Oral Presentation Australasian Extracellular Vesicles Conference 2020

SWATH-mass spectrometry as a biomarker discovery platform for comprehensive proteomic profiling of plasma-derived extracellular vesicles for glioma tumour monitoring. (#14)

Susannah Hallal 1 2 3 , Heng Wei 3 4 , Maggie Lee 3 4 , Hao-Wen Sim 5 6 7 , Brindha Shivalingam 1 3 , Michael E. Buckland 2 3 4 , Kimberley L. Kaufman 1 2 3 4
  1. Neurosurgery Department, Chris O'Brien Lifehouse, Sydney, NSW, Australia
  2. School of Medical Sciences, University of Sydney, Sydney, NSW, Australia
  3. BrainStorm Brain Cancer Research Group, Brain and Mind Centre, University of Sydney, Sydney, NSW, Australia
  4. Department of Neuropathology, Royal Prince Alfred Hospital, Sydney, NSW, Australia
  5. Department of Medical Oncology and NHMRC Clinical Trials Centre, Chris O'Brien Lifehouse, Sydney, NSW, Australia
  6. Central Clinical School, University of Sydney, Sydney, NSW, Australia
  7. The Kinghorn Cancer Centre, St Vincent’s Hospital, Sydney, NSW, Australia

Diffuse gliomas (grades II-IV) are the most frequent and devastating primary brain tumours of adults. The current clinical management for glioma patients involves a tissue biopsy for diagnostics and routine neuro-radiographic assessments, both of which have limitations for accurate clinical assessment. With efforts to improve the clinical management of glioma, comes a growing trend to design minimally-invasive liquid biopsies (i.e. blood tests) that can measure glioma-derived molecules in body fluids and allow for tumour evolution to be assessed in real-time. In this regard, extracellular vesicles (EVs; 30-1000 nm membranous particles) hold major promise as biomarker reservoirs. EVs encapsulate molecules from their cell-of-origin, play integral roles in glioma biology and cross the blood-brain-barrier into the periphery where they are stable and readily accessible. Despite their suitability as biomarkers, in-depth proteomic characterisation of circulating-EVs by traditional shot-gun proteomic methods has been hindered by the complexity of the blood. In this study, a data-independent acquisition (DIA) proteomics platform, sequential window acquisition of all theoretical fragment ion spectra (SWATH), was used in conjunction with a targeted data extraction strategy to profile circulating-EVs from glioma patients. EVs were isolated by size exclusion chromatography from the plasma of preoperative glioma II-IV patients (n=41) and controls (n = 11). Nanoparticle tracking and transmission electron microscopy confirmed the isolation of small-EV subtypes (< 200 nm). The plasma-EV peptides were sequenced by SWATH-MS, and the identities and absolute quantities of the proteins were extracted using a comprehensive custom spectral library comprised of 8662-protein species. A total of 4054 proteins were identified in the plasma-EVs of all sample groups (q-value < 0.05). Of these proteins, 463 changed significantly across the glioma and non-glioma cohorts (p < 0.05), and included proteins previously reported to have significance in GBM-EVs. Principal component analysis showed excellent discrimination between the patient groups, with samples observed to cluster with their respective glioma subtype/grade. Using SWATH mass spectrometry we describe the most comprehensive proteomic plasma-EV profiles for glioma reported to-date, for which future studies using larger longitudinal cohorts could define a set of circulating-EV biomarkers, capable of stratifying glioma patients and detecting recurrence, progression and treatment resistance.