Limitations of the dorsal skinfold window chamber model in evaluating anti-angiogenic therapy during early phase of angiogenesis

Abstract

Background: Angiogenesis is an essential process during tumor development and growth. The murine dorsal skinfold window chamber model has been used for the study of both tumor microvasculature and other vascular diseases, including the study of anti-angiogenic agents in cancer therapy. Hyperspectral imaging of oxygen status of the microvasculature has not been widely used to evaluate response to inhibition of angiogenesis in early tumor cell induced vascular development. This study demonstrates the use of two different classes of anti-angiogenic agents, one targeting the Vascular Endothelial Growth Factor (VEGF) pathway involved with vessel sprouting and the other targeting the Angiopoietin/Tie2 pathway involved in vascular destabilization. Studies evaluated the tumor microvascular response to anti-angiogenic inhibitors in the highly angiogenic renal cell carcinoma induced angiogenesis model.

Methods: Human renal cell carcinoma, Caki-2 cells, were implanted in the murine skinfold window chamber. Mice were treated with either VEGF pathway targeted small molecule inhibitor Sunitinib (100 mg/kg) or with an anti-Ang-2 monoclonal antibody (10 mg/kg) beginning the day of window chamber surgery and tumor cell implantation. Hyperspectral imaging of hemoglobin saturation was used to evaluate both the development and oxygenation of the tumor microvasculature. Tumor volume over time was also assessed over an 11-day period post surgery.

Results: The window chamber model was useful to demonstrate the inhibition of angiogenesis using the VEGF pathway targeted agent Sunitinib. Results show impairment of tumor microvascular development, reduced oxygenation of tumor-associated vasculature and impairment of tumor volume growth compared to control. On the other hand, this model failed to demonstrate the anti-angiogenic effect of the Ang-2 targeted agent. Follow up experiments suggest that the initial surgery of the window chamber model may interfere with such an agent thus skewing the actual effects on angiogenesis.

Conclusions: Results show that this model has great potential to evaluate anti-VEGF, or comparable, targeted agents; however the mere protocol of the window chamber model interferes with proper evaluation of Ang-2 targeted agents. The limitations of this in vivo model in evaluating the response of tumor vasculature to anti-angiogenic agents are discussed.

Keywords: Angiogenesis; Angiopoietin-2; Anti-angiogenic therapy; Dorsal skinfold window chamber model; Vascular endothelial growth factor.

Figure 1

Human renal cell carcinoma, Caki-2, tumor growth in the window chamber. (A) Titanium chambers are surgically implanted in nude mice and tumor cells injected (2 × 10⁴) subcutaneously into the window. (B) Tumor cell induced microvascular development in the window chamber over time. Hyperspectral imaging of hemoglobin saturation provide oxygenation status of tumor microvasculature.

Figure 2

VEGF inhibition in Caki-2 cell induced angiogenesis. Mice bearing window chambers with Caki-2 tumors were treated with VEGF inhibitor (Sunitinib). (A) Tumor volume and (B) tumor microvascular response to Sunitinib was evaluated compared to control. Median + 90/10 percentile; control (n = 8), Sunitinib (n = 7) (combination of two independent experiments). **, p < 0.01, Mann–Whitney U-Test.

Figure 3

Tumor vasculature after VEGF inhibition. Immunohistochemical analysis of tumors at study endpoint. Line, median; *, p < 0.05; Mann–Whitney U-Test. Representative images of the median of each group. Red, MECA. Images taken with Zeiss Axioplan Imaging2 microscope with 20× objective; scale bar = 140 μm.

Figure 4

Ang-2 inhibition in Caki-2 cell induced angiogenesis. Mice bearing window chambers with Caki-2 tumors were treated with Ang-2 inhibitor (monoclonal antibody). (A) Tumor volume and (B) tumor microvascular response to the Ang-2 inhibitor was evaluated compared to control. Median + 90/10 percentile; control (n = 8), anti-Ang-2 (n = 7) (combination of two independent experiments).

Figure 5

Tumor vasculature after Ang-2 inhibition. Immunohistochemical analysis of tumors at study endpoint. Line, median; *, p < 0.05; Mann–Whitney U-Test. Representative images of the median of each group. Red, MECA. Images taken with Zeiss Axioplan Imaging2 microscope with 20× objective; scale bar = 140 μm.

Figure 6

Vascular structure after Ang-2 inhibition. Immunohistochemical analysis of vascular structure at study endpoint. (A) Normal vasculature with pericyte coverage. (B) Tumor vasculature without pericyte coverage. Scale bar, 46 μm. (C) Ang-2 inhibition led to increased number of vessels that maintained pericyte coverage. Line, median; *, p < 0.05; Mann- Whitney U-Test. Representative images of each group. White arrows show pericyte covered vessels; yellow arrow shows vessels without pericyte coverage. Red, MECA (endothelium); green, NG2 (pericyte); blue, DAPI. Images taken with Zeiss Axioplan Imaging2 microscope with 20× objective; scale bar = 140 μm.

Figure 7

Ang-2 and VEGF inhibition in the intradermal assay. Mice were injected intradermally with Caki-2 renal cell carcinoma cells and treated with either the Ang-2 inhibitor (10 mg/kg) or Sunitinib (100 mg/kg) beginning the day prior to tumor cell inoculation. Tumor volume (A, C) and the number of tumor cell induced blood vessels (B, D) were determined at the end of a 7 day period. Bar, mean with SEM (n = 12); line, median (n = 12); **, p < 0.01; ***, p < 0.0001; Mann–Whitney U-Test.

Figure 8

Ang-2 serum levels in mice. Levels of Ang-2 in the circulation were determined for mice that underwent window chamber surgery -/+tumor cell injection and compared to basal levels (no surgery).