Single‐Cell Isolation Chip Integrated with Multicolor Barcode Array for High‐Throughput Single‐Cell Exosome Profiling in Tissue Samples

Abstract Exosomes, functional biomarkers involved in cancer progression, have gained widespread attention for promoting tumor formation, growth, and metastasis. Current bulk exosome detections in bodily fluids enable cancer functional analysis, but average secretion levels from cell populations, losing parent cell information and ignoring exosome heterogeneity from diverse cell subgroups, necessitating an effective platform for analyzing single‐cell exosome functional heterogeneity. Here, a high‐throughput platform is presented, capable of efficient single‐cell isolation and multi‐color exosome phenotype analysis, as well as quantifying trace exosomes secreted by single cells. Photothermal‐driven single‐cell chips achieve significant single‐cell isolation efficiency (≈97%) within 5 min, facilitating the ultra‐high throughput single‐cell exosome analysis. By conducting mass spectrometry and protein interaction of breast cancer exosome phenotypic proteins, key exosome phenotypes are identified. Tens of thousands of single cells from breast cancer cell lines, and clinical tissues are analyzed, revealing various subgroup differences. The study finds more CD44 and EGFR co‐expressing exosome subgroups in breast cancer cell lines, while immune‐evasion PD‐L1 high‐phenotype exosome subgroups are primarily presented in complex tumor microenvironments, especially in HER2‐positive tissues. This platform offers powerful single‐cell isolation, exosome quantification, and phenotypic analysis capabilities, making it a powerful tool for advancing single‐cell exosome analysis in cancer research.