Spatial single-cell isotope tracing reveals heterogeneity ofde novofatty acid synthesis in cancer cells

Abstract Metabolism has emerged as a key factor in homeostasis and disease including cancer. Yet, little is known about the heterogeneity of metabolic activity of cancer cells due to the lack of tools to directly probe it. Here, we present a novel method, 13 C-SpaceM for spatial single-cell isotope tracing of glucose-dependent de novo lipogenesis. The method combines imaging mass spectrometry for spatially-resolved detection of 13 C6-glucose- derived 13 C label incorporated into fatty acids composing cellular lipids with microscopy and computational methods for data integration and analysis. We validated 13 C-SpaceM on a spatially-heterogeneous normoxia-hypoxia model of liver cancer cells. We demonstrated the single-cell heterogeneity of acetyl-CoA pool labelling degree upon ACLY knockdown that is hidden in the bulk analysis and its effect on synthesis of individual fatty acids. Overall, 13 C-SpaceM provides a capacity to probe metabolic activity on the single-cell level.