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Singled Out: New Technology for Analyzing Single Extracellular Vesicles (EVs)
Singled Out: New Technology for Analyzing Single Extracellular Vesicles (EVs)

The Common Fund Extracellular RNA Communication (ERC) program aims to understand the fundamental biology of extracellular RNA (exRNA), as well as accelerate development of exRNAs as potential therapeutics and diagnostics. RNA, sometimes in the form of extracellular RNA or “exRNA,” can be released by cells and transported through the body, with the potential to influence a recipient cell. The current and final second stage of the ERC program focuses on tool and technology development to understand biological containers like Extracellular Vesicles (EVs) that carry exRNAs and other cargo through the body. Researchers from the ERC program recently piloted a new method called ‘Single Extracellular Vesicle Protein Analysis Using Immuno-Droplet Digital Polymerase Chain Reaction (PCR) Amplification.’ This method can ultra-sensitively detect rare proteins of interest carried by single EVs. Finding rare proteins in body fluids may be critical to detecting diseases much earlier than currently possible. This method could allow clinicians to identify rare but highly predictive tumor cell derived EVs (“tEVs”) and improve the performance of current EV cancer diagnostics. 

Because EV populations are highly varied, even more so than the cells from which they are derived, more research is needed to better understand them and their cargo. Detecting rare proteins contained in EVs – those that are in low quantities or in only select EVs – can be informative for determining if a disease is in very early stages. However, detecting rare proteins in EVs can be problematic because direct detection results in very low level signals that could be uninterpretable. The new method developed by ERC researchers uses a signal amplification step to overcome previous limitations of direct detection. This novel method also pairs signal amplification with a technique for isolating single EVs, letting researchers identify the protein cargo of individual EVs. Using multiple chemical tags to identify different proteins allows researchers to look for more than one protein at a time, which may offer insight into protein signatures contained in EVs. 

To verify the method, researchers first examined EVs containing the well characterized protein PD-L1. PD‐L1 levels can be predictive of response to immunotherapy. Using their new method coupled with flow cytometry, a technique to simultaneously analyze specific characteristics of thousands of individual cells, they directly determined the quantity and fraction of PD‐L1 containing EVs released by tumor cells. Better understanding of EV variation and of the RNA and non-RNA cargo they carry is necessary to determine which and how these carriers send messages to other cells. Not only is this important for understanding the role of EVs in intercellular signaling, but it also increases the translational potential to diagnose and treat diseases.

Ko, J., Wang, Y., Carlson, J. C. T., Marquard, A., Gungabeesoon, J., Charest, A., Weitz, D., Pittet, M. J., Weissleder, R., Single Extracellular Vesicle Protein Analysis Using Immuno‐Droplet Digital Polymerase Chain Reaction Amplification. Adv. Biosys. 2020, 1900307.

This page last reviewed on August 23, 2023