Ultra-Sensitive Automated Profiling of EpCAM Expression on Tumor-Derived Extracellular Vesicles

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.

Isolation and characterization of exosomes for cancer research

“…This technique meets the rigorous demands of separating exosomes that contain specific target membrane proteins. The conclusion that capture-based techniques involving the Ep-CAM biomarker represent the best approach for separating exosomes in comparison with other methods has been widely accepted due to comprehensive analyses of the efficiency of recycling exosomes [ 47 ]. Recently, a study revealed that an approach for isolating EVs from urine using the Vn96-peptide, which specifically binds to EVs containing a heat shock protein, is much faster than traditional methods in prostate cancer, such as UC [ 48 ].…”

Exosomes as prognostic biomarkers in pancreatic ductal adenocarcinoma—a systematic review and meta-analysis

“…Our search key identified 2224 records. Of the 18 articles eligible for qualitative synthesis 3,11,17,18,[20][21][22][23][24][25][26][27][28][29][30][31][32][33] , 11 were suitable for quantitative synthesis 3,11,17,18,[20][21][22][23][24][25][26] . The 904 patients included in our review comprise only nonoverlapping populations.…”

Exosome-Mediated Crosstalk Between Tumor and Tumor-Associated Macrophages

“…It also can be used for qualitative and quantitative determination of exosomes. Nevertheless, the storage conditions of exosomes obtained by immunoaffinity chromatography are relatively harsh and are not suitable for large-scale separation of exosomes ( Amrollahi et al, 2019 ). The method of polymer precipitation usually uses polyethylene glycol (PEG) as a medium to harvest exosomes under the centrifugation condition by reducing the solubility of the exosomes.…”