Focused ultrasound BBB opening yields distinct, tissue-specific extracellular vesicle profiles for glioblastoma liquid biopsy

Introduction: Extracellular vesicles (EVs) are highly information-dense liquid biopsy reservoirs carrying proteins, RNAs, lipids, and metabolites that reflect the real-time state of their parent cells. In glioblastoma (GBM), EVs offer a uniquely accessible window into an otherwise sequestered compartment, yet the blood brain barrier (BBB) restricts their enrichment and profile within the circulation. Focused ultrasound (FUS) with microbubbles is now a clinically burgeoning method for transient and precise local opening of the BBB, creating an opportunity to interrogate EV flux from tumor and surrounding brain. While FUS-mediated BBB opening (BBBO) has been shown to augment circulating nucleic acids via “sonobiopsy,” its effects on EV release and cargo remain poorly defined. Here, we evaluate how FUS-BBBO alters the abundance and composition of tumor- versus brain-derived EVs enriched from blood plasma in a high-fidelity GBM model. Methods: Orthotopic GBMs were established via intracranial SB28 implantation (n=24). Tumors were screened by contrast-enhanced MRI and randomized into volume-matched sham or FUS treatment groups. BBBO was performed using a neuronavigation-guided preclinical FUS system. Plasma was collected 30 min or 24 h post-treatment. EVs were isolated by ion-exchange chromatography and characterized by NTA and Western blot. Tumor- and brain-derived EVs were enriched using Tumor SPARCs™ and Neuro SPARCs™, respectively, and subjected to DIA-MS proteomic analysis. Results: FUS-BBBO did not significantly alter total plasma EV concentrations associated with either compartment; rather, it induced substantial remodeling of EV cargo. Across samples, >5,000 proteins were identified. Approximately 80% of differentially abundant proteins were unique to either the tumor- or brain-derived EV panel, indicating compartment-specific responses to FUS. Strikingly, FUS exposure yielded 222 and 290 unique proteins within brain-derived and tumor-derived circulating EV populations, respectively. Pathway analysis revealed enrichment in vesicle trafficking, neurovascular stress signaling, cytoskeletal remodeling, and immune regulatory pathways - potentially reflecting FUS-induced alterations in tumor microenvironmental stress, BBB dynamics, and neuro-immune cross-talk. Conclusions: FUS-BBBO drives robust, compartment-specific reprogramming of EV proteomes in GBM, revealing biomarker candidates absent under sham conditions. These findings position EV profiling as a sensitive approach for capturing FUS-induced tissue remodeling and may expand the biomarker repertoire available for spatially selective GBM liquid biopsy. Ongoing studies are assessing temporal dynamics and correspondence with parental tissue proteomes.