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Cell-Surface Proteins in Tumor Metastasis and Angiogenesis

R.T. Aimes, A. Zijlstra, J.D. Hooper, R. Mellor, T. Truong, K. Regazzoni, J.P. Quigley

We have established in vivo model systems that can recapitulate the major cellular and tissue events that occur in tumor metastasis and angiogenesis. The model systems allow quantitative measurements, submicroscopic analysis in real time, and molecular intervention. In addition, use of subtractive immunization, which is used to generate unique neutralizing monoclonal antibodies, in combination with functional proteomics enables us to identify molecules that are functionally active in metastasis and angiogenesis.

METASTASIS

Selected human tumor cells inoculated onto the chorioallantoic membrane of developing chick embryos form primary tumors on the membrane in 4-7 days. A small percentage of the cells in the primary tumor disseminate through the vasculature and within 3-4 days, arrest and proliferate in secondary organs of the embryo. Measuring a small number of early-arriving metastatic cells (<200) growing in the secondary organ has always been technically difficult. We use an approach in which unique regions of human DNA, known as Alu repeat sequences, are amplified by polymerase chain reaction from the total DNA extracted from various organs of the chick embryo.

Chicken DNA contains no Alu sequences, so any product generated by the polymerase chain reaction indicates that human tumor cells are present in the organ. We can detect as few as 50 human tumor cells present in the entire chick embryo lung and can measure the expansion of the metastatic cells by using this technology. We are using various screening procedures, including quantitative real-time polymerase chain reaction, to identify molecules that enhance, or conversely inhibit, the appearance of metastatic human tumor cells in organs of chick embryos.

We are also using a more standard method of monitoring human tumor metastasis in immunodeficient mice. However, in comparison with our chick embryo metastasis assay, this method is less quantitative, requires more time (3-5 weeks), and is more difficult to use for molecular intervention. We are using the mouse metastasis assay mostly to confirm the efficacy of effector molecules that initially are identified in the chick embryo assay.

Using subtractive immunization, we generated several unique antimetastatic monoclonal antibodies. One of the antibodies was used to probe a human cDNA expression library. The antigen recognized by the antibody was isolated, sequenced, and identified as PETA-3/CD151, a member of the tetraspanin family of proteins. Our results indicate that PETA-3 is a cell-surface protein that directly contributes to the metastatic phenotype. Another of the antimetastatic monoclonal antibodies was used as a tool in a functional proteomics approach to identify a novel tyrosine-phosphorylated glycoprotein that contributes to the metastatic process. This 135-kD cell-surface molecule is being characterized biochemically and mechanistically.

ANGIOGENESIS

One of the most commonly used in vivo assays for angiogenesis is the chick embryo chorioallantoic membrane assay. We recently developed a quantitative variation of this assay that allows detection and measurement of the newly sprouting blood vessels responding to an angiogenic stimulus. A highly specific collagen-cleaving matrix metalloproteinase, MMP-13, has been implicated in the tissue remodeling that occurs during the formation of the new blood vessels. We are characterizing this proteolytic event.

Recently, we developed an ex vivo model for angiogenesis, the aortic ring assay. In this assay, vascular aortic tissue from various genetically defined mice is embedded in collagen, and outgrowth of new blood vessels is monitored microscopically and biochemically. Using this assay, we again found that specific collagen-cleaving metalloproteinases are implicated in vessel outgrowth. A variety of defined metalloproteinase-deficient mice are being examined for outgrowth of blood vessels. Our results should indicate and define the rate-limiting proteolytic enzyme in angiogenic tissue remodeling.

We are also using an in vitro model system for the formation of tubes by endothelial cells to identify key regulatory molecules in angiogenesis. We examined a group of membrane-anchored serine proteases and secreted serine proteases for their differential mRNA expression in this vascular tube-forming assay and also in human tissue samples. A number of unexpected serine proteases (e.g., acrosin, neurotrypsin, epitheliasin) became circumstantially linked to angiogenesis and are now being cloned, expressed, and characterized. In addition, we are using subtractive immunization, which worked so well in the metastasis system, in the angiogenesis model system. Preliminary results indicate that this technique can be used to identify specific antigens that function in the formation of blood vessels.

PUBLICATIONS

Hooper, J.D., Zijlstra, A., Aimes, R.T., Liang, H., Claassen, G.F., Testa, J.E., Tarin, D., Quigley, J.P. Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135kDa cell surface phosphorylated glycoprotein antigen. Oncogene, in press.

Rozanov, D.V., Deryugina, E.I., Ratnikov, B.I., Monosov, E.Z., Marchenko, G.N., Quigley, J.P., Strongin, A.Y. Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP): the role of the cytoplasmic tail Cys(574), the active site Glu(240), and furin cleavage motifs in oligomerization, processing, and self-proteolysis of MT1-MMP expressed in breast carcinoma cells. J. Biol. Chem. 276:25705, 2001.

Zijlstra, A., Mellor, R., Panzarella, G., Aimes, R.T., Marchenko, N., Quigley, J.P. A quantitative analysis of the metastatic cascade of human tumor cells using human specific real-time PCR. Cancer Res., in press.