Positive and negative selection of extracellular vesicle-based biomarkers: insights from L1CAM immunocapture and emerging immunodepletion strategies

Extracellular vesicles (EVs) reflect their spatiotemporal cellular origins and, when released into the peripheral bloodstream, represent sources of biomarkers for processes occurring in inaccessible tissues, such as the brain. Positive selection by immunoaffinity isolation targeting a specific EV surface marker can be used to preferentially enrich for EVs from a particular cell type over other EVs in circulation. However, the case of L1CAM immunocapture for enrichment of brain-derived neuronal EVs has exemplified the biological and technical influences affecting the outcomes of positive selection, which are discussed here. These include findings from the wider (non-EV) literature showing that soluble L1CAM is a binding partner of the common EV marker CD9. Additional studies show that CD9 is not expressed by the vast majority of neurons in the brain, but it is highly expressed by neurons of the peripheral nervous system, which could affect the interpretation of studies demonstrating the co-localisation of L1CAM and CD9 on EVs to assert isolation of EVs derived from neurons in the brain. Next, emerging negative selection strategies are discussed which, when combined with positive selection, could overcome some of the challenges associated with enriching for EV-based biomarkers. These strategies include depleting EVs on a cell-by-cell type basis, as well as targeting common EV markers to simultaneously deplete diverse, undesired EV populations whilst selecting for the EVs of interest. An example is given of how CD9 depletion could enrich and select for brain-derived neuronal EVs. Additionally, studies that have used negative selection alone to select for EVs, and the advantages of this approach for biomarker detection, are highlighted. Finally, based on these insights, considerations for the choice of future positive and negative selection targets are discussed, including how understanding the nuances of EV heterogeneity could facilitate the process of EV-based biomarker discovery and their translation to the clinic.

Extracellular vesicles are miniscule parcels released by cells with functions that include communication. These parcels contain molecular messages in the form of proteins and RNA which can be read by other cells that receive the parcels. Since the messages reflect the status of the sending cell, they can also warn other cells about a disease affecting the sending cell.

The parcels are transported between cells by the movement of fluids in the body, including the bloodstream. Because of this, researchers in the lab can take a blood sample then use techniques to separate the parcels from the blood and read the molecular messages contained within. This is a useful approach for understanding diseases in inaccessible tissues such as the brain because the disease processes can be indirectly studied via the parcels that were released into the blood by brain cells.

A barrier to this approach is that most of the parcels found in the blood are derived from blood cells and these mask disease-specific signals coming from the brain. To overcome this, researchers can use an approach called positive selection to target a brain cell marker on the parcels’ surface and enrich brain-specific signals. However, in a large, complex structure such as the human body, many cells release parcels with overlapping markers which can again obscure brain-specific messages. This article looks at how issues such as these have been raised and addressed when targeting a protein called L1CAM to enrich parcels coming from neuronal cells in the brain. Negative selection is also discussed as an alternative approach, where the unwanted parcels are targeted for removal to enrich the desired parcels in the sample. Together, these approaches could clarify messages coming from the brain so that we can identify early warning signals (biomarkers) in disease diagnostics and design more effective treatments.