Shedding of extracellular membrane vesicles from both normal and tumor cells in culture

Tumor cells of different origins shed extracellular membrane vesicles (MVs), that contain angiogenetic- and pro-apoptotic-factors as well as matrix metalloproteases (MMPs). In addition, also neurons and astrocytes in culture produce VEGF- and FGF2- containing MVs, while oligodendroglioma (G26/24) cells release FasL-containing MVs that inhibit neurite sprouting and cause neuronal apoptosis. Starting from these observations, we have been analyzing composition of MVs produced by both normal and tumor cells in culture. We found that MVs from G26/24 cells contain TRAIL, Hsp70, and VEGF. We also traced the route of shed MVs, by adding vesicles that contain 35S-labeled proteins to unlabeled neurons, and found labeled proteins in neurons. Moreover, the ORFs encoding VEGF and TGFβ were cloned into the pEGFP-N2 plasmid, in order to produce the factors as fluorescent proteins in mammalian cells. We are now selecting stably transfected cell lines to be used for studying the fate of the corresponding proteins during the process of secretion and/or shedding of extracellular vesicles. Finally, by using as a model 8701-BC cells, derived from a ductal infiltrating breast cancer, we have been analyzing the proteomic pattern of vesicles, compared with the total cell lysates, in order to find out events of specific sorting of molecules to MVs. Vittorelli ML, 2003, Curr Topics Devel Biol 54: 411-32 Taverna S, et al. 2003, J Biol Chem. 278: 51911-9 D’Agostino S, et al. 2006, Int J Oncol. 29: 1075-85 Schiera G, et al. 2007, J Cell Mol Med. 11: 1384-94 Proia P, et al. 2008, Int J Mol Med.21: 63-7