Establishment of green fluorescent protein-expressing hepatocellular carcinoma cell lines with different metastatic potential: relevant models for in vivo monitoring of metastasis and angiogenesis

Abstract

Purpose: To establish stable green fluorescent protein (GFP)-expressing metastatic human hepatocellular carcinoma (HCC) cell lines with different metastatic potential for long-term in vivo studies of metastasis and angiogenesis.

Methods: The pIRES2-EGFP vector, which contains an enhanced GFP gene, was transfected into MHCC97-H and MHCC97-L, HCC cell lines with different metastatic potential. The stability of GFP expression, basic biological characteristics, invasion abilities in vitro, and spontaneous metastasis in vivo of the new cell lines (MHCC97-HG and MHCC97-LG) were studied. Microvessel density (MVD) of orthotopic implanted tumors was compared by anti-CD31 immunohistochemical staining, and real-time angiogenesis and metastasis of GFP-transfected tumors were detected by intravital fluorescent microscope.

Results: The GFP-transfected cell lines stably expressed green fluorescence in the absence of G418 over a 36-day period. Compared with the parental cell lines, they exhibited no distinct differences in biological characteristics. MHCC97-HG showed more aggressive invasion and spontaneous metastatic behavior than MHCC97-LG, and even its parental cell line, MHCC97-H (P<0.01). MVD levels induced by MHCC97-HG orthotopic implanted tumors were significantly higher than MHCC97-LG (P<0.01). Real-time angiogenesis and sequential steps of metastasis could be detected clearly under intravital fluorescent microscope.

Conclusions: These two stable GFP-expressing HCC cell lines with the same genetic background and different metastatic potential were established, which could be useful models for monitoring metastasis and angiogenesis of HCC.

Fig. 1A–D

A, B GFP Expression of MHCC97-HG/MHCC97-LG cells maintained in the absence of G418 after 36 days; and C,D their orthotopic implanted tumors, visualized by fluorescent microscopy

Fig. 2A–F

Invasion assay in vitro and spontaneous lung metastasis in vivo. A,B Showing MHCC97-HG/MHCC97-LG cells penetrating the artificial basement membrane. The cell numbers of MHCC97-HG were significantly more than MHCC 97-LG (light microscope, 200×); C,D,E,F Spontaneous lung metastasis 35 days after MHCC97-HG/MHCC97-LG orthotopic implantation. (C,D paraffin sections with HE staining, light microscope 400×; E,F cryostat sections without staining, light microscope 200×, the images on the right bottom show the same field under fluorescent microscopy. The arrows point to the metastasis foci)

Fig. 3A–D

Anti-CD31 immunohistochemistry and intravital fluorescent microscopy. A,B MHCC97-HG/MHCC97-LG, showing red fluorescence (CD31 staining for MVD, pointed to by arrows) among green fluorescence (tumors). The MVD levels of MHCC97-HG SOI tumors were significantly higher than MHCC97-LG tumors, fluorescent microscopy, 200×; C Intravital fluorescent microscopy showed the vessels (pointed to by arrows) appeared dark against the green fluorescence of the MHCC97-HG tumor tissue, 80×; D Small liver metastatic foci (pointed to by arrows) near MHCC97-HG orthotopic implanted tumor, 14 days after orthotopic implantation. 80×