Single-Cell Metabolic Profiling in a Glioblastoma Co-culture Model Using AP-MALDI-based Mass Spectrometry Imaging

Abstract Mass spectrometry imaging enables spatially resolved, label-free detection of metabolites in tissue and culture systems, providing insight into their metabolic landscape and spatial distribution. However, conventional approaches often lack the spatial resolution and specificity needed to investigate metabolic heterogeneity at the single-cell level, particularly in physiologically relevant models. Here, we present a single-cell ambient mass spectrometry imaging platform, enabling direct chemical mapping of metabolites at 10 μm resolution. This method integrates cell labelling, high resolution microscopy and AP-MALDI Orbitrap mass spectrometry imaging to achieve cell-type-specific metabolite profiling. To demonstrate its application, we applied this approach to glioblastoma (GBM), an aggressive adult brain tumour characterised by cellular heterogeneity, metabolic adaptation, and infiltrative growth within the tumour microenvironment. A co-culture model combining patient-derived glioblastoma invasive-margin cells with human cortical astrocytes was used to recapitulate the invasive niche. Distinct metabolic signatures emerged upon glioblastoma–astrocyte interaction, involving pathways related to nucleotide metabolism, phospholipid and sphingolipid turnover, and tryptophan and tyrosine metabolism. These findings suggest cell-type-specific metabolic activity and potential intercellular metabolic interplay. Overall, this workflow offers a broadly accessible and robust approach for investigating metabolic heterogeneity at cellular resolution, enabling insights into metabolic interactions of heterogenous cell types in both disease and non-disease settings.