TRD2 PROJECT SUMMARY/ABSTRACT Eleven of the 13 Collaborative Projects (CPs 1-10,13) that are the driving force behind this HMTRC renewal and 10 of the 12 Service Projects (SPs 1-4, 6, and 8-11) demonstrate a clear need for improved hyperpolarized (HP) probes and methods, realistic preclinical models, correlative pathology and molecular methodologies, and other synergistic imaging studies provided by TRD2. Over the last decade of funding, TRD2 has an outstanding record of productivity, having supported 117 peer-reviewed publications and 41 NIH grants, and during the prior funding period, we have leveraged preclinical advances to obtain FDA IND approval for two new HP probes – [2- 13C]pyruvate for improved imaging of the TCA cycle and in 2021 co-polarized [13C,15N2]urea and [1-13C]pyruvate for simultaneous metabolic and perfusion imaging. Based on the new needs of the CPs and SPs, the advice of our External Advisory Committee, and clinical consultants, this TRD2 renewal project will take several exciting innovative directions focused on increasing HP 13C probe sensitivity, in vivo performance, and clinical translatability through the development/optimization of HP probes, probe formulations and polarization approaches (Aim 1), optimizing more realistic preclinical models and methods for HP 13C MR studies (Aim 2) and obtaining correlative pathologic, molecular and multimodal imaging studies (Aim 3) to optimize and validate HP 13C MR biomarkers and methods for clinical translation. These research goals will be accomplished in synergy with TRD1 through implementing a new preclinical 9.4T MR scanner with 1H/13C MRI cryoprobe capabilities, a next generation higher field, lower temperature preclinical DNP polarizer and improved rf coil technologies for significantly improved preclinical HP 13C MR studies. With TRD3, we will implement more sensitive dynamic HP 13C MR pulse sequences (EPSI, EPI and bSSFP), with a focus on developing robust, standardized, and quantitative imaging protocols, and new on-line preclinical data analysis and display tools integrated into the free, open-source platforms. Although the HP MR probes and techniques will be specifically driven (push-pull) by the CPs, and utilized (pushed) in the SPs, they will greatly expand the scientific scope and preclinical applicability of HP 13C MR in general and significantly impact its clinical translation. The main deliverables of this project are the dissemination of optimized HP probe preparations and methods, new NMR-compatible cell and tissue culture bioreactor systems and preclinical GEM and PDX models optimized for HP 13C MR studies, correlative pathology, molecular and synergistic imaging approaches, and the associated dissemination and training for the biomedical community.