Abstract
Extracellular vesicles (EVs) are abundant in most biological fluids and considered promising biomarker candidates, but the development of EV biomarker assays is hindered, in part, by their requirement for prior EV purification and the lack of standardized and reproducible EV isolation methods. We now describe a far-field nanoplasmon-enhanced scattering (FF-nPES) assay for the isolation-free characterization of EVs present in small volumes of serum (< 5 µl). In this approach, EVs are captured with a cancer-selective antibody, hybridized with gold nanorods conjugated with an antibody to the EV surface protein CD9, and quantified by their ability to scatter light when analyzed using a fully automated dark-field microscope system. Our results indicate that FF-nPES performs similarly to EV ELISA, when analyzing EV surface expression of epithelial cell adhesion molecule (EpCAM), which has clinical significant as a cancer biomarker. Proof-of-concept FF-nPES data indicate that it can directly analyze EV EpCAM expression from serum samples to distinguish early stage pancreatic ductal adenocarcinoma patients from healthy subjects, detect the development of early stage tumors in a mouse model of spontaneous pancreatic cancer, and monitor tumor growth in patient derived xenograft mouse models of pancreatic cancer. FF-nPES thus appears to exhibit strong potential for the direct analysis of EV membrane biomarkers for disease diagnosis and treatment monitoring.
Highlights
Tumor biopsies remain the gold standard for diagnosis, but are limited by how a tumor can be resolved and how amenable it is to biopsy
Nanoparticle tracking analysis demonstrated that PANC-1 Extracellular vesicles (EVs) isolated by ultracentrifugation had a mean diameter of ~130 nm and primarily overlapped the size range attributed to exosomes (30 – 150 nm) (Supplementary Figure 1A), while transmission electron microscopy found that these particles demonstrated the cup-shaped morphology characteristic of exosomes (Supplementary Figure 1B) (György et al, 2011)
Western blot analysis found that EV lysates contained epithelial cell adhesion molecule (EpCAM) and the cytosolic phosphoprotein tumor susceptibility gene 101 (TSG101) but not the mitochondrial membrane protein voltage-dependent anion-selective channel 1 (VDAC1), used as negative control for exosome samples (Supplementary Figure 1C) (Lötvall et al, 2014)
Summary
Tumor biopsies remain the gold standard for diagnosis, but are limited by how a tumor can be resolved and how amenable it is to biopsy. Analysis of tumor-derived or tumor-associated factors (e.g. circulating tumor cells (CTCs) and DNA (ctDNA), extracellular vesicles (EVs), and various protein biomarkers) in plasma or serum offer an attractive alternative. Such approaches have attracted substantial interest over the past decade for their ability to evaluate cancerous lesions, including primary and metastatic tumors, and permit serial analysis of their response to treatment. In contrast to CTCs, ctDNA, and many proteins biomarkers, tumor-derived EVs are relatively abundant and stable in the circulation, contain factors that reflect the phenotype of their parent cell, and are secreted by all tumor cells and should reflect tumor heterogeneity (Kalluri, 2016)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
