Direct lipid analysis of exosomes in serum by online miniaturized asymmetrical flow field-flow fractionation and electrospray ionization-mass spectrometry: Application to extrahepatic cholangiocarcinoma

Exosomes are submicron-sized extracellular vesicles involved in immune regulation, tumor metastasis, and cellular communication. Their lipid composition, distinct from parental cells, plays a crucial role in diseases like cancer. However, lipidomic analysis of exosomes, particularly in complex samples like blood, requires advanced techniques. This study optimizes miniaturized flow field-flow fractionation (mFlFFF) coupled with electrospray ionization mass spectrometry (ESI-MS) for direct lipidomic analysis of exosomes in serum. The mFlFFF technique resolves exosomes for size-based lipid analysis without prior extraction. Lipidomic profiling of serum exosomes from patients with extrahepatic cholangiocarcinoma (eCCA) identified over 1000 lipid species, with 64 showing significant changes compared to healthy controls. Target lipids were analyzed by mFlFFF-ESI-MS, revealing 35 species that distinguish eCCA patients from controls, suggesting their potential as biomarkers. Elevated levels of lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol (PI) were observed in the eCCA group, indicating lipid alterations linked to cancer progression and inflammation. Notably, PI 38:4, involved in the release of arachidonic acid, highlights its role in inflammatory processes associated with cancer. This study demonstrates the potential of mFlFFF-ESI-MS for lipidomic analysis of exosomes and offers a non-invasive approach for cancer diagnosis, with future implications for therapeutic targeting of lipid pathways in cholangiocarcinoma.