Predictive Analysis in Oral Cancer Immunotherapy:\nProfiling Dual PD-L1-Positive Extracellular Vesicle Subtypes with\nStep-Wedge Microfluidic Chips

PD-L1-positive\nextracellular vesicles (PD-L1<sup>+</sup> EVs) play\na pivotal role as predictive biomarkers in cancer immunotherapy. These\nvesicles, originating from immune cells (I-PD-L1<sup>+</sup> EVs)\nand tumor cells (T-PD-L1<sup>+</sup> EVs), hold distinct clinical\npredictive values, emphasizing the importance of deeply differentiating\nthe PD-L1<sup>+</sup> EV subtypes for effective liquid biopsy analyses.\nHowever, current methods such as ELISA lack the ability to differentiate\ntheir cellular sources. In this study, a novel step-wedge microfluidic\nchip that combines magnetic microsphere separation with single-layer\nfluorescence counting is developed. This chip integrates magnetic\nmicrospheres modified with anti-PD-L1 antibodies and fluorescent nanoparticles\ntargeting EpCAM (tumor cell marker) or CD45 (immunocyte marker), enabling\nsimultaneous quantification and sensitive analysis of PD-L1<sup>+</sup> EV subpopulations in oral squamous cell carcinoma (OSCC) patients’\nsaliva without background interference. Analysis results indicate\nreduced levels of I-PD-L1<sup>+</sup> EVs in OSCC patients compared\nto those in healthy individuals, with varying levels of heterogeneous\nPD-L1<sup>+</sup> EVs observed among different patient groups. During\nimmunotherapy, responders exhibit decreased levels of total PD-L1<sup>+</sup> EVs and T-PD-L1<sup>+</sup> EVs, accompanied by reduced levels\nof I-PD-L1<sup>+</sup> EVs. Conversely, nonresponders show increased\nlevels of I-PD-L1<sup>+</sup> EVs. Utilizing the step-wedge microfluidic\nchip allows for simultaneous detection of PD-L1<sup>+</sup> EV subtypes,\nfacilitating the precise prediction of oral cancer immunotherapy outcomes.