Development of an integrated nanotechnology-based platform for the early diagnosis of cancer: assessment of circulating biomarkers in Her2-positive breast cancer and role of novel binders in biomarkers detection

Early detection of cancer plays a crucial role in determining disease prognosis. The major challenge consists in the ability of identifying the disease through the quantification of a set of specific biomarkers in tissues and/or, more interestingly, released in the bloodstream. Non-invasiveness, sensitivity, parallelization, low cost, are some of the most relevant keywords in this field. Hence the development of miniaturized devices for the early detection of cancer is at the core of nanodiagnostics, requiring the recognition and quantification of low amounts of specific disease biomarkers, through the development of sensitive diagnostic tools. In this context, we have developed a nanodiagnostic platform for the non-invasive quantification of cancer biomarkers circulating in the bloodstream. The assay, that relies on Atomic Force Microscopy (AFM), is based on molecular manipulation to create density-optimized functional spots of surface-immobilized binders and differential AFM topography. It is label-free, allows the parallel detection of different cancer biomarkers, entails a single binder per antigen and when implemented with fluorescence labelling/readout can be used for epitope mapping. The possibility to exploit DNA nanografting and subsequent immobilization of binders through DNA-directed immobilization confers robustness to the assay. We explored the feasibility of novel binders as camelid nanobodies and aptamers, to improve the quality of the functionalization, and therefore device sensitivity, with the added advantage of binders easy engineering. In this study we focused on a prospective, clinically-relevant circulating cancer biomarker, the extra-cellular domain (ECD) of Human Epidermal Growth Factor Receptor (Her2), whose shedding and release in the blood is related to the progression of Her2-positive tumors and response to anticancer therapies. By employing robust, easily engineered camelid nanobodies as binders, we measured ECD-Her2 concentrations in the range of the actual clinical cutoff value for Her2 positive breast cancer. The specificity for Her2 detection was preserved when measured in complex matrices as standardized human serum, and in parallel with other potential biomarkers, demonstrating the intended implementation of multiplexing analysis, strongly required to define the biological tumor subtype and to univocally refer specific molecular levels to tumor status and progression. A better understanding of the Her2 receptor biology, overexpression in tumor cell membranes and release of the ECD to the bloodstream is however required to interpret the measured levels of ECD-Her2 at best. At present, there are controversial studies and conflicting results about the correlation between the protein levels in serum and the attested Her2 status in tumor tissue, which make the clinical significance of circulating ECD-Her2 still uncertain. Therefore we developed a multi-integrated approach in order to elucidate Her2 overexpression, dimerization and ECD shedding mechanism and to fully validate its prognostic value; moreover we preliminarily studied some fundamental aspects of the relationship between rafts-mediated exosomes formation and Her2 integration on them in order to clarify its possible role in metastasis occurrence. This approach relies on different multi-scale techniques and enables to correlate information coming from advanced optical microscopies (membrane proteins localization), nanotechnology-based diagnostic tools (detection of protein and vesicle biomarkers) and novel super resolution fluorescence microscopies (quantification and co-localization of different biomarkers). This innovative platform will be instrumental in identifying and quantifying clinically useful biomarkers and to translate basic science results into the clinics to impact cancer diagnosis, prognosis, and therapy.