Abstract 4483: Detection of immunomodulatory markers in circulating exosomes
Authors: Sanchez Veronica , Daaboul George 
Published: September 2020

Abstract
Introduction: Cancer growth and progression are associated with immune suppression. Checkpoint blockade and CAR-T therapies have had success with improving cancer survival rate, yet some patients still respond poorly. This represents a challenge for oncologist when choosing personalized immunotherapies. Recent work has implicated changes in host immunology as a factor. Several biomarkers reflecting tumor immune microenvironment, such as PD-L1, can be used to predict treatment outcome; however invasive sampling deem this route difficult. Circulating biomarkers, have shown promising results as metric predictive of immunotherapy response as they can be non-invasively obtained and trended over time. Exosomes are a subclass of extracellular vesicles (EV) distributed widely in body fluids like urine and plasma. Exosomes can transport cell signals for communication in their cargo and surface, composed of proteins, nucleic acids, and other molecules. Emerging evidence has shown that exosomes can carry immune checkpoint molecules, whose expression has been correlated with anti-tumor immunity response. Here, we report the development of a non-invasive assay for detection of immunomodulatory markers in circulating exosomes.
Methods: Circulating PD-L1 expressing exosomes are captured on the ExoView® sensors containing different antibody clones against PD-L1. Samples were incubated on chips overnight to allow the antibody to bind the antigen on the EV, thus immobilizing it. After incubation, chips were washed and immunolabeled. Once immunolabeled the chips were washed to remove unbound antibody and scanned using the ExoViewer R100. Assay performance was evaluated with PDL-1 over-expressing exosomes and cancer cell line derived exosomes. The assay was then tested using human plasma from different cancer types.
Results: EVs were successfully captured by PD-L1, and antibodies against exosome markers. The specificity of each PD-L1 clone was studied with HEK293T-derived exosomes over-expressing PD-L1. Results demonstrated that all tested clones were able to capture PDL-1 positive exosomes versus negative control. However when tested with exosomes from several cancer cell lines, the different antibody clones captured different number of exosomes within the same sample, suggesting epitope accessibility might be a problem in some cases. When immunolabeling such EVs for CD9 and CD63 (exosome markers), some clones showed differential expression of these proteins, suggesting heterogeneity in PD-L1 protein being expressed by cancer cell lines. Lastly, we tested the ability of the assay in detecting PD-L1 positive exosome from plasma, and detected differences between control and patients with different types of cancer.
Conclusions: We successfully used exosomes to detect PD-L1 from human plasma. This assay could potentially be used for treatment outcome and monitoring, and combined with other markers to identify the source of PDL-1.
Citation Format: Veronica Sanchez, George Daaboul. Detection of immunomodulatory markers in circulating exosomes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4483.