Proteomic profiling of vesicles released by 8701-bc cells

Abstract

8701-BC cells were shown to release “membrane vesicles” playing a role in tumor progression mechanisms. On the other hand, production of “exosomes”, smaller vesicles known to be involved in immune response activation, had not been revealed. The first goal of this study was to separate different vesicle populations from 8701-BC cell conditioned medium. To this aim, the medium was differentially centrifuged. Western analysis revealed that the 15,000xg pelletted fraction contains β1-integrin, which had been shown to be clustered in membrane vesicles shed by 8701-BC cells, but not Hsc70, a protein found in exosomes. On the contrary, Hsc70 is detectable while β1-integrin is not present in the fraction obtained by a further centrifugation at 100,000xg from 15,000xg supernatant. Moreover, the absence of Cytochrom C in both fractions excludes the contamination with apoptotic vesicles.These results suggested that 8701-BC cells release both membrane vesicles and exosomes and that their separation can be achieved by differential centrifugation.Then, to analyze the whole protein content of the vesicle preparations, a proteomic approach was chosen. Protein 2D-PAGE analysis was performed and the gel images were analyzed in silico, using ImageMaster 2D Platinum software. The preliminary comparative proteomic analysis revealed a set of protein spots differently abundant in the vesicle fractions. These data strongly encourage for further investigation using 2D-PAGE coupled with MS-MALDI-TOF analysis which could highlight physiological roles of the two different kinds of vesicles.