Abstract
Early detection of malignant tumours and, especially, micrometastases and disseminated tumour cells is still a challenge. In order to implement highly sensitive diagnostic tools we demonstrate the use of nanoprobes engineered from nanobodies (single-domain antibodies, sdAbs) and fluorescent quantum dots (QDs) for single- and two-photon detection and imaging of human micrometastases and disseminated tumour cells in ex vivo biological samples of breast and pancreatic metastatic tumour mouse models expressing human epidermal growth factor receptor 2 (HER2) or carcinoembryonic antigen (CEA). By staining thin (5–10 µm) paraffin and thick (50 µm) agarose tissue sections, we detected HER2- and CEA-positive human tumour cells infiltrating the surrounding tissues or metastasizing to different organs, including the brain, testis, lung, liver, and lymph nodes. Compared to conventional fluorescently labelled antibodies the sdAb-HER2-QD and sdAb-CEA-QD nanoprobes are superior in detecting micrometastases in tissue sections by lower photobleaching and higher brightness of fluorescence signals ensuring much better discrimination of positive signals versus background. Very high two-photon absorption cross-sections of QDs and small size of the nanoprobes ensure efficient imaging of thick tissue sections unattainable with conventional fluorescent probes. The nanobody–QD probes will help to improve early cancer diagnosis and prognosis of progression by assessing metastasis.
Highlights
Detection of malignant tumours and, especially, micrometastases and disseminated tumour cells is still a challenge
We used antibody fragments derived from llama antibodies, often called “nanobodies”, which are much smaller than conventionally used full-size IgG antibodies, with a molecular weight of only 13 kDa. These single-domain antibodies are the smallest antibody fragments capable of binding their antigens with affinities comparable to conventional antibodies6,10. sdAbs are characterized by a low tendency to aggregate; they diffuse much better into tissues than full-size IgGs, and their size allows the labelling of thicker tissue segments than conventional IgGs do[11]
Representative haematoxylin and eosin (H&E) stained sections illustrate the primary tumour and the distorted architecture of the lymph nodes infiltrated by tumour cells
Summary
Detection of malignant tumours and, especially, micrometastases and disseminated tumour cells is still a challenge. In order to implement highly sensitive diagnostic tools we demonstrate the use of nanoprobes engineered from nanobodies (single-domain antibodies, sdAbs) and fluorescent quantum dots (QDs) for single- and two-photon detection and imaging of human micrometastases and disseminated tumour cells in ex vivo biological samples of breast and pancreatic metastatic tumour mouse models expressing human epidermal growth factor receptor 2 (HER2) or carcinoembryonic antigen (CEA). SdAbs are characterized by a low tendency to aggregate; they diffuse much better into tissues than full-size IgGs, and their size allows the labelling of thicker tissue segments than conventional IgGs do[11] Because of these advantages, we have recently conjugated QDs to nanobodies in a highly oriented fashion, with all antigen binding sites facing outwards, which considerably increases the nanoprobe sensitivity for diagnostic and possible therapeutic use in oncology[12,13] and demonstrated their advantages in breast and lung cancer cell imaging[14]. CEA is commonly used as a serum marker as part of both preoperative staging and follow-up of the response of cancer patients to surgery and chemotherapy[19]
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