title: In vivo breast cancer characterization imaging using two monoclonal antibodies activatably labeled with near infrared fluorophores.
abstract: BACKGROUND
  The gene expression profiles of cancer cells are closely related to their aggressiveness and metastatic potential. Antibody-based immunohistochemistry (IHC) of tissue specimens is a common method of identifying expressed proteins in cancer cells and increasingly inform treatment decisions. Molecular imaging is a potential method of performing similar IHC studies in vivo without the requirement for biopsy or tumor excision. To date, antibody-based imaging has been limited by high background levels related to slow clearance, making such imaging practical. However, optically activatable imaging agents, which are only fluorescent when bound to their cognate receptor, open the possibility of doing in vivo multi-color IHC.
  
  
  METHODS
  We describe the use of activatable, near infrared fluorescence-labeled AlexaFluor680 (Alexa680) conjugated panitumumab (Pan) targeted against human epidermal growth factor receptor (EGFR) (Pan-Alexa680) and Indocyanine Green (ICG) conjugated trastuzumab (Tra) targeted against human epidermal growth factor receptor type 2 (HER2) (Tra-ICG) were synthesized and evaluated in cells in vitro and in an orthotopic breast cancer mouse model in vivo.
  
  
  RESULTS
  Pan-Alexa680 (self-quenched; SQ) and Tra-ICG were initially quenched but demonstrated a 5.2- and 50- fold dequenching capacity under detergent treatment, respectively. In vitro microscopy and flow cytometry using MDA-MB-468 (EGFR+/HER2-) and 3T3/HER2 cells (EGFR-/HER2+), demonstrated specific fluorescence signal for each cell type based on binding to Pan-Alexa680(SQ) or Tra-ICG. An in vivo imaging study employing a cocktail of Pan- Alexa680(SQ) and Tra-ICG (each 50 μg) was injected into mice with orthotopic MDA-MB-468 and 3T3/HER2 tumors in the breast. Each probe visualized only the target-specific breast tumor.
  
  
  CONCLUSIONS
  Multi-color target-specific fluorescence breast cancer imaging can be achieved in vivo by employing two activatable fluorescent probes administered as a cocktail. The images allowed us to see a specific receptor expression in each breast tumor without post-image processing.
authors.affiliation: Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Building 10, RoomB3B69, 10 Center Dr. Bethesda, MD 0892-1088, USA.
authors.forename: Kohei
authors.initials: K
authors.lastname: Sano
authors.forename: Makoto
authors.initials: M
authors.lastname: Mitsunaga
authors.forename: Takahito
authors.initials: T
authors.lastname: Nakajima
authors.forename: Peter L
authors.initials: PL
authors.lastname: Choyke
authors.forename: Hisataka
authors.initials: H
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000276:immunology; D045744:Cell Line, Tumor; D003952:Diagnostic Imaging / Q000379:methods; D066246:ErbB Receptors / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007208:Indocyanine Green; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D000077544:Panitumumab; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D066246:ErbB Receptors; D005456:Fluorescent Dyes; D007208:Indocyanine Green; D000077544:Panitumumab; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; C512478:EGFR protein, human; C508053:ERBB2 protein, human