VEGFR2 heterogeneity and response to anti-angiogenic low dose metronomic cyclophosphamide treatment

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.

Angiogenesis in multiple sclerosis and experimental autoimmune encephalomyelitis

“…mitoxantrone, cyclophosphamide), used to revert the aggressive course of MS, also exert an anti-angiogenic activity [216,217]. A chemotherapeutic agent, cladribine, effective but unsafe in MS, decreases the level of angiogenic factors [218].…”

Endothelial Cell Heterogeneity

“…These properties differ between different tumor types, between blood vessels of a single tumor, and between different stages of tumor progression (reviewed in Aird 2009). For example, the percentage of tumor endothelial cells expressing Tie-2 and vascular growth factor receptor (VEGFR)-2 varies in different human tumors and mouse xenografts (Fathers et al 2005;Patten et al 2010). Although most genes that are expressed in tumor endothelial cells are also up-regulated in physiological angiogenic processes, there are important exceptions (Seaman et al 2007).…”

The impact of tumor receptor heterogeneity on the response to anti-angiogenic cancer treatment

“…(C) The angiogenic ratio (the ratio of proangiogenic complexes to anti-angiogenic complexes) is predicted to oscillate throughout the course of treatment. [28][29][30][31] We set the density of each receptor by sampling within two orders of magnitude of the baseline value. This variability in the receptor number influences the dynamics of free VEGF, free TSP1, and the angiogenic ratio, as shown in Fig.…”

“…29,30 Thus, understanding the effects of tumor receptor variability is clinically relevant. [28][29][30][31] Our model predicts that the metric of angiogenic receptor expression can serve as a predictive tissue biomarker to distinguish the patients in which the combination anti-angiogenic therapy will elicit a strong therapeutic response. Overall, we establish a new computational framework to predict the effects of anti-angiogenic therapies and understand clinical observations.…”

“…[24][25][26][27] Additionally, the VEGFR2 heterogeneity was shown to affect the response to an anti-angiogenic cyclophosphamide treatment in an in vitro experimental setting. 28 The patient-to-patient VEGFR1 and neuropilin variability has been associated with the response to bevacizumab treatment as intra-tumoral biomarkers. 29,30 Thus, understanding the effects of tumor receptor variability is clinically relevant.…”