For the past half-century, cell biology and molecular biology have been carried out on cells growing in dishes and by the extracellular analysis of cellular components including genes and proteins. The advent of green fluorescent protein (GFP) as reporter gene is enabling a paradigm change in cell biology and molecular biology. In the first phase of revolution, molecular processes such as gene expression and ion fluxes were visualized in living cells in vitro. Spectrally distinct fluorescent proteins, especially red fluorescent proteins (RFPs), are being isolated by groups throughout the world which enable simultaneous multi-color “rainbow imaging” of numerous process in the living cell both in vitro and in vivo.

We have developed imaging of tumors and metastases in mice by use of cancer cells expressing GFP. These mice present many new possibilities for research including real-time studies of tumor progression, metastasis dynamics, and drug–response evaluations. We introduced the GFP gene into a large series of human and rodent cancer-cell lines in vitro, which stably express GFP after transplantation to rodents. With this fluorescent tool, single cells can be imaged and tracked in vivo.

produced transgenic mice with GFP under the control of a chicken ?-actin promoter and cytomegalovirus enhancer. With the GFP host mouse, it became possible to visualize all the host cells that can interact with the tumor. All of the tissues from these transgenic mice, with the exception of erythrocytes and hair, fluoresce green.

We have developed a simple yet powerful technique for delineating morphological events of tumor–host interactions with dual-color fluorescence. The method clearly images implanted tumors and adjacent stroma, distinguishing unambiguously the host and tumor-specific components of the malignancy. Dual-color fluorescence imaging is effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice. This model shows with great clarity the details of tumor–stroma interactions, especially tumor-induced angiogenesis and tumor-infiltrating lymphocytes. The GFP-expressing tumor vasculature, both nascent and mature, is readily distinguished interacting with RFP-expressing cancer cells. GFP-expressing dendritic cells were observed contacting RFP-expressing cancer cells with their dendrites. GFP-expressing macrophages were observed engulfing RFP-expressing cancer cells. GFP lymphocytes were observed surrounding cells of the RFP tumor, which eventually regressed.

In order to develop a method to color-code cells in vivo, we have established stable, bright GFP- or RFP-expressing HT-1080 human fibrosarcoma clones. The implantation of mixtures of HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies. The lung metastases are visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of genes of interest.

In transgenic mice with GFP under the control of nestin regulatory sequences, nestin-expressing cells, marked by GFP fluorescence, appear in the population of hair follicle stem cells. The relatively small, oval-shaped, nestin-expressing cells in the bulge area surround the hair shaft and are interconnected by short dendrites. Expression of the unique protein, nestin, in neural stem cells, hair follicle stem cells as well as nascent blood vessels suggests their possible relation.

Intravital imaging has shown that carcinoma cells in a primary breast tumor can move at up to 10 times the velocity of cells in vitro. Metastatic cancer cells moving along linear paths in association with extracellular-matrix fibers had the highest velocity. Breast carcinoma cells showed solitary amoeboid movement. Cells in metastatic breast tumors were observed to be attracted to blood vessels. Cell polarity towards blood vessels was correlated with increased intravasation and metastasis.

C57/B6-GFP-expressing mice. Transgenic C57/B6-GFP mice were obtained from the Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. The C57/B6-GFP mice express GFP under the control of the chicken ?-actin promoter and cytomegalovirus enhancer. All tissues from this transgenic line, with the exception of erythrocytes and hair, fluoresce green under excitation light. The GFP gene, regulated as described above, was crossed into nude mice on a C57/B6 background.

Nestin is an intermediate filament gene that is a marker for central nervous system progenitor cells and neuroepithelial stem cells, hair follicle stem cells and blood vessels. Transgenic mice carrying enhanced GFP under the control of the nestin second-intron enhancer were also used as hosts to visualize nascent angiogenesis in transplanted RFP-expressing tumors.

The pLNCX₂ vectors were purchased from Clontech Laboratories (Palo Alto, CA, USA). The pLNCX₂ vector contains the neomycin resistance gene for antibiotic selection in eukaryotic cells. The RFP gene (DsRed2; Clontech Laboratories) was inserted in the pLNCX₂ vector at the Egl II and Not I sites.

For retroviral transduction, PT67, an NIH3T3-derived packaging cell line, expressing the 10 Al viral envelope, was purchased from Clontech Laboratories. PT67 cells were cultured in DME (Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gemini Bio-products, Calabasas, CA, USA). For vector production, PT67 cells at 70% confluence, were incubated with a precipitated mixture of DOTAP^TM reagent (Boehringer, Mannheim, Germany), and saturating amounts of pLNCX₂-DsRed2 plasmid for 18 h. Fresh medium was replenished at this time. The cells were examined by fluorescence microscopy at 48 h post-transfection. For selection of brightly fluorescing cells producing high titer retroviral supernatants, the RFP-expressing packaging cells were cultured in the presence of 500 ?g/ml-2000 ?g/ml G418 increased in a step-wise manner (Life Technologies, Grand Island, NY, USA) for seven 7 days to select a clone producing high amounts of RFP retroviral vector.

The pLEIN retroviral vector (Clontech Laboratories expressing GFP and the neomycin resistance gene on the same bicistronic message was used as a GFP expression vector in PT67 cells, as described above for RFP retroviral vector production.

For GFP or RFP gene transduction, 20% confluent cancer cells were incubated with a 1:1 precipitated mixture of retroviral supernatants of PT67 cells and RPMI 1640 or other culture media (Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gemini Bio-products) for 72 h. Fresh medium was replenished at this time. Cancer cells were harvested with trypsin/EDTA and subcultured at a ratio of 1:15 in a selective medium, which contained 50 ?g/ml G418. To select brightly fluorescent cells, the level of G418 was increased to 800 ?g/ml in a step-wise manner. Clones expressing GFP or RFP were isolated with cloning cylinders (Bel-Art Products, Pequannock, NJ, USA) using trypsin/EDTA and were amplified and transferred by conventional culture methods in the absence of selective agent (Yamamoto et al., 2003a,b).