Identification of causes of tumor hypoxia is important because hypoxia causes resistance to radiation and a number of drugs; it induces production of angiogenic factors; and may increase metastatic potential. The overall goal of this project is to quantitatively establish how tumor oxygenation depends on oxygen delivery and consumption, and to use this information to identify effective methods for controlling tumor hypoxia. Prior work by this group has focused on chronic hypoxia. Here, acute hypoxia will also be investigated with the goal of determining what fractions of tumor are either chronically or acutely hypoxic and, for acute hypoxia, to determine what the time scales are, under conditions of normal and modified oxygen supply and demand. In Specific Aim 1, three dimensional vascular arrangements in tumors in rat dorsal flap window chamber preparations will be determined using microscopic magnetic resonance imaging and serial histologic reconstruction. Intravital microscopy and O2 microelectrodes will be used to measure temporal changes in blood flow and perivascular 02 levels and if temporal variations in 02 consumption rates occur. Theoretical models will be used to predict the extent and character of chronic and transient hypoxia based on these observations. The goal of Specific Aim 2 is to examine the effectiveness of several approaches for modifying tumor oxygenation. Previous modeling studies predict that reduction in 02 consumption rate and/or increase in blood 02 content are most effective. Thus, we will determine how reduction in 02 consumption (induced by hypothermia, hyperglycemia, rotenone or metronidazole) or increase in blood 02 content (using carbogen, 100% 02, or hyperbaric 02) alone or in combination will alter microvascular function, 02 consumption, and oxygenation. The effects of hemoglobin-containing liposomes as 02 carriers will be investigated, since some tumor vessels are observed to carry plasma but not red cells. Nicotinamide is reported to reduce intermittent blow flow, and its effects will be tested, with and without carbogen. We will also study means to reduce tumor p02, in line with current strategies to exploit such conditions with hypoxic cytotoxins. Strategies that we will test will focus on methods to increase 02 consumption rate and decrease tumor blood flow. In Specific Aim 3, strategies for modifying tumor oxygenation will be further tested by polarographic measurements of tumor oxygenation in flank preparations and by regrowth delay experiments following tumor irradiation. Improved quantitative understanding of the determinants of tumor oxygenation and of methods to modify 02 delivery and consumption should form a basis for successful control of tumor hypoxia. The proposed studies will employ a unique combination of experimental and theoretical methods and expertise to gain such understanding of tumor oxygenation and its modification at the microcirculatory level.