Abstract A30: Chronic lymphocytic leukemia-derived extracellular vesicles mediate NFkB signaling and pro-inflammatory cytokine release in monocytes

Abstract

Abstract The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Extracellular vesicles (EV) released by CLL cells are taken up by non-malignant cells in the microenvironment and mediate major disease-related changes in recipient cells. In the current study, we characterized CLL EVs, especially focusing on vesicle-incorporated RNA transcripts, and defined the role of CLL EVs in changing the myeloid tumor microenvironment. EVs were isolated from blood plasma of CLL patients and healthy donors as well as from supernatant of the CLL cell line MEC-1 by a serial centrifugation protocol. Characterization of EVs by electron microscopy, Nanoparticle Tracking Analysis (NTA) and Western blotting revealed vesicles 30 to 350 nm in size, which are positive for various EV marker proteins such as Rab5a and Hsp70. Quantification of blood plasma-derived EVs indicated an enrichment of B-cell derived EVs in plasma of CLL patients compared to healthy donors, although absolute EV counts were not altered in CLL. Focusing on RNA analysis, an enrichment of small RNAs in EVs was observed. Subsequent small RNA sequencing revealed a unique microRNA signature of MEC-1 EVs, with CLL-relevant miRNAs such as miR-21, miR-155 and miR-146a being the most abundant miRNAs. Moreover, full length and 5'end fragment forms of Y RNAs, another class of small non-coding RNAs, were enriched in MEC-1 EVs and CLL plasma EVs. Further evaluating the functional relevance of CLL EVs within the tumor microenvironment, a rapid uptake of CLL cell-derived EVs by human monocytes and macrophages was observed. Uptake of CLL EVs in monocytes induced NFkB signaling and the release of multiple pro-inflammatory cytokines such as CCL2, CCL3, IL-6 and IL-8, which are also upregulated in plasma of CLL patients. In conclusion, tumor-derived EVs harbor a distinct set of non-coding RNAs. The uptake of EVs in recipient cells and the concomitant transfer of incorporated RNAs mediate substantial phenotypic changes in target cells. In the current study, this is exemplified for monocytes, which present several disease-relevant alterations upon EV uptake, including cellular activation and secretion of pro-inflammatory cytokines. Citation Format: Franziska Haderk, Laura Llao Cid, Etienne Moussay, Jerome Paggetti, Karolin Willmund, Jana Seiler, Sven Diederichs, Maria Goebel, Jan Duerig, Thorsten Zenz, Stephan Stilgenbauer, Marc Zapatka, Peter Lichter, Martina Seiffert. Chronic lymphocytic leukemia-derived extracellular vesicles mediate NFkB signaling and pro-inflammatory cytokine release in monocytes. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A30.