New approaches for the management of glioblastoma (GBM) are an urgent and unmet clinical need. Here, we illustrate that the efficacy of radiotherapy for GBM is strikingly potentiated by concomitant therapy with the arginine-depleting agent ADI-PEG20 in a non-arginine-auxotrophic cellular background (argininosuccinate synthetase 1 positive). Moreover, this combination led to durable and complete radiological and pathological response, with extended disease-free survival in an orthotopic immune-competent model of GBM, with no significant toxicity. ADI-PEG20 not only enhanced the cellular sensitivity of argininosuccinate synthetase 1–positive GBM to ionizing radiation by elevated production of nitric oxide (˙NO) and hence generation of cytotoxic peroxynitrites, but also promoted glioma-associated macrophage/microglial infiltration into tumors and turned their classical antiinflammatory (protumor) phenotype into a proinflammatory (antitumor) phenotype. Our results provide an effective, well-tolerated, and simple strategy to improve GBM treatment that merits consideration for early evaluation in clinical trials.

Tumour cells have a high demand for arginine. However, a subset of glioblastomas has a defect in the arginine biosynthetic pathway due to epigenetic silencing of the rate limiting enzyme argininosuccinate synthetase (ASS1). These tumours are auxotrophic for arginine and susceptible to the arginine degrading enzyme, pegylated arginine deiminase (ADI-PEG20). Moreover, ASS1 deficient GBM have a worse prognosis compared to ASS1 positive tumours. Since altered tumour metabolism is one of the hallmarks of cancer we were interested to determine if these two subtypes exhibited different metabolic profiles that could allow for their non-invasive detection as well as unveil additional novel therapeutic opportunities. We looked for basal metabolic differences using one and two-dimensional gas chromatography-time-of-flight mass spectrometry (1D/2D GC-TOFMS) followed by targeted analysis of 29 amino acids using liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS). We also looked for differences upon arginine deprivation in a single ASS1 negative and positive cell line (SNB19 and U87 respectively). The acquired data was evaluated by chemometric based bioinformatic methods. Orthogonal partial least squares-discriminant analysis (OPLS-DA) of both the 1D and 2D GC-TOFMS data revealed significant systematic difference in metabolites between the two subgroups with ASS1 positive cells generally exhibiting an overall elevation of identified metabolites, including those involved in the arginine biosynthetic pathway. Pathway and network analysis of the metabolite profile show that ASS1 negative cells have altered arginine and citrulline metabolism as well as altered amino acid metabolism. As expected, we observed significant metabolite perturbations in ASS negative cells in response to ADI-PEG20 treatment. This study has highlighted significant differences in the metabolome of ASS1 negative and positive GBM which warrants further study to determine their diagnostic and therapeutic potential for the treatment of this devastating disease.

Glioblastoma (GBM) cells require high levels of nicotinamide adenine dinucleotide (NAD) to fuel metabolic reactions, regulate their cell cycle and support DNA repair in response to chemotherapy and radiation. Inhibition of a key enzyme in NAD biosynthesis, NAMPT, has demonstrated significant anti-neoplastic activity. Here, we sought to characterise NAD biosynthetic pathways in GBM to determine resistance mechanisms to NAD inhibitors. GBM cells were treated with the NAMPT inhibitor FK866 with and without NAD precursors, and were analysed by qPCR, Western blot and proliferation assays (monolayer and spheroid). We also measured changes in the cell cycle, apoptosis, NAD/NADH levels and energy production. We performed orthoptic xenograft experiments in athymic nude mice to test the efficacy of FK866 in combination with temozolomide (TMZ). We show that the expression of key genes involved in NAD biosynthesis is highly variable across GBM tumours. FK866 inhibits proliferation, reduces NAD levels and limits oxidative metabolism, leading to G2/M cell cycle arrest; however, this can be reversed by supplementation with specific NAD precursors. Furthermore, FK866 potentiates the effects of radiation and TMZ in vitro and in vivo. NAMPT inhibitors should be considered for the treatment of GBM, with patients stratified based on their expression of key enzymes in other NAD biosynthetic pathways.

Background: We have retrospectively reviewed our series of brain tumor patients operated on using 3D IntraOperative UltraSound (IOUS) to report technical advantages and areas of improvement. Methods: Clinical and radiological data of patients with a diagnosis of high-grade glioma IV operated with and without IOUS were retrieved and analyzed. Results: We have found 391 patients operated using IOUS coupled with neuronavigation and 257 using neuronavigation standalone. We have selected a pool of 60 patients with a diagnosis of GlioBlastoma (GB), comparing two equally sized groups operated with and without IOUS, respectively. The average extent of resection (EOR) in the IOUS group was 93%, while in the control group, it was 80%. IOUS was significantly associated with improved EOR (P < 0.0004), even when accounting for other factors affecting EOR. The average overall survival (OS) was 13.4 months, and the average progression-free survival (PFS) was 7.4 months. The Cox proportional hazard model showed an advantage in OS on patients operated using the IOUS. No statistically significant effect was observed on PFS. Conclusion: Intraoperative ultrasound coupled with image guidance is associated with an improved EOR and possibly an improved OS. While we are aware of several limitations related to the present analysis, these data support the routine use of IOUS as a safe and reliable technology. Larger, prospective series with updated IOUS technology are desirable to verify the accuracy of these results.

Background: Glioblastoma (GB) is a particularly malignant brain tumour which carries a poor prognosis and presents limited treatment options. MRI is standard practice for differential diagnosis at initial presentation of GB and can assist in both treatment planning and response assessment. MRI radiomics allows for discerning GB features of clinical importance that are not evident by visual analysis, augmenting the morphological and functional tumour characterisation beyond traditional imaging techniques. Given that radiotherapy is part of the standard of care for GB patients, establishing a platform for phenotyping radiation treatment responses using non-invasive methods is of high relevance. Methods: In this study, we modelled the responses to ionising radiation across four orthotopic mouse models of GB using diffusion and perfusion radiomics. We have identified the optimal set of radiomic features that reflect tumour cellularity, microvascularity, and blood flow changes brought about by radiation treatment in these murine orthotopic models of GB, and directly compared them with endpoint histopathological analysis. Results: We showed that the selected radiomic features can quantify textural information and pixel interrelationships of tumour response to radiation therapy, revealing subtle image patterns that may reflect intra-tumoural spatial heterogeneity. When compared to GB patients, similarities in selected radiomic features were noted between orthotopic murine tumours and non-enhancing central tumour areas in patients, along with several discrepancies in tumour cellularity and vascularization, denoted by distinct grey level intensities and nonuniformity metrics. Conclusion: As the field evolves, radiomic profiling of GB may enhance the evaluation of targeted therapeutic strategies, accelerate the development of new therapies, and act as a potential virtual biopsy tool.