title: A Cystine Knot Peptide Targeting Integrin αvβ6 for Photoacoustic and Fluorescence Imaging of Tumors in Living Subjects.
abstract: Photoacoustic imaging is a nonionizing biomedical imaging modality with higher resolution and imaging depth than fluorescence imaging, which has greater sensitivity. The combination of the 2 imaging modalities could improve the detection of cancer. Integrin αvβ6is a cell surface marker overexpressed in many different cancers. Here, we report the development and evaluation of a dye-labeled cystine knot peptide, which selectively recognizes integrin αvβ6with high affinity, for photoacoustic and fluorescence imaging. The new dual-modality probe may find clinical application in cancer diagnosis and intraoperative imaging of integrin αvβ6-positive tumors.
  
  
  METHODS
  An engineered cystine knot peptide, R01, that recognizes integrin αvβ6was labeled with Atto 740 (A740) and evaluated for its specific cell uptake and its sensitivity threshold. A740-R01 was injected via the tail vein into nude mice xenografted with A431 (integrin αvβ6-positive) or 293T (integrin αvβ6-negative) tumors. Photoacoustic and fluorescence scans of tumors were acquired before and at 0.5, 1, 2, and 4 h after injection of A740-R01. Dynamic photoacoustic scans of various normal organs were also acquired. Ex vivo fluorescence imaging of tissues was performed 1 h after injection.
  
  
  RESULTS
  The A740-R01 demonstrated integrin αvβ6-dependent binding to A431 cells in culture. Sensitivity studies indicated that the probe may potentially detect lesions as small as 1 or 6 mm3by fluorescence or photoacoustic imaging, respectively. The photoacoustic and fluorescence signals of A431 xenografts at 1 h after injection were 1.87 ± 0.25 arbitrary units (AU) and 8.27 ± 0.87 AU, respectively. Target specificity was confirmed by low tumor uptake in 293T tumors at 1 h after injection (1.07 ± 0.15 AU and 1.10 ± 0.14 AU for photoacoustic and fluorescence signals, respectively). A740-R01 exhibited hepatobiliary clearance marked by high uptake in the liver, spleen, and intestine but low uptake in the kidneys.
  
  
  CONCLUSIONS
  A740-R01 specifically targeted integrin αvβ6with low nanomolar affinity. A740-R01 was able to detect integrin αvβ6both in vitro and in vivo by photoacoustic and fluorescence imaging. A740-R01 is able to detect αvβ6-positive tumors in living subjects and may have clinical application in cancer diagnosis and real-time image-guided surgery.
authors.affiliation: Department of Radiology, Molecular Imaging Program at Stanford, Canary Center for Cancer Early Detection, Stanford University, Stanford, California; and Department of Medical Ultrasound, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Radiology, Molecular Imaging Program at Stanford, Canary Center for Cancer Early Detection, Stanford University, Stanford, California; and.
authors.forename: Richard
authors.initials: R
authors.lastname: Kimura
authors.affiliation: Department of Radiology, Molecular Imaging Program at Stanford, Canary Center for Cancer Early Detection, Stanford University, Stanford, California; and.
authors.forename: Lotfi
authors.initials: L
authors.lastname: Abou-Elkacem
authors.affiliation: Department of Radiology, Molecular Imaging Program at Stanford, Canary Center for Cancer Early Detection, Stanford University, Stanford, California; and.
authors.forename: Jelena
authors.initials: J
authors.lastname: Levi
authors.affiliation: Department of Radiology, Molecular Imaging Program at Stanford, Canary Center for Cancer Early Detection, Stanford University, Stanford, California; and.
authors.forename: Lingyun
authors.initials: L
authors.lastname: Xu
authors.affiliation: Department of Radiology, Molecular Imaging Program at Stanford, Canary Center for Cancer Early Detection, Stanford University, Stanford, California; and sgambhir@stanford.edu.
authors.forename: Sanjiv Sam
authors.initials: SS
authors.lastname: Gambhir
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D045744:Cell Line, Tumor; D003553:Cystine; D005260:Female; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D061848:Optical Imaging / Q000379:methods*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D061088:Photoacoustic Techniques / Q000379:methods*; D011487:Protein Conformation; D021381:Protein Transport; D014018:Tissue Distribution
chemical_list: D000951:Antigens, Neoplasm; D016023:Integrins; D010455:Peptides; C073878:integrin alphavbeta6; D003553:Cystine
keywords: cystine knot; integrin αvβ6; photoacoustic fluorescence imaging