Chronic Lymphocytic Leukemia-Derived Extracellular Vesicles Contain a Distinctive Proteome, As Well As Specific Micro RNAs and Y RNAs

The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Of note, CLL cells themselves induce changes in surrounding cells, and extracellular vesicles (EVs) released by CLL cells represent a newly discovered mechanism of cell-cell communication. EVs are membrane enclosed nanoparticles 30 to 1000 nm in size and are able to reprogram recipient cells by transferring proteins and RNA molecules from their cell of origin. Thus, we aimed to analyze CLL cell-derived EVs present in blood plasma of CLL patients as well as those released by the CLL cell line MEC-1, in order to understand their role within the microenvironment.

EVs were isolated from blood plasma of CLL patients and healthy donors, as well as from MEC-1 cell culture supernatant by serial centrifugation and density-based separation. Characterization of EVs by electron microscopy and immunoblot analysis revealed vesicles 20 to 300 nm in size, which are positive for various EV marker proteins such as Rab5a and Hsp70. Proteome analysis via mass spectrometry indicated differences in the composition of plasma derived EVs and peripheral blood mononuclear cells (PBMCs) from the respective patient, as well as between plasma derived EVs from healthy donors compared to CLL patients. Regarding the later, CLL EVs were specifically enriched for proteins involved in antigen presentation, endocytosis signaling as well as integrin mediated signaling and leukocyte extravasation. RNA analysis by BioAnalyzer profiling indicated an enrichment of small RNAs in EVs compared to cells. Subsequent small RNA sequencing revealed a unique microRNA signature of MEC-1 EVs with the 5 most abundant miRNAs encompassing about 60% of all detected miRNAs. Among them, the CLL-relevant miR-21 and miR-155, as well as miR-146a were selectively enriched in EVs. Moreover, Y RNAs, another class of small non-coding, regulatory RNAs, were highly enriched in MEC-1 EVs and their presence was also observed in plasma EVs of CLL patients. We uncovered a rapid uptake of CLL cell-derived EVs by human monocytes and macrophages. Whether the identified proteins and RNA transcripts shown to be enriched in CLL EVs induce phenotypic changes in targeted cells is being investigated. Further, quantification of plasma EVs in a large cohort of CLL patients is currently conducted and differences in the amount of EVs in correlation to disease outcome are analyzed.

Harboring a distinct RNA and protein profile, EVs are potent vehicles for shuttling RNA and proteins to recipient cells and might be involved in the establishment of a supportive microenvironment in CLL. Functional analyses regarding possible effects of CLL EVs on target cells will broaden the knowledge of CLL pathogenesis and might help to identify new therapeutic options for CLL.