Single‐Cell Enzymatic Screening for Epithelial Mesenchymal Transition with an Ultrasensitive Superwetting Droplet‐Array Microchip

Abstract The phenotypic changes of circulating tumor cells (CTCs) during the epithelial–mesenchymal transition (EMT) have been a hot topic in tumor biology and cancer therapeutic development. Here, an integrated platform of single‐cell fluorescent enzymatic assays with superwetting droplet‐array microchips (SDAM) for ultrasensitive functional screening of epithelial–mesenchymal sub‐phenotypes of CTCs is reported. The SDAM can generate high‐density, volume well‐defined droplet (0.66 nL per droplet) arrays isolating single tumor cells via a discontinuous dewetting effect. It enables sensitive detection of MMP9 enzyme activities secreted by single tumor cells, correlating to their epithelial–mesenchymal sub‐phenotypes. In the pilot clinical double‐blind tests, the authors have demonstrated that SDAM assays allow for rapid identification and functional screening of CTCs with different epithelial–mesenchymal properties. The consistency with the clinical outcomes validates the usefulness of single‐cell secreted MMP9 as a biomarker for selective CTC screening and tumor metastasis monitoring. Convenient addressing and recovery of individual CTCs from SDAM have been demonstrated for gene mutation sequencing, immunostaining, and transcriptome analysis, revealing new understandings of the signaling pathways between MMP9 secretion and the EMT regulation of CTCs. The SDAM approach combined with sequencing technologies promises to explore the dynamic EMT plasticity of tumors at the single‐cell level.