The accumulation of lactate in tumors has been correlated with poor clinical outcomes (1,2), suggesting that measuring lactate concentration can potentially be used to assess the risk of metastasis and to monitor response to treatment. Hyperpolarized ¹³C-substrates, such as [1-¹³C]pyruvate, are being actively explored as a tool to image the pyruvate-to-lactate conversion by measuring hyperpolarized ¹³C lactate and pyruvate signals in animal models, as well as in prostate cancer patients (3,4). Since the hyperpolarization decays rapidly (~60s), the fate of the injected ¹³C-substrates are typically not known outside of this short window. In this study, we estimate the concentrations of total lactate and ¹³C₁- and ¹³C₃-lactate in rat tumor extracts and compare these values to the variables derived from hyperpolarized imaging acquisitions (e.g. pyruvate-to-lactate ratio) to better understand the ¹³C-lactate signal in cancer.Three male Rowett nude (RNU) rats with subcutaneous xenografts of MDA-MB-231 human breast cancer cell line co-injected with MS1 mouse endothelial cell line were injected with 2ml of pre-polarized 80mM [1-¹³C]pyruvate, co-polarized with 27mM HP001(bis-1,1-(hydroxymethyl)-[1-¹³C]cyclopropane-d8), over 12s. Spectral-spatial (SPSP) excitation pulses (TR/TE=56/24ms, 64x8x6 cm3) were applied to obtain lactate, pyruvate, and HP001 images. For anatomical reference, 2D fast spin echo (5s TR, 192x192 matrix, axial) images were acquired. All images were acquired on a 3T GE MR750 scanner. The region of interest (ROI) was selected based on the anatomical image to cover the entire tumor region for each rat (Fig. 1). The lactate and pyruvate signals from ROI was plotted and fitted to the linear line of best fit (Fig. 2). 80mM/2ml non-hyperpolarized [3-¹³C]pyruvate was injected over 12s following the imaging study (approximately 4 min after the initial [1-¹³C]pyruvate injection) and the tumors were harvested 1 min from the start of [3-¹³C]pyruvate injection. Metabolites were extracted from tumor samples (1852, 1642, 930 mg wet weight, respectively) in perchloric acid (5), lyophilized and re-dissolved in 400 to 450μl D₂O containing 10mM DMSO for NMR spectroscopic studies. Proton spectra were acquired with 2s water pre-saturation sequence (TR=60s, 32scans, sw=±2500Hz) to determine total lactate pool (Fig. 3A), using 10mM DMSO as the internal concentration reference. ¹³C spectra were acquired (Fig. 3B) using a proton-decoupled (WALTZ-16) sequence (flip angle=45°, TR=5.1s, 32768 points, 45052 scans, 10Hz exponential apodization). To enable concentration measurement, a 250mM ¹³C-urea sample was scanned before each ¹³C spectra acquisition to compensate for any signal instability. The signal from each peak was determined by calculating the area-under-the-peak. Only the signals with SNR > 2 were considered. ¹³C-lactate concentrations were determined by comparing lactate signals to signals from 1, 2, 3mM [3-¹³C]sodium pyruvate solutions acquired under the same condition. ¹³C₁-lactate signal was further corrected for using a short TR during the acquisition, assuming an actual T1 of 40s (6). All NMR data were acquired on a 7T NMR spectrometer.The range of pyruvate and lactate signals detected in the tumor region in each rat is shown in Fig. 2. The relationship between the corresponding lactate and pyruvate signals from each voxel within ROI is shown in the same figure. Based on the pyruvate and HP001 images (not shown), the tumor in Rat C appeared to be poorly perfused compared to the other rats. Nonetheless, ¹³C₁- and ¹³C₃-lactate peaks (184.2 and 21.8ppm, respectively) were both detectable in all rats. The estimates of ¹³C-lactate concentrations, as well as total lactate pool size, are summarized in Table 1. Both ¹³C₃-lactate concentration and pyruvate-to-lactate ratio were positively correlated with ¹³C₁-lactate concentration (R²=0.95 and 0.92, respectively, see Fig. 4). On the contrary, the estimated total lactate pool size had no obvious correlation to the other parameters measured.A measurable level of ¹³C lactate remained in the tumors following the hyperpolarized experiment, enabling ex vivo NMR measurements of labelled and unlabelled lactate. A strong positive correlation between hyperpolarized pyruvate-to-lactate ratio and the ex-vivo NMR measurements was observed. Surprisingly, the total lactate pool size from proton NMR did not correlate with the other parameters measured.