limited literature on cGB as this is the largest UK cGB series to date.

Craniectomy with soft tissue reconstruction for locally advanced non-melanoma skin cancer of scalp with calvarial invasion: The Nottingham experience.

INTRODUCTION: Locally advanced non-melanoma skin cancer (NMSC) involving the periosteum or calvarium poses a clinical challenge for patients who are unfit for immunotherapy due to medical comorbidities and/or frailty. This case series aims to investigate outcomes for patients undergoing craniectomy and soft tissue reconstruction. METHOD: Patients who underwent craniectomy and soft tissue reconstruction for invasive NMSC with calvarium or periosteal invasion between 2016 and 2022 were included. Data, including demographics, operative details, and clinical outcomes, were gathered from Nottingham University Hospitals' digital health record and the histopathology electronic database. RESULT: Eight patients (average age: 78.4 years, 3 females 5 males) with significant comorbidities and varying degrees of periosteal or bone invasion fulfilled the inclusion criteria. Diagnoses included four squamous cell carcinomas, two basal cell carcinomas, and two pleomorphic dermal sarcomas. Five patients had a history of prior incomplete deep margin excision. The median sizes for soft tissue defect, tumor and bone defect size were 51.83 cm2, 34.63 cm2 and 42.25 cm2, respectively. Intraoperative complications included one dural tear. Four patients underwent local flap reconstruction and with split-thickness skin grafting, four patients underwent free flap reconstruction. Adjuvant radiotherapy was administered to three patients. Complications comprised partial graft loss in two and complete graft loss in one. There was partial flap loss in one case. One patient required subsequent parotidectomy due to regional progression before achieving disease control. All patients achieved lasting locoregional disease control (average follow-up 29.7 months). CONCLUSION: Craniectomy with soft tissue reconstruction proves to be a safe and effective treatment option in advanced NMSC of the scalp in patients unsuitable for immunotherapy due to frailty or medical co-morbidity.

Total intravenous anaesthesia with propofol and remifentanil is associated with reduction in operative time in surgery for glioblastoma when compared with inhalational anaesthesia with sevoflurane.

BACKGROUND: Total intravenous anaesthesia (TIVA) is emerging as a preferred neuroanaesthetic agent compared with inhalational anaesthetic (IA) agents. We asked if TIVA with propofol and remifentanil was associated with shorter operative times compared to IA using sevoflurane in brain tumour surgery under GA. METHODS: We performed a retrospective analysis of all patients undergoing surgery for glioblastoma (GBM). We assessed choice of GA agent (TIVA or IA) with total time patient was under GA (anaesthetic time), operative time and time taken to recover fully from GA (recovery time). RESULTS: Over a two year period 263 patients underwent surgery under GA for their GBM including 188 craniotomy operations, 63 burr hole biopsy procedures and 12 open biopsy procedures. Of these, 79 operations took place under TIVA and 184 operations under IA. TIVA was associated with significantly reduced mean operative time including time taken to wake up in theatre (104 min with TIVA, 129 min with IA; p = 0.02). TIVA was also associated with trends toward shorter mean recovery time (118 min, versus 135 min with IA; p = 0.08) and shorter mean anaesthetic time (163 min, versus 181 min with IA; p = 0.07). There was no difference between TIVA and IA groups as regards duration of inpatient stay, readmission rates, complications or survival. CONCLUSIONS: TIVA with propofol and remifentanil may reduce anaesthetic, operative and recovery times in patients undergoing surgery for their GBM. These findings may be attributable to favourable effects on intracranial pressure and cerebral perfusion, as well as rapid recovery from GA. In addition to clinical advantages, there may be financial and logistical benefits.

Spatially resolved transcriptomic profiles reveal unique defining molecular features of infiltrative 5ALA-metabolizing cells associated with glioblastoma recurrence.

