Optical molecular imaging of hypoxic breast cancer - From prospect to preclinical practice

Abstract

Current imaging modalities for breast cancer diagnosis and therapy monitoring either lack sensitivity, specificity, make use of radiation and/or give images of limited resolution. Optical molecular imaging is a novel technique that detects light emitted by (breast)cancer-specific probes with a sensitive camera. As hypoxia is a common condition in solid tumors, proteins upregulated in hypoxic cells are of special interest as target for molecular imaging of breast cancer. In this thesis we reviewed expression levels of hypoxia markers carbonic anhydrase IX (CAIX), glucose transporter 1 (GLUT1), insulin-like growth factor 1 receptor (IGF1R) and C-X-C chemokine receptor type-4 (CXCR4). Then, we performed an immunohistochemistry (IHC) study to evaluate expression levels of hypoxia markers in human breast cancer, benign and normal tissues. These studies showed that hypoxia upregulated markers are expressed in less than half of all breast tumors, necessitating the targeting of novel markers or tumormarker combinations. In order to discover novel immunogenic hypoxia markers we selected binders against hypoxic HeLa cells by a modified phage display selection strategy. Immunoprecipitation and mass spectrometry revealed decay-accelerating factor (DAF) as hypoxia-upregulated membrane protein. After evaluation of hypoxia-specific tumor markers, we developed three types of NIR-labelled probes that target hypoxic breast tumors: a 0.13 kDa small molecule (2-nitroimidazole), 15 kDa nanobodies (anti-CAIX nanobody B9 and anti-DAF nanobody A2A11, 15 kDa) and 150 kDa IgG antibodies (anti-CAIX and anti-CD44v6). After in vitro evaluation of binding affinity and hypoxia specificity, probes were tested in a mouse model with orthotopically transplanted MCF10DCIS xenografts, mimicking pre-invasive ductal carcinoma in situ of the breast. Mice were imaged up to 4 days after injection. Tumor-to-normal tissue ratios (TNR) of fluorescent signals were calculated for each time point. High tumor contrast was observed between 24 and 96 hours after injection of 2-nitroimidazole and antibodies. As rapid imaging (less than 8h after injection) with both 2-nitroimidazole and antibodies resulted in limited contrast, we tested nanobodies B9 and A2A11 in the same animal model. This resulted in optimal contrast already 2 hours after injection. After imaging non-invasively, mice were sacrificed and tumor signals were measured after removing the skin, resulting in stronger fluorescent signals compared to the non-invasive imaging. Then, tumors were resected, mimicking image-guided surgery. After evaluation of these three probes in vivo, we concluded that nanobodies are useful as probes for rapid molecular imaging. Subsequently, in the same mouse model we tested combinations of nanobodies (anti-HER2, anti-CAIX and irrelevant control R2), injected simultaneously, to test if this would lead to higher contrast compared to single nanobody injections. We also evaluated receptor status determination of CAIX and HER2 simultaneously, in vivo and ex vivo, by labelling nanobodies with either 700 nm or 800 nm near-infrared (NIR)-dyes. Due to a relatively high background signal, we concluded that combinations of anti-CAIX and anti-HER2 nanobodies did not result in higher tumor contrast. However, by molecular fluorescence pathology we successfully showed different expression patterns of CAIX and HER2 in tumor sections, as confirmed by IHC.