Abstract 144: Leukemic exosomes stimulate cells from the microenvironment to promote chronic lymphocytic leukemia
Authors: Paggetti Jerome , Berchem Guy , Moussay Etienne 
Published: May 2015

Abstract
Bone marrow mesenchymal stem cells (MSC) and endothelial cells (EC) are known to provide anti-apoptotic stimuli to chronic lymphocytic leukemia (CLL) cells via direct contact or secretion of soluble factors. We recently reported the profiling of circulating miRNA and identified specific miRNAs present at higher level in CLL plasma compared to healthy donors (Moussay et al., PNAS, 2011). Originating from endosomes, exosomes are small extracellular vesicles (50-150 nm) known to efficiently transport and transfer mRNA, microRNA and proteins to target cells. Therefore, exosomes constitute a new component of intercellular communication and their eventual implication in CLL remains totally unknown.
We first observed that CLL B cells efficiently transfer vesicles to human MSC in vitro through 400 nm culture inserts. Therefore we isolated, purified and characterized exosomes derived from CLL cell lines, primary cells culture supernatants. Profiling of proteins and microRNAs contents revealed the presence of oncogenic molecules. We further showed that purified CLL-exosomes rapidly entered and transferred proteins to target cells (MSC and EC). Exosome miRNAs efficiently targeted cellular mRNA as confirmed by luciferase reporter assay. Exosomes could also be taken up by mouse bone marrow cells in vivo. We next identified PI3K, AKT, and MAPK signaling as the key molecular pathways triggered by CLL-exosomes. We detected a rapid phosphorylation of kinases after 5 min of incubation with CLL-exosomes, the subsequent phosphorylation of NF-kB pathway inhibitors, and p65 translocation to nucleus.
Interestingly, CLL-exosomes induced a dramatic actin remodeling suggesting a switch to a cancer-associated fibroblast (CAF) phenotype. We then observed that CLL-exosomes modulated gene expression in target cells among which cytokines (APRIL, BAFF, IL-6, and IL-8), chemokines (CCL2, CCL5, and CXCL1), and factors involved in cell adhesion and migration (ICAM-1 and MMP-1). These factors were also secreted in supernatants of MSC and EC. Some of the released cytokines supported CLL survival after 6 days of CLL culture. Finally, exosomes were also shown to increase CLL adhesion, MSC and EC proliferation, cell migration and to enhance EC angiogenic capabilities.
In conclusion, CLL-exosomes contain pro-oncogenic molecules and strongly affect key functions of MSC and EC which are critical components of the bone marrow microenvironment. Activation of these cells by CLL-exosomes led to release of cytokines/chemokines and oncogenic factors that could promote angiogenesis and also favor leukemic cells survival and migration. Our findings may lead to applications in both diagnosis and therapy development. Molecules identified at the surface or inside exosomes may be further used as cancer biomarkers.
Citation Format: Jerome Paggetti, Guy Berchem, Etienne Moussay. Leukemic exosomes stimulate cells from the microenvironment to promote chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 144. doi:10.1158/1538-7445.AM2014-144