Unravelling the distribution of extracellular vesicles in vivo using recombinant tetraspanins

Background:Extracellular vesicles (EVs) which were considered as garbage bags of cells came into view only a decade ago and are now increasingly recognized for their importance in cell-to-cell communication. Its their apparent natural ability to transfer cargo from donor cell to recipient cell thereby conferring messages in paracrine or endocrine manner. Over a decade, lot of research has been done to understand the omics, mode of secretion and uptake mechanisms. However, especially the trafficking of EVs in vivo is still poorly understood. Methods: We here generated the tetraspanins CD63 and CD81 C-terminally fused to a snorkel tag (1) that adds an additional transmembrane domain to the four existing ones to be able to attach further tags facing the extracellular space. Due to their extravesicular orientation, these tags can be used as a future tool to understand trafficking of EVs in vivo. As a first step, we aimed to give proof of principle that our constructs allow to track and isolate functional recombinant EVs from cultured cells. We therefore established a method to isolate functional EVs carrying our recombinant tetraspanins using a combination of antihemagglutinin affinity matrix and precission protease cleavage to isolate EVs without damaging the EV membrane and without losing the CLIP and FLAG tags which are preceding to precission protease site and HA tag. Results: Indeed, we were able to purify the EVs by this strategy. To further proof that these EVs are able to transfer intact and active cargo to recipient cells, we additionally loaded the EVs with Cre recombinase mRNA (2). Therefore, we stably expressed recombinant tetraspanins and Cre recombinase in donor HeLa cells and fluorescent colour switch LoxP system in recipient HEK293 cells (3). Indeed, snorkel tagged EVs were taken up in this experiment. Using an in vivo mimicking 3D cell culture model (4), we also observed a crosstalk from human dermal fibroblasts to keratinocytes with snorkel tag containing EVs. Summary/conclusion: Finally, we are currently testing if snorkel tag containing EVs from the stable HeLa cell line introduced into a xenograft mouse model can be isolated from plasma and tissues to understand the distribution of tumour derived EVs in different tissues. We therefore pave the ground for using snorkel-tagged EVs as a valuable tool to understand EV trafficking in vivo.