Functional in vivo optical imaging of tumor angiogenesis, growth, and metastasis prevented by administration of anti-human VEGF antibody in xenograft model of human fibrosarcoma HT1080 cells

Abstract

Angiogenesis plays a crucial role in cancer progression and metastasis. Thus, blocking tumor angiogenesis is potentially a universal approach to prevent tumor establishment and metastasis. In this study, we used in vivo and ex vivo fluorescence imaging to show that an antihuman vascular endothelial growth factor (VEGF) antibody represses angiogenesis and the growth of primary tumors of human fibrosarcoma HT1080 cells in implanted nude mice. Interestingly, administering the antihuman VEGF antibody reduced the development of new blood vessels and normalized pre-existing tumor vasculature in HT1080 cell tumors. In addition, antihuman VEGF antibody treatment decreased lung metastasis from the primary tumor, whereas it failed to block lung metastasis in a lung colonization experiment in which tumor cells were injected into the tail vein. These results suggest that VEGF produced by primary HT1080 cell tumors has a crucial effect on lung metastasis. The present study indicates that the in vivo fluorescent microscopy system will be useful to investigate the biology of angiogenesis and test the effectiveness of angiogenesis inhibitors.

Figure 1

Comparison of the angiogenic activity of distinct cancer cell lines by in vivo fluorescent imaging. (A) Quantitative real‐time RT‐PCR analysis of vascular endothelial growth factor (VEGF) mRNA levels in HT1080 cells and DU4475 cells. Each value is normalized to GAPDH expression. The increased VEGF mRNA levels in HT1080 cells compared to DU4475 cells are evident. Error bars represent SD. (B) In vivo imaging of tumor vasculature. Tumor vasculature was imaged 15 days after inoculation of DU4475 cells (left panels) or HT1080 cells (right panels) using AS‐IVM 680. Top panels show vasculature around the tumor imaged by the OV100 (arrowheads). Asterisks indicate AS‐IVM 680 diffusion in the tumor. Scale bar, 5 mm. Bottom panels show vasculature in the tumor imaged by the IV100. Scale bar, 500 μm. (C) The same tumors as in (B) after immunohistochemical staining for CD31. Left and right panels show DU4475 and HT1080 cell tumors, respectively. Scale bar, 30 μm.

Figure 2

Serial in vivo imaging and quantification of tumor vasculature in HT1080 cell tumors. (A) Serial images during the progression of angiogenesis. In vivo imaging of tumor vasculature was repeatedly performed at 1 day (left panels), 8 days (middle panels), and 14 days (right panels) post inoculation using AS‐IVM 680. Top panels show vasculature around the tumor imaged by the OV100 (arrowheads). Asterisks indicate AS‐IVM 680 diffusion in the tumor. Scale bar, 5 mm. Bottom panels show vasculature in the tumor imaged by the IV100. Scale bar, 500 μm. (B) Complementary in vivo imaging and immunohistochemical analysis at different time points. The same tumors were examined by the IV100 (top panels), and subsequently, immunostained for CD31 (bottom panels) at 10 days (left panels), 12 days (middle panels), and 15 days (right panels) after inoculation. White bar, 500 μm. Black bar, 30 μm. (C) Validation. The graph shows the correlation between the IV100 measurements of tumor vasculature and microvessel density (MVD) measurements (n = 9 different tumors). Blue, yellow, and red points respectively indicate measurements at 10 days, 12 days, and 15 days after inoculation. The Pearson’s correlation coefficient was 0.867 (P = 0.0021). Quantification of vasculature area and MVD was performed as described in the ‘Materials and Methods’.

