Abstract
Drugs that impair tumour angiogenesis, i.e. therapeutic antibody anti-vascular endothelial growth factor, bevacizumab (BEV), are becoming standard therapy for recurrent GBM, despite having no impact on overall survival times. Resistance to BEV is fatal, and mechanisms are largely unexplored. With access to exceedingly rare fresh-frozen serial GBM tumours, we performed comprehensive quantitative proteome analyses to identify important mechanisms of BEV escape and tumour recurrence. Tumour tissues from three patients [primary (n = 2), recurrent (n = 2) and post-BEV recurrent (n = 3)] were homogenised, clarified (1,000 x g, 4°C) and ultracentrifuged (100,000 x g, 4°C) to isolate the soluble (SOL) proteome supernatant from the microsomal (MEM) pellet. Digested SOL and MEM proteomes were analysed by two independent quantitative MS/MS approaches; Label-free quantitation performed on spectra obtained in triplicate using an Orbitrap Velos (Thermo Electron) and 4-plex iTRAQ-labelling coupled ERLIC-RP MS/MS analysis using a 5600 TripleTOF(®) (AB Sciex; single run for MEM; duplicate run for SOL). Spectra were processed using Mascot Distiller, Progenesis, Scaffold and ProteinPilot™ softwares. This multi-centre proteomics project has achieved a number of highly reproducible and comprehensive quantitative proteome datasets (average of 1760 MEM proteins and 2334 SOL proteins identified at 95% confidence levels) from precious serial GBM specimens. Significant differentially abundant proteins include those involved in Rho regulation of actin-based motility and cytoskeleton and endocytosis signalling. Bioinformatics analyses with captured whole exome sequencing data are underway to define novel mechanisms of evasive resistance to BEV in recurrent GBM.