A comprehensive proteomic SWATH-MS workflow for profiling blood extracellular vesicles: a new avenue for glioma tumour surveillance

Abstract

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.

Tasmanian devil facial tumor-derived extracellular vesicles reveal mesenchymal transition markers and adhesion molecules related to metastasis

“…Thus, the use of cell cultures enables the study of EVs derived from a single and well-defined cell type and the ability to manipulate conditions to reduce these body fluid contaminants (54). Further, we were able to optimize coverage of EV proteins in this study with DIA-MS techniques and targeted data analysis using EV protein spectral libraries (55,56). However, our findings must be considered in light of the challenges to extrapolate results from in vitro to in vivo models, and also corroborating them using functional assays.…”

Next-Generation Sequencing Comparative Analysis of DNA Mutations between Blood-derived Extracellular Vesicles and Matched Cancer Tissue in Patients with IV Grade Glioblastoma

“…Extracellular vesicles (EVs) have been found in biofluids and represent an intercellular mechanism of biomolecules transport (6,7) including by lipids (8), proteins (9) and nucleic acids (10). Glioblastoma cells release EVs in the local microenvironment and can be detected in the bloodstream, being able to trespass the blood brain barrier, which is often compromised in these patients, mediating angiogenesis, proliferation, immunomodulation, carcinogenesis, and invasiveness (11)(12)(13).…”