SUMMARY The NeuroAccess system represents a pioneering approach to address the unmet critical need for effective blood-based molecular characterization in brain cancer patients. By utilizing focused ultrasound and microbubbles to open the blood-brain barrier non-invasively, it allows biomarkers such as cell-free DNA (cfDNA) into the systemic circulation, facilitating liquid biopsy in conditions where biopsies with traditional techniques are difficult to obtain. The overall objective of this study is to ensure the optimized safety and efficacy of the NeuroAccess system in enabling liquid biopsies (LB) for brain tumor patients. The study comprises three aims: Firstly, to demonstrate the safety of repeated BBB opening (BBBO) in healthy rats with variable intra-treatment delays. Secondly, to optimize BBBO volume and transmit sequence in diseased rats, assessing the increase in mutant EGFR biomarkers in peripheral circulation. Finally, to optimize the sensitivity and specificity of the LB method by confirming BBBO and measuring biomarker levels in both healthy and tumor-bearing rats. Success criteria include statistically significant biomarker concentration increases and confirmed safe BBBO. Our long-term goal is to significantly improve the management and outcomes of brain tumor patients through reliable and non-invasive liquid biopsies. Tissue biopsies are difficult to obtain for brain tumor patients other than in an invasive manner and/or when surgery is performed. Liquid biopsies are not currently used because the blood-brain barrier prevents biomarkers from migrating from brain tissue into the circulation system. The NeuroAccess system addresses this challenge by enabling non-invasive liquid biopsies. Existing research including data from studies on humans, demonstrates the potential and feasibility of safe blood-brain barrier openings for the purposes of liquid biopsy, however the NeuroAccess device makes the approach feasible and practical. This project proposes innovative techniques to increase the reliability, sensitivity, and specificity of the focused ultrasound based liquid biopsy method. The proposed research is significant because it tackles the complexities of brain tumor diagnostics, aiming to bring substantial improvements in the detection and management of brain tumors. The successful implementation of the NeuroAccess system has the potential to shift the paradigm in brain tumor treatment, aligning with the urgent needs of patients. NCI Phase I SBIR Confidential Page 2 of 12 Aims Page and Research Plan Draft