It is now becoming well established that vesicles are released from a broad range of cell types and are involved in cell-to-cell communication, both in physiological and pathological conditions. Once outside the cell, these vesicles are termed extracellular vesicles (EVs). The cellular origin (cell type), subcellular origin (through the endosomal pathway or pinched from the cell membrane) and content (what proteins, glycoproteins, lipids, nucleic acids, metabolites) are transported by the EVs, and their size, all seem to be contributing factors to their overall heterogeneity. Efforts are being invested into attempting to block the release of subpopulations of EVs or, indeed, all EVs. Some such studies are focussed on investigating EV inhibitors as research tools; others are interested in the longerterm potential of using such inhibitors in pathological conditions such as cancer. This review, intended to be of relevance to both researchers already well established in the EV field and newcomers to this field, provides an outline of the compounds that have been most extensively studied for this purpose, their proposed mechanisms of actions and the findings of these studies. ARTICLE HISTORY

Background Cancer cells release heterogeneous populations of extracellular vesicles (EVs) that transmit aggressive phenotypic traits to recipient cells. We aimed to establish if the heterogenous EVs population or a sub-population is responsible, if we could block undesirable cell-to-cell communication by EVs, and, if some EVs continued to be released, would their undesirable influences on recipient cells continue. Methods Three triple-negative breast cancer (TNBC) cell lines were used. Non-toxic concentrations of calpeptin, Y27632, manumycin A, GW4869 and combinations thereof were tested to block EVs. Ultracentrifugation-based methods collected EVs, which were then characterised by nanoparticle tracking analysis, immunoblotting, and transmission electron microscopy. A quick screening flow cytometry method evaluated EVs in solution. The influences of EVs on recipient cells’ migration was investigated. Results All EV sub-populations were apparently involved in transmitting undesirable phenotypic characteristics. All compounds/combinations significantly (64–98%) reduced EVs’ release. Our quick screening broadly reflected our more comprehensive EVs analysis. The 2–36% of EVs that continued to be released caused less transmission to recipient cells, but not on a comparable scale to the reduction of EVs release achieved. Conclusion Up to 98% inhibition of EVs’ release was achieved. To prevent the transmission of undesirable phenotypic traits by EVs, their total inhibition may be necessary.