BACKGROUND: Spatiotemporal heterogeneity originating from genomic and transcriptional variation was found to contribute to subtype switching in isocitrate dehydrogenase-1 wild-type glioblastoma (GBM) prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) enables intraoperative visualization of infiltrative tumors outside the magnetic resonance imaging contrast-enhanced regions. The cell population and functional status of tumor responsible for enhancing 5ALA-metabolism to fluorescence-active PpIX remain elusive. The close spatial proximity of 5ALA-metabolizing (5ALA +) cells to residual disease remaining post-surgery renders 5ALA + biology an early a priori proxy of GBM recurrence, which is poorly understood. METHODS: We performed spatially resolved bulk RNA profiling (SPRP) analysis of unsorted Core, Rim, Invasive margin tissue, and FACS-isolated 5ALA + /5ALA - cells from the invasive margin across IDH-wt GBM patients (N = 10) coupled with histological, radiographic, and two-photon excitation fluorescence microscopic analyses. Deconvolution of SPRP followed by functional analyses was performed using CIBEROSRTx and UCell enrichment algorithms, respectively. We further investigated the spatial architecture of 5ALA + enriched regions by analyzing spatial transcriptomics from an independent IDH-wt GBM cohort (N = 16). Lastly, we performed survival analysis using Cox Proportinal-Hazards model on large GBM cohorts. RESULTS: SPRP analysis integrated with single-cell and spatial transcriptomics uncovered that the GBM molecular subtype heterogeneity is likely to manifest regionally in a cell-type-specific manner. Infiltrative 5ALA + cell population(s) harboring transcriptionally concordant GBM and myeloid cells with mesenchymal subtype, -active wound response, and glycolytic metabolic signature, was shown to reside within the invasive margin spatially distinct from the tumor core. The spatial co-localization of the infiltrating MES GBM and myeloid cells within the 5ALA + region indicates PpIX fluorescence can effectively be utilized to resect the immune reactive zone beyond the tumor core. Finally, 5ALA + gene signatures were associated with poor survival and recurrence in GBM, signifying that the transition from primary to recurrent GBM is not discrete but rather a continuum whereby primary infiltrative 5ALA + remnant tumor cells more closely resemble the eventual recurrent GBM. CONCLUSIONS: Elucidating the unique molecular and cellular features of the 5ALA + population within tumor invasive margin opens up unique possibilities to develop more effective treatments to delay or block GBM recurrence, and warrants commencement of such treatments as early as possible post-surgical resection of the primary neoplasm.

Stimuli-Responsive Multifunctional Nanomedicine for Enhanced Glioblastoma Chemotherapy Augments Multistage Blood-to-Brain Trafficking and Tumor Targeting.

Minimal therapeutic advances have been achieved over the past two decades for glioblastoma (GBM), which remains an unmet clinical need. Here, hypothesis-driven stimuli-responsive nanoparticles (NPs) for docetaxel (DTX) delivery to GBM are reported, with multifunctional features that circumvent insufficient blood-brain barrier (BBB) trafficking and lack of GBM targeting-two major hurdles for anti-GBM therapies. NPs are dual-surface tailored with a i) brain-targeted acid-responsive Angiopep-2 moiety that triggers NP structural rearrangement within BBB endosomal vesicles, and ii) L-Histidine moiety that provides NP preferential accumulation into GBM cells post-BBB crossing. In tumor invasive margin patient cells, the stimuli-responsive multifunctional NPs target GBM cells, enhance cell uptake by 12-fold, and induce three times higher cytotoxicity in 2D and 3D cell models. Moreover, the in vitro BBB permeability is increased by threefold. A biodistribution in vivo trial confirms a threefold enhancement of NP accumulation into the brain. Last, the in vivo antitumor efficacy is validated in GBM orthotopic models following intratumoral and intravenous administration. Median survival and number of long-term survivors are increased by 50%. Altogether, a preclinical proof of concept supports these stimuli-responsive multifunctional NPs as an effective anti-GBM multistage chemotherapeutic strategy, with ability to respond to multiple fronts of the GBM microenvironment.

Feasibility of Photodynamic Therapy for Glioblastoma with the Mitochondria-Targeted Photosensitizer Tetramethylrhodamine Methyl Ester (TMRM).

