Spatial single-cell isotope tracing reveals heterogeneity of de novo fatty acid synthesis in cancer.

While heterogeneity is a key feature of cancer, understanding metabolic heterogeneity at the single-cell level remains a challenge. Here we present 13 C-SpaceM, a method for spatial single-cell isotope tracing that extends the previously published SpaceM method with detection of 13 C 6 -glucose-derived carbons in esterified fatty acids. We validated 13 C-SpaceM on spatially heterogeneous models using liver cancer cells subjected to either normoxia-hypoxia or ATP citrate lyase depletion. This revealed substantial single-cell heterogeneity in labelling of the lipogenic acetyl-CoA pool and in relative fatty acid uptake versus synthesis hidden in bulk analyses. Analysing tumour-bearing brain tissue from mice fed a 13 C 6 -glucose-containing diet, we found higher glucose-dependent synthesis of saturated fatty acids and increased elongation of essential fatty acids in tumours compared with healthy brains. Furthermore, our analysis uncovered spatial heterogeneity in lipogenic acetyl-CoA pool labelling in tumours. Our method enhances spatial probing of metabolic activities in single cells and tissues, providing insights into fatty acid metabolism in homoeostasis and disease.