MEMBRANE-VESICLES SHED INTO THE EXTRACELLULAR MEDIUM BY HUMAN BREAST-CARCINOMA CELLS CARRY TUMOR-ASSOCIATED SURFACE-ANTIGENS

We have compared the pattern of surface antigen expression, as detecte d by monoclonal antibodies (mAbs), in plasma membranes vs shed membran e vesicles of two human breast carcinoma cell lines, MCF-7 and 8701-BC . Antigen expression was detected on cells by immunofluorescence (IF) analysis, whilst, due to their small dimensions, the same technique wa s not applicable to vesicles. For these structures dot-blot analysis a nd immunoelectron microscopy (IEM) were employed. When applicable, bot h cell membranes and membrane vesicles were immunoprecipitated and the precipitate (IP) was analyzed by SDS-PAGE. Cells of both lines expres sed HLA class I antigens, epithelial cytokeratins, beta 1 integrins, C EA and the glycoprotein detected by mAb 19.9, but only MCF-7 cells exp ressed Lewis Y, episialin and globo-H antigens and only 8701-BC cells expressed folate receptor. Membrane vesicles of both cell lines appear ed to be rich in beta 1, alpha 3 and alpha 5 integrin chains, expresse d HLA class I antigens and carried most of the plasma membrane antigen s found in the cell membranes. Overall we have analyzed 17 antigens on the two cell lines and on their vesicles. The results obtained for ce lls (IF and IP) and those for vesicles (dot-blot and IP) were generall y concordantly positive or concordantly negative. We obtained a total of 26 clearly concordant combinations on 34 analyses. In three cases w e found discordant results, whereas in the remaining combinations we o bserved slight reactivity and we found difficulties in determining con cordance. Discordant results concerned the expression of the following antigens: folate receptors, which were clearly expressed in 8701-BC c ells but not detected by dot-blot analysis or IEM on their shed membra ne vesicles; neu (c-erb-B2) receptor found in MCF-7 cell membranes but not in their vesicles; and the globo-H antigen recognized by mAb MBr1 , detected at low levels on 8701-BC plasma membranes but undetectable on their membrane vesicles. Like vesicles shed in vitro by cultured ce lls, the vesicles shed in vivo by human breast carcinoma cells could b e tagged with several antibodies against tumor-associated antigens. Th e vesicles shed in vivo were found in association with a fiber network . Some of the fibers had the characteristic fibrin periodicity. These data suggest that tumor markers detected in the circulation of carcino ma patients, at least in part, are carried by shed membrane vesicles. Moreover the observation that membrane vesicles carry both tumor-assoc iated antigens and HLA class I molecules indicate that these structure s could in principle present antigens to the immune system. Together w ith our previous demonstration that membrane vesicles shed by breast c arcinoma cells contain TGF-beta, these results suggest an important ro le for vesicles in the immunological escape of these cells. The presen ce in membrane vesicles of integrins, together with the previous obser vation that they are rich in gelatinolytic activities, also points to a possible role of these structures in the metastatic behavior of tumo r cells.