Improving outcomes for diffuse glioma patients requires methods that can accurately and sensitively monitor tumour activity and treatment response. Extracellular vesicles (EV) are membranous nanoparticles that can traverse the blood–brain-barrier, carrying oncogenic molecules into the circulation. Measuring clinically relevant glioma biomarkers cargoed in circulating EVs could revolutionise how glioma patients are managed. Despite their suitability for biomarker discovery, the co-isolation of highly abundant complex blood proteins has hindered comprehensive proteomic studies of circulating-EVs. Plasma-EVs isolated from pre-operative glioma grade II–IV patients (n = 41) and controls (n = 11) were sequenced by Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) and data extraction was performed by aligning against a custom 8662-protein library. Overall, 4054 proteins were measured in plasma-EVs. Differentially expressed proteins and putative circulating-EV markers were identified (adj. p-value < 0.05), including those reported in previous in-vitro and ex-vivo glioma-EV studies. Principal component analysis showed that plasma-EV protein profiles clustered according to glioma histological-subtype and grade, and plasma-EVs resampled from patients with recurrent tumour progression grouped with more aggressive glioma samples. The extensive plasma-EV proteome profiles achieved here highlight the potential for SWATH-MS to define circulating-EV biomarkers for objective blood-based measurements of glioma activity that could serve as ideal surrogate endpoints to assess tumour progression and allow more dynamic, patient-centred treatment protocols.

Extracellular vesicles (EVs) play key roles in glioblastoma (GBM; astrocytoma grade IV) biology and are novel sources of biomarkers. EVs released from GBM tumors can cross the blood-brain-barrier into the periphery carrying GBM molecules, including small non-coding RNA (sncRNA). Biomarkers cargoed in circulating EVs have shown great promise for assessing the molecular state of brain tumors in situ. Neurosurgical aspirate fluids captured during tumor resections are a rich source of GBM-EVs isolated directly from tumor microenvironments. Using density gradient ultracentrifugation, EVs were purified from cavitron ultrasonic surgical aspirate (CUSA) washings from GBM (n = 12) and astrocytoma II-III (GII-III, n = 5) surgeries. The sncRNA contents of surgically captured EVs were profiled using the Illumina^® NextSeq^TM 500 NGS System. Differential expression analysis identified 27 miRNA and 10 piRNA species in GBM relative to GII-III CUSA-EVs. Resolved CUSA-EV sncRNAs could discriminate serum-EV sncRNA profiles from GBM and GII-III patients and healthy controls and 14 miRNAs (including miR-486-3p and miR-106b-3p) and cancer-associated piRNAs (piR_016658, _016659, _020829 and _204090) were also significantly expressed in serum-EVs. Circulating EV markers that correlate with histological, neuroradiographic and clinical parameters will provide objective measures of tumor activity and improve the accuracy of GBM tumor surveillance.

There is a real need for biomarkers that can indicate glioma disease burden and inform clinical management, particularly in the recurrent glioblastoma (GBM; grade IV glioma) setting where treatment-associated brain changes can confound current and expensive tumour surveillance methods. In this regard, extracellular vesicles (EVs; 30-1000 nm membranous particles) hold major promise as robust tumour biomarkers. GBM-EVs encapsulate molecules that reflect the identity and molecular state of their cell-of-origin and cross the blood-brain-barrier into the periphery where they are readily accessible. Despite the suitability of circulating-EVs for GBM biomarker discovery, sample complexity has hindered comprehensive quantitative proteomic studies. Here, sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) was used in conjunction with a targeted data extraction strategy to comprehensively profile circulating-EVs isolated from plasma. Plasma-EVs sourced from pre-operative glioma II-IV patients ( n =41) and controls ( n =11) were sequenced by SWATH-MS, and the identities and absolute quantities of the proteins were extracted by aligning the SWATH-MS data against a custom glioma spectral library comprised of 8662 high confidence protein species. Overall, 4054 plasma-EV proteins were quantified across the cohorts, and putative circulating-EV biomarker proteins identified ( adjusted p-value<0.05) included previously reported GBM-EV proteins identified in vitro and in neurosurgical aspirates. Principle component analyses showed that plasma-EV protein profiles clustered according to glioma subtype and WHO-grade, and plasma-EV proteins reflected the extent of glioma aggression. Using SWATH-MS, we describe the most comprehensive proteomic plasma-EV profiles for glioma and highlight the promise of this approach as an accurate and sensitive tumour monitoring method. Objective blood-based measurements of glioma tumour activity will support the implementation of next-generation, patient-centred therapies and are ideal surrogate endpoints for recurrent progression that would allow clinical trial protocols to be more dynamic and adapt to the individual patient and their cancer.

