The role of extracellular vesicles in CLL pathogenesis

Extracellular vesicles (EVs) released by tumor cells represent a novel mechanism in cell communication which allows them to alter their microenvironment. EVs are membrane enclosed nanoparticles 30 to 1000 nm in size. By hijacking proteins and RNA molecules from their cell of origin, vesicles are able to reprogram target cells and thus might be involved in creating a pro-tumorigenic microenvironment. As we and others have observed that CLL cells are able to influence the differentiation of myeloid cells and thereby initiate a pro‐survival feedback in their favor, we hypothesized that EVs are involved in this process. Therefore, we established a protocol to isolate EVs from serum of CLL patients as well as healthy donors by serial centrifugation and density‐based separation. Characterization of isolated EVs by electron microscopy revealed a heterogeneous vesicle population enriched for structures of 30 to 300 nm in size with no major contamination of apoptotic bodies or cell debris (Haderk et al., 2013). Using immunogold stainings, the presence of the MHC class II protein HLA-DR on the surface of the EVs was detected. In addition, Western blot analyses of EV preparations and the corresponding whole cell lysates revealed an enrichment of EV marker proteins, i.e. Rab5a, Hsp70 and HLA-DR, in the vesicle preparations, confirming the isolation of EVs.
Proteomic profiling by mass spectrometry comparing lysates of blood plasma‐derived EVs versus the respective peripheral blood mononuclear cells from CLL patients or healthy donors revealed an EV-specific protein profile which is currently investigated in detail. Isolation and characterization of the RNA content of the EVs revealed an enrichment of small RNA species which are currently analyzed by next generation sequencing. In addition, functional studies with isolated EVs are ongoing, to evaluate their impact on target cells and therefore their potential role in CLL pathogenesis.

Isolation and characterization of EVs in CLL

Protocol for isolation of extracellular vesicles and their detection by electron microscopy and Western blot analysis using antibodies specific for known marker proteins (Haderk et al., 2013).

to top
powered by webEdition CMS