title: Characterizing breast cancer xenograft epidermal growth factor receptor expression by using near-infrared optical imaging.
abstract: BACKGROUND
  Epidermal growth factor receptor (EGFR) overexpression is associated with several key features of cancer development and growth. Therefore, EGFR is a very promising biological target for tumor diagnosis and anticancer therapy. Characterization of EGFR expression is important for clinicians to select patients for EGFR-targeted therapy and evaluate therapeutic effects.
  
  
  OBJECTIVE
  To investigate whether near-infrared (NIR) fluorescent dye Cy5.5-labeled anti-EGFR monoclonal antibody Erbitux can characterize EGFR expression level in MDA-MB-231 and MCF-7 breast cancer xenografts using an in vivo NIR imaging method.
  
  
  METHODS
  A fluorochrome probe was designed by coupling Cy5.5 to Erbitux through acidylation, and the fluorescence property of the Erbitux-Cy5.5 conjugate was characterized by fluorospectroscopy. Flow cytometry and laser confocal microscopy were used to test the EGFR specificity of the antibody probe in vitro. Erbitux-Cy5.5 was also injected intravenously into immune-deficient mice bearing MDA-MB-231 or MCF-7 tumors. Whole-body and region-of-interest fluorescence images were acquired and analyzed. The EGFR expression was also analyzed and confirmed by immunohistochemical assay.
  
  
  RESULTS
  The maximum excitation/emission wavelength for the Erbitux-Cy5.5 probe was 674/697 nm, similar to that of free Cy5.5 (674/712 nm). Confocal microscopy confirmed receptor-specific uptake in MDA-MB-231 and MCF-7 cells. In flow cytometry probe specificity assay, Erbitux-Cy5.5 showed a 9.32-fold higher affinity for MDA-MB-231 than MCF-7 cells. In vivo NIR imaging also indicated specific uptake in EGFR-positive tumors. Probe uptake rate and maximum intake dose in MDA-MB-231 were significantly higher than those in MCF-7 xenografts (P < 0.001). Immunohistochemical staining confirmed the in vivo imaging results, showing differentiated EGFR expression in MDA-MB-231 (+ + +) and MCF-7 (+) tumor tissues.
  
  
  CONCLUSIONS
  Erbitux-Cy5.5 may be used as a specific NIR contrast agent for the noninvasive characterization of EGFR expression level in breast cancer xenografts.
authors.affiliation: Department of Medical Imaging and Nuclear Medicine, Fourth Affiliated Hospital, Harbin Medical University, Harbin, China.
authors.forename: Kezheng
authors.initials: K
authors.lastname: Wang
authors.forename: Kai
authors.initials: K
authors.lastname: Wang
authors.forename: Weihual
authors.initials: W
authors.lastname: Li
authors.forename: Tao
authors.initials: T
authors.lastname: Huang
authors.forename: Renfei
authors.initials: R
authors.lastname: Li
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Baozhong
authors.initials: B
authors.lastname: Shen
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Chen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000737:chemistry; D001943:Breast Neoplasms / Q000378:metabolism*; D002232:Carbocyanines / Q000737:chemistry; D045744:Cell Line, Tumor; D000068818:Cetuximab; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D008819:Mice, Nude; D018613:Microscopy, Confocal; D009368:Neoplasm Transplantation; D012680:Sensitivity and Specificity; D019265:Spectroscopy, Near-Infrared / Q000379:methods*; D018709:Statistics, Nonparametric
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D066246:ErbB Receptors; D000068818:Cetuximab