Abstract 2131: Immunoaffinity isolation of EpCAM expressing exosomes utilizing high throughput microfluidic chip with IEDDA chemistry (EpCAM-TCOOncoBean Chip)
Authors: Onukwugha Nna-Emeka , Nagrath Sunitha , McEacheron Henry 
Published: April 2023

Abstract
The overall objective of this study is to investigate the microfluidic isolation of tumor derived exosome (TDEs) using the OncoBean Chip, functionalized with the unique inverse electron demand Diels-Alder (IEDDA) for downstream analysis of patient TDEs for therapeutic applications. This chemistry allows for capture and subsequent release of exosomes for immunomodulation and functional studies. We developed an approach for targeting TDEs in patient’s blood plasma by using the epithelial cell adhesion molecule (EpCAM) antibody, a highly expressed biomarker associated with non-small cell lung cancer.The OncoBean Chip is a high throughput radial-flow microfluidic device designed for the specific isolation of TDEs. The bean shaped micro-nodes allow for a varying shear profile to better capture nanosized particles by positioning them towards the middle curvature. We optimized the device using exosomes extracted from cell culture media by ultracentrifugation. First, the surface of the device was salinized with (3-Aminopropyl) triethoxysilane, as an anchor, following plasma activation of the surface. Then, bonded with 3,3'-dithiobis(sulfosuccinimidyl propionate) as a crosslinker, to facilitate the release. Finally, trans-cyclooctene (TCO) is attached as the exposed agent for capture of TDEs. The exosome sample is functionalized with an EpCAM antibody (Ab) conjugated to a tetrazine (Tz) molecule via incubation. The anti-EpCAM-Tz conjugate bonds to surface EpCAM on the TDEs creating a TDE-EpCAM-Tz complex for isolation. The TDEs are processed through the functionalized OncoBean Chip for Ab specific isolation. We tested and validated the EpCAM specific isolation and confirmed TDEs were forming complexes with the Ab-Tz conjugates and not arbitrarily sticking to the nodes. This was done by flowing a sample of TDE-Tz through a functionalized device where little to no capture was expected. Then we evaluated the isolation of EpCAM specific TDEs by using H1650 and A549 exosomes and comparing the capture with CD63, a common exosome marker. Limited capture is expected in the A549 group due to low EpCAM expression for the Ab-Tz conjugate to bind too.Additionally, EV isolation was confirmed and quantified by NTA which reveals capture efficiency up to 90% for the H1650 TDEs. Capture efficiency of the no Ab control was found to be at 30% and below while the A549 control, was found to be at about 20% and below. Hence, our OncoBean Chip with IEDDA chemistry has successfully demonstrated efficient and specific isolation of EpCAM and CD63 expressing TDEs from two different cell lines. Data from Western Blot analysis and scanning electron microscopy further confirm the physical characteristics and protein expression of the TDEs established by the International Society of Extracellular Vesicles (ISEV).
Citation Format: Nna-Emeka Onukwugha, Sunitha Nagrath, Henry McEacheron. Immunoaffinity isolation of EpCAM expressing exosomes utilizing high throughput microfluidic chip with IEDDA chemistry (EpCAM-TCOOncoBean Chip) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2131.