ABS 041: Detecting Tau in Glioblastoma Extracellular Vesicles via High-Sensitivity SiMoA-based Workflow

Julia R. Miller ¹, Jennifer Pollock ¹, Sarah Planchak, M.S. ¹, Anubhav Tripathi, Ph.D. ¹

¹ Center for Biomedical Engineering and School of Engineering Brown University, Providence, RI

The Van Wickle Journal (2026) Volume 2, ABS041

Introduction: Glioblastoma (GBM) is an aggressive, heterogeneous brain tumor that forms from glial cells and the most common malignant brain tumor in adults with a survival of 16-18 months post-diagnosis. Currently, glioblastoma patients are faced with limited and invasive treatment options and its clinical management suffers from a lack of early diagnostic tests. The diagnostic gold standard consists of an invasive tumor biopsy followed by expensive neuroimaging such as MRI. Recently, extracellular vesicles (EVs) have emerged as a potentially promising and noninvasive diagnostic marker for the disease due to their ability to carry disease markers across the blood-brain barrier (BBB) and into peripheral fluids. However, while brain-derived EVs are particularly useful for diagnosing and tracking the progression of CNS diseases like GBM, their low concentration in peripheral biofluids necessitates high-sensitivity detection methods
This study aims to develop an ultrasensitive immunoassay workflow to detect brain-derived (BD) tau in GBM-derived EVs. It builds on previous research that validated a new single-molecule array (SiMoA), which allows for the detection of concentrations as low as 108 EVs per milliliter, as well as the characterization of protein expression in both general and brain-derived EV populations. Previous research has shown tau to play an important role in GBM progression, acting as a regulator of tumor growth, cell organization, and migration.
This study builds on that knowledge by isolating EVs from GBM supernatant using ultracentrifugation (UC) targeting tetraspanins (CD9, CD63, and CD81) with antibody-coated Dynabeads. Eluted EV yields were quantified via nanoparticle tracking analysis (NTA). The vesicles were also lysed to conduct downstream analysis, such as assessing internal cargo by running an assay kit for brain-derived (BD) tau on the SiMoA HD-X. The overall goal was to determine whether BD-tau is detectable in EVs and whether it is surface-bound or internal.

Methods: EVs were isolated from pooled human plasma and conditioned media collected from primary human GBM spheroid cultures obtained under Rhode Island Hospital Institutional Review Board approval (IRB #418015). Samples were thawed at 37°C and centrifuged at 300 × g for 10 min at 4°C to remove cellular debris. Supernatants were subsequently centrifuged at 10,000 × g for 30 min to further eliminate larger particles and cellular debris. EVs were then isolated by ultracentrifugation at 100,000 × g for 4 h at 4°C. Following ultracentrifugation, EV pellets derived from plasma and conditioned media were resuspended in cold EV diluent and maintained at 4°C prior to same-day analysis. Brain-derived tau concentrations in EV lysates were quantified using the Simoa® BD-Tau Advantage PLUS Assay Kit (Quanterix, Lexington, MA; Lot #504275) according to the manufacturer’s protocol.

Results: We demonstrated that BD-tau is detectable and quantifiable in EVs isolated from GBM cell culture media using the ultrasensitive Simoa assay. BD-tau levels in GBM-derived EVs were several orders of magnitude above the assay detection limit and were elevated relative to healthy plasma EVs after normalization for EV count. These findings show that GBM-derived EVs carry measurable tau-associated signals and support EV-associated BD-tau as a potential liquid biopsy biomarker for GBM diagnosis and longitudinal disease monitoring.

Discussion: Our findings demonstrate that BD-tau is present in EVs derived from GBM cell culture media and can be sensitively quantified using the Simoa assay. Elevated BD-tau levels in GBM-derived EVs compared to healthy plasma EVs suggest that GBM cells release tau-associated signals through EVs, supporting their potential use as liquid biopsy biomarkers for GBM diagnosis and disease monitoring. However, ultracentrifugation may co-isolate non-EV contaminants, and the localization of BD-tau within or on EVs remains unclear. Future studies should isolate neural-specific EV populations, characterize phosphorylated tau species, and validate findings across additional patient-derived GBM samples clinically.