TOMOGRAPHIC/SPECTRAL IMAGING OF FGF-MED ANGIOGENESIS

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The overall objective of this proposal is to determine whether tumor angiogenesis constitutes a process that requires uni- or bi-directional signalling between tumor cells and endothelial cells comprising the intratumoral vasculature, and whether this communication is mediated by basic fibroblast growth factor (bFGF) and Fibroblast growth factor receptor (FGFR). To obtain an answer for this important but unanswered question, we will inject human melanomas, grown as subcutaneous tumors in nude mice, with vector constructs generating bFGF/FGFR antisense transcripts under the control of a human tyrosinase promoter and likewise, an RSV LTR promoter. Since tyrosinase is an essential gene of the pigmentation pathway, bFGF and FGFR antisense transcripts, generated under the control of this promoter, will be expressed in melanoma cells but not in tumor-interspersing vascular endothelial cells, In contrast, bFGF/FGFR antisense constructs under the control of the RSV LTR promoter will be expressed in the melanoma cells and the intratumoral vasculature. Thereupon, the tumors will be injected with different cyanine fluorochrome-conjugated antibodies specific for CD31, an antigen expressed on endothelial but not melanoma cells, and S100, an antigen present only melanoma cells. Using fluorescence tomography and AOTF (acousto-optic tunable filters) microscopy, we will then visualize, record and compare, in vivo, the fate of the endothelial and malignant cells within the tumors of the two groups of animals. This schedule of intratumoral inoculations and tumor imaging will be continued until we observe growth-arrest and regression of the bFGF/FGFR antisense-targeted melanomas. After sacrificing the animals and removing their tumors or regressed nodules, we will perform multi-parameter immunohistochemistry and in situ hybridization of tissue sections prepared from the specimens. As the final step of these investigations, the stained melanoma sections will be subjected to qualitative and quantitative microscopic Spectral Imaging to determine the expression or lack thereof, of angiogenesis, proliferation and apoptosis-specific markers.