Abstract
Inactivating mutations including both germline and somatic mutations in the adenomatous polyposis coli (APC) gene drives most familial and sporadic colorectal cancers. 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. Intestinal organoids have gained widespread popularity in studying colorectal cancer and chemotherapies, because their 3D structure more accurately mimics an in vivo environment. Here, we aimed to investigate intra-cellular lipid disturbances induced by APC gene mutations in intestinal organoids using a reversed-phase ultra-high-performance liquid 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-CreERT2 Apcfl/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. HexCer(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, resulting 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.
Highlights
Colorectal cancer is the third most common cancer and the fourth most common cause of cancer deaths worldwide [1]
The quantity control (QC) dilution series samples approached the QCs cluster, as their concentration increased. Both Principal components analysis (PCA) score plots showed the same outlier, a sample from APCfl/fl m862 donor, which was due to a sample loss during the sample extraction, this sample was excluded from the subsequent analyses
PCA was carried out based on the data from the WT and Apcfl/fl organoids and a clear separation between the two groups was observed along the first principal component in both electrospray ionisation (ESI)+ and ESI- modes (Figure 1 and Supplementary Figure S3)
Summary
Colorectal cancer is the third most common cancer and the fourth most common cause of cancer deaths worldwide [1]. Approximately 5% of cases are associated with hereditary conditions, such as familial adenomatous polyposis (FAP). The patients with FAP have one mutated copy of the APC gene and carry a near 100% lifetime risk of developing colorectal cancer [3]. Even in cases of sporadic colorectal cancer, loss of functional APC is typically the first mutational event to occur [4]. APC mutations have been found in over 80% of colonic tumours [4]. Given its prevalence, uncovering the wider effects of an inactivating APC mutation is important to fully understand colorectal cancers and aid the development of more effective personalised treatments. The APC gene encodes the tumour suppressor protein APC, which regulates the degradation of proto-oncogene protein β-catenin
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