Optical imaging of pre-invasive breast cancer with a combination of VHHs targeting CAIX and HER2 increases contrast and facilitates tumour characterization

Abstract

BackgroundOptical 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.MethodsWe 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.ResultsApplication 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-016-0166-y) contains supplementary material, which is available to authorized users.

Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics

“…Other studies have assessed carbonic anhydrase IX (CAIX) and HER2-CAIX targeting for optical imaging ( 21 ). Notably, the HER2-CAIX combination synergistically enhanced the T/B ratio and could also detect lung metastases ( 22 ). Additionally, 89 Zr-HER3 ( 23 ), 18 F-HER2 ( 24 ), and 68 Ga-NOTA-CD20 ( 25 ) nanobodies have demonstrated success in various tumor models.…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…Periplasmic produced biotinylated nanobodies were exploited for the functionalization of microbubbles to use as ultrasound contrast agent [76], for the diagnostic of H3N2 and swine influenza, and for the identification of ochratoxin A contaminations in cereals [77][78][79][80]. Periplasmic expression was also successfully exploited to produce nanobodies suitable for reacting with maleimide fluorophores [81], fusions between VHHs and nanoluciferase to use as bioluminescent immunoreagents [82], for recovering correct-folded nanobody-intein fusion proteins suitable for thioester reactions and finally for the fabrication of immunomicelles [83]. This expression route was also preferred for producing a hybrid molecule in which an anti-lysozyme nanobody was inserted into a solvent-exposed loop of β-lactamase [84].…”

Nanobody-Based Delivery Systems for Diagnosis and Targeted Tumor Therapy

“…In 2016, Kijanka et al ( 147 ) reported a combination of optical conjugations based on two different Nbs against two different breast tumor markers, for an improved tumor detection: Nb B9 against CAIX, which targets the peri-necrotic regions of tumors, and Nb 11A4 against HER2. This dual-spectral imaging strategy accomplishes successfully the optical molecular imaging of CAIX and HER2-positive DCIS xenografts in vivo , under conditions mimicking surgical settings.…”

“…This dual-spectral imaging strategy accomplishes successfully the optical molecular imaging of CAIX and HER2-positive DCIS xenografts in vivo , under conditions mimicking surgical settings. This strategy is assumed to facilitate a faster detection of tumor markers, and it is highly promising to utilize the dual-spectral imaging strategy for the early diagnosis, treatment program planning, and monitoring the treatment response ( 147 ).…”