(592c) Immunoaffinity Isolation of EpCAM Expressing Exosomes Utilizing High Throughput Microfluidic Chip with Iedda Chemistry (EpCAM-TCOOncoBean Chip)

Liquid biopsy in cancer diagnosis typically lacks specificity and progression status of the tumor, but the incorporation of exosome as a biomarker offers a more expansive and informed view of the tumor landscape ^[1]. Henceforth, we present this study with the overall objective of investigating the microfluidic isolation of tumor derived exosome (TDEs) using the OncoBean Chip. Previously, our device had been functionalized with a Neutravidin and Biotin paired chemistry for isolation of CD63 expressing TDEs but the chemistry included interfering release and TDEs expressing a tumor biomarker were deemed more relevant to tumor characterization. Therefore, we adapted a 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.

Introduction:

Cancer has been defined as the world’s second leading cause of death, with non-small lung cancer (NSCLC) being the most prevalent among cancer related deaths. Factors contributing to the high mortality rate stem from poor prognosis technology and late-stage detection to racial and socioeconomic disparities. Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein highly expressed in rapidly growing epithelial tumors, henceforth is identified as a tumor-associated antigen. Previously, EpCAM levels have been used in conjunction with the CellSearch system to correlate circulating tumor cell (CTC) expression of EpCAM to patient overall survival (OS) rate in breast, prostate, and non-small lung cancer patients ^[2]. However, CTCs are increasingly rare in blood samples, about 1-10 CTC/mL for metastatic patients and even less for non-metastatic patients. The scarcity of EpCAM expressed CTCs makes current isolation techniques tedious for prognostic applications. Recently, exosomes have been identified as a fitting alternative for CTC biomarkers, primarily due to their large abundance in various bodily fluids. These exosomes are a class of extracellular vesicle (EVs) (50-250nm) secreted from the membrane of almost all cells. Due to their biogenesis, these EVs contain bioactive constituents specific to the parent cell. Specially, tumor derived exosomes (TDE) have been shown to play an essential role in the formation and progression of cancer, while encapsulating significant bioactive material from the tumor cell. These TDE operate similarly to a seed-and-soil relationship within the body, where the bioactive material act as a seed for the tumor by prepping the microenvironment for the flourishment of a new tumor. Additionally, these bioactive constituents can act as promising biomarkers for cancer diagnosis and prognosis. Henceforth, we propose an immunoaffinity microfluidic device for the isolation and release of these EpCAM expressing TDE for downstream characterization as a plausible prognosis approach for NSCLC.

Material & Methods:

The device we propose is the OncoBean chip, it 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 TDEs extracted from cell culture media by ultracentrifugation. Contradictory to original chemistry of the Oncobean chip, this device utilizes a two-part functionalization with both the surface of the device and the sample being functionalized. 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 of captured TDEs through the reduction of the disulfide bond. Finally, trans-cyclooctene (TCO) is attached as the exposed agent for capture of TDEs. The exosome sample is functionalized with an EpCAM or CD63 antibody (Ab) conjugated to a tetrazine (Tz) molecule via incubation. The anti-^TzEpCAM conjugate bonds to surface EpCAM on the TDEs creating a ^EPCAM-TzTDE complex for isolation. The TDEs are processed through the functionalized OncoBean chip for Ab specific isolation through the powerful binding of TCO and the ^EPCAM-TzTDE conjugate. We tested and validated the EpCAM specific isolation and confirmed TDEs were forming complexes with the ^TzAb conjugates and not arbitrarily sticking to the nodes. This was done by flowing a sample of TDES and 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.

Results and Discussion:

We have demonstrated the successful capture of majority EpCAM expressed TDE from H1650 and A549 cell lines. The radial flow profile of the OncoBean coupled with the IEDDA chemistry allows for high throughput processing of samples. After device functionalization, sample ^Tz-AbTDE were processed through the OncoBean chip. Device effluent was collected for capture efficiency using nanoparticle tracking analysis (NTA). Additionally, TDE isolation was confirmed and quantified by NTA which reveals capture efficiency up to 90% for the H1650 ^EpCAM-TzTDEs conjugates. Capture efficiency of the no Ab control was found to be at 20% and below while the A549 control, was found to be at about 40% 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).

1. Yu, W., et al., Exosome-based liquid biopsies in cancer: opportunities and challenges. Ann Oncol, 2021. 32(4): p. 466-477.
2. Nicolazzo, C., et al., EpCAM(low) Circulating Tumor Cells: Gold in the Waste. Dis Markers, 2019. 2019: p. 1718920.