RNA modifications are diverse, dynamic, and reversible transcript alterations rapidly gaining attention due to their newly defined RNA regulatory roles in cellular pathways and pathogenic mechanisms. The exciting emerging field of ‘epitranscriptomics’ is predominantly centred on studying the most abundant mRNA modification, N6-methyladenine (m⁶A). The m⁶A mark, similar to many other RNA modifications, is strictly regulated by so-called ‘writer’, ‘reader’, and ‘eraser’ protein species. The abundance of genes coding for the expression of these regulator proteins and m⁶A levels shows great potential as diagnostic and predictive tools across several cancer fields. This review explores our current understanding of RNA modifications in glioma biology and the potential of epitranscriptomics to develop new diagnostic and predictive classification tools that can stratify these highly complex and heterogeneous brain tumours.

(1) Background: MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation remains an important predictive biomarker in high-grade gliomas (HGGs). The influence of necrosis on the fidelity of MGMT promoter (MGMTp) hypermethylation testing is currently unknown. Therefore, our study aims to evaluate the effect of varying degrees of necrosis on MGMTp status, as determined by pyrosequencing, in a series of primary and recurrent HGGs; (2) Methods: Within each case, the most viable blocks (assigned as ‘true’ MGMTp status) and the most necrotic block were determined by histopathology review. MGMTp status was determined by pyrosequencing. Comparisons of MGMTp status were made between the most viable and most necrotic blocks. (3) Results: 163 samples from 64 patients with HGGs were analyzed. MGMTp status was maintained in 84.6% of primary and 78.3% of recurrent HGGs between the most viable and necrotic blocks. A threshold of ≥60% tumor cellularity was established at which MGMTp status was unaltered, irrespective of the degree of necrosis. (4) Conclusions: MGMTp methylation status, as determined by pyrosequencing, does not appear to be influenced by necrosis in the majority of cases at a cellularity of at least 60%. Further investigation into the role of intratumoral heterogeneity on MGMTp status will increase our understanding of this predictive marker.

Tangential flow filtration (TFF) is an effective method for scalable EV isolation. Here, we describe and validate a reference protocol for TriPort-TFF, a laboratory-assembled TFF system featuring a three-port reservoir design enabling sample circulation, retentate return, and diafiltration. Constructed from individually sourced, commercially available components with detailed assembly and operating parameters, TriPort-TFF can be built at a lower cost than commercially integrated TFF platforms. We evaluated the system using conditioned media (CM) from human MDM cultured with EV-depleted human serum, human cerebral organoids in serum-free medium, and mouse BMDM cultured with EV-depleted foetal bovine serum (FBS) unstimulated or LPS-stimulated. The system reproducibly isolated EVs from all three sources, and biological replicates of BMDM showed distinct proteomic clusters between LPS-stimulated and unstimulated conditions. The requirement for subsequent size-exclusion chromatography (SEC) after TFF depended on the sample’s serum-presence. In serum-containing CM, TFF alone co-concentrated serum proteins alongside EVs, subsequent SEC improved the particle-to-protein ratio, reduced inter-replicate variability and improved proteomic depth. TFF alone in serum-free organoid CM already yielded higher particle-to-protein ratios than TFF-only preparations from serum-containing CM, with SEC further improving this ratio. TriPort-TFF offers an accessible system for EV-isolation that can be combined with SEC for diverse sample types.