A virtual-droplet system for sensing MMP9 activity of single suspended and adhered cancer cells

Abstract Cancer cells’ MMP9 activity plays vital roles in tumor development. Tumor cells are in an adhesion profile during cell-cell and cell-matrix interaction. However, it is challenging for current platforms (e.g., typical droplet systems) to measure MMP9 secreted by single cells in an adhesion profile and retrieve the cells of interest for downstream analysis. Herein, we report a single-cell protease sensor (SCPS) based on ‘virtual droplet’ that can fulfill the above two requirements. SCPS can form 648 virtual droplets within one minute as a one-cell-per-droplet profile, which offers an isolated space with a substrate to support cells’ adhesion and enables MMP9 measurement for hundreds of single adhered cells within two hours. In addition to high cellular heterogeneity of cancer cells in MMP9 production, we also found that adhered MDA-MB-231 cells produced relatively more MMP9 than that done by suspended cells. Furthermore, the cells producing high MMP9 and low MMP9 were retrieved and mRNA-sequence was performed on them. The results demonstrated that some KEGG pathways associated with cell migration and invasion were enriched and a few related genes (e.g., FYN, MAPK3, RAF) were upregulated in high-MMP9 cells. This SCPS system provides a promising strategy for measuring single-cell’s protease activity.