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 communicationis due to the release of signalling molecules from both tumor cells and surrounding stromal cells. Several secretedproteins lack the N-terminal signal peptides and, therefore, they are secreted by alternative unconventional processessuch as secretion mechanism mediated by vesicle shedding in the extracellular matrix. Actually, a certain number ofproteins, 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 instromal fibroblasts and endothelial cells as well as in tumor cells themselves [1]. Moreover, MMPs vehicled bytumor shed vesicles in association with Integrin beta1 induce the adhesion to and the degradation of extracellularmatrix, thus helping cell migration and tumor invasion [2]. Notably vesicles carry many proangiogenic growthfactors, such as VEGF [3] or FGF-2 [4] and enzymes as well as SphK1 [5], which act synergistically on endothelialcells to promote neoangiogenesis. Therefore, in cancer state extracellular shed vesicles play a role in tumorprogression, 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 amechanism similar to virus budding, and their diameter ranges between 100nm-1 μm, while exosomes are smallervesicles (10-100nm diameter) deriving from the endosomal pathway. Both kinds of vesicles are involved intransporting signal molecules to the extracellular milieu, but distinct roles are not yet been clarified. As reported byMuralidharan-Chari [6] the two vesicle populations can be separated by differential centrifugation: membranevesicles are sedimented at relatively low speed centrifugation while exosomes require a 100.000gultracentrifugation. Since the extensive characterization of thosevesicles can enlighten their role in cancer progression,especially for what concerns their interaction with thesurrounding connective tissue, this work was focused on theproteomic characterization of vesicles shed in vitro by MDAMB 231, a continuous cell line derived from an invasive breastcarcinoma 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 wascentrifuged at 15000g for 30 minutes to sediment membranevesicles. The resulting supernatant was ultracentrifuged at100.000g for 90 minutes to sediment exosomes. Both pellets wereanalyzed through a proteomic approach, in parallel with the total cell lysate. This was the first study in which15000g pellet undergoes proteomic analysis, separately from 100000g pellet. MDA-MB 231 proteome wascharacterized by comparison with the reference map of 8701 BC cells, another cell line derived from ductal invasivebreast cancer which had been extensively characterized [8]. The 2D gels obtained from the 15.000g and the100.000g sediments were compared with the one of MDA MB 231 total cell lysate. Both proteomic profiles differfrom the one of cell lysate and some differences are also observed between each other. In particular, the pellet at15.000g, which in some other studies was considered as containing cell debris, differs from the proteomic profile ofcell lysate in some protein component. The analysis by Image Master software showed also the enrichment invesicles and exosomes of prot