Cell Surface Labeling by Engineered Extracellular Vesicles

Abstract

Deoxycytidine [3]). Extracellular vesicles (EVs) are increasingly recognized as a new class of multivalent messengers involved in cell-cell communication, both locally and at distance. [4-7] EVs are membranebound particles released by virtually every cell type, with stressed and transformed cells secreting the highest amounts. [8-11] Investigations of EVs' roles in different pathophysiological conditions are thwarted by biases introduced during isolation and reinfusion procedures. In particular, these biases manifest at different levels: (i) certain EV subsets may be excluded, [6] (ii) operator judgment is involved in deciding how many EVs to reinfuse, and (iii) an assumption that EV concentration is highest in blood, whereas several studies have shown that EV biodistribution occurs primarily via lymph fluid. [12-15] To circumvent these issues, the EV-releasing cells of interest can be genetically engineered to express membrane-bound reporters (or other functional biomolecules) in vitro or in vivo. [16] Although this approach is still in its infancy, it avoids the above limitations and to unbiasedly investigate untouched, endogenously released EVs. Although a detailed description of how EVs signal between cells is still missing, two nonexclusive mechanisms have been proposed: (i) membrane fusion between EVs and target cells, thereby releasing EV cargo (e.g., microRNAs and proteins, including oncogenes) directly in the cytoplasm of targeted cells; and (ii) surface binding with subsequent ligand-receptor type signaling. Membrane fusion (also known as horizontal transfer) has been described using several in vitro models (see Pucci and Pittet [17] and references therein), but proved more challenging to demonstrate in vivo. Recently, horizontal transfer of tumor-derived EVs was tested in mice using a sensitive and stringent approach based on the CRE-Lox reporter system. [15,18] Although CRE recombinase has an efficiency of 55%, [19] horizontal transfer was shown to happen mainly between tumor cells in vivo, but not between tumor cells and host cells. [15,18] These results have been further confirmed using other genetic strategies, [20,21] suggesting that surface binding may represent the main mechanism of EV-mediated tumor-host communications in vivo. A major limitation in the study of native EV signaling to host cells in vivo derives from their small size, which restricts the amount of reporter molecules carried by a single EV. As a consequence, only target cells binding the highest levels of EVs [12,14,15] will accumulate enough reporter molecules to be detected and investigated. Nonetheless, many host cell types Extracellular vesicles (EVs) can mediate local and long-range intercellular communication via cell surface signaling. In order to perform in vivo studies of unmanipulated, endogenously released EVs, sensitive but stringent approaches able to detect EV-cell surface interactions are needed. However, isolation and reinfusion of EVs can introduce biases. A rigorous way to study EVs in vivo is by genetically eng...

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Engineered exosomes for studies in tumor immunology

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