Lipid profiling of mouse intestinal organoids for studying APC mutations

Inactivating mutations including both germline and somatic mutations in the adeno-matous polyposis coli (APC) gene drives most familial and sporadic colorectal can-cers. Understanding the metabolic implications of this mutation will aid to establish its wider impact on cellular behaviour and potentially inform clinical decisions. However, to date, alterations in lipid metabolism induced by APC mutations remain unclear. In-testinal organoids have gained widespread popularity in studying colorectal cancer and chemotherapies, because their 3D structure more accurately mimics an in vivo environ -ment. Here, we aimed to investigate intra-cellular lipid disturbances induced by APC gene mutations in intestinal organoids using a reversed-phase ultra-high-performance liq-uid chromatography mass spectrometry (RP-UHPLC-MS)-based lipid profiling method. Lipids of the organoids grown from either wild-type (WT) or mice with APC mutations (Lgr5-EGFP-IRES-CreER(T2) Apc(fl/fl)) were extracted and analysed using RP-UHPLC-MS. Levels of phospholipids (e.g. PC(16:0/16:0), PC(18:1/20:0), PC(38:0), PC(18:1/22:1)), ceramides (e.g. Cer(d18:0/22:0), Cer(d42:0), Cer(d18:1/24:1)) and hexosylceramides (e.g. Hex-Cer(d18:1/16:0), HexCer(d18:1/22:0)) were higher in Apcfl/fl organoids, whereas levels of sphingomyelins (e.g. SM(d18:1/14:0), SM(d18:1/16:0)) were lower compared with WT. These observations indicate that cellular metabolism of sphingomyelin was up-regulated, result-ing in the cellular accumulation of ceramides and production of HexCer due to the absence of Apcfl/fl in the organoids. Our observations demonstrated lipid profiling of organoids and provided an enhanced insight into the effects of the APC mutations on lipid metabolism, making for a valuable addition to screening options of the organoid lipidome.