Project Summary Glioblastoma multiforme is the most common primary brain tumor and one of the deadliest of human cancers. Early diagnosis and sensitive therapeutic monitoring remain major challenges in the treatment of this disease. Monitoring of tumor development and therapeutic interventions largely relies on clinical evaluation and MRI. However, both clinical examination and MRI are insensitive measures of disease status. For example, MRI resolution is limited to the order of millimeters, which translates to a significant delay before a tumor can be detected. While biopsies are valuable, they are invasive and can result in significant morbidity. Less invasive platforms for early diagnostic and therapeutic monitoring are desperately needed. Within the past two decades, extracellular vesicles have emerged as important regulators of a diverse range of biological processes in dif- ferent pathologies including brain cancer. These vesicles are a family of membrane-enveloped nanoparticles with diverse biological function, diagnostic potential, and therapeutic applications. These vesicles are typically <1000 nm in size and are present in blood at concentrations of up to 1000 vesicles/mL in cancer patients. They have been shown to contain tumor-specific nucleic acids, proteins, and lipid cargoes and appear highly en- riched with cancer-specific miRNA. Extracellular vesicles as circulating biomarkers are one of the most promis- ing new diagnostic and therapeutic paradigms in cancer research. Extracellular vesicles include two major frac- tions (exosomal and microparticles) differing by size, cargoes, and functional role. Both fractions can serve as potential biomarkers for diagnosis, staging, and monitoring of therapeutic interventions. A major limitation of using extracellular vesicles for diagnosis and monitoring of brain tumors is the relatively low proportion of tu- mor-specific extracellular vesicles, in plasma samples in particular large size extracellular vesicles fraction (mi- croparticles) due to the presence of blood-brain barrier (BBB) and blood-tumor barrier (BTB). To address this challenge, we will use ultrasound bursts combined with circulating microbubbles to temporarily disrupt these barriers and unique approach to analyze a presence of all extracellular vesicles fractions including larger size microparticle fraction in the plasma. This procedure, which can be performed using ultrasound devices that ac- curately and safely focus an ultrasound beam through the human skull, has a great potential as a noninvasive and targeted method to disrupt BBB/BTB for drug delivery. Here will instead use it to increase the presence of extracellular vesicles large-size fractions in the patient's plasma. Combined with analysis of the tumor-specific extracellular vesicles in plasma samples, this technology could be a powerful tool for both the diagnosis and treatment of brain tumors – a noninvasive biopsy. To reach this goal, we will perform extensive studies in mu- rine glioma models to optimize the procedure and evaluate it during different tumor stages. If the proposed ex- periments are successful, we will have compelling evidence that may lead to human trials.

Project Narrative The detection of circulating extracellular vesicles fractions in the blood plasma is a promising noninvasive tool for monitoring brain tumor development, but its sensitivity is relatively poor and especially limited for large size extracellular vesicles (microparticles). We propose to use focused ultrasound together with innovative ultra- sensitive analysis methods to increase the number and range of circulating extracellular vesicles and allow combined analysis of all extracellular vesicles fractions for tumor biomarkers. 1