Our hypothesis is that a tumor can be considered as an entity composed of a variety of cell subpopulations. Each subpopuIation may have its own inherent properties and intrinsic sensitivities to therapies. These different sensitivities can arise from environmental or genetic factors or both. The overall tumor response to anticancer therapies then is a reflection of the responses of the individual cell subpopulations. The central goal of this research proposal continues to be the investigation of the various critical cell subpopulations comprising solid tumors. We will use cell separation techniques including centrifugal elutriation and fluorescence activated cell sorting to isolate and characterize cell subpopulations dispersed from rodent tumors, human tumor xenografts or clinical samples. Emphasis will be placed on determining the biological characteristics of hypoxic cells. There is evidence to suggest that tumors may contain cells which may be chronically or transiently hypoxic. Such cells would result from different mechanisms, have different characteristics, and have different implication for therapies. We plan to probe various rodent and human tumor models for the presence of such cells using tracer dyes combined with histological and flow cytometric techniques as well as radiolabeled sensitizers. Quiescent (Q) cells may represent another cell subpopulation of considerable relevance in clinical anticancer therapy. Experiments are proposed to study the response of such cell to different chemotherapeutic agents. Q cells in rodent tumor models will be characterized by acridin orange (AO) staining and flow cytometry analysis and isolated by centrifugal elutriation. In human tumor cells, FlTC conjugated anti-Ki-67 and anti-p105 antibodies and flow cytometry will be used to determine the percentage of proliferating cells. In parallel with the AO staining and Ki-67 or plO5 analyses, 3H-TdR labelling will be performed. Experiments extending Q cell determinations to human tumor biopsies also are proposed. Since changes in the quiescent cell population, if they occur during the course of therapy, could have a major impact on therapy, it is also our goal to attempt to establish techniques for measuring Q cell fractions in treated tumors. Finally, experiments are designed to gain a better understanding of the biological basis and underlying mechanisms for tumor response to altered fractionation schemes. Ultimately we hope that these approaches can be reliably developed to an extent where the use of human tumor biopsy material may lead to radiotherapy treatments more tailored to the individual patient.
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