One of the most challenging problems in the treatment of glioblastoma (GBM) is the highly infiltrative nature of the disease. Infiltrating cells that are non-resectable are left behind after debulking surgeries and become a source of regrowth and recurrence. To prevent tumor recurrence and increase patient survival, it is necessary to cleanse the adjacent tissue from GBM infiltrates. This requires an innovative local approach. One such approach is that of photodynamic therapy (PDT) which uses specific light-sensitizing agents called photosensitizers. Here, we show that tetramethylrhodamine methyl ester (TMRM), which has been used to asses mitochondrial potential, can be used as a photosensitizer to target GBM cells. Primary patient-derived GBM cell lines were used, including those specifically isolated from the infiltrative edge. PDT with TMRM using low-intensity green light induced mitochondrial damage, an irreversible drop in mitochondrial membrane potential and led to GBM cell death. Moreover, delayed photoactivation after TMRM loading selectively killed GBM cells but not cultured rat astrocytes. The efficacy of TMRM-PDT in certain GBM cell lines may be potentiated by adenylate cyclase activator NKH477. Together, these findings identify TMRM as a prototypical mitochondrially targeted photosensitizer with beneficial features which may be suitable for preclinical and clinical translation.

CovidNeuroOnc: A UK multicenter, prospective cohort study of the impact of the COVID-19 pandemic on the neuro-oncology service.

BACKGROUND: The COVID-19 pandemic has profoundly affected cancer services. Our objective was to determine the effect of the COVID-19 pandemic on decision making and the resulting outcomes for patients with newly diagnosed or recurrent intracranial tumors. METHODS: We performed a multicenter prospective study of all adult patients discussed in weekly neuro-oncology and skull base multidisciplinary team meetings who had a newly diagnosed or recurrent intracranial (excluding pituitary) tumor between 01 April and 31 May 2020. All patients had at least 30-day follow-up data. Descriptive statistical reporting was used. RESULTS: There were 1357 referrals for newly diagnosed or recurrent intracranial tumors across 15 neuro-oncology centers. Of centers with all intracranial tumors, a change in initial management was reported in 8.6% of cases (n = 104/1210). Decisions to change the management plan reduced over time from a peak of 19% referrals at the start of the study to 0% by the end of the study period. Changes in management were reported in 16% (n = 75/466) of cases previously recommended for surgery and 28% of cases previously recommended for chemotherapy (n = 20/72). The reported SARS-CoV-2 infection rate was similar in surgical and non-surgical patients (2.6% vs. 2.4%, P > .9). CONCLUSIONS: Disruption to neuro-oncology services in the UK caused by the COVID-19 pandemic was most marked in the first month, affecting all diagnoses. Patients considered for chemotherapy were most affected. In those recommended surgical treatment this was successfully completed. Longer-term outcome data will evaluate oncological treatments received by these patients and overall survival.

Biomedical engineering approaches to enhance therapeutic delivery for malignant glioma.

We review the challenges of next-generation therapeutics for both systemic and localised delivery to brain tumours and discuss how recent engineering advances may be used to enhance brain penetration of systemic delivery therapies. The unmet clinical need which drug delivery seeks to address is discussed with reference to the therapy obstacles that the intra-tumour heterogeneity of glioma present. The unmet chemistry and biomedical engineering challenge to develop controlled release therapeutics is appraised, with commentary on current success/failures in systemic carrier-mediated delivery, including receptor-targeted, cell-based, blood-brain-barrier disrupting and MRI-guided focused ultrasound. Localised therapeutic delivery is a relatively under-studied research avenue and is discussed with reference to existing technologies in preclinical development. These include convection-enhanced delivery, alternative catheter delivery, and neuro-surgically applied delivery systems such as polymeric hydrogels and interstitial spray. A myriad of nano-scale therapeutic delivery systems is emerging as potential future medicines for malignant brain tumours. Such biomedically-engineered systems will increasingly feature in next-generation neuro-oncological clinical trials to deliver repurposed and experimental therapeutics, aimed at achieving therapeutic drug concentrations in the brain, with associated mortality and morbidity benefits for patients.

Etoposide and olaparib polymer-coated nanoparticles within a bioadhesive sprayable hydrogel for post-surgical localised delivery to brain tumours.

