Proteomic analysis of exosome-like vesicles derived from breast cancer cells.

The phenomenon of membrane vesicle-release by neoplastic cells is a growing field of interest in cancer research, due to their potential role in carrying a large array of tumor antigens when secreted into the extracellular medium. In particular, experimental evidence show that at least some of the tumor markers detected in the blood circulation of mammary carcinoma patients are carried by membrane-bound vesicles. Thus, biomarker research in breast cancer can gain great benefits from vesicle characterization.Conditioned medium was collected from serum starved MDA-MB-231 sub-confluent cell cultures and exosome-like vesicles (ELVs) were isolated by ultracentrifugation. Ultrastructural analysis of ELVs was performed by transmission electron microscopy (TEM) and the purity of fraction was confirmed by western blotting assays. Proteomic profile of ELVs was carried out by 2 D-PAGE and protein identification performed by MALDI-ToF Mass Spectrometry.On the basis of ultrastructural and immunological characterization, the isolated vesicles have been classified as exosome-like vesicles (ELVs). The proteomic investigation showed a distinctive protein profile of the ELVs, in comparison to the whole cell lisates (WCL) proteome, which could be instrumental for cancer progression. The proteins were clustered into functional categories, according to the current bioinformatics resources and a Venn diagram was constructed based on these clusters.It is reasonable to assume that vesicle production allows neoplastic cells to exert different effects, according to the possible acceptor targets. For instance, vesicles could potentiate the malignant properties of adjacent neoplastic cells or activate non-tumoral cells. Moreover, vesicles could convey signals to immune cells and surrounding stroma cells. The present study may significantly contribute to the knowledge of the vesiculation phenomenon, which is a critical device for trans cellular communication in cancer.