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RESEARCH PRODUCT

Abstract 5135: Exosomes released by K562 chronic myeloid leukemia cells promote endothelial cell tubular differentiation through uptake and cell-to-cell transfer

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Abstract We hypothesized that exosomes were a venue through which to transfer pro-angiogenic stimuli into and between endothelial cells during endothelial cell tubular differentiation. Exosomes are microvesicles of endocytic origin released by most normal and tumor cells that play an important role in cell-to-cell communication. Angiogenesis is recognized to be a factor in progression of chronic myeloid leukemia (CML). However, the mechanism through which this happens has not been elucidated. We first optimized and characterized secretion of exosomes from CML K562 cells, showing expected selective enrichment of exosomal markers CD63, CD81 and Tsg101 in exosomes compared to the K562 whole cell lysate. Next, these exosomes were stained with PKH, a fluorescent plasma membrane dye, and incubated with human umbilical vein endothelial cells (HUVECs) on Matrigel for measurement of exosome uptake under semi-physiologic conditions. We found that K562 exosomes were internalized by HUVECs during tubular differentiation, and that exosome uptake increased and exosomes redistributed over the time. Moreover, we observed a two-fold increase in cumulative length of vascular tubes formed on Matrigel in HUVEC exposed to K562 exosomes under limiting growth factor medium, with a maximum effect obtained with an exosome concentration of 10ug/ml (p=0.007). Next, we incubated HUVECs on Matrigel with PKH-labeled exosomes to dissect intracellular exosome movement and exosome-cell interactions during tube formation, using multi-dimensional confocal microscopy. Exosomes were perinuclear early in tubulogenesis; whereas, in later stages, exosomes were more peripheral in the cytoplasm and sub-membranous areas. Over time, the peripheral exosomes moved towards and then into nanotubular structures that were part of the tubulogenesis process. Nanotubes met and merged, connecting neighboring cells. Actin and tubulin staining was used to show the localization of exosomes in the nanotubes. Three-dimensional reconstruction of confocal images demonstrated that the actin and tubulin were in a loose network without clear organization. This analysis also revealed exosome presence within and movement through the nanotubes. The use of dual color PKH stain allowed demonstration that exosomes were shared along the nanotubular network between connected HUVEC cells, confirming cell-cell exosomal exchange. Time-lapse confocal microscopy further documented nanotubular exosome transfer. These results show that CML exosomes traffic into and between endothelial cells and stimulate tube formation in a physiologic in vitro model, suggesting they may play an important role in leukemia stimulation of bone marrow angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5135. doi:10.1158/1538-7445.AM2011-5135