Glioblastoma is a malignant brain tumour with a median survival of 14.6 months from diagnosis. Despite maximal surgical resection and concurrent chemoradiotherapy, reoccurrence is inevitable. To try combating the disease at a stage of low residual tumour burden immediately post-surgery, we propose a localised drug delivery system comprising of a spray device, bioadhesive hydrogel (pectin) and drug nanocrystals coated with polylactic acid-polyethylene glycol (NCPPs), to be administered directly into brain parenchyma adjacent to the surgical cavity. We have repurposed pectin for use within the brain, showing in vitro and in vivo biocompatibility, bio-adhesion to mammalian brain and gelling at physiological brain calcium concentrations. Etoposide and olaparib NCPPs with high drug loading have shown in vitro stability and drug release over 120 h. Pluronic F127 stabilised NCPPs to ensure successful spraying, as determined by dynamic light scattering and transmission electron microscopy. Successful delivery of Cy5-labelled NCPPs was demonstrated in a large ex vivo mammalian brain, with NCPP present in the tissue surrounding the resection cavity. Our data collectively demonstrates the pre-clinical development of a novel localised delivery device based on a sprayable hydrogel containing therapeutic NCPPs, amenable for translation to intracranial surgical resection models for the treatment of malignant brain tumours.

Identifying the spatial and temporal dynamics of molecularly-distinct glioblastoma sub-populations.

Glioblastomas (GBMs) are the most aggressive primary brain tumours and have no known cure. Each individual tumour comprises multiple sub-populations of genetically-distinct cells that may respond differently to targeted therapies and may contribute to disappointing clinical trial results. Image-localized biopsy techniques allow multiple biopsies to be taken during surgery and provide information that identifies regions where particular sub-populations occur within an individual GBM, thus providing insight into their regional genetic variability. These sub-populations may also interact with one another in a competitive or cooperative manner; it is important to ascertain the nature of these interactions, as they may have implications for responses to targeted therapies. We combine genetic information from biopsies with a mechanistic model of interacting GBM sub-populations to characterise the nature of interactions between two commonly occurring GBM sub-populations, those with EGFR and PDGFRA genes amplified. We study population levels found across image-localized biopsy data from a cohort of 25 patients and compare this to model outputs under competitive, cooperative and neutral interaction assumptions. We explore other factors affecting the observed simulated sub-populations, such as selection advantages and phylogenetic ordering of mutations, which may also contribute to the levels of EGFR and PDGFRA amplified populations observed in biopsy data.

Defective heart chamber growth and myofibrillogenesis after knockout of adprhl1 gene function by targeted disruption of the ancestral catalytic active site.

ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibian Xenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of the adprhl1 gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lacking Adprhl1 exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.

Using Light for Therapy of Glioblastoma Multiforme (GBM).

: Glioblastoma multiforme (GBM) is the most malignant form of primary brain tumour with extremely poor prognosis. The current standard of care for newly diagnosed GBM includes maximal surgical resection followed by radiotherapy and adjuvant chemotherapy. The introduction of this protocol has improved overall survival, however recurrence is essentially inevitable. The key reason for that is that the surgical treatment fails to eradicate GBM cells completely, and adjacent parenchyma remains infiltrated by scattered GBM cells which become the source of recurrence. This stimulates interest to any supplementary methods which could help to destroy residual GBM cells and fight the infiltration. Photodynamic therapy (PDT) relies on photo-toxic effects induced by specific molecules (photosensitisers) upon absorption of photons from a light source. Such toxic effects are not specific to a particular molecular fingerprint of GBM, but rather depend on selective accumulation of the photosensitiser inside tumour cells or, perhaps their greater sensitivity to the effects, triggered by light. This gives hope that it might be possible to preferentially damage infiltrating GBM cells within the areas which cannot be surgically removed and further improve the chances of survival if an efficient photosensitiser and hardware for light delivery into the brain tissue are developed. So far, clinical trials with PDT were performed with one specific type of photosensitiser, protoporphyrin IX, which tends to accumulate in the cytoplasm of the GBM cells. In this review we discuss the idea that other types of molecules which build up in mitochondria could be explored as photosensitisers and used for PDT of these aggressive brain tumours.

Overall Survival in Malignant Glioma Is Significantly Prolonged by Neurosurgical Delivery of Etoposide and Temozolomide from a Thermo-Responsive Biodegradable Paste.

