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 three-dimensional 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 wildtype (WT) or mice with Apc mutations (Lgr5-EGFP-IRES-CreERT2Apcfl/fl) were extracted and analysed using RP-UHPLC-MS. Concentrations 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 hexosylceramide (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 to WT. These observations indicate that cellular metabolism of sphingomyelin was upregulated, 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.

中文翻译：

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用于研究 APC 突变的小鼠肠道类器官的脂质分析。

包括腺瘤性结肠息肉 (APC) 基因中的种系和体细胞突变在内的失活突变驱动大多数家族性和散发性结直肠癌。了解这种突变的代谢影响将有助于确定其对细胞行为的更广泛影响，并可能为临床决策提供信息。然而，迄今为止，由 APC 突变引起的脂质代谢改变仍不清楚。肠道类器官在研究结直肠癌和化疗方面已广受欢迎，因为它们的三维结构更准确地模拟了体内环境。这里，我们旨在使用基于反相超高效液相色谱质谱 (RP-UHPLC-MS) 的脂质分析方法研究由肠道类器官中 APC 基因突变引起的细胞内脂质紊乱。使用 RP-UHPLC-MS 提取和分析从野生型 (WT) 或具有 Apc 突变 (Lgr5-EGFP-IRES-CreERT2Apcfl/fl) 的小鼠生长的类器官的脂质。磷脂浓度（例如 PC(16:0/16:0)、PC(18:1/20:0)、PC(38:0)、PC(18:1/22:1)）、神经酰胺（例如 Cer (d18:0/22:0)、Cer(d42:0)、Cer(d18:1/24:1)) 和己糖神经酰胺（例如 HexCer(d18:1/16:0)、HexCer(d18:1/22) :0)) 在 Apcfl/fl 类器官中较高，而与 WT 相比，鞘磷脂水平（例如 SM(d18:1/14:0)、SM(d18:1/16:0)）较低。这些观察结果表明鞘磷脂的细胞代谢被上调，由于类器官中没有 Apcfl/fl，导致神经酰胺的细胞积累和 HexCer 的产生。我们的观察结果证明了类器官的脂质谱，并提供了对 APC 突变对脂质代谢影响的深入了解，为类器官脂质组的筛选选项提供了有价值的补充。