1.0 Summary Cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular fatty acids (FA) and de novo synthesis. Given that oxidation of de novo synthesised FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates - however hitherto FAs were considered part of a common pool. To probe FA metabolic partitioning, cancer cells were supplemented with stable-isotope labelled FAs. Structural analysis of the resulting glycerophospholipids revealed that labelled FAs from uptake were largely incorporated to canonical ( sn -)positions on the glycerol backbone. Surprisingly, labelled FA uptake disrupted canonical isomer patterns of the unlabelled lipidome and induced repartitioning of n -3 and n -6 polyunsaturated-FAs into glycerophospholipid classes. These structural changes evidence differences in the metabolic fate of FAs derived from uptake or de novo sources and demonstrate unique signalling and remodelling behaviours usually hidden to conventional lipidomics. Highlights Lipid isomers reveal discrete metabolic compartmentalisation in cancer FAs derived from uptake and de novo synthesis have different metabolic fates Stearate uptake signals for PUFA ( n -3 and n -6) repartitioning between lipid classes sn -positional isomers are a marker for aberrant lipid metabolism

Isomeric lipid signatures reveal compartmentalised fatty acid metabolism in cancer