Abstract
BackgroundTargeting tumor vasculature is a strategy with great promise in the treatment of many cancers. However, anti-angiogenic reagents that target VEGF/VEGFR2 signaling have met with variable results clinically. Among the possible reasons for this may be heterogeneous expression of the target protein.MethodsDouble immunofluorescent staining was performed on formalin-fixed paraffin embedded sections of treated and control SW480 (colorectal) and WM239 (melanoma) xenografts, and tissue microarrays of human colorectal carcinoma and melanoma. Xenografts were developed using RAG1-/- mice by injection with WM239 or SW480 cells and mice were treated with 20 mg/kg/day of cyclophosphamide in their drinking water for up to 18 days. Treated and control tissues were characterized by double immunofluorescence using the mural cell marker α-SMA and CD31, while the ratio of desmin/CD31 was also determined by western blot. Hypoxia in treated and control tissues were quantified using both western blotting for HIF-1α and immunohistochemistry of CA-IX.ResultsVEGFR2 is heterogeneously expressed in tumor vasculature in both malignant melanoma and colorectal carcinoma. We observed a significant decrease in microvascular density (MVD) in response to low dose metronomic cyclophosphamide chemotherapy in both malignant melanoma (with higher proportion VEGFR2 positive blood vessels; 93%) and colorectal carcinoma (with lower proportion VEGFR2 positive blood vessels; 60%) xenografts. This reduction in MVD occurred in the absence of a significant anti-tumor effect. We also observed less hypoxia in treated melanoma xenografts, despite successful anti-angiogenic blockade, but no change in hypoxia of colorectal xenografts, suggesting that decreases in tumor hypoxia reflect a complex relationship with vascular density. Based on α-SMA staining and the ratio of desmin to CD31 expression as markers of tumor blood vessel functionality, we found evidence for increased stabilization of colorectal microvessels, but no such change in melanoma vessels.ConclusionsOverall, our study suggests that while heterogeneous expression of VEGFR2 is a feature of human tumors, it may not affect response to low dose metronomic cyclophosphamide treatment and possibly other anti-angiogenic approaches. It remains to be seen whether this heterogeneity is partly responsible for the variable clinical success seen to date with targeted anti-VEGFR2 therapy.
Highlights
Targeting tumor vasculature is a strategy with great promise in the treatment of many cancers
Conserved VEGFR2 Expression Patterns between Xenograft and Human Clinical Tumors The VEGFR2 status of vessels in colorectal carcinoma and melanoma was compared between human clinical samples and mice xenografted with human cancer cells of the same type, and there were no significant differences in vascular phenotype between xenografts and clinical cases within a cancer type (Table 1) (p < 0.05)
We consistently found that colorectal carcinoma xenograft tumors had significantly fewer VEGFR2 positive blood vessels than malignant melanoma xenografts
Summary
Targeting tumor vasculature is a strategy with great promise in the treatment of many cancers. Anti-angiogenic reagents that target VEGF/VEGFR2 signaling have met with variable results clinically. Solid tumors rely on a robust vascular supply for growth and spread to distant sites, blocking tumor growth by disrupting angiogenesis is a rational anti-cancer strategy and several targeted therapies are currently approved or are in clinical trial [1]. Administration of anti-angiogenic drugs should induce a reduction in tumor vessel density, due to destruction of unstable vessels and/or prevention of new sprouting. While LDM cyclophosphamide did not induce significant blood vessel normalization in the RIP1-Tag pancreatic insulinoma model [14], it is not known whether vessels recruited to xenografted human cancer will be normalized by this therapeutic approach
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