This project has focused on development and application of technology to evaluate signaling events in the laboratory and in clinical and preclinical samples, as related to invasion and angiogenesis. We have worked to optimize the TLA for application to angiogenesis and also to clinical core biopsies. Measurement of CD31/PECAM was successful on lysates of cultured endothelial cell but required over 300mm2 of archival tumor tissue, a quantity not feasible for successful application to core needle biopsies. Other measures of angiogenesis activation, phospho(p)-VEGFR-2, activated endothelial nitric oxide synthase, and other endpoints are being tested. Related to this are studies to optimize and validate the TLA for further application to clinical samples. The focus of the last year has remained quality assurance and control of the TLA. We analyzed the effects of different tissue solubilization buffers on frozen ovarian tumor samples in order to identify the best signal intensity dynamic range, reproducibility, tissue solubility, and signal consistency. We have used the previously determined optimal pixelation and analytic approaches for these studies. Biopsies from our ongoing clinical trial are being prepared for array analysis. We have been studying the role of progranulin and secretory leukocyte protease inhibitor (SLPI) in invasion and metastasis of ovarian cancer xenografts. Following our demonstration that SLPI is a prosurvival protein for ovarian cancer and that this did not depend upon the protease inhibitor activity, we investigated the behavior of cells overexpressing SLPI and/or its protease inhibitor mutants. Increased aggressiveness, invasive behavior and metastatic dissemination were observed. This has led to investigation of the role of SLPI in the biology of invasion and metastasis with mechanistic studies ongoing. Further, a neutralizing monoclonal antibody is available for collaborative advancement for clinical application. In addition to understanding why this gene is amplified and its protein product overexpressed, our study of SLPI in ovarian cancer provides a novel clinical translational objective for the laboratory. Studies with CAI remain collaborative with a prior member of the Section. Imatinib-resistant chronic myelogenous leukemia have been shown to be selectively susceptible to CAI therapy. A reduction in proliferative capacity, increase in apoptosis, and reduction in activation of the bcr-abl fusion protein and its related pathway have been shown. These data may be a step from which to consider CAI for testing in this select subset of imatinib-resistant CML patients. Use of CAI for ophthalmologic disease is under consideration for licensure and is being developed by collaborators. CAI will be tested therapeutically for benefit in macular degeneration. This comprehensive approach to angiogenesis and microenvironment study in the laboratory and patient samples will provide opportunity for proof of principle of molecularly targeted agents in stromal therapy and will complement our continuing clinical work with signal transduction inhibition therapy.
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