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 …