Extracellular vesicles (EVs) released from cancer cells contribute to various malignant phenotypes of cancer, including metastasis, cachexia, and angiogenesis. Although DNA, mRNAs, miRNAs, and proteins contained in EVs have been extensively studied, the function of metabolites in EVs remains unclear. In this study, we performed a comprehensive metabolomic analysis of pancreatic cancer cells, PANC-1, cultured under different oxygen concentrations, and small EVs (sEVs) released from them, considering the fact that hypoxia contributes to the malignant behavior of cells in pancreatic cancer, which is a poorly diagnosed cancer. sEVs were collected by ultracentrifugation, and hydrophilic metabolites were analyzed using capillary ion chromatography-mass spectrometry and liquid chromatography-mass spectrometry, and lipids were analyzed by supercritical fluid chromatography-tandem mass spectrometry. A total of 140 hydrophilic metabolites and 494 lipids were detected in sEVs, and their profiles were different from those in cells. In addition, the metabolomic profile of sEVs was observed to change under hypoxic stress, and an increase in metabolites involved in angiogenesis was also detected. We reveal the hallmark of the metabolites contained in sEVs and the effect of tumor hypoxia on their profiles, which may help in understanding EV-mediated cancer malignancy.

Many previous studies have reported the various proteins specifically secreted as inducers in the dorsal or ventral regions in vertebrate gastrula. However, little is known about the effect on cell fate of small molecules below 1000 Da. We therefore tried to identify small molecules specifically expressed in the dorsal marginal zone (DMZ) or ventral marginal zone (VMZ) in vertebrate gastrula. Small intracellular and secreted molecules were detected using explants and supernatant samples. Hydrophilic metabolites were analyzed by capillary ion chromatography–mass spectrometry and liquid chromatography–mass spectrometry, and lipids were analyzed by supercritical fluid chromatography–tandem mass spectrometry. In total, 190 hydrophilic metabolites and 396 lipids were identified. The DMZ was found to have high amounts of glycolysis- and glutathione metabolism-related metabolites in explants, and the VMZ was richer in purine metabolism-related metabolites. We also discovered some hydrophilic metabolites and lipids differentially contained in the DMZ or VMZ. Our research would contribute to a deeper understanding of the cellular physiology that regulates early embryogenesis.

Gas chromatography/mass spectrometry (GC/MS) is widely employed in metabolomics owing to its high resolution, reproducibility, and compatibility with volatile and semi-volatile compounds. While helium has traditionally been used as the carrier gas, increasing costs and global shortages have prompted the exploration of hydrogen gas as an alternative. Hydrogen offers faster analysis times and reduced ion source contamination but presents challenges, including ion source reactions, which can change compound fragmentation patterns. To address this, a hydrogen-deactivated electron ionization (EI) source has been developed to minimize undesired chemical interactions and improve spectral integrity. In this study, we systematically evaluated the effect of extraction lens sizes (3, 6, and 9 mm) under hydrogen carrier gas conditions using n-alkane and human metabolomic standard solutions. The hydrogen-deactivated ion source equipped with a 3 mm extraction lens provided superior peak symmetry and the highest signal intensities, with acceptable relative standard deviation (RSD) values (i.e., below 30%). In addition, the reproducible fragmentation patterns as those observed with helium carrier gas were obtained for phosphorylated metabolites. Using this optimized configuration, we applied hydrogen gas-based GC/MS to metabolomic profiling of the human colon cancer cell line HCT116 with mutant isocitrate dehydrogenase 1 (IDH1). Key metabolic alterations, including increased levels of the known biomarker 2-hydroxyglutaric acid, were detected in IDH1-mutant cells compared with wild-type cells. These findings establish hydrogen gas-based GC/MS with a hydrogen-deactivated ion source as a robust and reliable platform for metabolomics, offering an effective alternative to helium-based systems.