Background:Exosomes are released from cells and circulated into the blood. Thus, exosomes are regarded as an attractive biological resource for the development of new cancer marker for liquid biopsy diagnosis. Exosomes generally covered with glycocalyx, which are controlled by a host cell glyco-synthetic machinery as similar to secreted and plasma membrane glycoproteins. Several exosomal subpopulations classified by tetraspanins have been investigated in relationship to diseases. However, their comparative analysis has never been attempted in terms of glycomics. In this study, we demonstratecomparative glycomic analysis between exosomes, secreted, and cell membrane glycoproteins derived from pancreatic cancer cells using lectin microarray system.
Methods:Pancreatic cancer culture cells derived secreted, plasma membrane glycoproteins, and exosomes were prepared, respectively. Exosomes were isolated and purified using commercially available exosome isolation kits. Further each CD antigens-positive exosomes were fractionated from total exosomes using specific antibodies against exosome marker (CD9, CD63, CD81) and were subjected to the comparative glycan profiling with lectin microarray.
Finding:As the result, it was found that the total exosome-derived glycoproteins contain abundant glycoproteins with specific glycan structures compared with secreted and membrane glycoproteins. Moreover, the multivariable analysis of the glycan profiling of the antigen positive exosomes indicated that each exosome fraction showed specific lectin signal patterns. The results suggest that the surface glycan structures vary depending on the exosome subpopulations.
Conclusion:In this study, we succeeded in establishing the protocol for rapid glycan profiling on the surface of small particles such as exosomes. This method is expected to be useful for the discovery of tumor-associated exosomal glycosylation.