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Molecular & Cellular Analysis Technologies
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The molecular complexity and in vivo inaccessibility of most tumor cells within solid tumors can greatly limit genomic- and proteomic-based discovery of useful targets for tumor-specific imaging and therapeutic agents in vivo. To overcome endothelial cell (EC) barriers and achieve more effective targeting and penetration into solid tumors, we shift analytical focus from the tumor cell to the vascular EC surface and its caveolae in direct contact with the circulating blood. To reduce data complexity to a meaningful subset of targetable proteins expressed on the EC surface, we will use tissue sub-cellular fractionation, novel multimodal mass spectrometric analysis, in silico subtraction, and bioinformatics interrogation of structure and function to unmask, from the >100,000 proteins in the tissue, those few intravenously accessible proteins differentially expressed on vascular endothelium in human renal tumors. This technology and overall approach has been validated in rodent solid tumors whereby new vascular targets have been uncovered permitting tumor-specific imaging, penetration, and effective radio immunotherapy (Nature, 429:629-35, 2004). But, currently very little is known about the expression of proteins in tumor neovascular endothelium, especially in human tissue. We now wish to apply our new technology to map comprehensively the proteome of luminal EC surfaces and caveolae in human renal tumors in vivo. It is likely that human tumors will express a different constellation of proteins on tumor neovasculature not yet uncovered or induced in animal models. To this end, we propose the following specific aims: 1) To use novel tissue sub fractionation and proteomic analytical approaches to map comprehensively vascular EC surfaces and caveolae in human renal tumors vs. matched normal renal tissue to unmask candidate tumor-induced/associated vascular proteins. 2) To create new antibodies to newly discovered human renal tumor EC targets and to use antibodies as probes to validate the expression of tumor-induced/associated proteins at the EC surface and its caveolae in human tissues and thereby to assess the degree of target specificity for the neovasculature of human solid tumors. Such mapping may also elucidate the effects of the tumor on the developing vascular endothelium and yield important tumor-specific vascular targets for improving noninvasive diagnostic imaging and therapy as well as yield new diagnostic and prognostic markers for the molecular classification of tumor biopsies