Verotoxin sensitivity of ECV304 cells in vitro and in vivo in a xenograft tumour model: VT1 as a tumour neovascular marker.

Abstract

Binding of verotoxin 1 (VT1) via its receptor (globotriaosylceramide/Gb3) to both endothelial and tumour cell subsets suggests that VT1 may have both antineoplastic and antiangiogenic potential. We investigated this potential using the ECV304 cell line, which, although identified as a bladder carcinoma cell line, displays some endothelial characteristics, including tubule formation (differentiation) following appropriate stimulation. Differentiated ECV304 cells retained Gb3 expression/VT1 sensitivity. VT1 internalization and retrograde transport through the Golgi to the ER was observed. ECV304 xenografts in immunocompromised mice were invasive but surprisingly poorly vascularized. Intratumoural VT1 injection significantly reduced ECV304 xenograft growth and enhanced mouse survival. Gb3 expression was decreased in residual tumour, likely due to cell cycle arrest. Untreated ECV304 xenograft sections bound VT1 throughout the tumour. Anti-von Willebrand factor (vWF) antibody staining for neovasculature showed only morphologically atypical, indistinct structures, unlabelled by VT1. Human bladder carcinoma samples were, in contrast, highly vascular and blood vessels were 100% co-labelled by anti-vWF antibody and VT1, with no extravascular staining. These results suggest that ECV304 xenografts are not characteristic of bladder carcinoma in terms of Gb3 expression, and that VT1 staining may provide a new reliable index of tumour neovasculature. We conclude that ECV304 cells are not an appropriate in vivo model of either tumour angiogenesis or bladder carcinoma. These studies, nevertheless, further demonstrate the in vivo antineoplastic and antiangiogenic potential of VT1, and show that Gb3 is expressed in cells undergoing in vitro 'vascular' differentiation, and in the neovasculature of human bladder carcinomas.