Detection of clustered circulating tumour cells in early breast cancer.

Circulating tumour cell (CTC) clusters have been proposed to be major players in the metastatic spread of breast cancer, particularly during advanced disease stages. Yet, it is unclear whether or not they manifest in early breast cancer, as their occurrence in patients with metastasis-free primary disease has not been thoroughly evaluated. In this study, exploiting nanostructured titanium oxide-coated slides for shear-free CTC identification, we detect clustered CTCs in the curative setting of multiple patients with early breast cancer prior to surgical treatment, highlighting their presence already at early disease stages. These results spotlight an important aspect of metastasis biology and the possibility to intervene with anti-cluster therapeutics already during the early manifestation of breast cancer.

Antibody purification-independent microarrays (PIM) by direct bacteria spotting on TiO2-treated slides.

The preparation of effective conventional antibody microarrays depends on the availability of high quality material and on the correct accessibility of the antibody active moieties following their immobilization on the support slide. We show that spotting bacteria that expose recombinant antibodies on their external surface directly on nanostructured-TiO(2) or epoxy slides (purification-independent microarray - PIM) is a simple and reliable alternative for preparing sensitive and specific microarrays for antigen detection. Variable domains of single heavy-chain antibodies (VHHs) against fibroblast growth factor receptor 1 (FGFR1) were used to capture the antigen diluted in serum or BSA solution. The FGFR1 detection was performed by either direct antigen labeling or using a sandwich system in which FGFR1 was first bound to its antibody and successively identified using a labeled FGF. In both cases the signal distribution within each spot was uniform and spot morphology regular. The signal-to-noise ratio of the signal was extremely elevated and the specificity of the system was proved statistically. The LOD of the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4ng/mL and 10µg/mL. The microarrays prepared with bacteria exposing antibodies remain fully functional for at least 31 days after spotting. We finally demonstrated that the method is suitable for other antigen-antibody pairs and expect that it could be easily adapted to further applications such as the display of scFv and IgG antibodies or the autoantibody detection using protein PIMs.