(13)C-SpaceM: Spatial single-cell isotope tracing reveals heterogeneity of de novo fatty acid synthesis in cancer.

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)C(6)-glucose-derived (13)C label incorporated into esterified fatty acids 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. Investigating cultured cells, we revealed single-cell heterogeneity of lipogenic acetyl-CoA pool labelling degree upon ACLY knockdown that is hidden in the bulk analysis and its effect on synthesis of individual fatty acids. Next, we adapted (13)C-SpaceM to analyze tissue sections of mice harboring isocitrate dehydrogenase (IDH)-mutant gliomas. We found a strong induction of de novo fatty acid synthesis in the tumor tissue compared to the surrounding brain. Comparison of fatty acid isotopologue patterns revealed elevated uptake of mono-unsaturated and essential fatty acids in the tumor. Furthermore, our analysis uncovered substantial spatial heterogeneity in the labelling of the lipogenic acetyl-CoA pool indicative of metabolic reprogramming during microenvironmental adaptation. Overall, (13)C-SpaceM enables novel ways for spatial probing of metabolic activity at the single cell level. Additionally, this methodology provides unprecedented insight into fatty acid uptake, synthesis and modification in normal and cancerous tissues.