Fluorescent imaging of angiogeneosis and spontaneous metastasis in transplanted hepatoma:experiment with mice

Abstract

To establish stable enhanced green fluorescent protein (GFP) -expressing human adenoid cystic carcinoma (ACC-M-GFP) cell lines with high lung metastatic potential, and to study the value and influential factor of in vivo monitoring of metastasis and angiogenesis by fluorescent imaging. Human adenoid cystic carcinoma cell line ACC-M-GFP labeled with enhanced GFP was established. ACC-M-GFP cells of the dose 10(7)/ml were injected subcutaneously into two nude mice. Once the subcutaneous tumor reached 0.5 cm in diameter, it was removed, cut into 1 mm3 pieces, and implanted into the left hepatic lobe of 8 nude mice. The animals were observed by whole-body optical imaging system and stereofluorescent microscope each two weeks from the fourth week after transplantation. One mouse was killed and autopsied after each examination. The livers, lungs, and adjacent organs, such as diaphragm and lymph nodes were excised, and fluorescent imaging of different organs separately was studied. Hepatoma was cryostat sectioned and angiogenesis was visualized under fluorescence microscope. The findings of fluorescent imaging were compared with those of HE staining on routine paraffin sections. Liver tumors were transplanted successfully in all 8 nude mice. By whole-body optical system, the fluorescent signal from the liver tumor was detected through a skin-flap window after 6 - 8 weeks. Tumors were visualized directly through skin after 8 - 10 weeks. The hepatoma became enormous after 12 - 14 weeks, when the fluorescent signal of abdominal metastasis was detected. Metastasis of lung and lymph node was found by single organ imaging after 16 -18 weeks. By stereofluorescent microscope, microvessels were detected after 4 weeks, and a large quantity of tumor vessels like black branch stripes were found after 12 - 14 weeks. The tumor vessels appeared as dark tubiform network against the green fluorescence of the implanted tumor in section. Fluorescent imaging provides a method to monitor tumor growing, infiltrating, metastasis and angiogenesis in real time in vivo.