Single- and two-photon imaging of human micrometastases and disseminated tumour cells with conjugates of nanobodies and quantum dots.

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.

IL-13 but not IL-4 signaling via IL-4Rα protects mice from papilloma formation during DMBA/TPA two-step skin carcinogenesis.

Interleukin 4 (IL-4) was shown to be tumor-promoting in full carcinogenesis studies using 3-methylcholanthrene (MCA). Because heretofore the role of IL-4 in DMBA/TPA (9,10-dimethyl-1,2-benz-anthracene/12-O-tetradecanoylphorbol-13-acetate) two-stage carcinogenesis was not studied, we performed such experiments using either IL-4(-/-) or IL-4Rα(-/-) mice. We found that IL-4Rα(-/-) but not IL-4(-/-) mice have enhanced papilloma formation, suggesting that IL-13 may be involved. Indeed, IL-13(-/-) mice developed more papillomas after exposure to DMBA/TPA than their heterozygous IL-13-competent littermate controls. However, when tested in a full carcinogenesis experiment, exposure of mice to 25 µg of MCA, both IL-13(-/-) and IL-13(+/-) mice led to the same incidence of tumors. While IL-4 enhances MCA carcinogenesis, it does not play a measurable role in our DMBA/TPA carcinogenesis experiments. Conversely, IL-13 does not affect MCA carcinogenesis but protects mice from DMBA/TPA carcinogenesis. One possible explanation is that IL-4 and IL-13, although they share a common IL-4Rα chain, regulate signaling in target cells differently by employing distinct JAK/STAT-mediated signaling pathways downstream of IL-13 or IL-4 receptor complexes, resulting in different inflammatory transcriptional programs. Taken together, our results indicate that the course of DMBA/TPA- and MCA-induced carcinogenesis is affected differently by IL-4 versus IL-13-mediated inflammatory cascades.