Development of an Extracellular Vesicle Microfluidic Affinity Purification (EV-MAP) In Vitro Assay for Breast Cancer Management

Abstract

Breast cancer is the second-most prevalent cancer worldwide and most commonly occurring cancer type in women with roughly 2 million new cases diagnosed in 2018. Current breast cancer management is a multidisciplinary approach which uses imaging for detection and monitoring, and tissue biopsy for diagnosis. Limitations within these technologies shifted focus toward liquid biopsies which are minimally invasive, allow for frequent sampling with a simple blood draw, and represent the heterogeneity of the cancer. One biomarker recently discovered for use in liquid biopsies are extracellular vesicles (EVs). Their ability to stably carry mRNA through circulation makes them a potentially suitable marker for mRNA expression profiling, a diagnostic method currently implemented in breast cancer management. Herein, an EV microfluidic affinity purification (EV-MAP) device was developed for selective isolation of disease-associated EVs for mRNA expression analysis. The EV-MAP assay produced a 99 ± 1% specificity for the gene panel used in this study through the implementation of 1% PVP-40/1% BSA blocking and 0.2% Tween 20® washing agents . A proteolytic digestion strategy using proteinase K and subsequent sonication was developed to remove EVs from the device surfaces and produced a release efficiency of 99 ± 1%. EV-MAP also captured two distinct populations of disease-associated EVs through orthogonal markers either selecting for mesenchymal or epithelial-derived EVs, with one population showing gene expression profiling differences from the cells of origin while the mesenchymal populations resulted in similar mRNA expression profiles to their host cells. Finally, healthy controls and breast cancer patient samples were processed through the EV-MAP assay. ddPCR of these samples resulted in several genes showing copy numbers below the limit of detection of ddPCR, thereby highlighting the mass limitations associated with analyzing mRNA in EVs using the EV-MAP gene panel.