AbstractHyperpolarised magnetic resonance imaging (HP ¹³C-MRI) is an emerging clinical technique to detect [1-¹³C]lactate production in prostate cancer (PCa) following intravenous injection of hyperpolarised [1-¹³C]pyruvate. Here we differentiate clinically significant PCa from indolent disease in a low/intermediate-risk population by correlating [1-¹³C]lactate labelling on MRI with the percentage of Gleason pattern 4 (%GP4) disease. Using immunohistochemistry and spatial transcriptomics, we show that HP ¹³C-MRI predominantly measures metabolism in the epithelial compartment of the tumour, rather than the stroma. MRI-derived tumour [1-¹³C]lactate labelling correlated with epithelial mRNA expression of the enzyme lactate dehydrogenase (LDHA and LDHB combined), and the ratio of lactate transporter expression between the epithelial and stromal compartments (epithelium-to-stroma MCT4). We observe similar changes in MCT4, LDHA, and LDHB between tumours with primary Gleason patterns 3 and 4 in an independent TCGA cohort. Therefore, HP ¹³C-MRI can metabolically phenotype clinically significant disease based on underlying metabolic differences in the epithelial and stromal tumour compartments.