PROJECT SUMMARY This competitive renewal application focuses on continued development of cancer cell-specific nanobubble (NB) ultrasound contrast agents for real-time guidance of prostate biopsies using transrectal ultrasound (TRUS). In the current clinical workflow, prostate cancer (PCa) biopsies are almost always performed using TRUS guidance. However due to poor soft tissue contrast of the B-mode ultrasound scans, the delineation of tumors within the prostate using TRUS is not clear. Because only 1% of the prostate tissue is sampled with a typical 8-12 core biopsy, a lack of direct guidance of the biopsy to suspicious lesions has led to high false negative rates and rising morbidity from current standard of care. The development of a new tool to accurately depict cancer within the prostate in real-time using TRUS is thus urgently needed to aid in biopsy guidance. To provide a practical tool for clear identification of potential malignancies during prostate biopsies, we have developed a nanoparticle-based ultrasound contrast agent (called a nanobubble) targeted to the prostate specific membrane antigen (PSMA) which is significantly over-expressed in most prostate cancers. The PSMA-NBs, are similar in structure to clinically used microbubbles (MB) and are clearly visible on clinical US. In contrast to MB, which remain in the vasculature, the small size, deformable shell and gas core, enable NBs extravasate and directly bind to cancer cells. This results in highly specific accumulation of contrast at the tumor itself leading to better resolution and detection of PCa. During our initial award, we developed an entirely new platform NB formulation which has been rigorously vetted and tested extensively in animal models of PCa. The objective of the next phase of research proposed in this renewal is to continue working toward clinical translation by performing IND-enabling studies. Here, we will scale up the NB formulation, test batch-to-batch reproducibility, conduct toxicology studies and engineer viable long-term storage techniques. Concurrently we will develop multiparametric US imaging biomarkers unique to NBs and acquisition pulse sequences optimized for use with the NBs to further enhance the sensitivity and specificity of the PSMA-NB technology; there are currently no NB specific image acquisition or processing tools for contrast enhanced ultrasound. We will then test the sensitivity and specificity of PSMA-NB enabled TRUS biopsies in a large animal model of prostate cancer in dogs, which can accommodate clinical TRUS transducers and a near-identical workflow to clinical standard of care. These steps are critical to the advancement of the proposed imaging techniques to clinical use. The proposed research feasibility is supported by strong preliminary data generated by an integrated research team with complementary expertise in NB formulation and PSMA (Exner, Basilion), bubble-US interactions (Kolios), image processing (Wilson), and clinical p...