Tumor Growth, Local Invasion, Micrometastasis, and Lymph Node Metastasis of Oral Squamous Cell Carcinoma Visualized in Live Tissue by Green Fluorescent Protein Expression

We report the in vitro establishment of a highly stable green fluorescent protein (GFP)-expressing transfectant of a highly-invasive human tongue squamous cell carcinoma (HTSCC) cell line, SAS-H1. The fluorescent cells permitted the visualization of tumor growth, local invasion, micrometastasis and cervical lymph node metastasis after submucosal injection into the tongues of nude mice. SAS-H1 cells were transfected with the pEGFP-N1 expression vector containing the GFP and neomycin resistance genes. Stable SAS-H1 clones expressing high levels of GFP were selected stepwise in vitro in levels of geneticin (G418) of up to 3,500 μg/ml. Subsequent early stages of local invasion and micrometastasis were visualized by GFP fluorescence in a primary tumor of the tongue. Furthermore, lymph node metastasis was confirmed for all of the orthotopic transplants in mice. However, no distant metastases, including those of lung and liver, were observed. Thus, this model should be useful for studying the metastatic process and for evaluating anti-metastasis agents in pre-clinical trials.