Our goal in year 2 was to use the tissue slice (TSC) NMR compatible bioreactor optimized in year 1 to identify hyperpolarized metabolic biomarkers of prostate cancer presence and aggressiveness. To accomplish this goal my group finished the engineering of a 5 mm bioreactor and acquired hyperpolarized [1-13C]pyruvate data indicating that similar signal to noise and quality data can be achieved with 4 to 5 prostate tissue slices in the 5 mm bioreactor as was acquired from 30-40 tissue slices in the prior 10 mm bioreactor. The time course of viability of the tissue slices prior to and after the bioreactor 5 mm bioreactor study was also established using a live-dead tissue assays performed by the co-PI s Peehl and Ronen on this proposal. We subsequently utilized this optimized 5 mm bioreactor in hyperpolarized [1-13C] MR studies to begin correlating hyperpolarized [1-13C] pyruvate metabolism with pathologic grade (aim 2) and observed a grade dependent increase in the flux of hyperpolarized [1-13C]pyruvate to [1-13C]lactate, with higher levels of lactate in higher pathologic grade cancers. More cancer TSCs need to be studied to demonstrate biologic significance.