title: Molecular imaging with a fluorescent antibody targeting carbonic anhydrase IX can successfully detect hypoxic ductal carcinoma in situ of the breast.
abstract: Ductal carcinoma in situ (DCIS) of the breast is difficult to remove completely during surgery as it is not palpable and can therefore require re-excision. Real-time visualization of DCIS using near-infrared fluorescent probes could help the surgeon during surgery as well as the pathologist post-operatively to distinguish the tumor from healthy tissue. As hypoxia-induced necrosis is a common phenomenon in DCIS, we investigated the molecular imaging of DCIS using a fluorescent antibody targeting a hypoxia marker, carbonic anhydrase IX (CAIX), in a preclinical mouse model. A monoclonal antibody against human CAIX was fluorescently labeled with the near-infrared dye IRDye800CW and characterized in vitro. An in vivo study was performed in SCID/Beige mice that were orthotopically transplanted with human breast cancer cells mimicking human DCIS (MCF10DCIS) and MCF10DCIS stably expressing CAIX. A clinically approved fluorescence imaging system was used to monitor probe uptake and to determine tumor-to-normal tissue ratios (TNR). Mean in vivo TNR of CAIX-transduced (CAIX+) tumors was 7.5 ± 0.5. Mean in vivo TNR of DCIS tumors with hypoxic areas reached a plateau level at 48 h after injection of 2.1 ± 0.1 (mean ± SEM) compared to 1.7 ± 0.1 in DCIS without hypoxic areas. Mean intra-operative TNR of DCIS tumors with necrotic regions was higher than that of DCIS tumors without necrotic regions 96 h after injection-2.9 ± 0.1 and 1.5 ± 0.1, respectively-while the TNR of CAIX+ tumors was 11.2 ± 1.0. Specific tumor uptake of MabCAIX-IRDye800CW was confirmed by a biodistribution assay, and immunofluorescence imaging on tumor sections showed specific uptake in hypoxic tumor regions, with higher contrast than conventional chromagen-based immunohistochemistry. Molecular fluorescence imaging with MabCAIX-IRDye800CW can be successfully used to detect hypoxic DCIS before and during surgery to facilitate radical resection. Furthermore, it allows for sensitive CAIX-specific immunofluorescence microscopy of tumor sections, thereby introducing the concept of molecular fluorescence pathology.
authors.affiliation: Division of Cell Biology, Department of Biology, Science Faculty, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
authors.forename: Aram S A
authors.initials: AS
authors.lastname: van Brussel
authors.forename: Arthur
authors.initials: A
authors.lastname: Adams
authors.forename: Jeroen F
authors.initials: JF
authors.lastname: Vermeulen
authors.forename: Sabrina
authors.initials: S
authors.lastname: Oliveira
authors.forename: Elsken
authors.initials: E
authors.lastname: van der Wall
authors.forename: Willem P Th M
authors.initials: WP
authors.lastname: Mali
authors.forename: Paul J
authors.initials: PJ
authors.lastname: van Diest
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen En Henegouwen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology; D014408:Biomarkers, Tumor / Q000276:immunology; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology / Q000601:surgery; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000276:immunology; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology / Q000601:surgery; D015687:Cell Hypoxia / Q000276:immunology; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases