Proteomic analysis of extracellular vesicles shed in vitro by MDA MB 231 breast carcinoma cells

Cross talk between tumor cells and connective tissue plays a key role in tumor progression. The communication is due to the release of signalling molecules from both tumor cells and surrounding stromal cells. Several secreted proteins lack the N-terminal signal peptides and, therefore, they are secreted by alternative unconventional processes such as secretion mechanism mediated by vesicle shedding in the extracellular matrix. Actually, a certain number of proteins, playing roles in some aspects of tumor progression, have been found in shed vesicles. For example, EMMPRIN, carried out in vesicles shed by tumor cells, stimulates matrix metalloproteinase (MMP) production in stromal fibroblasts and endothelial cells as well as in tumor cells themselves [1]. Moreover, MMPs vehicled by tumor shed vesicles in association with Integrin beta1 induce the adhesion to and the degradation of extracellular matrix, thus helping cell migration and tumor invasion [2]. Notably vesicles carry many proangiogenic growth factors, such as VEGF [3] or FGF-2 [4] and enzymes as well as SphK1 [5], which act synergistically on endothelial cells to promote neoangiogenesis. Therefore, in cancer state extracellular shed vesicles play a role in tumor progression, through several mechanisms that are elicited in the connective tissue. Two main kind of vesicles, named membrane ones and exosomes, were described; membrane vesicles are thought to be produced by a mechanism similar to virus budding, and their diameter ranges between 100nm-1 μm, while exosomes are smaller vesicles (10-100nm diameter) deriving from the endosomal pathway. Both kinds of vesicles are involved in transporting signal molecules to the extracellular milieu, but distinct roles are not yet been clarified. As reported by Muralidharan-Chari [6] the two vesicle populations can be separated by differential centrifugation: membrane vesicles are sedimented at relatively low speed centrifugation while exosomes require a 100.000g ultracentrifugation. Since the extensive characterization of those vesicles can enlighten their role in cancer progression, especially for what concerns their interaction with the surrounding connective tissue, this work was focused on the proteomic characterization of vesicles shed in vitro by MDA MB 231, a continuous cell line derived from an invasive breast carcinoma which release vesicles also in serum-free medium [7]. 24 hours conditioned serum-free medium was collected and, after two low speed centrifugations to remove cell debris, it was centrifuged at 15000g for 30 minutes to sediment membrane vesicles. The resulting supernatant was ultracentrifuged at 100.000g for 90 minutes to sediment exosomes. Both pellets were analyzed through a proteomic approach, in parallel with the total cell lysate. This was the first study in which 15000g pellet undergoes proteomic analysis, separately from 100000g pellet. MDA-MB 231 proteome was characterized by comparison with the reference map of 8701 BC cells, another cell line derived from ductal invasive breast cancer which had been extensively characterized [8]. The 2D gels obtained from the 15.000g and the 100.000g sediments were compared with the one of MDA MB 231 total cell lysate. Both proteomic profiles differ from the one of cell lysate and some differences are also observed between each other. In particular, the pellet at 15.000g, which in some other studies was considered as containing cell debris, differs from the proteomic profile of cell lysate in some protein component. The analysis by Image Master software showed also the enrichment in vesicles and exosomes of proteins which were not detectable in the cell lysate. Data from in silico analysis are now subjected to LC-MS analyses in order to confirm presumed identification of proteins observed also in the cell lysate and to identify unknown proteins observed in shed vesicles and exosomes. Those molecules could have important roles regarding the vesicle functions and their characterization could help in elucidating the pathways involved in connective tissue-shed vesicle interactions during cancer progression.