Cell-Resolved Mass Spectrometry Imaging Integrated with Isotope Tracing Elucidates Macrophage Polarization-Specific Metabolic Reprogramming

Tumor-associated macrophages, pivotal regulators of antitumor immunity, exert dual functions through their tumoricidal M1 and tumor-promoting M2 phenotypes, which are closely linked to their metabolic states. While conventional mass spectrometry imaging (MSI) can characterize the metabolic features of macrophages, it fails to capture dynamic metabolic activity and real-time substrate utilization within individual cells. In this research, we present an integrated approach that couples cell-resolved matrix-assisted laser desorption/ionization (MALDI)-MSI with stable isotope tracing to visualize dynamic metabolic heterogeneity across individual macrophage phenotypes in situ . Using isotopically labeled fatty acids as metabolic tracers, we revealed that M1 macrophages exhibit significantly enhanced synthesis of phospholipids, including phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidic acid (PA), compared to M2 macrophages, highlighting a polarization-specific metabolic signature linked to their antitumor function. Moreover, we observed that coculture with tumor cells markedly downregulated the levels of newly labeled phospholipids in M1 macrophages. Critically, the pharmacological inhibition of cPLA2, a key enzyme in the phospholipid metabolic pathway, significantly impaired the antitumor efficacy of M1 macrophages. These findings collectively demonstrate the functional importance of phospholipid metabolism in sustaining macrophage-mediated antitumor immunity. We envision that this spatially resolved metabolic tracing strategy will open new avenues for investigating cell-resolved metabolic crosstalk in complex biological environments.