PURPOSE: The proliferation and subsequent metastasis of tumorigenic melanocytes is highly dependent on the proximity of supplying blood vessels. Recently, the mitogenic characteristics of angiotensin II type 2 (AT2) receptors have been suggested to inhibit angiogensis and tumor cell growth. The purpose of this study is to analyze tumor progression of B16-F1 mouse melanoma xenografts implanted into the right dorsal flank of C57BL/6 mice over a two-week time period in presence or absence of selective AT2 receptor activation regarding tumorigenic potential, e.g. tumor extension size, capillary density and immunogenic responses.
METHODS: The study included a total of twenty (n=20) C57BL/6J mice. A control group of ten (n=10) animals that received physiological saline and an experimental group of ten (n=10) animals that received 100 ng/kg/min CGP-42112A (CGP; Sigma-Aldrich, St.Louis MO) subcutaneously using ALZET 1002 osmotic mini pumps (Durect Corporation,Cupertino, CA) for fifteen days following B16-F1 melanoma xenograft implantation into the right dorsal flank. The animals were kept in a controlled environment and given food and water ad lib. Daily animal weights were recorded. Tumor growth was analyzed weekly by digital photography in combination with tumor area extension measurement using NIH ImageJ. After 15 days animals were sacrificed and tumors were excised and weighed. Cryostatic sections of the tumors were examined for the expression of AT1A and AT2 receptors, endothelial cell marker PECAM-1 (CD31) to examine capillary density, and macrophage infiltration as a sign of immune response
RESULTS: Average tumor area extension demonstrated no significant differences between saline (control) and CGP treatment during the first twelve days 68.62±14.58mm2; control and 79.24±15.6mm2; CGP. In contrast, average tumor area extension following 15 days CGP treatment demonstrated a significant proportional 2-fold increase (p<0.05) compared to saline treatment with 119.6±21.97mm2; control and 234.3±50.67mm2; CGP. Excised tumors following 15days CGP treatment demonstrated a proportional 1.6 fold increase in average tumor weight (p=0.26) with 1.02±0.27g compared to saline treated tumors (0.62±0.24g). Immunoblotting using a PECAM-1 (CD31) specific antibody that specifically recognizes endothelial cells as well as some monocytes, neutrophiles, platelets and T-Lymphocytes demonstrated AT2 receptor dependent inhibition of vessel growth. A proportional 5.0 percent down regulation of PECAM-1 expression (p=0.14) was observed following CGP treatment with 94.41±2.86 percent expression compared to 100±2.09 percent expression in the control group. We also observed a 15 percent reduction (p=0.15) in macrophage marker expression following CGP treatment with 85.12±3.73 percent expression compared to the saline control (100±9.63).
CONCLUSION: The presence of AT1 and AT2 receptors in tissue and blood vessels of B16-F1 melanoma xenografts suggests that angiotensinergic drugs can effectively target tumors. Against our expectation, the selective AT2 agonist CGP yielded proportional 1.5 to 2.0 fold increase in tumor weight and tumor area extension, respectively. Immunoblotting indicated a proportional 5% reduction in PECAM-1 expression representative of vascular growth in correlation with a proportional 15% reduction in macrophage marker detection representative of macrophage infiltration. Although the exact mechanism is not fully understood, we suggest that the observed CGP-dependent increase in tumor growth is due to reduced macrophage infiltration and, accordingly, reduced activation of immunologic responses in melanoma tissue. The reduced macrophage infiltration may be caused by CGP binding to vascular AT2 receptors and subsequent inhibition of angiogenesis indirectly reducing immune cell infiltration. Alternatively, direct binding of CGP to macrophage surface AT2 receptors may cause a direct down regulation of macrophages.