PURPOSE: High-grade glioma (HGG) treatment is limited by the inability of otherwise potentially efficacious drugs to penetrate the blood-brain barrier. We evaluate the unique intracavity delivery mode and translational potential of a blend of poly(DL-lactic acid-co-glycolic acid; PLGA) and poly(ethylene glycol; PEG) paste combining temozolomide and etoposide to treat surgically resected HGG. EXPERIMENTAL DESIGN: To prolong stability of temozolomide prodrug, combined in vitro drug release was quantitatively assessed from low pH-based PLGA/PEG using advanced analytic methods. In vitro cytotoxicity was measured against a panel of HGG cell lines and patient-derived cultures using metabolic assays. In vivo safety and efficacy was evaluated using orthotopic 9L gliosarcoma allografts, previously utilized preclinically to develop Gliadel. RESULTS: Combined etoposide and temozolomide in vitro release (22 and 7 days, respectively) was achieved from a lactic acid-based PLGA/PEG paste, used to enhance stability of temozolomide prodrug. HGG cells from central-enhanced regions were more sensitive to each compound relative to primary lines derived from the HGG-invasive margin. Both drugs retained cytotoxic capability upon release from PLGA/PEG. In vivo studies revealed a significant overall survival benefit in postsurgery 9L orthotopic gliosarcomas, treated with intracavity delivered PLGA/PEG/temozolomide/etoposide and enhanced with adjuvant radiotherapy. Long-term survivorship was observed in over half the animals with histologic confirmation of disease-free brain. CONCLUSIONS: The significant survival benefit of intracavity chemotherapy demonstrates clinical applicability of PLGA/PEG paste-mediated delivery of temozolomide and etoposide adjuvant to radiotherapy. PLGA/PEG paste offers a future platform for combination delivery of molecular targeted compounds.

The Invasive Region of Glioblastoma Defined by 5ALA Guided Surgery Has an Altered Cancer Stem Cell Marker Profile Compared to Central Tumour.

Glioblastoma, a WHO grade IV astrocytoma, is a highly aggressive and heterogeneous tumour that infiltrates deeply into surrounding brain parenchyma, making complete surgical resection impossible. Despite chemo-radiotherapy, the residual cell population within brain parenchyma post-surgery causes inevitable recurrence. Previously, the tumour core has been the focus of research and the basis for targeted therapeutic regimes, which have failed to improve survival in clinical trials. Here, we focus on the invasive margin as defined by the region with 5-aminolevulinic acid (5ALA) (GliolanTM) fluorescence at surgery beyond the T1 enhancing region on magnetic resonance imaging (MRI). This area is hypothesized to constitute unique microenvironmental pressures, and consequently be molecularly distinct to tumour core and enhancing rim regions. We conducted hematoxylin and eosin (H&E), array real time polymerase chain reaction (PCR), and immunohistochemistry staining on various intra-tumour regions of glioblastoma to determine molecular heterogeneity between regions. We analyzed 73 tumour samples from 21 patients and compared cellular density, cell proliferation, and the degree of vascularity. There is a statistically significant difference between the core, invasive margin and other regions for cell density (p < 0.001), cell proliferation (p = 0.029), and vascularity (p = 0.007). Aldehyde dehydrogenase 1 (ALDH1) and Nestin immunohistochemistry were used as a measure of stem-like properties, showing significantly decreased Nestin expression (p < 0.0001) in the invasive margin. Array PCR of the core, rim, and invasive regions showed significantly increased fibroblast growth factor (FGF) and ALDH1 expression in the invasive zone, with elevated hypoxia inducing factor 1-alpha (HIF1α) in the rim region, adjacent to the hypoxic core. The influence of varying microenvironments in the intra-tumour regions is a major key to understanding intra-tumour heterogeneity. This study confirms the distinct molecular composition of the heterogeneous invasive margin and cautions against purported therapy strategies that target candidate glioblastoma stem-like genes that are predominantly expressed in the tumour core. Full characterization of tumour cells in the invasive margin is critical, as these cells may more closely resemble the residual cell population responsible for tumour recurrence. Their unique nature should be considered when developing targeted agents for residual glioblastoma multiforme (GBM).

The Molecular and Phenotypic Basis of the Glioma Invasive Perivascular Niche.

