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Molecular imaging with a fluorescent antibody targeting carbonic anhydrase IX can successfully detect hypoxic ductal carcinoma in situ of the breast

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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.

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Acknowledgments

We would like to thank A. Martens for the pLV-CMV-LUC2-IRES-GFP vector and P.C. Pearlman for writing the algorithm in Matlab. We are indebted to P.W.B. Derksen and S.G. Elias for their suggestions concerning the mouse model. Also, we would like to thank the animal facility of the University Utrecht and the biobank of the University Medical Center Utrecht for their support. This research was supported by the Center for Translational Molecular Medicine—Mammary Carcinoma Molecular Imaging for Diagnosis and Therapeutics (CTMM—MAMMOTH, Project 203)—and by an unrestricted educational grant from Aegon to P.J.vD.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Division of Cell Biology, Department of Biology, Science Faculty, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands

    Aram S. A. van Brussel, Sabrina Oliveira & Paul M. P. van Bergen en Henegouwen

  2. Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands

    Aram S. A. van Brussel, Jeroen F. Vermeulen, Sabrina Oliveira & Paul. J. van Diest

  3. Department of Radiology, University Medical Center Utrecht, Utrecht, The Netherlands

    Arthur Adams & Willem P. Th. M. Mali

  4. Division of Internal Medicine and Dermatology, University Medical Center Utrecht, Utrecht, The Netherlands

    Elsken van der Wall

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  1. Aram S. A. van Brussel
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Correspondence to Paul M. P. van Bergen en Henegouwen.

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Aram S. A. van Brussel and Arthur Adams have contributed equally to this work.

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van Brussel, A.S.A., Adams, A., Vermeulen, J.F. et al. Molecular imaging with a fluorescent antibody targeting carbonic anhydrase IX can successfully detect hypoxic ductal carcinoma in situ of the breast. Breast Cancer Res Treat 140, 263–272 (2013). https://doi.org/10.1007/s10549-013-2635-6

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