Objective: To investigate the invasion and metastasis of glioma cell in vivo by implanting C6 glioma cells with the enhanced green fluorescent protein (EGFP) gene into the brain of SD rats.
Methods: C6 glioma cells were transfected with a plasmid vector (pEGFP-N3) containing the EGFP gene. Stable EGFP-expressing clones were isolated and examined by flow cytometry and electron microscopy. EGFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later, the rats were killed and consecutive brain sections were examined using fluorescence microscopy after adjacent sections were examined by immunohistochemistry or routine hematoxylin and eosin staining for the visualization and detection of tumor cell invasion. Xenograft was primarily cultured to determine the existence of the EGFP gene in vivo.
Results: There was no obvious difference in cell cycle and ultrastructure between transfected cells and untransfected cells. EGFP-transfected C6 glioma cells maintained stable high-level EGFP expression in the central nervous system during their growth in vivo. EGFP fluorescence clearly demarcated the primary tumor margins and readily allowed for the visualization of distant micrometastasis and local invasion on the single-cell level. Small locally invasive foci, including those immediately adjacent to the leading invasive edge of the tumor, were virtually undetectable by routine hematoxylin and eosin staining and immunohistochemistry.
Conclusion: EGFP-transfected C6 glioma cells can be visualized by fluorescence microscopy after intracranial implantation. This model is an excellent experimental animal model in the research of glioma invasion and metastasis.