Multi‐Phenotypic Exosome Secretion Profiling Microfluidic Platform for Exploring Single‐Cell Heterogeneity

Abstract Cellular phenotypic and functional heterogeneities have advanced cancer evolution and treatment resistance. Although exosome‐bound proteins reflect cellular functions, single‐cell exosomes are rarely profiled owing to the lack of effective platforms. Herein, the authors developed an integrated microfluidic platform consisting of a single‐cell trapping chip and a spatially coded antibody barcode chip for the multiplexed outline of exosome secretion by single cells. Using this platform, five phenotypic exosomes of over 1 000 single cells are simultaneously profiled, in addition to inflammatory factor secretion from the same single cell. Also, a robust analysis workflow for single‐cell secretion profiling is proposed to explore the intercellular heterogeneity, which integrated unsupervised clustering and linear clustering. When applied to the tumor cell lines of epithelial‐origin and normal epithelial cell lines, the strategy identifies functionally heterogeneous subpopulations with unique secretion patterns. Notably, special functional cell subsets for unique phenotypic exosomes (HSP70 + , EPCAM + ) are found within ovarian tumor cells. The strategy proposed offers a new analysis approach for cellular differential exosome secretion at single‐cell resolution using inflammatory factors, ultimately reinforcing the understanding of cell‐to‐cell heterogeneity and tumor landscape, and providing a valuable universal platform for single‐cell biomarker exploration in biological and clinical research.