Pazopanib Inhibits Tumor Growth, Lymph-node Metastasis and Lymphangiogenesis of an Orthotopic Mouse of Colorectal Cancer

Abstract

Background/aim: Pazopanib (PAZ) can inhibit tumor progression, but whether PAZ inhibits lymph node metastasis and lymphangiogenesis in colorectal cancer is still unknown. The aim of the present study was to determine the efficacy of PAZ on tumor growth, lymph node metastasis and lymphangiogenesis in an orthotopic nude mouse model in colorectal cancer.

Materials and methods: CT-26-green fluorescence protein (GFP)-expressing mouse colon cancer cells were injected into nude mice to establish a subcutaneous colorectal cancer model and were treated with saline and PAZ. Additionals subcutaneous tumors were harvested and cut into 5 mm³ fragments, then tumor fragments were implanted orthotopically in the cecum to establish an orthotopic colorectal-cancer nude mouse model. Orthotopic mice were randomized into two groups for the treatment with saline and PAZ, respectively. Tumor width, length and mouse body weight was measured twice a week. The Fluor Vivo imaging system was used to image the GFP. Hematoxylin & eosin staining and immunohistochemical staining was used for histological analysis.

Results: PAZ inhibited the growth of subcutaneous colorectal cancer, as wells as orthotopic transplanted colorectal cancer tumors. PAZ suppressed lymph node metastasis and lymphangiogenesis in the orthotopic colon cancer model. No significant changes were observed in the body weight between the control and the mice treated with PAZ.

Conclusion: PAZ can inhibit the growth of colorectal cancer and inhibit lymph node metastasis and lymphangiogenesis in orthotopic colon cancer nude mouse models.

Keywords: Pazopanib; colorectal cancer; lymph node metastasis; lymphangiogenesis; nude mice; orthotopic tumor.

Figure 1. PAZ inhibited the growth of subcutaneously implanted CT26-GFP in nude mice in a model of colorectal cancer. (A) photographs of representative CT26 subcutaneous tumors of colorectal cancer in the PAZ and control groups at treatment day 14. Arrows indicate the margins of the tumors. (B) Photographs (upper panel) and fluorescence images (black and white; lower panel) of subcutaneous tumors from the control and the PAZ groups. (C) Mouse body weight. Bar graphs show mouse body weight in the PAZ and in the control groups at pre- and post-treatment. (D) Line graphs indicate the relative subcutaneous tumor volume (ratio of tumor volume at each time point to volume at the start of the treatment) for the PAZ and the control group. (N=5, *p<0.05; ***p<0.001).

Figure 2. Longitudinal non-invasive in vivo fluorescence imaging of tumor progression over time in nude mice treated with PAZ in the CT26-GFP orthotopic colorectal cancer model. Fluorescence imaging was performed on days: i) 1, ii) 8, and iii) 14 after the initiation of the treatment. (A) Control group (N=5) (B) PAZ group (N=5).

Figure 3. Effect of PAZ on the body weight of CT-26-GFP orthotopic colorectal cancer mouse models. Bar graphs show body weight in the control and the PAZ groups at pre-treatment (A) and 2 weeks post initiation of treatment (B). There were no significant differences between the two groups (N=5 in each group, p>0.05).

Figure 4. Photographs and fluorescence images of orthotopic CT-26-GFP colorectal cancer nude-mouse model at autopsy in the control and PAZ groups. (A-C) Representative photographs of the control group (upper panels) and corresponding fluorescence images (lower panels). (A) Nude mice have bloody ascites, and tumors show GFP fluorescence. (B) After removal of the transplanted tumor, many metastatic lymph nodes can be seen, particularly in the mesentery (C). (D-F) Representative photographs of the PAZ group (upper panels) and corresponding GFP images (lower panels). (D) Nude mice do not show bloody ascites in the PAZ group. (E) Following removal of the transplanted tumor, images show metastatic lymph nodes expressing GFP, with very few metastatic lymph nodes in the isolated mesentery (arrows in F).

Figure 5. Effect of PAZ on relative tumor volume and tumor weight in the orthotopic CT-26-GFP colorectal cancer nude mouse model at autopsy. (A) Bar graph shows relative tumor volume (tumor volume/100) in the control and PAZ group. (B) Bar graph shows tumor weight in the control and PAZ group. (n=5 in each group, *p<0.05, **p<0.01).

Figure 6. Histology of the CT26-GFP orthotopic colorectal-cancer mouse model. (A) H&E stained section of the control group. (B) H&E stained sections of the PAZ group. (C-E) H&E stained sections of the control tumor invading the intestine. (F-H) H&E stained sections of metastatic lymph nodes in the control mice. A, B, E, H: Magnification 200×; C, F: magnification, 40×; D, G: magnification, 100×.

Figure 7. PAZ inhibits lymphangiogenesis of the CT26-GFP orthotopic-colorectal cancer mouse model. (A) Representative lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1) stained in the control tumor (red arrows) and the PAZ group (B; red arrows). (C) The graph shows the corresponding lymphatic vessel density in the control and PAZ group. Magnification 200×; **p<0.01.