The goals of this project are to quantify oxygen and photosensitizer distributions in tumors, describe the effects of photodynamic therapy (PDT) on these distributions, and measure the consequences on tumor response. Depletion of tumor oxygen by the illuminating light for PDT has been identified as potentially therapy-limiting. Methods of studying this oxygen depletion have included polarographic needle probe measurement of tissue PO(2) and spectroscopic determination of blood oxygen concentration. Tumor-averaged measurements of oxygen concentration indicate that PDT can create severe tumor hypoxia, but these methods lack the spatial resolution to detect gradients in oxygen distribution. We hypothesize that tumor responses to PDT will be determined by the spatial distribution of oxygen relative to targets of damage (e.g. the vascular endothelium) and photosensitizer biodistribution. The description of oxygen and sensitizer distributions during PDT could suggest reasons for treatment failure and facilitate the development of more effective treatment protocols. A fluorinated series of 2-nitroimidazole hypoxic markers, including the drugs EF3 and EF5, has been developed within our laboratories. Hypoxic markers are a unique means to investigate the regional effects of PDT on tumor oxygenation. EF3 will be used to quantify the oxygenation of murine tumors through immunohistochemistry of frozen sections and flow cytometry of cell suspensions. Patterns and intensities of hypoxic marker binding will be compared to those of photosensitizer distribution (determined by their inherent fluorescence), tumor vascularity (labeled by antibodies), tumor perfusion (labeled by injected fluorescent dyes), and apoptosis (detected by commercial kits). Gradients in tumor oxygenation, which may exist as a function of distance from the blood vessels, will be quantified. The consequences of oxygen maintenance or depletion at the blood vessels will be examined in terms of PDT-associated vascular damage, including the development of necrosis and apoptosis. The manipulation of fluence rate and drug dose to control local oxygen depletion and improve tumor response will be examined. Investigations will be carried out using three clinically relevant photosensitizers, Photofrin, Foscan and Lutex.
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