Characterization of cell uptake and intracellular trafficking of exosomes by quantitative live cell imaging: towards biomimetic delivery vehicles of therapeutic RNA

Exosomes are biological nanoparticles which play a role in long distance cell-to cell communication. These 40-100 nm sized vesicles are released by virtually all cells and derive from the multivesicular bodies within their parent cells. They modulate their target cell fate by induction of cell signaling as well as RNA and protein cargo transfer. Exosomes have also moved into the spotlight of clinical research, with potential use as biomarkers or next generation therapeutic delivery agents.
Exosomes are thought to be highly efficient intercellular messengers but quantitative characterization is lacking. Also, their routes of cell uptake and subcellular fate within recipient cells remain elusive. This work introduces an in depth and quantitative characterization of exosome cargo, physicochemical properties, labeling, isolation and their recipient cell interaction at the single cell – single vesicle level.
Basic protocols for exosome purification were revisited in order to allow for isolation of exosomes with sufficient yields and in as native state as possible to enable functional studies. Since exosome integrity and recovery yields after differential ultracentrifugation (UC), the most commonly used protocol for exosome isolation, turned out to be poor and unreproducible, we describe an alternative protocol based on ultrafiltration (UF) with subsequent gel filtration (GF) for recovering exosomes relatively selectively, with intact biophysical and functional properties and significantly higher yields.
Next we establish methods for specific exosome labeling using fluorescent marker proteins transiently expressed in parent cells, which led to a focus on FP tagged CD63 constructs. CD63-emGFP labeled exosomes were extensively characterized and showed identical properties compared to unlabeled exosomes based on sucrose density gradient, CryoTEM microscopy and proteomics analysis. Furthermore, we successfully adapted fluctuation correlation spectroscopy for characterization of fluorescently labeled exosomes.
In another part of this work we describe a high content screen for exosome uptake which we use to provide a first systematic and quantitative profiling of exosome uptake across a panel of exosome parent recipient cells, including HEK293, Huh7, B16F10 as parental cells and additional primary fibroblasts, primary keratinocytes, iPS derived motor neurons and HUVEC primary human endothelial cells as recipient cell lines. These quantitative profiling data reveals preferences in exosome internalization by different cell types and suggests that specific receptor ligand interactions may determine tissue specificity.
Finally, we address one of the fundamental questions in the field of cellular communication: how exosomes released by one cell enter and interact with their recipient cell. Our data quantifies for the first time the cell uptake dynamics of exosomes at the single vesicle and single cell level and reveals a quantitative efficiency paralleling that of infective pathogens rather than artificial delivery vehicles. We demonstrate that exosome uptake is largely mediated by active recruitment and surfing on filopodia to reach endocytic hotspots for their internalization at the filopodia base. This provides a cell biological explanation for the remarkably high efficiency of exosomes in targeting recipient cells and discovers a new parallel to some viruses and other pathogens. We propose that the process of filopodia surfing may have evolved as a highway for exosomes into the cell, being hijacked by certain pathogens for host cell interaction. This data does not support the previously reported exosome uptake by vesicle fusion with the plasma membrane or cargo release by endosomal escape. Instead we observe intact exosome uptake to enter endocytic vesicles, which then scan along the endoplasmic reticulum (ER) and end up in lysosomes. Our data suggest a model of controlled cargo delivery to defined subcellular localizations like the ER, rather than vesicle fusion and free release into the cytoplasm.