Visualization of the metastatic process by green fluorescent protein expression.

We demonstrate here the visualization of the cancer metastatic process in live tissue in vivo by green fluorescent protein (GFP) expression. The human lung adenocarcinoma cell-line Anip 973 was transfected with the humanized GFP-S65T cDNA and stable high-level GFP-expressing transfectants were established. GFP transfectants were initially inoculated subcutaneously in nude mice. Five weeks after transplantation, when the tumor had reached 1.2 cm in diameter, fragments of subcutaneously tumor were implanted onto the visceral pleura of nude mice by surgical orthtopic implantation (SOI) as a spontenous metastatic model. GFP expressing cells were injected intravenously in nude mice as an experimental hematogenous metastasis model. Mice were sacrificed four and eight weeks after treatment. At eight weeks, SOI-treated mice had lymphogenous (3/4 mice) and direct seeding (3/4) metastasis in the pulmonary hilum, cervical lymph nodes, the mediastinum and contralateral pleural cavity as detected by GFP expression in live tissue. All intravenously injected mice had metastases in the lung (4/4) and some of them had metastases in the brain (2/4) and other organs (1/4) as detected by GFP expression in fresh tissue. Some of the lung metastases produced by intravenous injection remained as dormant small colonies even eight weeks after treatment. These different metastatic patterns after SOI and intravenous injection visualized by GFP expression indicates that initial steps of the metastatic cascade influence the subsequent progression of metastasis.