There is strong rational for studying how hypoxia develops in tumor tissue because O2 is an important modifier of therapeutic effects. However, the degree of tumor hypoxia varies greatly among individuals. Thus, understanding the mechanisms creating hypoxia could lead to methods of recognizing and overcoming it that have not been previously considered. Fractionated radiotherapy (RT) may partially reverse hypoxia but the process may be incomplete in patients who have local treatment failure. Thus knowledge of mechanisms of reoxygenation is also needed. Our multidisciplinary approach has incorporated experimental results with mathematical models of tumor microcirculation. Observations relevant to the long-term goals of the project are: (1) perivascular pO2 can be quite low, suggesting that conditions conducive to altered rheology are present, which can be reversed with the calcium antagonist, flunarizine. (2) Using a Green's function model of O2 transport, we have found the vascular geometry of tumors alters tissue oxygenation in a way not predicted by Krogh cylinder-type calculations. (3) Bradykinin, a mediator of inflammation, increases blood flow in normal tissue while decreasing tumor blood flow to the point of intermittency; followed by preferential leukocyte-endothelial (L/E) cell adhesion in normal tissues. RT up- regulates adhesion and flow in normal tissues within 1-hour post- exposure, suggesting inflammatory pathways similar to those observed with bradykinin. (4) L/E cell adhesion is greatly depressed in our tumor model, as compared with normal tissues under control or stimulated states, suggesting that the tumor is less susceptible to stimulation of inflammation - probably because of aberrancies in the adhesion molecules. Thus, we propose a continuation of this grant, with the following specific aims: (1) microelectrode measurements of tissue O2 tension in a dorsal flap window chamber will test whether the O2 consumption rates are higher in tumor periphery than in adjacent normal tissue center. (2) A histologic marker of hypoxia (nitroimidazole) and bioluminescence measurements of ATP, glucose, and lactate in adjacent slices will be used to distinguish between anaerobic or aerobic glycolysis and respiration in situ. (3) Mathematical models of O2 transport will be expanded to include 3-dimensional vascular architecture, in situ measurements of O2 consumption, and interdependence of O2 and glucose consumption using data obtained from Specific Aims 1 and 2. (4) Mechanisms behind radiation- induced regulation of L/E interactions in tumor and normal tissues will be explored by using agonists and antagonists of pathways of inflammation. Reasons for decreased interaction in tumors will be tested by examining alterations in fatty acid metabolism and/or nitric oxide synthesis. L/E cell interactions in non-metastatic and metastatic variants of two tumor lines (R3230 Ac and Dunning) will be performed. (5) The process of reoxygenation will be studied by performing intravital hemoglobin saturation measurements that can be repeated in vascular segments before and after RT of heat. These proposed studies are unique in scope and are likely to produce new information regarding metabolic and physiologic processes leading to heterogeneity in tumor oxygenation, as well as insights into the regulatory factors involved in changes in normal tissue and tumor blood flow after radiation and heat.
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