Proteomic profiling of vesicles released by 8701-BC cells

Tumor cells were shown to release, both in vitro and in vivo, “vesicles” that vehicle several proteins involved in cell-cell and cell-matrix interactions. An interesting challenge is the characterization of the protein content of such vesicles that may increase the understanding of their potential roles in vivo. 8701-BC, a continuous line of breast carcinoma cells, was shown to release “membrane vesicles” with a diameter ranging from 100 to 1000 nm and 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, after two low speed centrifugations performed to remove cells and cell debris, the medium was differentially centrifuged. Western analysis, carried out using specific antibodies, revealed that the 15,000xg pelletted fraction contains ␣1-integrin, a protein which had been shown to be clustered in membrane vesicles shed by 8701-BC cells1, but not Hsc70, a protein found in exosomes2, 3. On the contrary, Hsc70 is detectable while ␣1-integrin is not present in the fraction obtained by a further centrifugation at 100,000xg of 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 of the different fractions was performed and the resulting 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 help to elucidate physiological roles of the two different kinds of vesicles. 1. Dolo V, Ginestra A, Cassarà D, Violini S, Lucania G, Torrisi MR, Nagase H, Canevari S, Pavan A, Vittorelli ML. (1998) Cancer Res. 58(19): 4468-74. 2. Hegmans J, Bard M, Hemmes A, Luider T, Kleijmeer M, Prins JB, Zitvogel L, Burgers S, Hoogsteden H, and Lambrecht B. (2004) Amer. J. Pathol. 164, 1807- 1815. 3. Choi DS, Lee JM, Park GW, Lim HW, Bang JY, Kim YK, Kwon KH, Kwon HJ, Kim KP, and Gho YS. (2007). J Prot Res. 6, 4646-4655.