Membrane vesicles shed into the extracellular medium by human breast carcinoma cells carry tumor-associated surface antigens

Abstract

We have compared the pattern of surface antigen expression, as detected by monoclonal antibodies (mAbs), in plasma membranes vs shed membrane 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 was not applicable to vesicles. For these structures dot-blot analysis and immunoelectron microscopy (IEM) were employed. When applicable, both cell membranes and membrane vesicles were immunoprecipitated and the precipitate (IP) was analyzed by SDS-PAGE. Cells of both lines expressed HLA class I antigens, epithelial cytokeratins, beta 1 integrins, CEA and the glycoprotein detected by mAb 19.9, but only MCF-7 cells expressed Lewis Y, episialin and globo-H antigens and only 8701-BC cells expressed folate receptor. Membrane vesicles of both cell lines appeared to be rich in beta 1, alpha 3 and alpha 5 integrin chains, expressed HLA class I antigens and carried most of the plasma membrane antigens found in the cell membranes. Overall we have analyzed 17 antigens on the two cell lines and on their vesicles. The results obtained for cells (IF and IP) and those for vesicles (dot-blot and IP) were generally concordantly positive or concordantly negative. We obtained a total of 26 clearly concordant combinations on 34 analyses. In three cases we found discordant results, whereas in the remaining combinations we observed slight reactivity and we found difficulties in determining concordance. Discordant results concerned the expression of the following antigens: folate receptors, which were clearly expressed in 8701-BC cells but not detected by dot-blot analysis or IEM on their shed membrane 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 cells, the vesicles shed in vivo by human breast carcinoma cells could be tagged with several antibodies against tumor-associated antigens. The 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 carcinoma patients, at least in part, are carried by shed membrane vesicles. Moreover the observation that membrane vesicles carry both tumor-associated antigens and HLA class I molecules indicate that these structures could in principle present antigens to the immune system.(ABSTRACT TRUNCATED AT 400 WORDS)

The Rho/ROCK pathway for lysophosphatidic acid-induced proteolytic enzyme expression and ovarian cancer cell invasion

“…These GTPases are monomeric 20-30 kDa GTPbinding proteins and function as molecular switches by altering between active GTP-bound and inactive GDPbound forms (Bar-Sagi and Hall, 2000). One of these Rho GTPases, RhoA, is upregulated in peritoneal metastatic lesions compared with primary tumors of advanced ovarian carcinoma (Dolo et al, 1995) and has an important role in the peritoneal dissemination of ovarian cancer cells (Horiuchi et al, 2008). RhoA has been known to regulate ovarian cancer cell migration and invasion through focal adhesion assembly (Sawada et al, 2002;Mukai et al, 2003).…”

Shedding of the Matrix Metalloproteinases MMP-2, MMP-9, and MT1-MMP as Membrane Vesicle-Associated Components by Endothelial Cells

“…23,24 In tumor cell-derived vesicles it was suggested that this integrin promoted the interaction of the vesicles with the extracellular matrix, helping localize the degradation by proteases. 18 In this case also the presence of this integrin might indicate an association of the protease-containing vesicles with matrix components resulting in focal degradation of the matrix. Vesicle shedding has been described in transformed and normal cells.…”

“…18 Conditioned medium obtained as above was centrifuged at 600 ϫ g for 15 minutes and then at 1500 ϫ g for 15 minutes to remove cells and large debris. The supernatants were centrifuged at 100,000 ϫ g for 1 hour at 4°C.…”

“…18 Because ␤1 integrins mediate endothelial cell interaction with the surrounding extracellular matrix and are important in angiogenesis 23,24 we tested whether HUVEC-derived vesicles contained this integrin chain. The ␤1 integrin was detected in Western blot analysis (Figure 4), suggesting also that in this cellular model the vesicles might be associated with the extracellular matrix.…”

Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells

“…MP are also shed by both hematological malignant cells, solid tumors in vitro and in vivo and have been implicated in tumor cell invasiveness, 43 escape from immune surveillance, 44 angiogenesis 45 and contribute to the hypercoagulable state observed in many malignancies. 43,44,46,47 Our findings for a role of MP in the acquisition and spread of P-gp mediated MDR, thus defines a new clinical significance for MP in cancer-cell biology.…”