Microfluidic Replication and Phenotypic Profilingof Extracellular Vesicles from the Tumor Microenvironment Using Dual-SwitchAptamer Logic Gates

The phenotypic profiling of extracellular vesicles (EVs) within the tumor microenvironment (TME) provides critical insights into the intercellular communication mechanisms of EVs underlying tumor physiology. However, conventional methods typically isolate EVs from the extracellular space through tissue fragmentation, which compromises tissue viability, and neglects the spatial organization of the tissue and the dynamic nature of EV secretion. Herein, we introduce an innovative microfluidic platform to cultivate intact tumor tissues while preserving their spatial architecture and facilitating natural EV secretion. This system enables the direct replication of EVs onto the chip for high-fidelity phenotypic analysis. Utilizing a combinatorial-aptamer-induced dual-switch logic gate methodology, this approach allows for the precise subtyping of EVs derived from both tumor cells and immune cells within the TME. Specifically, aptamers targeting EpCAM and PD-L1, along with the connector probe, were employed to induce a dual-switch signal to identify distinct EV populations. This strategy enables noninvasive, real-time capture and phenotypic profiling of EVs directly within the microfluidic environment. Furthermore, our findings indicate that immunotherapy with PD-1 antibodies significantly enhances the secretion of EVs by immune cells within the TME, underscoring the potential role of EVs as mediators of therapeutic responses. Overall, we have developed a robust, noninvasive method for the phenotypic profiling of EVs in the TME, offering a powerful tool for investigating the biological functions and implications of EVs in tumor pathophysiology.