title: VEGFR2 heterogeneity and response to anti-angiogenic low dose metronomic cyclophosphamide treatment.
abstract: BACKGROUND
  Targeting 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.
  
  
  METHODS
  Double 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.
  
  
  RESULTS
  VEGFR2 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.
  
  
  CONCLUSIONS
  Overall, 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.
authors.affiliation: Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON Canada N1G 2W1.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Patten
authors.forename: Una
authors.initials: U
authors.lastname: Adamcic
authors.forename: Kristen
authors.initials: K
authors.lastname: Lacombe
authors.forename: Kanwal
authors.initials: K
authors.lastname: Minhas
authors.forename: Karolina
authors.initials: K
authors.lastname: Skowronski
authors.forename: Brenda L
authors.initials: BL
authors.lastname: Coomber
mesh_terms: D000199:Actins / Q000378:metabolism; D020533:Angiogenesis Inhibitors / Q000008:administration & dosage*; D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000378:metabolism; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000098:blood supply / Q000188:drug therapy* / Q000378:metabolism; D003520:Cyclophosphamide / Q000008:administration & dosage*; D003893:Desmin / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D008545:Melanoma / Q000098:blood supply / Q000188:drug therapy* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D055806:Microvessels / Q000187:drug effects* / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000517:prevention & control*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D013997:Time Factors; D046888:Tissue Array Analysis; D047368:Tumor Burden; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D000199:Actins; D020533:Angiogenesis Inhibitors; D000951:Antigens, Neoplasm; D003893:Desmin; C497442:HIF1A protein, human; D018398:Homeodomain Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C064658:RAG-1 protein; D003520:Cyclophosphamide; D040301:Vascular Endothelial Growth Factor Receptor-2; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases