Application of Nanotechnology for the Discovery of Circulating Proteins as Novel Biomarkers of Breast Cancer

Resumo

Breast cancer is one of the most common cancers in women and accounts for about 14% of cancer-related deaths in females around the world. Breast cancer is a heterogeneous disease that presents a wide variety of molecular and clinical characteristics, as well as variability in clinical progression. For the treatment choice, patients are classified according to intrinsic biological subtypes within the breast spectrum, using clinical-pathological criteria, i.e. the recognition of amplification and/or overexpression of the human epidermal growth factor receptor 2 (HER2) oncogene, the immunohistochemical classification of the estrogen receptor (ER) and the progesterone receptor (PR), and Ki-67 labelling index. This classification allows for a more personalized approach to medical treatments, with favorable results. However, despite that, almost 10-15% of these patients still experience local or distant recurrences in the first 5 years from diagnosis. Classification of breast cancer might be markedly improved if new biomarkers identified with the use of high-throughput “omics” approaches could support diagnosis based on histopathological patterns. In this way, nanomaterials have been introduced into the field of proteomics to establish a new and rapidly evolving research area termed nanoproteomics. It is well known that the dispersion of a nanomaterial in physiological fluid results in the formation of a protein shell named “protein corona” (PC). PC varies depending on the characteristics of the biological media, the physical (size, shape, curvature) and chemical properties (composition, surface charge/chemistry, hydrophobicity/hydrophilicity) of the nanomaterial, and the incubation time. Disease-associated biomarkers comprise less than 1% of serum proteins. In this way, through the formation of the PC, nanoparticles could act as sorbent materials for the enrichment of low-abundance peptides/proteins presented in serum samples before the biomarker identification by mass spectrometry (MS) analysis. Importantly, otherwise undetectable changes in protein concentration at an early stage of the disease (as breast cancer), after any treatment (chemotherapy, immunotherapy) or surgery could be detected analyzing the PC composition. Thus, characterization of the PC around NPs offers distinct advantages over sole proteomic approaches and increases the success of identifying molecular targets. Firstly, this thesis aims to optimize the formation of the bio-corona formed around the surface of gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), platinum nanoparticles (PtNPs) and magnetic nanoparticles (MNPs) after their interaction with proteins present in human serum. After that, it was developed an exhaustive qualitative and quantitative analysis of composition of the PC through electrophoretic separation (SDS-PAGE) in combination with liquid chromatography tandem-mass spectrometry (LC-MS/MS). This methodology was applied for the identification of serum biomarkers of the different breast cancer subtypes. This study shows that nanoproteomics is a valuable tool that can facilitate comprehensive and systematic identification of the serum proteome and the molecular classification of breast cancer.