CBMT-17. NOVEL APPROACH OF UTILISING SERUM/PLASMA EV AND CELL-FREE RNA FOR TREATMENT MONITORING IN GLIOBLASTOMA PATIENTS

INTRODUCTION: Glioblastoma (GBM) is a malignant primary brain tumour with dismal prognosis. Treatment monitoring remains a challenge in clinical routine, since brain imaging cannot reliably differentiate between true progression and treatment-associated changes. In this project, we evaluate different methods of extracellular vesicles (EV) purification, in order to specifically isolate GBM-EVs from human serum/plasma and introduce EVs, as well as cell-free RNA as possible biomarkers for treatment monitoring in GBM patients. METHODS: EVs from primary GBM cells and the Gaussia luciferase expressing Gli36-GLuc cells were isolated via size-exclusion chromatography (SEC) and ultracentrifugation. EV-surface markers were evaluated by flow cytometry. Gli36-GLuc EVs containing GLuc mRNA were spiked in healthy plasma. Thereafter, plasma EVs were isolated via ultracentrifugation, SEC and immunoprecipitation. Subsequently, RNA was isolated from vesicles and evaluated for GLuc levels via qRT-PCR. Total cell-free RNA from serum of GBM patients was tested for different mRNAs and micro-RNAs at different disease stages. RESULTS: EVs from GBM cells expressed high levels of CD29 and CD44, when compared to EVs from healthy donor plasma. Gli36-GLuc EVs spiked in healthy plasma were more effectively isolated with CD44-based immunoprecipitation than with ultracentrifugation or SEC, as shown by higher GLuc RNA levels in the corresponding vesicles. When compared to total cell-free RNA extracted from this plasma, RNA from EVs exhibited a higher GLuc yield. In cell-free RNA from GBM patients, MGMT levels alone were not capable of detecting progressive disease. CONCLUSIONS: 1. CD44 could serve as a novel, promising target for GBM-EV and be utilised for immunoprecipitation-based EV capturing. 2. Using the appropriate EV purification method possibly affects their potential as biomarkers for GBM. 3. MGMT levels alone in cell-free RNA of GBM patients did not correlate with disease status contrary to previous reports.