Figure 1.
Summary of novel methodology for establishment of OL0825 CTC line.
Subcutaneous implantation of 1×106 BNL 1ME A.7R.1 cells into the flank of a Balb/c mouse resulted in tumor development in the flank. Humane euthanization of mouse involved CO2 asphyxiation and intracardiac exsanguination. 500–1000 µl of whole blood was obtained. After centrifugation, buffy coat layer was subjected to red blood cell lysis, washed in PBS and cultured in medium in a humidified incubator at 37°C with air and 5% CO2. Tumor cell proliferation was observed within 48 hours. The cell line was designated OL0825 and is still very viable after 25 passages and repeated freezing and thawing. Phase contrast image of OL0825 was taken at ×40 magnification (objective lens).
Figure 2.
Circulating tumor cells have increased tumorigenicity.
1×106 BNL 1ME A.7R.1 or OL0825 cells were subcutaneously implanted into 7 separate Balb/c mice or surgically implanted into the liver of 3 separate Balb/c mice. A. Tumors from OL0825 had significantly greater volume than those from BNL 1ME A.7R.1. B. OL0825 cells resulted in tumor development twice as fast as BNL 1ME A.7R.1 cells in the survival surgical intrahepatic implantation model. C. Representative images of orthotopic tumor in the liver and metastatic lesions in the lung from implantation of OL0825 into liver of immune competent wild type Balb/c mice. Images of H&E-stained slides were acquired at ×40 (objective lens). Results presented as mean ± SEM; P<0.01.
Figure 3.
Circulating tumor cells have increased mesenchymal characteristics.
Analysis of gene and protein expression of markers of epithelial-mesenchymal transition were performed using qPCR and Western blotting respectively. There is significantly decreased E-cadherin gene expression (A), increased fibronectin gene expression (B), increased collagen I gene expression (C) and increased vimentin gene expression (D) by OL0825 than BNL 1ME A.7R.1 cells. E. OL0825 demonstrate increased fibronectin, collagen I, vimentin and decreased E-cadherin protein expression in comparison to BNL 1ME A.7R.1 cells. Relative densitometry is indicated below the bands. Results presented as mean ± SEM; P<0.05; P<0.01; N = 3.
Figure 4.
HGF and c-Met overexpression in HCC CTCs.
Analysis of gene expression of HGF and c-Met was performed by qPCR. c-Met protein expression analysis was performed by Western blotting. Analysis of HGF protein expression and secretion were performed by Western blotting and ELISA respectively. Gene expression of HGF (A) and c-Met (C) is increased in OL0825 in comparison to BNL 1ME A.7R.1. B. HGF protein secretion is significantly higher in OL0825 cells than in BNL 1ME A.7R.1 cells. D. OL0825 has increased protein expression of HGF, c-Met and phosphorylated c-Met (p-c-Met) in comparison to BNL 1ME A.7R.1. Relative densitometry is indicated below the bands. Results presented as mean ± SEM; P<0.05; P<0.01; N = 3 (for qPCR); N = 6 (for HGF ELISA).
Figure 5.
HGF induces EMT in BNL 1ME A.7R.1 cells.
BNL 1ME A.7R.1 cells were cultured in 6-well plates and treated with 25 ng/ml mouse HGF (R & D Systems) for 5 days. Control cells were untreated. RNA and protein extraction were performed and qPCR and Western blotting were performed as described in “Experimental procedures” section. HGF-treated BNL 1ME A.7R.1 cells demonstrated increased fibronectin gene and protein expression (A, D), increased collagen I gene and protein expression (B, D) and loss of E-cadherin gene and protein expression (C, D). Relative densitometry is indicated below the bands (D). Results presented as mean ± SEM; P<0.01; N = 3.
Figure 6.
DNA methylation analysis of HGF and c-Met promoters.
A. Analysis of bisulfite DNA from BNL 1ME A.7R.1 cells indicates that HGF has 35% of CpGs methylated at two sites. B. Analysis of bisulfite DNA from OL0825 CTC line shows that the HGF has methylation percentages of 35% and 34% detected at the first and second sites respectively. C. Statistical analysis of the percentage of CpGs methylated in the HGF amplicon (including data from a third CpG site analyzed in Figure S6) shows that there is a small difference between the methylation of BNL 1ME A.7R.1 and OL0825 cells. D. Methylation analysis of c-Met from BNL 1ME A.7R.1 cells indicates the presence of methylation in 6 CpGs including the first 2 CpGs (which are close to 2 putative transcriptional start sites). The percentages of methylation at these first 2 sites are 12% and 13% respectively. E. Methylation analysis of c-Met in OL0825 cells indicates that c-Met has lower methylation than that of BNL 1ME A.7R.1 cells, with the first 2 CpGs being methylated at only 7% and 5% respectively. F. Statistical analysis of the differences between methylation levels in BNL 1ME A.7R.1 and OL0825 cells shows that the data are highly significant. Results presented as mean ± SEM; N = 3 for HGF and N = 6 for c-Met.
Figure 7.
Summary of potential role of HGF/c-Met in hematogenous dissemination of HCC.
Schematic diagram illustrating the role of HGF/c-Met in hematogenous dissemination of HCC cells. Primary tumor cells express HGF and c-Met with partial promoter demethylation. This is associated with good expression of E-cadherin and limited expression of fibronectin, collagen I and vimentin. However, when HCC cells escape from the liver and start circulating in the blood-stream, increased expression of HGF and c-Met associated with increased promoter demethylation is observed. Circulating HCC cells also display evidence of EMT: loss of E-cadherin, increased fibronectin, increased collagen I and increased vimentin expression. We conclude that increased HGF and c-Met may induce EMT in CTCs to sustain hematogenous dissemination.