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.

Keywords: Breast cancer; CAIX; Fluorescence pathology; HER2; Nanobody or VHH; Optical molecular imaging.

Fig. 1

Use of a combination of two tumour-specific VHHs for optical imaging results in higher T/B ratios. a Optical imaging of mice xenografted with MCF10DCIS cells in time upon injection of the combination 11A4-800 and B9-800 (upper panel) and the control combinations, 11A4-800 and R2-800 (middle panel) or B9-800 and R2-800 (bottom panel). Arrows point to tumours, bl depicts bladder. b T/B ratios in time (n = 7 per group). Graph represents mean ± SEM. These were calculated from ROIs drawn around each tumour and non-tumour area. c Comparison of overall T/B ratios and their SEM obtained at all time points for animals injected with B9-800 and 11A4-800 and the controls: B9-800 and R2-800 or 11A4-800 and R2-800. The p values were determined, and statistical significance is indicated by * when p < 0.05, ** when p < 0.01 and *** when p < 0.0001

Fig. 2

Dual spectral molecular imaging using tumour-specific VHHs allows for simultaneous expression status determination of two different tumour markers, namely CAIX (targeted by VHH B9) and HER2 (targeted by VHH 11A4). Irrelevant VHH R2-700 was used as a negative control. Mice xenografted with MCF10DCIS cells were imaged in time upon injection of the following: a tumour-specific combination consisting of 11A4-680 and B9-800, b control combination consisting of 11A4-800 and R2-700 and c control combination consisting of B9-800 and R2-700. The observed yellow colour present in the overlays in each of the bottom panels originates from the overlay of green and red signals

Fig. 3

Rapid increase of T/B ratio in the first 6 h p.i. allows fast imaging upon injection of the following: a B9-800 and 11A4-680 (n = 8), b 11A4-800 and R2-700 (n = 8) and c B9-800 and R2-700 (n = 7). Graphs represent mean ± SEM. At 6 h p.i, the p values were determined and statistical significance is indicated by * when p < 0.05 and *** when p < 0.0001

Fig. 4

Dual-spectral imaging with two tumour-specific VHHs allows simultaneous tumour molecular status determination in an invasive, mimicked intra-operative setting. a Five hours p.i. of 11A4-680 and B9-800 (left), 11A4-800 and R2-700 (middle) or B9-800 and R2-700 (right) mice were sacrificed by cervical dislocation and their skin removed to mimic image-guided surgery setting. b–d T/B ratios were calculated for tumours imaged in the mimicked intra-operative setting. Bars represent mean, n = 4 mice in groups 11A4-800 and R2-700 and B9-800 and 11A4-680, n = 6 mice in group B9-800 and R2-700. Statistical significance is indicated by * when p < 0.05 and *** when p < 0.0001

Fig. 5

Optical molecular imaging with two tumour-specific VHHs allows detection of lung metastasis ex vivo. Optical imaging of metastasized lungs 5 h p.i. of B9-800 and 11A4-680 combination (upper panel) or the control R2-800 and R2-700 (middle panel). Optical imaging of healthy lungs 5 h p.i. of B9-800 and 11A4-680 (lower panel). Yellow colour in the overlay panel corresponds to the overlay of green (B9-800) and red (11A4-680) colours

Fig. 6

Dual-spectral fluorescence molecular pathology allows simultaneous determination of HER2 and CAIX expression status. Detection of fluorescence on tumour sections obtained from mice 5 h p.i. of the combinations a 11A4-680 and B9-800, b 11A4-800 and R2-700 and c B9-800 and R2-700. The fluorescence detected with the odyssey scanner is depicted in green for channel 800 nm (IRDye800CW) and in red for channel 700 nm (IRDye680RD and IRDye700DX). H&E staining of the corresponding section is depicted, where an asterisk indicates necrotic area. d IHC on sections of tumour xenografts depicting expression of HER2 and CAIX