VISUALIZATION AND CHARACTERIZATION OF EXOSOMES IN BREAST CANCER CELLS

Abstract

Objectives Exosomes are extracellular vesicles of endocytic origin with a size range of 40-150 nm and a lipid bilayer membrane. Though exosomes are known as dynamic mediators of intercellular communication, its characteristics and function have not been fully studied. In this report, we used a metabolic labeling method to prepare fluorescent exosomes to investigate the characteristics and the movement of exosomes derived from various breast cancer cells. Findings MCF-7 and MDA-MB-231 cells were treated with three types of azido sugars, Ac4ManNAz, Ac4GalNAz, or Ac4GlcNAz (50 mM) for 3 days to produce the azide (-N3) containing exosomes through metabolic glycosylation. It is confirmed that the azido sugar decorated exosomes were able to maintain their original characteristics such as size, lipid bilayer morphology, and protein profile. The exosomes prepared with Ac4ManNAz have shown the highest labeling efficiency with ADIBO-Cy3 fluorescence dye in MCF-7, MDA-MB-231, BT-549 and MDA-MB-468 cells at 17.2%, 14.8%, 13.2% and 14.9% respectively. To study their uptake capability, the labeled exosomes from different origins were incubated with a panel of breast cancer cells, including MCF-7, MDA-MB-231, MDA-MB-468 and BT-549, and normal NIH/3T3 fibroblast cells for 24 hours. The cells were then evaluated by fluorescence microscopic imaging and flow cytometry. It was observed that exosomes from different origins have a different uptake efficiency, suggesting that each exosome may have its unique navigation systems. Furthermore, the cells which have aggressive metastatic potential, such as MDA-MB-231 showed a better pickup of all exosomes. In contrast, the exosomes released by MDA- MB-468 showed higher loading in five kinds of cells. Conclusions Our development has demonstrated an effective labeling method that can facilitate exosome research by providing a new way of quantification and tracking in vitro and potentially in vivo studies.