Antiangiogenic cancer therapy: monitoring with molecular US and a clinically translatable contrast agent (BR55).
OBJECTIVE
To develop and test human kinase insert domain receptor (KDR)-targeted microbubbles (MBs) (MB(KDR)) for imaging KDR at the molecular level and for monitoring antiangiogenic therapy in a human colon cancer xenograft tumor model in mice.


METHODS
Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. A heterodimeric peptide that binds to human KDR with low nanomolar affinity (K(D) = 0.5 nmol/L) was coupled onto the surface of perfluorobutane-containing lipid-shelled MBs (MB(KDR)). Binding specificity of MB(KDR) to human KDR and cross-reactivity with murine vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) were tested in cell culture under flow shear stress conditions (at 100 sec(-1)). In vivo binding specificity of MB(KDR) to VEGFR2 was tested in human LS174T colon cancer xenografts in mice with a 40-MHz ultrasonographic (US) transducer. Targeted contrast material-enhanced US imaging signal by using MB(KDR) was longitudinally measured during 6 days in tumors with (n = 6) and without (n = 6) antiangiogenic treatment (anti-VEGF antibody). Ex vivo VEGFR2 staining and microvessel density analysis were performed. Significant differences were evaluated (t, Mann-Whitney, or Wilcoxon test).


RESULTS
Cell culture experiments showed four times greater binding specificity of MB(KDR) to human KDR and cross-reactivity to murine VEGFR2 (P < or = .01). In vivo imaging signal was more than three times higher (P = .01) with MB(KDR) compared with control MBs and decreased significantly (approximately fourfold lower, P = .03) following in vivo receptor blocking with anti-VEGFR2 antibody. One day after initiation of antiangiogenic therapy, imaging signal was significantly decreased (approximately 46% lower, P = .02) in treated versus untreated tumors; it remained significantly lower (range, 46%-84% decreased; P = .038) during the following 5 days. Microvessel density was significantly reduced (P = .04) in treated (mean, 7.3 microvessels per square millimeter +/- 4.7 [standard deviation]) versus untreated tumors (mean, 22.0 microvessels per square millimeter +/- 9.4); VEGFR2 expression was significantly decreased (>50% lower, P = .03) in treated tumors.


CONCLUSIONS
Human MB(KDR) allow in vivo imaging and longitudinal monitoring of VEGFR2 expression in human colon cancer xenografts.
Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, 300 Pasteur Dr, Room H1307, Stanford, CA 94305, USA.
Marybeth A
MA
Pysz
Kira
K
Foygel
Jarrett
J
Rosenberg
Sanjiv S
SS
Gambhir
Michel
M
Schneider
Jürgen K
JK
Willmann
D020533:Angiogenesis Inhibitors / Q000008:administration & dosage*; D000818:Animals; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000000981:diagnostic imaging* / Q000188:drug therapy*; D003287:Contrast Media; D016503:Drug Delivery Systems / Q000379:methods; D006801:Humans; D051379:Mice; D045423:Microbubbles; D015336:Molecular Probe Techniques; D010455:Peptides; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014463:Ultrasonography / Q000379:methods*
D020533:Angiogenesis Inhibitors; D003287:Contrast Media; D010455:Peptides