Chronic Lymphocytic Leukemia-Derived Extracellular Vesicles Mediate NFκB Signaling and Pro-Inflammatory Cytokine Release in Monocytes

Abstract

Abstract Chronic lymphocytic leukemia (CLL)is a B-cell malignancy associated with an inflammatory milieu and impairedanti-tumor immunity. This study aimed at characterizing CLL-derived extracellularvesicles (EVs) and their role in the tumor microenvironment. EVs were isolated from blood plasma or culture supernatant of the CLL cell line MEC-1 by a serialcentrifugation protocol. Characterization of EVs by electron microscopy, Nanoparticle Tracking Analysis (NTA) and Western blotting revealed vesicles 30to 350 nm in size, which were positive for various EV marker proteins. Quantificationof EVs by NTA and ELISA indicated an enrichment of B-cell derived EVs in plasmaof CLL patients compared to healthy controls, although absolute EV counts were not different. As we observed an accumulation of small RNAs in EVs, small RNAsequencing was performed which revealed a unique microRNA signature of MEC-1 EVs, with CLL-relevant miRNAs such as miR-21, miR-155 and miR-146a being the mostabundant. Moreover, full length and 5′ end fragment forms of Y RNAs, another class of small non-coding RNAs, were enriched in MEC-1 and CLL plasma EVs. In vitro and in mice, CLL EVs were rapidly taken up by monocytes and macrophages which resulted in NFκB signaling and the release of multiple pro-inflammatory cytokines such as CCL2, CCL3, IL-6 andIL-8, proteins known to be elevated in CLL plasma. Moreover, several surface markers on monocytes were altered upon EV uptake, including an increase of the immune checkpoint molecule PD-L1 as well as downregulation of the chemokine receptorCCR2. Of interest, liposomes containing Y RNA transcripts induced a similar response in respective assays suggesting a novel role for these molecules in the tumor microenvironment of CLL.