Dissecting Cellular Diversity in Paracrine Signaling with Single-Cell Secretion Profiling

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Paracrine signaling is a pivotal biological process facilitating intercellular communication; however, the absence of methodologies enabling concurrent profiling of heterogeneous paracrine mediators with single-cell resolution hampers the mechanistic understanding of this regulatory paradigm. Here, we developed a spatially patterned antibody barcode microchip for high-throughput mapping of paracrine mediators, including cytokines, chemokines, and extracellular vesicles (EVs), facilitating systematic decoding of homotypic and heterotypic paracrine interaction networks. Applying this platform, we investigated cell-number-dependent secretion dynamics in three human cell models: THP-1 macrophages, HMC3 microglia, and SH-SY5Y neurons. The results revealed paracrine-mediated attenuation of secretory function in homotypic systems, whereas exosome secretion was largely cell-number-independent. Notably, macrophages and microglia exhibited divergent effects on neurons, with macrophages suppressing neuron's secretion while microglia enhancing it; both effects were strongly dependent on paracrine cell number. Furthermore, we identified previously undifferentiated heterogeneity in secretions from single cells, revealing significant variations among cell populations. This study demonstrates the mapping of different signaling mediators from paracrine single-cell pairs, providing insights into how cells communicate with neighboring cells through complex paracrine signaling networks.