Dynamic release of extracellular particles after opening of the blood-brain barrier predicts glioblastoma susceptibility to paclitaxel.

Liquid biopsies hold promise to improve the diagnosis, assessment of response to therapy, and ultimately guide the management of cancer patients. However, implementation of this approach in brain tumors has proven challenging due to the limited passage of molecules across the blood-brain barrier (BBB). We recently reported results from a phase I clinical trial in which the BBB was transiently opened in glioblastoma (GBM) patients using skull-implantable low-intensity pulsed ultrasound combined with microbubbles (LIPU/MB). In this study, treatment and BBB opening was performed every 3 weeks with paclitaxel administration until disease progression or up to 6 cycles (NCT04528680). As an exploratory objective of this trial, here we investigate extracellular vesicles and particles (EVPs/EPs) released into circulation in the context of tumor cell death as a potential biomarker for response to treatment. We develop and validate a microfluidic device designed to capture tumor-derived EVPs in glioblastoma patients (GlioExoChip). This approach leverages GBM-based expression of phosphatidylserine and Annexin-V chemistry that is traditionally used to measure apoptosis. EVPs are characterized using nanoparticle tracking analysis, proteomics, western blot, and scanning electron microscopy. Proteomic analysis of circulating EVPs isolated from GBM patients reveals distinct expression patterns to that of healthy individuals, and scRNA-seq analysis of these genes supported their tumoral origin within the GBM microenvironment. In vitro, paclitaxel-susceptible glioma cells treated with this drug exhibit apoptosis and dose-dependent EVP release. In concordance, we find changes in EVP release following the initiation of paclitaxel with LIPU/MB correlated with overall survival in GBM patients. Thus, our study introduces an efficient microfluidic platform for the capture of circulating GBM EVPs and demonstrates that release upon BBB opening is predictive of outcomes following paclitaxel treatment. This approach represents a real-time surrogate biomarker for treatment response for a disease where imaging-based assessment of response has not been shown to be reliable. Future prospective validation is warranted.