Abstract 3897: Isolation and molecular characterization of exosomes from glioblastoma patients using a microfluidic device after ultrasound-based opening of the blood-brain barrier
Authors: Kumari Abha , Youngblood Mark , Gould Andrew , Kang Yoon-Tae , Chen Li , Habashy Karl , Amidei Chris , Ward Rachel , Gomez Cristal , Bouchoux Guillaume , Canney Michael , Stupp Roger , Sonabend Adam M. , Nagrath Sunitha 
Published: March 2024

Abstract
Glioblastoma (GBM) is a highly aggressive tumor of the brain. Current techniques of detection like magnetic resonance imaging and tissue biopsy are inaccurate or highly invasive, requiring alternatives like liquid biopsy. Exosomes are nanosized vesicles that contain cargo, reflective of their cellular origin and have been used for early diagnosis of cancer. However, in GBM, the blood-brain barrier (BBB) leads to a paucity of pathological information in blood. This study investigates the effect of opening the BBB on circulating exosomes and their potential for early detection of GBM using a microfluidic platform (GlioExoChip). In 20 patients enrolled in a clinical trial NCT03744026, a pulsed ultrasound emitter- Sonocloud-9 was implanted into their brains to open the BBB. We show that GlioExoChip isolates GBM-specific exosomes with a high capture efficiency using phosphatidylserine and Annexin-V chemistry. Further, the amount of circulating EVs increases twofold after opening the BBB. Finally, the correlation between tumor growth rate and the exosome concentrations is evaluated longitudinally.
Methods: GlioExoChip is a microfluidic platform with a polydimethylsiloxane top and a glass bottom, functionalized with neutravidin-biotinylated annexin-V. Experiments are performed with GBM cell-line derived exosomes, followed by patient plasma. Isolated exosomes are characterized using Nanoparticle tracking analysis (NTA), scanning electron microscopy, Bicinchoninic acid (BCA) assay and western blot. Statistical analyses are performed using Student’s t-test and Pearson correlation.
Results: The microfluidic device GlioExoChip has a high capture efficiency of exosomes derived from GBM cell lines, i.e., 93.03±3.31% and 96.77±0.72% for GBM6 and U87, respectively. NTA and BCA showed that 15 times more exosomes were captured from patient plasma when compared with healthy. Further, western blot proved the presence of a glial protein and Flotillin-1 in exosomes isolated from patients. Exosome concentrations varied with different time points after sonication and had an average twofold increase from the BBB opening. In our longitudinal study with 8 patients, an association between the volumetric enhancement of the tumor and exosome concentrations after sonication shows that the average Pearson correlation among short progression-free survival (PFS) patients was 0.73 (avg. p=0.39), while no correlation was seen among long PFS patients (avg. r value=0.10; avg. p-value=0.54).
Conclusions: A microfluidic approach was used to investigate the role of circulating exosomes using phosphatidylserine-annexin V chemistry in GBM. BBB opening via sonication leads to an increase in the average GBM-specific exosome concentrations in plasma. Thus, exosomes are promising biomarkers for early detection in GBM.
Citation Format: Abha Kumari, Mark Youngblood, Andrew Gould, Yoon-Tae Kang, Li Chen, Karl Habashy, Chris Amidei, Rachel Ward, Cristal Gomez, Guillaume Bouchoux, Michael Canney, Roger Stupp, Adam M. Sonabend, Sunitha Nagrath. Isolation and molecular characterization of exosomes from glioblastoma patients using a microfluidic device after ultrasound-based opening of the blood-brain barrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3897.