Rapid Access of Antibodies to A5B1 Integrin Overexpressed on the Luminal Surface of Tumor Blood Vessels

Integrin A5B1 is overexpressed on endothelial cells of tumor vessels and is uniformly and rapidly accessible to antibodies in the bloodstream. Here, we determined whether antibodies rapidly gain access to integrin overexpressed on the abluminal (basolateral) surface of endothelial cells through vascular leakiness or whether the rapid accessibility results instead because the integrin is overexpressed on the luminal (apical) surface of endothelial cells due to loss of cell polarity. Using tumors in RIP-Tag2 transgenic mice as a model, we first compared the binding pattern of intravascular anti-A5B1 integrin antibody with the leakage pattern of nonspecific IgG. The distributions did not match: anti-A5B1 integrin antibody uniformly labeled the tumor vasculature, but IgG was located in patchy sites of leakage. We next injected an antibody to fibrinogen/fibrin, which resulted in patchy labeling of tumors that matched the leakage of IgG and the overall distribution of fibrin in tumors. Similarly, injected antibodies to the basement membrane protein fibronectin, a ligand of A5B1 integrin, or type IV collagen produced patchy sites of leakage instead of uniform labeling of vascular basement membrane. Differences in the kinetics of labeling, which forA5B1 integrin antibody was near maximal by 10 minutes but for the other antibodies gradually increased over 6 hours, indicated differences in accessibility of their respective targets. Isosurface rendering of confocal microscopic images was consistent with antibody binding to A5B1 integrin on the luminal surface of endothelial cells. Together, these findings indicate that the rapid accessibility of A5B1 integrin in RIP-Tag2 tumors results from overexpression of the integrin on the luminal surface of tumor vessels. (Cancer Res 2005; 65(7): 2712-21)