Regionally limited biopsy specimens (fine needle aspirates, core biopsies) are increasingly used for cancer diagnosis, treatment decisions and prognoses. Yet, heterogeneity in the structure and organization of cancer tissues is universally accepted. What are the ramifications of regional sampling in the face of such heterogeneity: This is critically important because techniques utilizing these sampling methods are being developed to evaluate cell cycle, tumor genetics, hypoxia, etc. Little is known about the accuracy of the regional sampling methods which are typically used to determine the extent, distribution and survival implications of tumor heterogeneity. Our goal is to evaluate the ability of regionally limited sampling methods to predict tumor heterogeneity and to determine how this heterogeneity influences cell survival following radiation. Heterogeneity of hypoxia, cell cycle and histopathological appearance will be stressed. To model heterogeneity, we have selected 3 tumor cells types (Morris 7777 hepatoma, 9L glioma, EMT6 mammary carcinoma) and 2 growth patterns (subcutaneous vs. tissue isolated epigastric implant). The subcutaneous and tissue isolated models should result in markedly different vascular architectures (central vs. peripheral distribution, response to pharmacologic manipulations, etc.) due to the centrally located host vessels in the tissue isolated tumor model. We will use the tumors at a consistent size (1-1.3 gm) and stage of vascular development (determined by number of days post implantation). Both rat tumors, 9L glioma and Morris 7777 hepatoma, are radioresistant, but their histopathologic appearance (necrosis, tumor cords) and biologic characteristics (growth and metabolic rate, hypoxic fraction) differ. The murine EMT6 is radioresistant with heterogeneous hypoxic areas in a tumor cord distribution. Growing these different tumor types in different vascular environments will allow us to model various aspects of human tumor morphology. We will assess hypoxia with nitroimidazole binding as measured by fluorescent activated cell sorting (FACS). The nitroimidazole to be used is pentafluorinated etanidazole (EF5); a monoclonal antibody (ELK3-51) has ben raised against its adducts. ELK-51 is conjugated to the fluorochrome, Cy3 for detection by FACS (and other) techniques. The results of these studies will be compared to histopathologic evaluations, cell cycle analyses and in vivo-in vitro plating efficiency assays. Data on regional tumor sampling methods can be used to investigate therapies where modifications of tumor heterogeneity are sought, especially as related to hypoxia's influence on radiation response . These clinically relevant techniques will eventually be applied in spontaneous (dog and cat) tumor models, with which the PI has extensive experience. The work proposed herein will ultimately lead to improved ability to objectively assess heterogeneity within individual tumors.