Figure 3

Effect of antihuman vascular endothelial growth factor (VEGF) antibody treatment on tumor angiogenesis and growth. (A) Serial images of tumor vasculature during treatment of antihuman VEGF antibody. Tumor vasculature of control mice (left panels) or antibody‐treated mice (right panels) was imaged using AS‐IVM 680. These images were acquired 3 days post inoculation, that is before therapy initiation, (top panels) and 10 days post inoculation, that is after 1 week of therapy, (bottom panels). Vasculature around the tumor was imaged by the OV100 (arrowheads). Asterisks indicate AS‐IVM 680 diffusion in the tumor. Scale bar, 5 mm. Vasculature in the tumor was imaged by the IV100. Scale bar, 500 μm. (B) Quantification of tumor angiogenesis. Values show the tumor vasculature area at 10 days post inoculation minus that at 3 days post inoculation. There was a statistically significant decrease in tumor angiogenesis in mice treated with antihuman VEGF antibody compared to control mice (P = 0.000196). The boxplots show the smallest observation, lower quartile, median (the bar in the box), upper quartile, and largest observation. (C) Serial images of the same vasculatures at the different time points during treatment of the antibody. Vasculature of untreated tumor (left panels) or tumor (middle panels) and ear (right panel) of treated mice was imaged by the IV100 using AS‐IVM 680. These images were acquired at 3 days post inoculation, that is before therapy initiation, (top panels) and 11–12 days post inoculation, that is after 1 week of therapy, (bottom panels). The boxed area indicates the same vasculatures at the different time points. Scale bar, 500 μm. (D) Mean tumor growth curves for antihuman VEGF antibody‐treated mice and control mice. Systemic treatment with antihuman VEGF antibody significantly inhibited tumor growth compared to untreated mice (P < 0.0001). (E) The effects of antihuman VEGF antibody on the cell growth of HT1080 cells. Cultured HT1080 cells were incubated with (+) or without (−) antihuman VEGF antibody for 72 h, and cell numbers were counted. Error bars represent SD.

Figure 4

Effect of antihuman vascular endothelial growth factor (VEGF) antibody treatment on spontaneous lung metastasis. (A) Complementary ex vivo imaging and immunohistochemical analysis. Imaging and analysis were performed 24 days after inoculation of GFP‐labeled HT1080 cells. Left panel shows fluorescent tumor micrometastases detected by the OV100. Asterisk indicates tissue autofluorescence. Scale bar, 1 mm. Right panel shows H&E staining of the boxed area in the left panel with arrows indicating metastatic foci. Scale bar, 60 μm. (B) Ex vivo images of fluorescent lung micrometastases in control mice (left panel) or antihuman VEGF antibody‐treated mice (right panel). Ex vivo imaging was performed 24 days after inoculation (i.e. after 3 weeks of treatment) by the OV100. Arrowheads indicate metastatic foci. Asterisks indicate tissue autofluorescence. Scale bar, 1 mm. (C) Quantification of spontaneous lung micrometastasis. The graph shows the spontaneous lung micrometastatic efficacy in antibody‐treated mice. The boxplots show the smallest observation, lower quartile, median (the bar in the box), upper quartile, and largest observation.

Figure 5

Effect of antihuman vascular endothelial growth factor (VEGF) antibody treatment on experimental lung metastasis. (A) Ex vivo images of fluorescent lung metastases of control mice (left panel) or antihuman VEGF antibody‐treated mice (right panel). Ex vivo imaging was performed 13 days after inoculation of GFP‐labeled HT1080 cells (i.e. after 2 week of treatment) by the OV100. Scale bar, 1 mm. (B) Quantification of experimental lung metastasis. The plots show the percentage of GFP‐positive foci within an area in control mice and antihuman VEGF antibody‐treated mice. Quantification of GFP‐positive foci within an occupied area was performed. The boxplots show the smallest observation, lower quartile, median (the bar in the box), upper quartile, and largest observation.

Figure 6

Correlation between tumor angiogenesis, tumor growth and spontaneous lung metastasis. (A) The correlation between the IV100 measurements of tumor vasculature area and tumor volume (P < 0.00001). (B) The correlation between the IV100 measurements of tumor vasculature area and the number of spontaneous lung metastasis (P = 0.00198). (C) The correlation between the tumor volume and the number of spontaneous lung metastasis (P < 0.0001).