Identification and proteomic profiling of biliary exosomes

Background & Aims: In previous studies the biliary proteome has been analyzed, showing a distinct set of proteins to be abundantly present in the bile fluid. Within these proteins there was a considerable amount of membranous and cytosolic proteins indicating a vesicular form of protein excretion within the biliary tract. We aimed to identify the possible secretion of exosomes into the bile fluid and characterize their biochemical and proteomic constitution.

Methods: Bile fluid of patients admitted to ERC due to the presence of choledocholithiasis in absent of clinical and laboratory signs of cholangitis were collected during routine ERC. We isolated microvesicles by using gradual centrifugation and further purification by floating onto a sucrose gradient. Isolated vesicles were further characterized by Western Blotting, Electron microscopy, Nanoparticle Tracking Analysis and mass spectrometry.

Results: In bile fluid of microvesicles with a size of 50 – 90nm could be regularly detected. In these vesicles exosomal markers like CD9, ADAM10 and HSP70 could be detected. Furthermore electron microscopy revealed cup shaped vesicles indicating the presence of exosomes in human bile fluid. The proteomic analysis of biliary exosomes showed more than 400 non-redundant proteins, of which more than 100 have not been detected in exosomes before as listed by the exosome database ExoCarta. Among these proteins are e.g. Annexin 10, CEACAM6, CEACAM7, as well as tetraspanins and mucins.

Conclusion: Microvesicles isolated from human bile fluid display any characteristics of exosomes. Their hepatobiliary origin afford them as a new diagnostic tool to gain further insight into the pathogenesis of biliary and cholestatic diseases.

Corresponding author: Rupp, Christian

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