CLL-Derived Exosomes Educate Endothelial Cells to Become CLL-Supportive Cells

Background: CLL is characterized by a gradual accumulation of mature appearing long-lived lymphocytes. Several pro-survival pathways that protect CLL cells from apoptosis are activated in these cells. For example, IL-6 dependent phosphorylation of the signal transducer and activator of transcription 3 (STAT3) provides CLL cells with a survival and a proliferative advantages. What is the source of IL-6 is currently unknown. Within lymphoid organs CLL cells engage in complex molecular interactions. In these sites, CLL cells are not simply the seeds that grow on the supportive soil of the microenvironment but play an active role in shaping its surrounding. Secreted by all types of cells, exosomes are nano-scaled particles that travel in blood and function as stable intercellular transport vehicles that deliver their cargo to cells that engulf them. For example, CLL-derived exosomes (herein CLL-exosomes) are taken up by mesenchymal stromal cells, transforming them to cancer associated fibroblasts. Given the appropriate stimulation, endothelial cells produce IL-6. Therefore, we hypothesized that CLL-exosomes actively recruit endothelial cells to become IL-6-secreting cells.Methods: CLL cells from 37 patients were included in this study. CLL-exosomes were isolated by ultracentrifugation. Electron microscopy, NanoSight tracking analysis, flow cytometry and Western immunoblotting were used to characterize CLL exosomes. Exosomal uptake by HUVECs was assessed by flow cytometry and fluorescent microscopy. The phosphor-protein profiling of exposed HUVECs was analyzed by mass spectrometry. RT-PCR and Western immunoblotting were used to determine the expression profile of HUVEC-exposed cells. HUVECs were transfected with β-catenin containing plasmid using DNA transfection reagent. Cytokine levels were determined by ELISA and CHIP assay was used to identify activated transcription factors .Results: First, we isolated CLL-exosomes from 37 treatment naïve patients. For that purpose we grew CLL cells and collected the secreted exosomes after 72 hours by ultracentrifugation. By NanoSight tracking system we identified large amount of exosomes and verified the presence of the typical cap shaped vesicles by electron microscopy. Western immunoblotting confirmed the presence of CD63 and CD81 exosomal markers and by flow we detected CD19/CD5 on these particles. Next, we exposed HUVECs to an increasing amount of CLL-exosomes and verified by two distinct methods that these particles are up taken by HUVECs in a dose- and time- dependent manner. By mass spectrometry we found 53 phosphorproteins that were at least 2 folds upregulated and none that were downregulated in HUVEC-exposed cells. Pathway analysis unraveled the central position of β-catenin. Immunoprecipitation studies verified that levels of phosphor-β-catenin are upregulated while levels of total β-catenin remained unchanged, suggesting that CLL-exosomes induced phosphorylation rather than the generation of newly formed β-catenin and leaving us wondering whether upregulation of phosphor β-catenin is somehow beneficial to their parental neoplastic cells. Because the IL-6 promoter binds 3 transcription factors that are activated by β-catenin we assumed that by activating the β-catenin pathway, endothelial cells become "micro factories" for the production of humoral IL-6. To verify the role of β-catenin in promoting production of IL-6 molecules in endothelial cells we transfected HUVECs with β-catenin containing plasmid. By Western immunoblotting we verified that the protein levels of β-catenin were upregulated. Then, by ChIP assay we found that 3 different transcription factors, namely LEF/TCF, CEBP and NFkB increased their binding to the IL-6 promoter region by 1.5 to 12 folds in HUVECs that were transfected with β-catenin ORF. Finally we show that intracellular STAT3 of CLL cells that were grown on this IL-6-rich medium are phosphorylated on tyrosine residues and that the rate of CLL cells in active apoptosis was markedly decreased. Conclusions: CLL cells shape their own fate. They do so by sending exosomes that activate the β-catenin axes. In this way CLL cells reprogram endothelial cells to become IL-6 producing cells and IL-6 contributes to CLL cells' survival.