Cancer is the leading cause of death in the United States, despite the fact that great advances have been made in its prevention and therapy. The growth of blood vessels in tumors is essential for their growth and metastasis. An attractive strategy for cancer therapy is to target cytotoxic agents to the endothelial cells lining tumor blood vessels rather than to the tumor cells themselves. One key advantage of this vascular targeting approach is that the endothelial cells are very accessible to agents injected into the blood whereas the tumor cells frequently are not. Genetically engineered tumor models that artificially induce selective marker expression on the tumor endothelium indicate that vascular targeting with immunotoxins and immuno-coagulants causes significant tumor regression. In order to implement this strategy, specific target sites need to be identified on the tumor endotheliurn. Tumor microenvironmental factors are expected to alter endothelial cell surface protein expression. Moreover, our recent work helps establish that specific transport vesicles, called caveolae, located on the endothelial cell surface contain tissue-specific proteins that when immunotargeted in vivo permit selective transport into and across the endothelial cell barrier. With these issues in mind, we believe that the vascular endothelium and caveolae in tumors are worthy of investigation at the molecular level. In this proposal, we focus on the effects of the tumor on the endothelium with the purpose of identifying caveolar proteins induced by the tumor that can be used as molecular targets for diagnostic and therapeutic probes. Proteins associated with the caveolae of the endothelium from normal tissue and from tumors will be resolved by utilizing novel membrane-isolation technique that isolate caveolae directly from tissues. The proteins in these caveolae will be comparatively analyzed and characterized. For the in vivo studies we will utilize a rat tumor model using rat and human breast adenocarcinoma cells. So far, we have identified several proteins in caveolae that are induced in tumors. Antibodies will be produced to these proteins in order to develop probes specific for the tumor blood vessels. These proteins will be analyzed by mass spectroscopy and if necessary, purified for amino acid microsequencing to determine their identity or novelty. The antibodies will be used to detect tumor growth and to immunolocalize the antigens in various normal and neoplastic tissues. Those antibodies specific for the tumor endothelium will be used not only to target the tumor vasculature in vivo but also to investigate their transport by caveolae in the tumor tissue. Because the tumor vasculature is critical for tumor growth, metastasis, and survival, a non-invasive, vector-mediated drug delivery system to the tumor will provide not only an accessible therapeutic modality but also a specific ablative approach to primary and metastatic cancer.