Gliomas are devastating brain cancers that have poor prognostic outcomes for their patients. Short overall patient survival is due to a lack of durable, efficacious treatment options. Such therapeutic difficulties exist, in part, due to several glioma survival adaptations and mechanisms, which allow glioma cells to repurpose paracrine signalling pathways and ion channels within discreet microenvironments. These Darwinian adaptations facilitate invasion into brain parenchyma and perivascular space or promote evasion from anti-cancer defence mechanisms. Ultimately, this culminates in glioma repopulation and migration at distances beyond the original tumour site, which is a considerable obstacle for effective treatment. After an era of failed phase II trials targeting individual signalling pathways, coupled to our increasing knowledge of glioma sub-clonal divergence, combinatorial therapeutic approaches which target multiple molecular pathways and mechanisms will be necessary for better treatment outcomes in treating malignant gliomas. Furthermore, next-generation therapy which focuses on infiltrative tumour phenotypes and disruption of the vascular and perivascular microenvironments harbouring residual disease cells offers optimism for the localised control of malignant gliomas.

Endothelial-like malignant glioma cells in dynamic three dimensional culture identifies a role for VEGF and FGFR in a tumor-derived angiogenic response.

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.

RHPS4 G-quadruplex ligand induces anti-proliferative effects in brain tumor cells.

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.

Adjuvant chemotherapy for brain tumors delivered via a novel intra-cavity moldable polymer matrix.

INTRODUCTION: Polymer-based delivery systems offer innovative intra-cavity administration of drugs, with the potential to better target micro-deposits of cancer cells in brain parenchyma beyond the resected cavity. Here we evaluate clinical utility, toxicity and sustained drug release capability of a novel formulation of poly(lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles. METHODS: PLGA/PEG microparticle-based matrices were molded around an ex vivo brain pseudo-resection cavity and analyzed using magnetic resonance imaging and computerized tomography. In vitro toxicity of the polymer was assessed using tumor and endothelial cells and drug release from trichostatin A-, etoposide- and methotrexate-loaded matrices was determined. To verify activity of released agents, tumor cells were seeded onto drug-loaded matrices and viability assessed. RESULTS: PLGA/PEG matrices can be molded around a pseudo-resection cavity wall with no polymer-related artifact on clinical scans. The polymer withstands fractionated radiotherapy, with no disruption of microparticle structure. No toxicity was evident when tumor or endothelial cells were grown on control matrices in vitro. Trichostatin A, etoposide and methotrexate were released from the matrices over a 3-4 week period in vitro and etoposide released over 3 days in vivo, with released agents retaining cytotoxic capabilities. PLGA/PEG microparticle-based matrices molded around a resection cavity wall are distinguishable in clinical scanning modalities. Matrices are non-toxic in vitro suggesting good biocompatibility in vivo. Active trichostatin A, etoposide and methotrexate can be incorporated and released gradually from matrices, with radiotherapy unlikely to interfere with release. CONCLUSION: The PLGA/PEG delivery system offers an innovative intra-cavity approach to administer chemotherapeutics for improved local control of malignant brain tumors.

Recapitulation of tumor heterogeneity and molecular signatures in a 3D brain cancer model with decreased sensitivity to histone deacetylase inhibition.

INTRODUCTION: Physiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS). METHODS: CNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat. RESULTS: Macroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches. CONCLUSIONS: Our comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system.

CD105 (Endoglin) exerts prognostic effects via its role in the microvascular niche of paediatric high grade glioma.

Paediatric high grade glioma (pHGG) (World Health Organisation astrocytoma grades III and IV) remains poor prognosis tumours, with a median survival of only 15 months following diagnosis. Current investigation of anti-angiogenic strategies has focused on adult glioblastoma multiforme (GBM) with phase III trials targeting vascular endothelial growth factor continuing. In this study we investigated whether the degree of vascularity correlated with prognosis in a large cohort of pHGG (n = 150) and whether different vessel markers carried different prognostic value. We found that CD105 (endoglin) had a strongly significant association with poor prognosis on multivariate analysis (p = <0.001). Supervised hierarchical clustering of genome wide gene expression data identified 13 genes associated with differential degrees of vascularity in the cohort. The novel angiogenesis-associated genes identified in this analysis (including MIPOL-1 and ENPP5) were validated by realtime polymerase chain reaction. We also demonstrate that CD105 positive blood vessels associate with CD133 positive tumour cells and that a proportion of CD105 positive vessel cells demonstrates co-positivity for CD133, suggesting that the recently described phenomenon of vasculogenic mimicry occurs in pHGG. Together, the data suggest that targeting angiogenesis, and in particular CD105, is a valid therapeutic strategy for pHGG.