Microbubbles coated with biotinylated rabbit anti-mouse vascular endothelial growth factor receptor 2 (VEGFR-2) monoclonal antibody

Angiogenesis, the development of new vasculature from pre-existing blood vessels (for details see Carmeliet and Jain (1)), is essential for the development, maintenance, progression, and metastasis of neoplastic tumors (2). Therefore, angiogenesis is considered to be the hallmark of cancerous tumors, and early detection of this process can facilitate the initiation of treatment and management of the disease (3). Although there are several known pro-angiogenic biomarkers, among these only vascular endothelial growth factor receptor 2 (VEGFR-2) (4), αβ integrin (5), and endoglin (6) are well characterized and are overexpressed in many cancerous tumors, such as those of the breast, ovaries, and the pancreas (3). The VEGFR-2 mediates its effects through a family of receptor tyrosine kinases that promote the mitogenesis, survival, differentiation, migration, and vascular permeability of endothelial cells (7). The αβ integrins are heterodimeric cell adhesion molecules that can bind several different endogenous ligands such as fibronectin, von Willebrand factor, fibrinogen, etc., and assist with the survival and migration of cancerous cells, which increases the invasive potential of these cells, and promotes angiogenesis in tumors (8). Endoglin (CD105) is a co-receptor of the transforming growth factor beta (TGF-β) and co-modulates the different activities, including angiogenesis, of the activated TGF-β receptor (9). Little information is available regarding the expression of the different angiogenic markers during the progression of a tumor from a small size to a larger size. Noninvasive visualization of angiogenic markers that are overexpressed during initial stages of the neoplasia can facilitate early detection and treatment of the disease (3). For this, Deshpande et al. developed a series of microbubble (MB; perfluorocarbon gas enclosed within spherical lipid shells harboring streptavidin moieties to bind biotinylated monoclonal antibodies (mAb) directed toward specific targets) based contrast agents that were coated with specific antibodies targeted to αβ integrin VEGFR 2 (MB), (MB), and endoglin (MB), respectively (3). The targeted MBs were then used with ultrasound imaging to determine the expression of the angiogenic biomarkers during the growth of human ovarian (SKOV3 cells), human breast (MDA-MB-361 cells), and human pancreatic (MiaPaCa2 cells) cell line xenograft tumors in mice. This chapter describes the studies performed with MB. Studies performed with MB and MB are described in separate chapters of MICAD (www.micad.nih.gov) (10, 11).