Extracellular vesicles in circulation in the identification of immune checkpoint associated with lung cancer

Introduction:

Immunotherapy has been one of the significant advances in recent years for the treatment of advanced tumors. This work aim to detect and block ICPs in patients with Non-small cell lung cancer (NSCL) cancer. We recently developed a new methodology allowing the identification of several ICPs at the surface of EVs in a single liquid-biopsies. First, we will profile ICP in NSCL cancer patients treated by immunotherapy to track for the emergence of resistance due to overexpression of ICPs. In a second part of the project, having identified novel EV-derived immune checkpoint candidates, we will determine if EVs can be used for immune checkpoint blockade.

Method :
EVs were isolated from plasma by ultracentrifugation method and characterized (NTA, western blotting, and DLS). ICP will be identified via an analysis of EV surface makers using the MAGPIX platform. To obtain ICP blockage; endothelial EVs were electroporated with siRNA targeting programmed death ligand 1 (PD-L1). Effects on pro-tumoral properties were assessed through several functional tests (proliferation, migration, adhesion, survival, invasion) and in vivo using the TC1-xenograft mice model.

Results :
As a proof of concept, we designed our assay to detect of 6 ICP-EV in the bloodstream of patients (LAG-3, PD-1, PD-L1, TIM-3, TIGIT, VISTA). We will use this technology to monitor the ICP-EV profile in patients undergoing immunotherapeutic treatment at three different time points (before treatment, after 3 and 6 months of treatment) in different cohorts. We have studied the effects of blocking PD-L1 in cancer cells. We confirmed PD-L1 inhibition in the targeted cells with the CARGO-EV treatment. PD-L1 downregulation affects pro-tumoral properties of lung cancer models in vitro and in vivo.

Summary/Conclusions:
We present a new method for circulating ICP-EV characterization in lung cancer patients. This study also shows that modified endothelial EV impairs tumor growth in the TC-1 lung cancer model.
Funding: This work is supported by ULIEGE, the CHU and the walloon region.