Development of a Non-Invasive Proteomic Approach to Profiling Molecular Changes in the Microenvironment to Investigate Stages of Breast Health

Early detection of breast cancer is critical for increasing survival rates. However, currently available screening strategies provide ambiguous results, leaving invasive tissue biopsy procedures necessary for definitive diagnosis. Considerable efforts have investigated using nipple aspirate fluid (NAF), a liquid biopsy rich in proteins representative of the breast microenvironment, as a non-invasive source of early detection biomarkers. However, by using traditional two-dimensional discovery proteomic approaches, many technical challenges of using NAF have limited analysis of large sample sizing: such as low expressed volume (<10µL) or insufficient analytical material (<200µg protein). Following non-invasive collection by manual massage, we developed a one-dimensional sample preparation workflow that reduced sample handling steps, minimised sample losses and increased sample throughput to 96-samples by using a PVDF-membrane based system, which was ideally suited to the NAF proteome. Samples were prepared within a single working day, and results correlated significantly with conventional in-solution protocols. Proteins typically associated with the dysregulation of innate immune response and haemostatic pathways had a significantly altered proteome profile in response to breast cancer. Overall, our new workflow will allow future studies to take a more high-throughput approach, revealing biomarkers for breast cancer early detection, and providing a real impact.

URI

http://hdl.handle.net/10454/19058

Type

Thesis

Qualification name

MPhil