Monocarboxylate transporters (MCTs) are transmembrane proteins that mediate the bi-directional transport of monocarboxylic acids such as lactate, pyruvate and ketones. MCT1 and MCT4 are key modulators of lactate homeostasis in tumours and represent promising targets for molecular cancer therapeutics. The MCT1 inhibitor AZD3965 is now in early phase clinical trials, thus understanding the impact of this drug on cancer cell metabolism may enable the discovery of pharmacodynamic biomarkers of target inhibition that will support the clinical development of such agents. Since MCT1 mediates the flux of lactate and pyruvate in and out of cells, here we use magnetic resonance spectroscopy (MRS) to investigate the effect of AZD3965 on a) intracellular (Lactate-I) and extracellular lactate (lactate-E) levels and b) hyperpolarised ¹³C-pyruvate-lactate exchange, as potential biomarkers for MCT1 inhibition in human cancer cells with varying MCT4 expression levels, a predictor of resistance to AZD3965.¹Human Raji (MCT4-) and Hut78 (MCT4 low (+)) lymphoma as well as HT29 (MCT4 high (+++)) colon carcinoma cells were treated with either 5nM or 25nM AZD3965 for 24h and levels of lactate in cell extracts (Lactate-I) or growth media (lactate-E) determined by ¹H MRS on a 500MHz Bruker spectrometer. Spectra were processed on MestRe-C v4.9 (University of Santiago de Compostela) and lactate levels normalized to an internal standard and to cell number. For ¹³C-pyruvate-lactate exchange studies, Raji, Hut78 and HT29 cells were treated as above for 24h or 1h (Raji cells only). Cells were then incubated at 37°C in FBS-free medium and dynamic ¹³C MR spectra acquired for 4 minutes with 2s intervals immediately after the addition of 10mM hyperpolarised [1-¹³C]pyruvic acid and 10mM unlabelled lactate. The ratio of the area under the curve for the summed lactate and pyruvate signals (Lactate_AUC/Pyruvate_AUC) was determined, corrected for cell number and used to estimate pyruvate-lactate exchange, as previously shown.² Data represent mean±SE.24h exposure to AZD3965 led to increased Lactate-I in MCT4- Raji to 2.65-fold and 12-fold relative to controls following exposure to 5nM and 25nM AZD3965, respectively (p<0.05). Similar effects were observed in MCT4+ Hut78 human lymphoma cells with Lactate-I increasing to ~10-fold with 5nM and maintaining this level at 25nM AZD3965 (p=≤0.02); however, in MCT4+++ HT29 human colon carcinoma cells Lactate-I showed only a trend towards an increase which did not reach statistical significance (167±30% to 204±34%, p≥0.056). AZD3965 treatment also led to a fall in Lactate-E in MCT4- Raji cells to ~60% and ~50% with 5nM and 25nM AZD3965, respectively (p≤0.05) but its effects on Lactate-E levels at both concentrations were insignificant in Hut78 (92±8% to104±12% of controls, p≥0.34) and HT29 cells (111±12% to126±17% of controls, p≥0.2), in line with the presence of an alternative transporter for lactate efflux, MCT4. Hyperpolarised ¹³C MRS analyses showed a significant decrease in Lactate_AUC/Pyruvate_AUC to 31±6% in 5nM and 19±2% in 25nM AZD3965-treated Raji cells relative to controls at 24h (p<0.001), an effect that was also observed at 1h following treatment with 25nM AZD396 (down to 27±7% of controls, p=0.002) indicating that drug-induced inhibition of ¹³C-pyruvate-lactate exchange is immediate. Reduced Lactate_AUC/Pyruvate_AUC was also observed in MCT4+ Hut78 cells and MCT4+++ HT29 cells treated with AZD3965, however these effects appeared to be MCT4-dependent with MCT4- Raji displaying the greatest fall in pyruvate-lactate exchange at 5nM AZD3965 and MCT4+++ HT29 showing the lowest reduction at 25nM AZD3965 with MCT4+ Hut78 falling in the middle (Figure 1). These results are consistent with MCT1-mediated transport of ¹³C-pyruvate being a rate limiting step in the ¹³C MRS-observed pyruvate-lactate exchange,³ although additional factors appear to be implicated.Our data show that AZD3965 triggers inhibition of lactate efflux leading to a MRS-detectable increase in Lactate-I in human cancer cells with modest or no MCT4 expression. Further, we show that AZD3965 inhibits ¹³C-pyruvate-lactate exchange (via blockade of ¹³C-pyruvate uptake) in human cancer cells with MCT4- cells showing the largest effect.Lactate-I and hyperpolarised ¹³C-pyruvate-lactate exchange are therefore promising non-invasive metabolic imaging biomarkers for monitoring the action of AZD3965 and potentially other MCT1 inhibitors.