CLL-Derived Exosomes Turn Endothelial Cells into IL-6 Producing Cells
Authors: Uziel Orit , Lipshtein Lian , Sarsor Zinab , Beery Einat , Bogen Shaked , Lahav Meir , Kliminski Vitali , Sharan Roded , Gervits Asia , Signorini Lorenzo , Shimony Shai , Raanani Pia , Rozovski Uri 
Published: November 2021

Abstract
CLL is characterized by gradual accumulation of mature appearing long-lived lymphocytes that travel in blood and reside in lymph nodes, spleen and bone marrow. In these sites, pro inflammatory humoral factors support the survival and proliferation of the neoplastic cells. Previous studies showed that levels of the proinflammatory cytokine IL-6 are at least 10 folds higher in patients with CLL compared with healthy individuals. Yet, which cells produce and secrete IL-6 and what triggers this cellular activity in CLL is unknown.
Secreted by all types of cells, exosomes are nano-scaled particles that travel in blood and carry a cargo that at least partially reflects the molecular makeup of its cell of origin. Exosomes, including those originating from neoplastic cells, function as stable intercellular transport vehicles that deliver their cargo to cells that engulf them. For example, CLL-derived exosomes are taken up by mesenchymal stromal cells, transforming them to cancer associated fibroblasts.
Given the appropriate stimulation, endothelial cells produce IL-6 which provides CLL cells with a survival advantage. Therefore, we hypothesized that CLL-exosomes turn endothelial cells into "IL-6-secreting cells".
To test this hypothesis, we transfected vein-derived (HUVECs) and arterial-derived (HAOEC) endothelial cells with exosomes that we isolated from the peripheral blood of 45 treatment naïve patients. We found that endothelial cells take-up CLL-exosomes in a dose- and time- dependent manner. Since CLL cells are protected from apoptosis in IL-6 rich environment, we wondered whether CLL-exosomes turn endothelial cells into IL-6-producing cells. To test this, we exposed endothelial cells to CLL-exosomes and found 50% increase in IL-6 levels, suggesting that the endothelial-exposed cells produced and secreted IL-6. Subsequently, we filtered out this growth medium and added CLL cells to this IL-6 enriched medium. After 15 minutes, STAT3 became phosphorylated and there was 40% decrease in the rate of apoptosis, indicating that IL-6 activated STAT3-dependent anti-apoptotic pathway. Phosphor-proteomics analysis of endothelial cells that were loaded with CLL-exosomes revealed 23 phosphor-proteins that were upregulated. Network analysis unraveled the central role of phosphor-b-catenin. To test whether b-catenin induces IL-6 in these cells, we transfected HUVECs with a b-catenin containing plasmid. We found by ELISA 30% increase in the levels of IL-6 in the culture medium and by chromatin immunoprecipitation assay an increased binding of 3 transcription factors (NFkB, LEF/TCF, and CEBP) to the IL-6 promoter. Taken together, we found that CLL cells communicate with endothelial cells through exosomes that they release. Once these exosomes are taken up by endothelial, they turn them into IL-6 producing cells, which in turn contributes to CLL cells' survival.

Disclosures
No relevant conflicts of interest to declare.