Lipids enable compartmentation and coordinate membrane-localized signaling events in cells, and dysregulation of lipid metabolism is linked to many disease states. However, limited tools are available for quantifying metabolic fluxes across the lipidome. To measure fluxes encompassing lipid homeostasis in cells and tissue slices, we apply stable isotope tracing, liquid chromatography-high-resolution mass spectrometry, and network-based isotopologue modeling to non-small cell lung cancer (NSCLC) models. Lipid metabolic flux analysis (Lipid-MFA) enables quantitation of fatty acid synthesis, elongation, headgroup assembly, and salvage reactions within virtually any biological system. Using Lipid-MFA, we observed decreased fatty acid synthase and very long-chain fatty acid (VLCFA) elongation fluxes, along with increased sphingolipid recycling, in p53-deficient versus liver kinase B1 (LKB1)-deficient NSCLC tumors using precision-cut lung slice culture. We also apply Lipid-MFA to demonstrate the unique trafficking of ceramides with distinct n-acyl chain lengths, highlighting the utility of this approach in elucidating molecular mechanisms in lipid homeostasis. • Lipid-MFA measures synthesis and salvage fluxes that support lipid homeostasis • Precision-cut lung slice culture enables Lipid-MFA in the tumor microenvironment • Loss of p53 or LKB1 tumor suppressors distinctly impacts sphingolipid flux in NSCLC • LC- versus VLC-ceramides are differentially trafficked to complex sphingolipids Wessendorf-Rodriguez et al. develop a pipeline for applying metabolic flux analysis to quantify lipid homeostasis. Lipid-MFA elucidates differences in synthesis and salvage fluxes in precision-cut lung slices harboring tumors with common driver mutations of lung adenocarcinoma. Application of Lipid-MFA reveals distinct trafficking of ceramide species into the sphingolipidome.
Wessendorf-Rodriguez et al. (Sun,) studied this question.