title: Optical imaging of pre-invasive breast cancer with a combination of VHHs targeting CAIX and HER2 increases contrast and facilitates tumour characterization.
abstract: BACKGROUND
  Optical molecular imaging is an emerging novel technology with applications in the diagnosis of cancer and assistance in image-guided surgery. A high tumour-to-background (T/B) ratio is crucial for successful imaging, which strongly depends on tumour-specific probes that rapidly accumulate in the tumour, while non-bound probes are rapidly cleared. Here, using pre-invasive breast cancer as a model, we investigate whether the use of combinations of probes with different target specificities results in higher T/B ratios and whether dual-spectral imaging leads to improvements in tumour characterization.
  
  
  METHODS
  We performed optical molecular imaging of an orthotopic breast cancer model mimicking ductal carcinoma in situ (DCIS). A combination of carbonic anhydrase IX (CAIX)- and human epidermal growth factor receptor 2 (HER2)-specific variable domains of the heavy chain from heavy-chain antibodies (VHHs) was conjugated either to the same fluorophore (IRDye800CW) to evaluate T/B ratios or to different fluorophores (IRDye800CW, IRDye680RD or IRDye700DX) to analyse the expression of CAIX and HER2 simultaneously through dual-fluorescence detection. These experiments were performed non-invasively in vivo, in a mimicked intra-operative setting, and ex vivo on tumour sections.
  
  
  RESULTS
  Application of the CAIX- and HER2-specific VHH combination resulted in an increase of the T/B ratio, as compared to T/B ratios obtained from each of these single VHHs together with an irrelevant VHH. This dual tumour marker-specific VHH combination also enabled the detection of small metastases in the lung. Furthermore, dual-spectral imaging enabled the assessment of the expression status of both CAIX and HER2 in a mimicked intra-operative setting, as well as on tumour sections, which was confirmed by immunohistochemistry.
  
  
  CONCLUSIONS
  These results establish the feasibility of the use of VHH 'cocktails' to increase T/B ratios and improve early detection of heterogeneous tumours and the use of multispectral molecular imaging to facilitate the assessment of the target expression status of tumours and metastases, both invasive or non-invasively.
authors: Division of Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands. Kijanka.Marta@gmail.com.
authors: Marta M
authors: MM
authors: Kijanka
authors: Division of Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands. AramvBrussel@hotmail.com.|Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands. AramvBrussel@hotmail.com.
authors: Aram S A
authors: AS
authors: van Brussel
authors: Division of Internal Medicine and Dermatology, University Medical Center Utrecht, Utrecht, The Netherlands. E.vanderWall@umcutrecht.nl.
authors: Elsken
authors: E
authors: van der Wall
authors: Department of Radiology, University Medical Center Utrecht, Utrecht, The Netherlands. W.Mali@umcutrecht.nl.
authors: Willem P T M
authors: WP
authors: Mali
authors: Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands. P.J.vanDiest@umcutrecht.nl.
authors: Paul J
authors: PJ
authors: van Diest
authors: Division of Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands. P.vanBergen@uu.nl.
authors: Paul M P
authors: PM
authors: van Bergen En Henegouwen
authors: Division of Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands. s.oliveira@uu.nl.|Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands. s.oliveira@uu.nl.
authors: Sabrina
authors: http://orcid.org/0000-0002-6011-2122
authors: S
authors: Oliveira
keywords: Breast cancer; CAIX; Fluorescence pathology; HER2; Nanobody or VHH; Optical molecular imaging