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

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.