Dissolution DNP provides a four orders of magnitude enhancement to carbon-13 nuclei. Coupled with MR’s ability to resolve both substrate and metabolites, dissolution DNP of ¹³C substrates has been used extensively for metabolic imaging in both pre-clinical¹ and proof-of-concept clinical studies² to non-invasively assess metabolic conversion. In addition to the Warburg Effect, many cancers - such as malignant renal cell carcinoma³ and prostate cancer⁴ - overexpress MCT4. This monocarboxylate transporter is primarily responsible for lactate efflux, resulting in acidification of the extracellular space and conferring a poor prognosis⁴. Because of structural differences in the intra and extra-cellular microenvironments, diffusion weighted imaging (DWI) of hyperpolarized pyruvate and lactate may provide unique information on lactate efflux and microstructure, potentially providing insight into MCT4 expression and therapeutic changes in a rapid, non-invasive manner. In this work, we explore the utility of hyperpolarized ADC mapping of pyruvate and lactate to assess lactate efflux and MCT4 expression in a transgenic mouse model of prostate cancer (TRAMP).48μL aliquots of [1-¹³C]pyruvate (Cambridge Isotopes, Cambridge, Massachusetts, USA) were polarized for 90 minutes in a HyperSense polarizer (Oxford Instruments). Samples were rapidly thawed and neutralized to a physiologic pH with a 4.5g solution comprised of 160mM NaOH, Tris buffer and EDTA. Animals were initially separated into two cohorts (early or late stage) by tumor size and animal age. TRAMP mice were injected with 300μL of neutralized pyruvate via tail-vein injection over 15s. 30s after the start on injection, metabolites were excited with a single-band spectral-spatial RF pulse, followed by a single-shot, double spin-echo flyback EPI prescribed for a 12mm thick slice and 2 x 2 mm in-plane resolution. Four b-values (25, 300, 600, 1000 s/mm²) were acquired per metabolite, with a constant flip angle of 30°. Data were corrected for RF utilization and fit voxel-wise to a monoexponential decay $$$ S(b)=S_{0,corr}\exp(-bD)$$$ to extract ADC maps for each metabolite. Following ¹³C imaging, a high-resolution ADC map was acquired from a dedicated ¹H coil using a spin-echo sequence (b = 25, 188, 331, 515 s/mm²), with TR/TE = 1.2s/20ms, an in-plane resolution of 0.31 x 0.31 mm and twenty-four 1 mm thick slices.Representative multiparametric early and late stage TRAMP data can be seen in Figure 1. ADC data are summarized in Figure 2. In agreement with prior work, ¹H ADC decreases for late stage TRAMP tumors due to increasing cellularity⁵, recapitulating the progression of human prostate cancer. Interestingly, the pyruvate ADC remains unchanged (0.95 ± 0.12 vs. 1.03 ± 0.19 x 10^-3 mm²/s) as the disease progresses, despite being a predominantly extracellular metabolite⁶. Conversely, the lactate ADC significantly increases (p < 0.05, t-test) by more than 40% for late stage tumors (0.68 ± 0.09 vs. 0.46 ± 0.05 x 10^-3 mm²/s), in agreement with increased lactate efflux. Disease progression was characterized by heterogeneity, highlighting the importance of spatial localization in the ¹³C data. This is borne out in the histograms of the lactate and pyruvate ADC (Fig. 3) for a representative early stage, late stage, and an additional mixed stage tumor that contains both early and late stage disease. The distributed lactate ADC for the mixed stage provides evidence for underlying heterogeneity in disease state and MCT4 expression, later confirmed by histopathology. These results are supported by activity and expression for LDH and MCT4 (Fig. 4), indicating increased LDH activity and overexpression of LDHA and MCT4 in late stage prostate cancer. Despite the small sample size, these initial results provide evidence that DWI of hyperpolarized substrates can identify changes in MCT4 expression.Hyperpolarized imaging has the potential to provide non-invasive measures of enzyme kinetics and transporter expression. We show that diffusion weighted imaging of hyperpolarized metabolites is sensitive to changes in MCT4 expression and lactate efflux, providing a novel way to assess metabolite compartmentalization and microstructural changes in the prostate.