Anti‐human CD 9 antibody Fab fragment impairs the internalization of extracellular vesicles and the nuclear transfer of their cargo proteins

The intercellular communication mediated by extracellular vesicles ( EV s) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell‐derived EV s play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EV s, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV ‐associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD 9 both in EV s and recipient cells strongly decreased the endocytosis of EV s and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti‐ CD 9 monoclonal antibody (referred hereafter as CD 9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EV s containing CD 9‐green fluorescent protein fusion protein and various melanoma cell lines and bone marrow‐derived mesenchymal stromal cells as recipient cells. Interestingly, CD 9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD 9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD 9 Fab‐mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti‐melanoma therapeutic strategies.