Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assays

Interesting technical feasability study looking at using PEA for profiling extracellular vesicles (EVs) such as exosomes and their potential as clinical biomarkers. EVs are released by many cells, can be found in bodily fluids and may exhibit different surface and internal components depending on their origin, which may make them potentially interesting as biomarkers (e.g. exosomes from cancer cells may transport tumor antigens). This study used 3 commercial Olink panels plus 3 experimental intracellular panels to examine protein biomarker expression in both EVs and source cells. In the first stage they examined whether PEA was suitable to detect proteins in exosome preparations from different cell lines, and how that related to the source cell lysates. Using the Onc 1v2 panel, 58 markers were detected in cell lysates, and 37 in the exosomes. Statistical analysis showed a distinct clustering of proteins in cell lysates with their respective exosomes for each of the cell lines examined, suggesting that this technique could be used to identify the cellular origin of exosomes. This was verified using additional cell lines and all six of the panels – all samples showing a clear clustering according to cell type for cell lysate vs exosome. A few proteins were detected in all types of exosome examined (e.g. ICAM1, U-PAR, EpCAM), but many were specific to individual cell lines and their corresponding exosomes. To examine the utility of these in vitro observations, EVs were isolated from human milk (exosomes) and seminal plasma (prostasomes) and analyzed for protein content compared to all the cell lines used in the study. Milk exosomes clustered closest to a human breast cancer cell line, whereas the prostasomes clustered with two prostate cancer-derived cell lines – suggesting that cellular origin may be determined for EVs present in bodily fluids. In addition to clustering with their source cell-type, the two types of exosomes also displayed very distinct proteomic profiles from each other. The conclusion was that PEA is a highly promising tool for classifying and tracing the tissue origin of circualting EVs, and that this could provide valuable diagnostic, prognostic and therapeutic avenues in the clinical setting.