Extracellular Vesicle Measurements with Nanoparticle Tracking Analysis: A Different Appreciation of Up and Down Secretion.

ABSTRACT

As is the case with most eucaryotic cells, cancer cells are able to secrete extracellular vesicles (EVs) as a communication means towards their environment and surrounding cells. EVs are represented by microvesicles and smaller vesicles called exosomes, which are known for their involvement in cancer aggressiveness. The release of such EVs requires the intervention of trafficking-associated proteins, mostly represented by the RAB-GTPases family. In particular, RAB27A is known for its role in addressing EVs-to-be secreted towards the the plasma membrane. In this study, shRNAs targeting RAB27A were used in colorectal (CRC) and glioblastoma (GB) cell lines in order to alter EVs secretion. To study and monitor EVs secretion in cell lines' supernatants, nanoparticle tracking analysis (NTA) was used through the NanoSight NS300 device. Since it appeared that NanoSight failed to detect the decrease in the EVs secretion, we performed another approach to drop EVs secretion (RAB27A-siRNA, indomethacin, Nexihnib20). Similar results were obtained i.e., no variation in EVs concentration. Conversely, NTA allowed us to monitor EVs up-secretion following rotenone treatment or hypoxia conditions. Therefore, our data seemed to point out the insufficiency of using only this technique for the assessment of EVs secretion decrease.