title: Ultrasound Molecular Imaging of the Breast Cancer Neovasculature using Engineered Fibronectin Scaffold Ligands: A Novel Class of Targeted Contrast Ultrasound Agent.
abstract: Molecularly-targeted microbubbles (MBs) are increasingly being recognized as promising contrast agents for oncological molecular imaging with ultrasound. With the detection and validation of new molecular imaging targets, novel binding ligands are needed that bind to molecular imaging targets with high affinity and specificity. In this study we assessed a novel class of potentially clinically translatable MBs using an engineered 10(th) type III domain of human-fibronectin (MB-FN3VEGFR2) scaffold-ligand to image VEGFR2 on the neovasculature of cancer. The in vitro binding of MB-FN3VEGFR2 to a soluble VEGFR2 was assessed by flow-cytometry (FACS) and binding to VEGFR2-expressing cells was assessed by flow-chamber cell attachment studies under flow shear stress conditions. In vivo binding of MB-FN3VEGFR2 was tested in a transgenic mouse model (FVB/N Tg(MMTV/PyMT634Mul) of breast cancer and control litter mates with normal mammary glands. In vitro FACS and flow-chamber cell attachment studies showed significantly (P<0.01) higher binding to VEGFR2 using MB-FN3VEGFR2 than control agents. In vivo ultrasound molecular imaging (USMI) studies using MB-FN3VEGFR2 demonstrated specific binding to VEGFR2 and was significantly higher (P<0.01) in breast cancer compared to normal breast tissue. Ex vivo immunofluorescence-analysis showed significantly (P<0.01) increased VEGFR2-expression in breast cancer compared to normal mammary tissue. Our results suggest that MBs coupled to FN3-scaffolds can be designed and used for USMI of breast cancer neoangiogenesis. Due to their small size, stability, solubility, the lack of glycosylation and disulfide bonds, FN3-scaffolds can be recombinantly produced with the advantage of generating small, high affinity ligands in a cost efficient way for USMI.
authors: 1. Department of Radiology, Molecular Imaging Program at Stanford, Stanford University, School of Medicine, Stanford, California, USA;
authors: Lotfi
authors: L
authors: Abou-Elkacem
authors: 1. Department of Radiology, Molecular Imaging Program at Stanford, Stanford University, School of Medicine, Stanford, California, USA;
authors: Katheryne E
authors: KE
authors: Wilson
authors: 2. Department of Chemical Engineering and Materials Science, University of Minnesota, Minnesota, USA;
authors: Sadie M
authors: SM
authors: Johnson
authors: 1. Department of Radiology, Molecular Imaging Program at Stanford, Stanford University, School of Medicine, Stanford, California, USA;
authors: Sayan M
authors: SM
authors: Chowdhury
authors: 1. Department of Radiology, Molecular Imaging Program at Stanford, Stanford University, School of Medicine, Stanford, California, USA;
authors: Sunitha
authors: S
authors: Bachawal
authors: 2. Department of Chemical Engineering and Materials Science, University of Minnesota, Minnesota, USA;
authors: Benjamin J
authors: BJ
authors: Hackel
authors: 3. Department of Health, Research & Policy, Stanford University, Stanford, California, USA.
authors: Lu
authors: L
authors: Tian
authors: 1. Department of Radiology, Molecular Imaging Program at Stanford, Stanford University, School of Medicine, Stanford, California, USA;
authors: Jürgen K
authors: JK
authors: Willmann
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000000981:diagnostic imaging*; D003287:Contrast Media / Q000008:administration & dosage*; D005353:Fibronectins / Q000008:administration & dosage; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D045423:Microbubbles; D057054:Molecular Imaging / Q000379:methods*; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging*; D011485:Protein Binding; D014463:Ultrasonography / Q000379:methods*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000032:analysis*
chemical_list: D003287:Contrast Media; D005353:Fibronectins; C000588866:KDR protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: Ultrasound molecular imaging, breast cancer, vascular endothelial growth factor receptor type 2, fibronectin scaffold, protein engineering; yeast display.