title: Tumor angiogenic marker expression levels during tumor growth: longitudinal assessment with molecularly targeted microbubbles and US imaging.
abstract: OBJECTIVE
  To evaluate the use of molecularly targeted microbubbles (MBs) and ultrasonography (US) in the noninvasive assessment of the level of expression of three angiogenic markers, α(v)β(3) integrin, endoglin, and vascular endothelial growth factor receptor (VEGFR) 2, on tumor vascular endothelial cells in vivo during tumor growth.
  
  
  METHODS
  All procedures using laboratory animals were approved by the Institutional Administrative Panel on Laboratory Animal Care. Binding specificity of three types of targeted MBs (MB(Integrin), MB(Endoglin), MB(VEGFR2)) was tested in cell culture under flow shear stress conditions. In vivo targeted contrast material-enhanced US imaging signal using the three MB types was measured at three tumor stages (small, medium, large) in three subcutaneous cancer xenografts (breast, ovarian, pancreatic cancer) in mice (n = 54). In vivo US imaging signal was correlated with ex vivo angiogenic marker expression. Significant differences were evaluated by using the Student t, analysis of variance, Wilcoxon, and Tukey Honest Significant Difference tests.
  
  
  RESULTS
  Cell attachment of all three MB types was significantly (P = .016) higher compared with control MBs, and this attachment could be significantly (P = .026) decreased by blocking antibodies. Angiogenic marker-expressing cells bound significantly (P = .003) more targeted MBs than negative control cells, and MB attachment significantly (P < .001) correlated with marker expression levels on cells (ρ = 0.87). In early stage breast and ovarian cancers, in vivo targeted contrast-enhanced US demonstrated significantly (P ≤ .04) higher endoglin expression than both α(v)β(3) integrin and VEGFR2 expression, whereas in early stage pancreatic cancer, marker expressions were not significantly different (P ≥ .07). There was good correlation (ρ ≥ 0.63; P ≤ .05) between in vivo targeted contrast-enhanced US imaging signals using the three MB types and ex vivo immunoblotting results regarding expression levels of the three angiogenic markers. Immunofluorescence confirmed expression of α(v)β(3) integrin, endoglin, and VEGFR2 on tumor vascular endothelial cells.
  
  
  CONCLUSIONS
  Targeted contrast-enhanced US imaging allows noninvasive in vivo assessment of the expression levels of α(v)β(3) integrin, endoglin, and VEGFR2, which vary during tumor growth in subcutaneous cancer xenografts.
authors.affiliation: Molecular Imaging Program at Stanford, Department of Radiology, Stanford University School of Medicine, 300 Pasteur Dr, Room H1307, Stanford, CA 94305-5621, USA.
authors.forename: Nirupama
authors.initials: N
authors.lastname: Deshpande
authors.forename: Ying
authors.initials: Y
authors.lastname: Ren
authors.forename: Kira
authors.initials: K
authors.lastname: Foygel
authors.forename: Jarrett
authors.initials: J
authors.lastname: Rosenberg
authors.forename: Jürgen K
authors.initials: JK
authors.lastname: Willmann
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism; D003287:Contrast Media / Q000378:metabolism; D000071063:Endoglin; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D016023:Integrins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008325:Mammary Neoplasms, Experimental / Q000098:blood supply / Q000000981:diagnostic imaging* / Q000378:metabolism; D051379:Mice; D045423:Microbubbles; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging*; D010051:Ovarian Neoplasms / Q000098:blood supply / Q000000981:diagnostic imaging* / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000098:blood supply / Q000000981:diagnostic imaging* / Q000378:metabolism; D018709:Statistics, Nonparametric; D014463:Ultrasonography; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D003287:Contrast Media; D000071063:Endoglin; C502004:Eng protein, mouse; D016023:Integrins; D047908:Intracellular Signaling Peptides and Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2