Single-cell lipidomics of adherent gut epithelial cell lines Caco-2 and HT29-MTX reveals a metabolic switch

In vitro cell models of the gut epithelium, particularly those based on the Caco-2 and HT29-MTX cell lines, play an important role in studying the uptake and metabolism of nutrients and pharmaceuticals. Previous studies have shown a distinctive lipidome signature for these cells, alone and in co-culture, using mass spectrometry imaging; however, only limit information on lipid identities was obtained. A novel method employing limited trypsinolysis for sampling live, adherent individual cell using and automated capillary extraction workflow was developed. The lipidomes of the cell samples was then mapped using LC-MS/MS. Approximately ~ 150 lipid were identified per live-cell sample which confirmed the distinctive lipidomes of the Caco-2 and HT29-MTX cell lines. Cell-to-cell heterogeneity was observed, especially in the Caco-2 cells, perhaps indicating a different degree of differentiation in the confluent cultures. Metabolic pathway analysis confirmed the differences related to the increased glycerol-3-phosphate pathway activity involved in di- and tri-glyceride synthesis in Caco-2 cells, consistent with their enterocyte-like characteristics. Conversely, HT29-MTX cells exhibited a more active phosphatidylcholine metabolism, related to their mucus-secreting capability. These differences validate the approach taken to single cell analysis of these adherent cell lines.