CLL-Derived Exosomes Reprogram the Phosphoprteomic Landscape of Endothelial Cells.

CLL cells travel between blood, bone marrow and lymphatic tissues. In the lymph nodes CLL cells receive pro-survival signals coming from neighboring cells. We hypothesized that CLL cells shape their own fate by sending "floating" messages that are taken up by endothelial cells. These messages subsequently reprogram the protein repertoire of the recipient cells to support their survival.

Exosomes are cellular-derived nano-sized particles, secreted by different type of cells including tumor cells, carrying a cargo of proteins and nucleic acids. They are taken up by other cells and alter their phenotype.

Our goal was to characterize the modifications that endothelial cells undergo in response to CLL- derived exosomes to transform them into CLL-supportive cells.

CLL cells were isolated from patients and cultured in exosome free medium for 72h. Thereafter, exosomes were isolated by ultracentrifugation and quantified by NanoSight tracking system. The uptake of exosomes by HUVEC (Human Umbilical Vein Endothelial Cells) was quantified by flow cytometry and fluorescent microscopy. The phosphoprotein profiling of exposed and unexposed cells was analyzed by Mass spectrometry and validated by Q-PCR. The levels of beta catenin and IL-6 were assessed by Western blotting and ELISA, respectively. To annotate the proteomics data we used DAVID (Database for Annotation, Visualization and Integrated Discovery) and ANAT (Advanced Network Analysis Tool).

We found that HUVECs uptake exosomes in a dose and time dependent manner, the pick uptake being 24h from exposure. 53 peptides were significantly more phosphorylated after cells exposure to CLL-derived exosomes. Q-PCR validated the phosphoproteomic results. Numerous of these proteins are involved in processes conferring a survival advantage to the cells.

Among these 53 proteins is beta catenin which was previously shown to induce the secretion of IL-6. Furthermore, IL-6 induces STAT3 phosphorylation, thus increasing CLL cells` viability. We confirmed that phosphor-beta catenin is several fold higher in exosomal-treated cells and that these cells secrete IL-6 to the cultured media after exosomal exposure and over expression of beta catenin.

By secreting exosomes which carry a cargo of mRNAs and proteins CLL cells reprogram endothelial cells to activate various pathways. In this way the crosstalk between CLL cells and endothelial cells may contribute to CLL cells` survival at lymph nodes where CLL cells survive and proliferate.