PS1129 CLL‐ DERIVED EXOSOMES REPROGRAM RESIDENT CELLS TO BECOME CLL SUPPORTIVE CELLS
Authors: Uziel O. , Lipshtein L. , Sarsur Z. , Beery E. , Lahav M. , Raanani P. , Rozovski U. 
Published: June 2019

Background:CLL cells travel between blood, bone marrow and lymphatic tissues. In the lymph nodes, CLL cells receive pro‐survival signals coming from resident cells. Why these cells provide a supportive environment to CLL cells is unknown. Likely, CLL cells actively recruit these cells to serve their needs. Exosomes are nanoscaled vesicles secreted by different types of cells including tumor cells and travel in body fluids. These lipid rafts carry a cargo of proteins and nucleic acids. Exosomes are up taken by bystander cells in their vicinity and alter their phenotype. We hypothesized that CLL cells shape their own fate by sending ”floating“ massages via exosomes that are taken up by resident cells. These massages subsequently reprogram signaling pathways of the recipient cells to support the survival of the neoplastic cells. Specifically, since CLL cells are in close vicinity to endothelial cells during their travel, we sought to explore the effect of CLL‐derived exosomes on these cells.Aims:To describe whether and how CLL derived exosomes recruit endothelial cellsto become ”tumor supportive“ cells.Methods:CLL derived exosomes were isolated by ultracentrifugation. HUVEC cells were the source of endothelial cells. The uptake of exosomes was quantified by flow cytometry. The phosphoproteomics profile of HUVEC cells was analyzed by MASS spectrometry and the RNA exosomal content by RNA‐seq. Western blotting of selected proteins and quantitative real‐time PCR validated the results of the ”OMICS“ studies. To annotate our findings we used DAVID (Database for Annotation, Visualization and Integrated Discovery).Results:To show that CLL exosomes are taken up by endothelial cells we exposed HUVEC cells to CLL‐derived exosomes. We found that exosomes are taken up by endothelial cells in a dose and time deponent manner. Since phosphorylation‐switch controls the activity of many intracellular signaling pathways, we asked how did the phosphorylation status of intracellular proteins changes after exposure to CLL exosomes. In endothelial cells we found significantly increased phosphorylation in 52 peptides and in none of the peptide tested decreased phosphorylation. The MASS spectrometry findings were subsequently validated in 3 of those proteins by Western immune blotting and annotation analysis mapped these proteins to the ”cell to cell adhesion“ pathway which is involved in trafficking and retention of CLL cells in bone marrow and lymphatic organ and the ”mRNA pathway“, a targetable pathway in CLL. Beta‐Catenin was one of these 52 proteins. The levels of the phosphorylated isoform of beta‐catenin, was 3.5 increased after exposure to CLL exosomes. Because beta‐catenin induces the transcription of IL‐6 we wondered whether CLL exosomes induces a beta‐catenin‐mediated increase in IL‐6. IL‐6, in turn, induces STAT3 phosphorylation, and provide the cell with a survival advantage. Chromatin immunoprecipitation assay confirmed that beta‐catenin binds the IL‐6 promoter. Subsequently, levels of IL‐6 in the culture media significantly increased. Furthermore, when CLL cells were added, to this culture media levels of phosphorylated STAT3 significantly increased and the rates of spontaneous apoptosis significantly decreased.Summary/Conclusion:In this study, we describe how the exosomal cargo of CLL‐derived exosomes recruit endothelial cells to produce and secrete IL‐6. This cytokines subsequently binds the IL‐6 receptor and induces STAT3 phosphorylation. pSTAT3 provides the cells with a relative protection from apoptosis and ultimately a survival advantage.