Oligosaccharide Expression and Modification Defines Novel Targets within the Vascular Tree: Selective Chondroitin Sulfate C Expression in the Tumor Microenvironment Generates a Novel Target of Otherwise Constitutive Receptors.

The expression of certain proteoglycans or modification of proteins within the vascular tree, dependent on the microenvironment, is critical for elucidating the biology of endothelium specificity and development. It also facilitates the targeting of physiologic and therapeutic agents to different addresses within the vascular map. Using in vivo panning, we have identified a truncated heparin-binding domain (HBDt) that recognizes its target selectively in tumor vasculature. Here we show that it localizes selectively to the endothelial cells of intra-tumoral blood vessels of various murine tumor models, such as CT26, LLC, N202, and Tramp-L1. The HBDt, as a part of the VEGF heparin-binding domain, is conserved throughout evolution and is known to bind the VEGFR-2/Npn-1 complex. Although the VEGFR-2/Npn-1 complex is expressed elsewhere in the vascular tree, this domain only localizes to a target in tumor vasculature. We have analyzed the basis of this selectivity in vitro and in vivo. In vitro analysis has shown that chondroitin sulfates are the most potent inhibitors of HBDt binding to heparin. We also show, using Western blot and confocal microscopy analyses, that VEGFR-2 and Npn-1, although expressed in different organs, are only recognized by HBDt when coexpressed with chondroitin sulfate C (C6S) in the tumor vasculature. The HBDt colocalized with VEGFR-2, Npn-1, and C6S but with not bFGFR or heparan sulfates in the intravasculature of different tumor models. Furthermore, the selective expression of C6S oligosaccaharide, in conjunction with VEGFR-2 and Npn-1, during the angiogenesis of tumor endothelium defines the target for the HBDt but not during aortic angiogensis. Therefore, our data demonstrate that the expression of C6S, as part of the HBDt receptor, is an example of the tumor microenvironment conditioning, which imparts association of a novel target on endothelium surfaces of tumors.