Establishment of a cascaded microfluidic single cell analysis system for molecular and functional heterogeneity analysis of circulating tumor cells

Single cell heterogeneity analysis of circulating tumor cells (CTCs) is expected to reveal important tumor biological information for the evaluation and prediction of tumor stage and metastatic progression. Although various methods have been proposed to isolate and detect CTCs, consecutive CTCs sorting and in-situ multi-dimensional heterogeneity analysis in a cascaded system remain challenging. Here, a cascaded microfluidic chip was constructed, successively integrating a spiral structure for size-based removal of blood cells and a single cell array structure for the subsequent capture of CTCs, complementing the advantages of high throughput and high separation purity for both structures. Importantly, the molecular and functional heterogeneity analysis of CTCs can be in-situ realized and real-time observed at the single-cell level. Using this system, we captured CTCs and situ-analyzed the heterogeneity of single CTCs from different types of cancer patients, finding that mesenchymal phenotype is present in more CTCs at the advanced stage, and CTCs show active motility and division ability in metastatic patients. Therefore, the cascaded microfluidic chip offers an effective strategy for studying the molecular and functional heterogeneity of single CTCs, providing critical information for the evaluation of tumor progression and metastasis.