CBM-14GLIOBLASTOMA CELLS EXPOSED TO 5-ALA RELEASE PROTOPORPHYRIN IX CONTAINING EXTRACELLULAR VESICLES DETECTABLE BY HIGH-RESOLUTION FLOW CYTOMETRY

Abstract

INTRODUCTION: Gliolan or 5-aminolevulinic acid (5-ALA) is used for fluorescence-guided neurosurgery in glioblastoma multiforme (GBM) patients. 5-ALA is part of the heme synthesis pathway. In GBM cells, accumulation of fluorescent protoporphyrin IX (PpIX) is observed after oral intake, providing the neurosurgeon with visual guidance during surgery. Extracellular vesicles (EVs) or exosomes are nano-sized (50-1000 nm) lipid membrane-enclosed vesicles released by cells. We hypothesized that EVs from fluorescent GBM cells can contain PpIX and are thus fluorescent as well, allowing for tumor derived extracellular vesicle detection by high-resolution flow cytometry. METHODS: U87-MG/EGFRvIII cells were cultured for 24-hours in presence of 500uM 5-ALA. Extracellular vesicles were isolated from cell culture supernatant by sequential (ultra)centrifugation and analysed using high-resolution flow cytometry. Fluorescent EVs were spiked into platelet-free plasma from healthy donors at different concentrations. Subsequently, plasma samples from GBM patients treated with 5-ALA were studied using high-resolution flow-cytometry. RESULTS: GBM cells treated with 5-ALA released fluorescent extracellular vesicles, which could be detected by high-resolution flow cytometry (488 nm excitation). These fluorescent EVs could be reliably quantified when spiked into healthy donor platelet-free plasma. In platelet-free plasma from GBM patients treated with 5-ALA, fluorescent extracellular vesicles could be observed. CONCLUSION: Here we provide a novel method to identify tumor-derived extracellular vesicles using high-resolution flow cytometry of PpIX positive particles. Current research focuses on detection of these particles in GBM patient derived bodily fluids and their potential relation to tumor volume and response to therapy.