The up-regulation of aerobic glycolysis and lactate production and efflux is an adaptation of cancer cells that aids in tumor survival, growth and metastasis1. Prior publications have consistently demonstrated significantly elevated hyperpolarized (HP) [1-¹³C]lactate levels in cancer2, however the relative amounts of intra- and extracellular HP lactate that contributes to the observed in vivo signal has not been determined. Aggressive renal cell carcinomas (RCCs) are tumors that exemplify the Warburg effect with both high lactate production and rapid lactate export. The goal of this study was to determine if diffusion weighted (DW) HP ¹³C MR, a clinically translatable technique^1-3, could provide an estimate of the amount of extracellular versus intracellular HP ¹³C lactate based on its apparent diffusion coefficient (ADC) in a RCC murine model. This was accomplished using a combination of ex vivo MR compatible 3D cell culture bioreactor and in vivo orthotopic murine model studies of the same RCC cell line. UOK262 cells, a model of metastatic RCCs, were used as described before³. Ex vivo: alginate-encapsulated UOK262 cells in bioreactor were studied on the 14.1T Varian INOVA with 100 G/cm gradients. A pulsed gradient double spin echo sequence was used for all hyperpolarized ¹³C diffusion experiments with δ=10ms, Δ=30ms, and a diffusion weighting of 3 to 15,000 s/mm² (Gx or Gy). Dynamic data (30° pulses, 3s interval for 300s) after HP [1-13C]pyruvate infusion (16µmols) were acquired. In vivo: Immune compromised mice implanted with UOK262 cells under the renal capsule were used for DW imaging with a spectrally and spatially selective 30° RF pulse for 8mm thick slice and 2x2mm in–plane resolution with 4 b-values (25-4000 s/mm²). Respiratory gating (two b-values/breath) was used to minimize signal loss from bulk motion.Ex vivo: Figure.1a shows a plot of HP [1-¹³C]pyruvate signal versus b-value (0-1500 s/mm²) from two MR compatible bioreactor studies. In the first study the bioreactor contains cell-free alginate microspheres (blue squares), and co-polarized [1-¹³C]pyruvate and [1-¹³C]lactate are injected. In the second study, the bioreactor contains UOK262 cell-laden alginate microspheres (red squares) and only hyperpolarized pyruvate is injected. The HP pyruvate signal from the bioreactor containing UOK262 cells reveals a bi-exponential signal decay, with the fast decay constant being the same as that of the cell-free bioreactor study, and the slow decay constant being attributed to restricted intracellular diffusion. Figure.1b shows representative HP ¹³C MR spectra obtained from the same bioreactor experiments at 3 increasing b-values (left to right). In the cell free study, we observe a complete loss of HP lactate and pyruvate signal at the highest b-value, whereas in the UOK262 cell study, HP and pyruvate signals are still visible. This is attributed to the shorter intracellular ADC. The extra- and intracellular ADC calculated for lactate and pyruvate in cell-free alginate microspheres and UOK262 cells, respectively, are listed in Table 1. Figure.2 shows a representative bioreactor study containing UOK262 cells after injection of HP pyruvate in which alternating low and high b-values (2.4 and 3863 s/mm²) were applied over time. This allowed for the detection of total HP lactate pool (low b-value) and the predominately intracellular pool (high-b value), and the calculation of the extra cellular pool (difference). In vivo: Mice with orthotopic UOK262 tumors were imaged by DW HP pyruvate MRI (4 b-values) when tumor volumes reached at least 0.2cc. DW HP signals were corrected for RF utilization and fit voxel-wise to a monoexponential decay S(b)=S₀e^-bD to compute the ADC map of lactate. We observe a lactate ADC in the tumor of 0.709 ± 0.15, (n=4). Discussion and Conclusion: We show that, in the bioreactor, intracellular and the extracellular HP lactate ADC can be measured simultaneously and dynamically. The intracellular ADC value of lactate determined using HP ¹³C is similar to that measured thermally using proton spectroscopy⁵. Additionally, the dynamic measurement of the lactate production and efflux was feasible using HP ¹³C DW spectroscopy in the bioreactor. These studies demonstrated that lactate efflux occurred almost instantaneously with lactate production, similar to recent detection of extracellular HP ¹³C lactate using chemical shift separation approach^6,7. The dynamically resolved intra- and extracellular lactate pools obtained after injection of HP pyruvate in the UOK262 cell bioreactor studies demonstrated that ~60% of the total HP lactate signal was extracellular during the time frame of the hyperpolarized MR acquisition. Based on the ex vivo intra- and extracellular lactate ADC values, and the measured in vivo ADC value of ≈ 0.7 mm2/s, ≈ 60% of the HP lactate signal arose from extracellular lactate in UOK262 orthotopic tumors.