Dynamic Tracking of Lipid Metabolism in Live Tumor Spheroids by Bioorthogonal Stimulated Raman Scattering Microscopy

Altered lipid metabolism has emerged as a critical hallmark of chemoresistance. However, interrogating the dynamic metabolic responses within biologically relevant three-dimensional (3D) tumor models remains challenging. Here, we present a bioorthogonal stimulated Raman scattering (SRS) imaging strategy to in situ, real-time track the spatiotemporal dynamics of fatty acid metabolism in live patient-derived 3D tumor spheroids. By simultaneously detecting carbon-deuterium (C-D) vibrational signals from incorporated exogenous fatty acids and intrinsic CH2 signals from pre-existing lipids, we performed longitudinal tracking of metabolic flux and quantified the subcellular lipid distribution in cisplatin-sensitive and -resistant ovarian cancer spheroids. Our results revealed substantial spatiotemporal heterogeneity in fatty acid incorporation, including preferential peripheral enrichment and dynamic edge-to-core gradients within tumor spheroid. Notably, cisplatin-resistant spheroids exhibited increased fatty acid uptake and preferential storage within lipid droplets (LDs), indicating a distinct lipid handling strategy associated with chemoresistance. Overall, our study showcases bioorthogonal SRS imaging as a powerful approach for resolving metabolic heterogeneity in live 3D tumor models and provides new insights into the lipid metabolic adaptations associated with chemoresistance.