Hypoxia-Targeting Fluorescent Nanobodies for Optical Molecular Imaging of Pre-Invasive Breast Cancer

Mol Imaging Biol. 2016 Aug;18(4):535-44. doi: 10.1007/s11307-015-0909-6.


Purpose: The aim of this work was to develop a CAIX-specific nanobody conjugated to IRDye800CW for molecular imaging of pre-invasive breast cancer.

Procedures: CAIX-specific nanobodies were selected using a modified phage display technology, conjugated site-specifically to IRDye800CW and evaluated in a xenograft breast cancer mouse model using ductal carcinoma in situ cells (DCIS).

Results: Specific anti-CAIX nanobodies were obtained. Administration of a CAIX-specific nanobody into mice with DCIS xenografts overexpressing CAIX showed after 2 h a mean tumor-to-normal tissue ratio (TNR) of 4.3 ± 0.6, compared to a TNR of 1.4 ± 0.2 in mice injected with the negative control nanobody R2-IR. In DCIS mice, a TNR of 1.8 ± 0.1 was obtained. Biodistribution studies demonstrated an uptake of 14.0 ± 1.1 %I.D./g in DCIS + CAIX tumors, 4.6 ± 0.8 %I.D./g in DCIS tumors, while 2.0 ± 0.2 %I.D./g was obtained with R2-IR.

Conclusions: These results demonstrate the successful generation of a CAIX-specific nanobody-IRDye800CW conjugate that can be used for rapid imaging of (pre-)invasive breast cancer.

Keywords: Breast cancer; Carbonic anhydrase IX; Molecular fluorescence pathology; Nanobody; Optical imaging; VHH.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carbonic Anhydrase IX / metabolism
  • Carcinoma, Intraductal, Noninfiltrating / pathology
  • Cell Hypoxia
  • Cell Surface Display Techniques
  • DNA / metabolism
  • Female
  • HeLa Cells
  • Humans
  • Hypoxia / pathology*
  • Immunization
  • Immunohistochemistry
  • Mammary Neoplasms, Animal / pathology*
  • Mice
  • Molecular Imaging / methods*
  • Neoplasm Invasiveness
  • Optical Imaging / methods*
  • Single-Domain Antibodies / metabolism*
  • Tissue Distribution
  • Xenograft Model Antitumor Assays


  • Single-Domain Antibodies
  • DNA
  • Carbonic Anhydrase IX