The basis of this application arises from new data suggesting the hypothesis that cells at intermediate oxygen concentrations (centered around approximately 1%) dominate the therapeutic response and biology of tumors. Our hypothesis originated from in vivo studies utilizing a quantitative method for determining tissue hypoxia. This method, developed in our laboratory, measures binding of the 2-nitroimidazole hypoxia marker EF5 (2-(2-nitro-1H-imidazol-1-yl)-N- (2,2,3,3,3-pentafluoropropyl)acetamide). The bound EF5 adducts are measured using fluorescent monoclonal antibodies and a fluorescence scale calibrated to biological measures of actual cellular pO2. Use of this method predicts the radiation response in individual rat tumors of two types studied to date (9L glioma and Morris 7777 hepatoma). There have been two models proposed for the production of tumor hypoxia: in the first, blood flow is in a steady state leading to diffusion limited hypoxia whereas in the second, blood flow cycles on and off leading to perfusion limited hypoxia. Which of these models is most relevant, in either rodent or human tumors, remains an important unresolved question. Despite having many biological and therapeutic ramifications, it has previously been addressed solely by indirect methods. Since the two models of hypoxia are predicted to form different patterns of hypoxia with respect to the tumor vasculature, we propose a direct investigation of this question by detailed 3-dimensional image analysis, using subcutaneous and orthotopic human xenograft tumors in nude rats. To support the in vivo image analysis studies, quantitative assessments of the biochemical and molecular consequences of steady state vs. cycling hypoxia will be made in vitro. These studies will emphasize those molecular markers known to be modified by the tumor microenvironment and presently under consideration as clinical prognostic markers (e.g. HIF-1alpha, Carbonic Anhydrase IX, Glut-1 and VEGF). Studies in humans continue to support the importance of hypoxia in limiting treatment response - thus new sensitization approaches are required. A new perfluorochemical emulsion (PFC) made by the Sonus Company (Seattle) will be employed to determine whether hypoxic tumor cells (both intermediate and severe hypoxia) can be targeted therapeutically. This emulsion (DDFP) has the remarkable property of forming stable, sub-micron sized gas bubbles above 28 degrees. The DDFP bubbles, like former liquid PFC emulsions, serve as high-capacity oxygen carriers but, unlike all previous formulations, do so at much lower concentrations. DDFP may thus be the first PFC emulsion practical and safe for use in multifraction clinical regimens.