An extensive study of potential inhibitors of extracellular vesicles release in triple-negative breast cancer

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.