Introduction: Immunotherapy has revolutionized cancer treatment. However, not all patients show effective responses to the established selection criteria. This study aims to determine whether extracellular vesicles (EVs) are appropriate tools for detecting and blocking immune checkpoint proteins (ICPs). We developed a new methodology allowing the identification of several ICPs at the surface of EVs in single liquid biopsies. Based on this technology development, we aim to profile ICPs in lung cancer patients treated by immunotherapy. Having identified novel EVs-derived immune checkpoint candidates, we will determine the efficiency of modified EVs as an immune checkpoint blockade.

Method: We isolated and characterized EVs from plasma by ultracentrifugation method. ICPs were identified through EVs surface markers analysis using the MAGPIX platform. Modified endothelial EVs were generated with siRNA targeting programmed death ligand 1 (PD-L1). Effects on pro-tumoral properties were assessed through several functional tests and in vivo using the TC1-xenograft mice model.

Results: As a proof of concept, we designed our assay to detect 6 ICPs-EVs in the bloodstream of patients (LAG-3, PD-1, PD-L1, TIM-3, TIGIT, VISTA). We highlighted PD-L1 downregulation affects the pro-tumoral properties in vitro and in vivo. 

Conclusions: We present a new method for circulating ICPs-EVs characterization in lung cancer patients. We aim to use this technology to monitor the ICPs-EVs profile in patients undergoing immunotherapeutic treatment. Furthermore, we confirmed that modified endothelial EVs impair TC-1 tumor model growth.