title: A comparison of non-biologically active truncated EGF (EGFt) and full-length hEGF for delivery of Auger electron-emitting 111In to EGFR-positive breast cancer cells and tumor xenografts in athymic mice.
abstract: BACKGROUND
  EGFt is a truncated form of human epidermal growth factor (hEGF) that is non-biologically active but retains binding and internalization into EGFR-positive cells. Our aim was to compare EGFt and hEGF for delivery of (111)In to human breast cancer (BC) cells and tumors and evaluate its cytotoxicity against EGFR-positive BC cells, mediated by the Auger electron emissions of (111)In.
  
  
  METHODS
  The binding, internalization and nuclear localization of EGFt and hEGF in MDA-MB-468 human BC cells were first assessed by confocal fluorescence microscopy. Subcellular fractionation was then used to quantify the cellular and nuclear uptake of (111)In-EGFt and (111)In-hEGF in MDA-MB-468 cells. The effect of exposure in vitro to (111)In-EGFt or (111)In-hEGF on the clonogenic survival of MDA-MB-468 (10(6) EGFR/cell) or MCF-7 cells (10(4) EGFR/cell) was determined. The pharmacokinetics and tumor and normal tissue biodistribution of (111)In-EGFt was compared to (111)In-hEGF in CD-1 athymic mice with s.c. MDA-MB-468 and MCF-7 tumors. Nuclear importation in MDA-MB-468 tumors was determined ex vivo by subcellular fractionation.
  
  
  RESULTS
  Fluorescently-labeled EGFt and hEGF were bound, internalized and localized in the nucleus of MDA-MB-468 cells. Binding of (111)In-EGFt to MDA-MB-468 cells was 8-fold lower than (111)In-hEGF, but nuclear importation as a proportion of cell-bound (111)In was 3.6-fold greater than (111)In-hEGF. Nuclear uptake of (111)In-EGFt was lower than (111)In-hEGF when differences in cell binding were taken into account. The cytotoxicity of (111)In-EGFt (1.0MBq/mL; 10 nmols/L) against MDA-MB-468 cells was 9-fold lower than (111)In-hEGF but only 2-fold lower at a higher concentration (1.85 MBq/mL; 40 nmols/L). (111)In-EGFt and (111)In-hEGF exhibited greater cytotoxicity against MDA-MB-468 cells than MCF-7 cells. (111)In-EGFt was eliminated more slowly from the blood of tumor-bearing mice and exhibited lower liver uptake but higher kidney accumulation. Uptake of (111)In-EGFt in MDA-MB-468 tumors was 2.2-fold lower than (111)In-hEGF, and was blocked by anti-EGFR monoclonal antibody, nimotuzumab. Nuclear uptake into MDA-MB-468 tumor cells was higher for (111)In-EGFt than (111)In-hEGF, but when the lower tumor uptake of (111)In-EGFt was considered, there were no overall differences.
  
  
  CONCLUSIONS
  We conclude that the absence of biological activity of EGFt makes it attractive for delivery of Auger electron-emitting (111)In to EGFR-overexpressing BC, but its lower cellular and tumor uptake would limit its effectiveness compared to (111)In-hEGF.
  
  
  CONCLUSIONS
  (111)In-EGFt may reduce the adverse effects previously observed in patients administered (111)In-hEGF since it is not biologically active, but its lower uptake by BC cells and tumors would limit its effectiveness for treatment of breast cancer.
authors.affiliation: Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi, Girona, Catalunya, Spain.
authors.forename: Clara
authors.initials: C
authors.lastname: Panosa
authors.affiliation: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada.
authors.forename: Humphrey
authors.initials: H
authors.lastname: Fonge
authors.affiliation: Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi, Girona, Catalunya, Spain.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Ferrer-Batallé
authors.affiliation: Metabolism & Cancer Group, Translational Research Laboratory, Catalan Institute of Oncology (ICO), Girona, Catalunya, Spain; Girona Biomedical Research Institute (IDIBGI), Girona, Catalunya, Spain.
authors.forename: Javier A
authors.initials: JA
authors.lastname: Menéndez
authors.affiliation: Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi, Girona, Catalunya, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Massaguer
authors.affiliation: Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi, Girona, Catalunya, Spain. Electronic address: rafael.llorens@udg.edu.
authors.forename: Rafael
authors.initials: R
authors.lastname: De Llorens
authors.affiliation: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada; Department of Medical Imaging, University of Toronto, Toronto, ON, Canada; Toronto General Research Institute and Joint Department of Medical Imaging, University Health Network, Toronto, ON, Canada. Electronic address: raymond.reilly@utoronto.ca.
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Reilly
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology / Q000532:radiotherapy*; D049109:Cell Proliferation / Q000528:radiation effects; D016503:Drug Delivery Systems; D004583:Electrons; D004815:Epidermal Growth Factor / Q000008:administration & dosage / Q000737:chemistry*; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D006801:Humans; D007205:Indium Radioisotopes / Q000627:therapeutic use*; D051379:Mice; D008819:Mice, Nude; D057054:Molecular Imaging / Q000379:methods*; D019275:Radiopharmaceuticals / Q000627:therapeutic use*; D047368:Tumor Burden; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D007205:Indium Radioisotopes; D019275:Radiopharmaceuticals; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: Breast cancer; EGF; EGFR; Indium-111; Radiopharmaceuticals; Theranostics