Abstract

Novel Anticancer Therapies Based on Interference with Uptake and Intracellular Distribution of Extracellular Vesicles in Recipient Cells

Aurelio Lorico, Professor of Pathology, Touro University Nevada School of Medicine

The intercellular communication mediated by extracellular vesicles (EVs) plays an important role in tumor progression. In particular, cancer cell-derived EVs participate in the transformation of tumor microenvironment. Interfering with the mechanisms regulating this cellular process might find therapeutic application particularly in oncology. Here, two potentially therapeutic strategies based on interference with EV uptake and intracellular distribution will be presented. The first is based on our recent finding that silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the uptake of EVs. Analogously, monovalent Fab fragments derived from 5H9 anti-CD9 monoclonal antibody (referred hereafter as CD9 Fab) partially blocked uptake of melanoma EVs in recipient cells. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9-green fluorescent protein fusion protein and various melanoma cell lines and bone marrow-derived mesenchymal stromal cells as recipient cells. CD9 Fab considerably reduced EV uptake in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. The second strategy is based on our previous report of a new intracellular pathway where a fraction of endocytosed EV-associated proteins and nucleic acids is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into nucleoplasmic reticulum. A tripartite protein complex, containing the endoplasmic reticulum-localized protein VAP-A, the cytoplasmic oxysterol-binding protein ORP3 and late endosome-associated small GTPase Rab7 orchestrates the specific localization of late endosomes into nucleoplasmic reticulum. Silencing of VAP-A or ORP3 abrogated the association of late endosomes with nuclear envelope invaginations and the transport of endocytosed EV-derived components to the nucleoplasm of recipient cells. We now report that silencing of ORP3 or VAP-A, but not its homologue VAP-B, reverses the pro-metastatic changes induced by EVs isolated from highly metastatic cells on their non-metastatic counterpart. By impeding intercellular communication in the tumor microenvironment. CD9 Fab-mediated inhibition of EV uptake or inhibition of the EV nuclear pathway, combined with direct targeting of cancerous cells, could lead to the development of novel anti-cancer therapeutic strategies.