title: Ultrasonic imaging of tumor angiogenesis using contrast microbubbles targeted via the tumor-binding peptide arginine-arginine-leucine.
abstract: Endothelial cells (EC) of angiogenic tumor vasculature are characterized by altered expression of molecular markers on their surface. Numerous peptides have been identified that specifically bind tumor angiogenic endothelium, including the tripeptide arginine-arginine-leucine (RRL). We hypothesized that ultrasound contrast microbubbles (MB) targeted via linkage with RRL would specifically adhere to tumor angiogenic endothelium versus normal myocardium, and that this selective adhesion could be detected ultrasonically. Microbubbles were conjugated to cyclic peptides containing either RRL (RRL-MB) or a glycine control sequence (control-MB). As measured in a parallel plate flow chamber, in vitro adhesion of RRL-MBs was three times greater to cultured tumor-derived ECs than to normal ECs (P < 0.01), demonstrating selective binding of RRL-MBs to tumor endothelium. Mice bearing s.c. Clone C or PC3 tumors were given i.v. injections of fluorescent RRL to show in vivo localization to tumor vasculature or were ultrasonically imaged following i.v. injections of targeted contrast MBs. Ultrasound images showed strong RRL-MB contrast enhancement within the tumors but not the control tissue myocardium. Control-MBs caused minimal enhancement in either tissue. Quantitative acoustic videointensity was significantly greater for the tumors than the hearts (5 +/- 1 versus 0.5 +/- 1 intensity units; P = 0.001). These data show that ultrasound contrast MBs targeted to tumor vasculature via RRL preferentially adhere to tumor versus normal vasculature and that this selective adherence can be detected with ultrasound. Targeted microbubbles may thus offer a noninvasive contrast-enhanced ultrasound imaging technique for the functional imaging of tumor neovascularization, and may have further implications for therapeutic tumor targeting.
authors.affiliation: Department of Bioengineering and McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: Gregory E R
authors.initials: GE
authors.lastname: Weller
authors.forename: Michael K K
authors.initials: MK
authors.lastname: Wong
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Modzelewski
authors.forename: Erxiong
authors.initials: E
authors.lastname: Lu
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Klibanov
authors.forename: William R
authors.initials: WR
authors.lastname: Wagner
authors.forename: Flordeliza S
authors.initials: FS
authors.lastname: Villanueva
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000098:blood supply* / Q000378:metabolism; D004452:Echocardiography / Q000379:methods; D004730:Endothelium, Vascular / Q000000981:diagnostic imaging / Q000378:metabolism; D019793:Fluorescein / Q000493:pharmacokinetics; D006801:Humans; D007930:Leucine / Q000378:metabolism; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008819:Mice, Nude; D045423:Microbubbles; D009206:Myocardium / Q000378:metabolism; D041681:NIH 3T3 Cells; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging* / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D014183:Transplantation, Heterologous
chemical_list: D009842:Oligopeptides; D001120:Arginine; D007930:Leucine; D019793:Fluorescein