McCann Thomas E, Kosaka Nobuyuki, Choyke Peter L, Kobayashi Hisataka
Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD, USA.
Methods Mol Biol. 2012;872:191-204. doi: 10.1007/978-1-61779-797-2_13.
Target-specific imaging probes represent a promising tool in the molecular imaging of human cancer. Fluorescently-labeled target-specific probes are useful in imaging cancers because of their ability to bind a target receptor with high sensitivity and specificity. The development of probes relies upon preclinical testing to validate the sensitivity and specificity of these agents in animal models. However, this process involves both conventional histology and immunohistochemistry, which require large numbers of animals and samples with costly handling. In this chapter, we describe a novel validation tool that takes advantage of genetic engineering technology, whereby cell lines are transfected with genes that induce the target cell to produce fluorescent proteins with characteristic emission spectra, thus enabling their easy identification as cancer cells in vivo. Combined with multicolor fluorescence imaging, this can provide rapid validation of newly-developed exogenous probes that fluoresce at different wavelengths. For example, the plasmid containing the gene encoding red fluorescent protein (RFP) was transfected into cell lines previously developed to either express or not express specific cell surface receptors. Various antibody-based or ligand-based optical-contrast agents, with green fluorophores were developed to concurrently target cancer cells and validate their positive and negative controls, such as the β-D: -galactose receptor, HER1, and HER2 in a single animal/organ. Spectrally-resolved multicolor fluorescence imaging was used to detect separate fluorescence emission spectra from the exogenous green fluorophore and RFP. Here, we describe the use of "co-staining" (matching the exogenous fluorophore and the endogenous fluorescent protein to the positive control cell line) and "counter-staining" (matching the exogenous fluorophore to the positive control and the endogenous fluorescent protein to the negative control cell line) to validate the sensitivity and specificity of target-specific probes. Using these in vivo imaging techniques, we are able to determine the sensitivity and specificity of target-specific optical contrast agents in several distinct animal models of cancer in vivo, thus exemplifying the versatility of our technique, while reducing the number of animals needed to conduct these experiments.
靶向特异性成像探针是人类癌症分子成像中一种很有前景的工具。荧光标记的靶向特异性探针可用于癌症成像，因为它们能够以高灵敏度和特异性结合靶受体。探针的开发依赖于临床前测试，以验证这些试剂在动物模型中的灵敏度和特异性。然而，这个过程涉及传统组织学和免疫组织化学，这需要大量动物和样本，处理成本高昂。在本章中，我们描述了一种利用基因工程技术的新型验证工具，通过该技术将基因转染到细胞系中，诱导靶细胞产生具有特征发射光谱的荧光蛋白，从而使其在体内易于被识别为癌细胞。结合多色荧光成像，这可以快速验证新开发的在不同波长下发出荧光的外源性探针。例如，将含有编码红色荧光蛋白（RFP）基因的质粒转染到先前开发的表达或不表达特定细胞表面受体的细胞系中。开发了各种基于抗体或配体的带有绿色荧光团的光学造影剂，以同时靶向癌细胞并验证其阳性和阴性对照，如单个动物/器官中的β-D-半乳糖受体、HER1和HER2。光谱分辨多色荧光成像用于检测来自外源性绿色荧光团和RFP的单独荧光发射光谱。在这里，我们描述了使用“共染色”（将外源性荧光团和内源性荧光蛋白与阳性对照细胞系匹配）和“反染色”（将外源性荧光团与阳性对照匹配，将内源性荧光蛋白与阴性对照细胞系匹配）来验证靶向特异性探针的灵敏度和特异性。使用这些体内成像技术，我们能够在几种不同的体内癌症动物模型中确定靶向特异性光学造影剂的灵敏度和特异性，从而证明了我们技术的多功能性，同时减少了进行这些实验所需的动物数量。
