Abstract A030: Rapid extraction and detection of extracellular vesicle-derived PD-L1 in a microfluidic platform

Abstract

Abstract Background. Extracellular vesicles (EV) are emerging as new biomarkers for cancer diagnostics and therapeutic monitoring1. For example, in patients that receive PD1/PD-L1 immune checkpoint inhibitors (a key pillar of cancer immunotherapy), circulating EV-derived PD-L1 is gaining attention as a non-invasive biomarker for therapeutic efficacy. Standard methodologies to measure EV-derived PD-L1 requires ultracentrifugation to first separate EVs and then quantify those that express PD-L1 using nano flow cytometry. These are tedious processes that require trained technologists in centralized facilities, which is a key barrier to frequently monitoring EV-based biomarkers. Yet, frequent monitoring is crucial to quickly identify and stop an ineffective therapy while more fruitful options are still available. Method. To bridge the gap, we develop a proof-of-concept digital microfluidic (DMF) device that can separate EVs directly from biofluids and quantify PD-L1+ EVs automatedly. Briefly, DMF device supports automatedly droplet handling, including droplet transport, splitting and mixing, based on electrowetting principles2. Immunomagnetic beads were used to capture EVs from biofluids, and EVs were then detected by electrochemical sensors inserted into the device. These tips of the electrochemical sensors were modified with gold nanoparticle self-assembled layers to enhance target binding, and CD9 antibodies were immobilized on the tips to bind to EVs. We then used anti-PD-L1 as secondary antibodies for target detection. Differential Pulse Voltammetry (DPV)3 were performed to obtain the results. Results. We used the DMF device to extract 109- 1011 #/mL EVs secreted by a human breast cancer cell line (MDA-MB-231). Briefly, 10 mL of cell culture media was loaded into the DMF device, and the sample was mixed and incubated with a 10 mL droplet of immunomagnetic beads to capture EVs. After 5 min, EVs were eluted in 20 mL of elution buffer, and the beads were discarded. We estimated the number of EVs using Nanoparticle Tracking Analysis; the number of EVs extracted on-chip was comparable with those that were extracted using standard in-tube method. We also verified the purity of the EVs extracted on-chip using Western Blot. Our results showed that the electrochemical sensors can detect as low as 104 #/mL PD-L1+ EVs, with a linear range of 104 – 107 #/mL (R2=0.9701). The entire process can be completed within 1 hour. Conclusion. We integrated automated EV extraction and their surface marker detection in a single DMF device. Our next step is to extract and detect EV-based biomarkers from plasma samples. This platform holds promise for the regular monitoring of EV-based biomarkers that are indicators of therapeutic responses in patients that receive cancer immunotherapies.