TOMOGRAPHIC/SPECTRAL IMAGING OF FGF-MED ANGIOGENESIS

该提案的总体目标是确定肿瘤血管生成是否构成一个需要肿瘤细胞和构成肿瘤内脉管系统的内皮细胞之间单向或双向信号传导的过程，以及这种通讯是否由碱性成纤维细胞生长因子（bFGF）和成纤维细胞生长因子受体（FGFR）介导。为了获得这个重要但尚未解答的问题的答案，我们将注射人类黑色素瘤，在裸鼠中作为皮下肿瘤生长，载体构建体在人类酪氨酸酶启动子和同样的RSV LTR启动子的控制下产生bFGF/FGFR反义转录本。 由于酪氨酸酶是色素沉着途径的必需基因，在该启动子控制下产生的bFGF和FGFR反义转录物将在黑色素瘤细胞中表达，但不在肿瘤散布的血管内皮细胞中表达。相反，在RSV LTR启动子控制下的bFGF/FGFR反义构建体将在黑色素瘤细胞和肿瘤内血管系统中表达。随后，肿瘤将被注射不同的花青荧光染料结合的抗体，这些抗体对 CD31（一种在内皮细胞但不表达黑色素瘤细胞的抗原）和 S100（一种仅存在于黑色素瘤细胞上的抗原）具有特异性。 然后，我们将使用荧光断层扫描和AOTF（声光可调滤光片）显微镜，在体内观察、记录和比较两组动物肿瘤内内皮细胞和恶性细胞的命运。这种瘤内接种和肿瘤成像的计划将继续进行，直到我们观察到 bFGF/FGFR 反义靶向黑色素瘤的生长停滞和消退。 处死动物并去除肿瘤或消退结节后，我们将对标本制备的组织切片进行多参数免疫组织化学和原位杂交。 作为这些研究的最后一步，染色的黑色素瘤切片将进行定性和定量显微光谱成像，以确定血管生成、增殖和凋亡特异性标记物的表达或缺乏。

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.