The use of fluorescent proteins for developing cancer-specific target imaging probes

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.