3] Green fluorescent protein to visualize cancer progression and metastasis

Abstract

Publisher Summary Chinese hamster ovary cells and the human lung adenocarcinoma cell lines ANIP 973 and H-460 are transfected with the dicistronic expression vector containing the humanized green fluorescent protein (GFP) cDNA. Stable GFP-expressing clones are selected in 1.5 μM methotrexate in vitro and injected subcutaneously in nude mice. Stable, high-level expression of GFP is maintained in the subcutaneously growing tumors. To utilize GFP expression for metastasis studies, fragments of subcutaneously growing tumor that are composed of GFP-expressing cells are implanted by surgical orthotopic implantation (SOI) in the ovary and lung, respectively, of nude mice. Subsequent micrometastases are visualized in systemic organs by GFP fluorescence in the lung, liver, brain, skeleton, and other organs down to the single-cell level. With this fluorescence tool, for the first time tumor cells were detected and visualized at the microscopic level in fresh viable tissue in their normal host organ. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance in the visualization and study of lung cancer metastasis in process. Lung tissue seeded with GFP-expressing ANIP 973 human lung carcinoma cells is incubated in three-dimensional sponge-gel matrix-supported histoculture. Tumor progression is continuously visualized by GFP fluorescence in the same individual cultures over a 52-day period, during which time the tumors spread throughout the histocultured lung.Histoculture tumor colonization is selective for the growth of lung cancer cells on lung tissue, as no growth occurs on histocultured mouse liver tissue, as also observed in vivo. The ability to support selective organ colonization in histoculture and visualize tumor progression by GFP fluorescence allows the in vitro study of tumor progression in situ.