Single‐Vesicle Functional Nanophenotyping Reveals Heterogeneous PD‐L1 Accessibility in Triple‐Negative Breast Cancer

The functional accessibility of membrane proteins is a fundamental determinant of biological activity but cannot be accurately inferred from protein abundance alone. Current extracellular vesicle (EV) analyses primarily quantify expression levels, providing limited information on whether surface ligands are physically accessible for molecular interactions. Here, we establish high-speed atomic force microscopy (HS-AFM) as a label-free, real-time approach for directly visualizing and quantifying ligand accessibility on individual small extracellular vesicles (sEVs) under near-physiological conditions. Using programmed death-ligand 1 (PD-L1) as a model immune checkpoint, we characterize antibody binding dynamics on sEVs derived from luminal breast cancer cells, triple-negative breast cancer (TNBC) cells, and patient-derived TNBC cells. PD-L1-high TNBC sEVs exhibit frequent, stable antibody docking with prolonged dwell times, whereas PD-L1-low luminal sEVs display predominantly transient interactions despite detectable PD-L1 expression. Patient-derived sEVs show intermediate binding dynamics, revealing functional heterogeneity that is not captured by bulk protein measurements. By integrating binding frequency and interaction dwell time, we establish a quantitative framework for nanoscale functional phenotyping of individual vesicles. These findings identify ligand accessibility as a distinct biophysical property complementary to protein abundance and establish HS-AFM as a platform for investigating membrane protein function at single-vesicle resolution.