The multidimensional signature of oxysterols: Comparative RPLC-HRMS-based profiling of healthy and pancreatic tumour cell lines

Oxysterols (OS) are emerging as active drivers of cancer, shaping aggressive, immunosuppressive tumour niches rather than acting as passive cholesterol byproducts. Here, we developed and applied an advanced LC-MS strategy for the comprehensive qualitative and quantitative profiling of the total (free and esterified) OS amount in healthy human pancreatic epithelial nestin-expressing (HPNE) cells and pancreatic ductal adenocarcinoma (PANC-1) cells. Both cell types were cultured under normoxic and hypoxic conditions to elucidate the impact of oxygen availability on OS metabolism. OS separation was achieved using two complementary stationary phases, i.e., cyanopropyl (ES-CN) and pentafluorophenyl (F5), providing orthogonal chromatographic selectivity. OS identification relied on retention matching with analytical standards across both columns and was further confirmed by the evaluation of diagnostic in-source fragmentation patterns. This multidimensional analytical framework ensured the unambiguous screening of up to 14 OS in cells and their extracellular vesicles (EVs). The tumour cell phenotype, together with hypoxic conditions, was associated with a marked accumulation of ring-oxidized OS, including 3β,5α,6β-cholestantriol, 7-ketocholesterol, and 7-hydroxycholesterol epimers, suggesting either a metabolic rewiring or an increased intracellular oxidative environment. In contrast, side-chain OS showed distinct dependencies on cell type and oxygen availability: 24(S)-hydroxycholesterol was reduced in tumour cells while the amount of 26(R/S)-hydroxycholesterol was increased when both cell types were grown in normoxic conditions. EVs exhibited a unique OS signature dominated by 25-hydroxycholesterol and 5β,6β-epoxycholesterol. This study proposes a robust, orthogonal LC-MS workflow for OS profiling and provides new insights into the regulation of OS levels in pancreatic cancer, paving the way for future fundamental biochemical investigations.