28 Toward an automated microfluidic platform for monitoring immune response to immunotherapy

BackgroundPD1/PD-L1 checkpoint inhibitors are at the forefront of cancer immunotherapies, yet, the overall response rate is only 10–30%.1 Even in initial responders, drug resistance evolves. Circulating biomarkers, including peripheral blood mononuclear cells (PBMCs), soluble and extracellular vesicles-derived PD-L1 are increasingly recognized as non-invasive biomarkers for evaluating the immune-mediated therapeutic responsiveness.2–4 Measuring these markers requires standard assays including flow cytometry, Western Blot and ELISA often found in core facilities. This creates logistic challenges in closely monitoring patients’ immune responsiveness. Yet, close monitoring is critical as it can help avoid prolonged use of a futile therapy which can otherwise lead to the loss of time, and financial burden on patients, healthcare and economy. It would be ideal to have a sensitive tool that can be easily used to frequently monitor patients’ immune responsiveness.MethodsWe integrate electrochemical microsensors and immunomagnetic beads into an automated digital microfluidic (DMF) platform5 to rapidly detect PBMCs, soluble and EV-derived PD-L1 in microliters of sample (figure 1A). This device can handle droplets on electrode arrays automatedly without complex setup (figure 1B). We use magnetic beads to extract EVs (150 nm – 2 mm) on-chip and detect PD-L1-positive ones. We also integrate hydrogel 3D matrix on the microsensors to increase the sensitivity for detecting soluble PD-L1 (~55 kDa, <10 nm).ResultsFirst, we detected as low as 5 cells in a 5 uL droplet PBMC sample (commercial, 103 – 5104 cells/mL) with PBS as control.6 The cyclic voltammetry (CV) characterization and the calibration curve (figure 2A) show an increased electrical signal at higher concentrations and good linearity (R2=0.995) fitted in a logarithmic regression equation. Second, we isolated EVs from E0771 breast cancer cell line culture medium and performed 10–104X dilution. We then captured those Evs with magnetic beads and labeled them with detection antibody with HRP. The CV characterization showed the 51ncreaseed electrical current with higher EV concentration (lower dilution rate) and a good linearity (figures 2B and 3). Last, soluble PD-L1 of 10 pg/mL – 10 ng/mL was measured. The differential pulse voltammetry results showed the sensor response to varied concentrations with good linearity (R2=0.9605) (figure 4).ConclusionsIn this platform, the turnaround time is < 2 hours without costly and bulky infrastructure. We showed the feasibility of the device to 1) quantify as low as 5 PBMCs in 5 uL sample; 2) extract and detect EV-derived PD-L1 on-chip; 3) detect the nanoscale soluble PD-L1 with 10pg/mL detection limit.References1. Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell 2017;168(4):707–23. 2. Wei C, Wang M, Gao Q, Yuan S, Deng W, Bie L, Ma Y, Zhang C, Li S, Luo S. Dynamic peripheral blood immune cell markers for predicting the response of patients with metastatic cancer to immune checkpoint inhibitors. Cancer Immunology, Immunotherapy. 2022:1–15. 3. Chen G, Huang AC, Zhang W, Zhang G, Wu M, Xu W, Yu Z, Yang J, Wang B, Sun H. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature. 2018;560(7718):382–6. 4. Wei W, Xu B, Wang Y, Wu C, Jiang J, Wu C. Prognostic significance of circulating soluble programmed death ligand-1 in patients with solid tumors: a meta-analysis. Medicine. 2018;97(3). 5. Zhang Y, Liu Y. Advances in integrated digital microfluidic platforms for point-of-care diagnosis: a review. Sensors & Diagnostics. 2022. 6. Zhang Y, Liu Y. A digital microfluidic device integrated with electrochemical impedance spectroscopy for cell-based immunoassay. Biosensors. 2022;12(5):330.