Our long-term goal in this competing renewal application continues to be the improved treatment of human solid tumors. As in the initial three years of this project, we will explore combination chemotherapy- radiotherapy treatments of human tumor xenografts, but with approaches and goals that are, to our knowledge, unique to this laboratory: using conventional chemotherapeutic agents to modulate tumor blood flow and/or cellular metabolism, thereby Increasing the radiosensitivity of tumor cell subpopulations. During the requested funding interval, we intend to determine the extent and nature of hypoxia and other microenvironmental anomalies in several types of xenografted human tumors growing in immunocompromised mice, and to test four common chemotherapeutic agents (carboplatin, CCNU, cyclophosphamide, and 5-fluorouracil) as radiation modifiers. We hypothesize that appropriate time and dose schedules will produce radiosensitization, altered blood flow, metabolic perturbations leading to reoxygenation, and/or preferential cytotoxicity. The therapeutic potential of the combination treatments will be assessed using fluorescence-activated cell sorting techniques to isolate and differentially determine the clonogenic potential of hypoxic versus aerobic cell subpopulations from the xenografts. These studies will be complemented by variations of the comet assay aimed at determining the growth fraction, and the hypoxic fraction of the tumors and isolated subpopulations. Concurrently, drug-induced changes in blood flow will be quantified at the macroscopic level with laser Doppler flowmetry, and at the microregional level with image analysis techniques. The potential and reliability of a new flow cytometric method for quantifying transient blood flow, based on dual labeling with thymidine analogues, will also be determined. Oxygen utilization rates will be measured for cells in situ using an innovative fluorescent staining procedure. We anticipate that our integrated results will, at the very least, provide novel information concerning the nature and role of hypoxia in the in vivo action and interaction of carboplatin, CCNU, cyclophosphamide and 5- fluorouracil with radiation; at best, new treatment schemes entirely feasible for immediate clinical testing will be suggested.
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