An extracellular vesicle multiplexed immunoassay to profile the immune checkpoint landscape in plasmatic samples

Immunotherapy based on immune checkpoint blockade has shown significant success in the treatment of advanced cancers, particularly solid tumors. However, only a subset of patients experiences long-term clinical benefits. One proposed mechanism of resistance is re-expression of immune checkpoint proteins (ICPs). Although more than 40 ICPs have been identified, monitoring their levels in the bloodstream remains challenging. Recent evidence indicates that various cell types within the tumor microenvironment can release extracellular vesicles (EVs) that display ICPs on their surface. Based on these observations, we developed a technique for detecting the ICP levels on the surface of EVs (EV-ICPs) in plasma samples. This multiplex array enabled the detection of EV-ICPs isolated by ultracentrifugation, using minimal volumes of plasma from both mice and human specimens. Notably, in tumor-bearing mice treated with anti-PD​L1 antibodies, we observed an increase in the abundance of several EV-ICPs. Furthermore, analysis of the plasma from patients with breast cancer revealed a generally elevated presence of EV-ICPs. The use of 8 EV-ICP markers as a signature could significantly discriminate patients with breast cancer from healthy controls. Thus, our EV-ICP multiplex array provides a promising, minimally invasive liquid biopsy approach for tracking dynamic changes in ICP expression during tumor growth and offers a new perspective for monitoring cancer progression.