Single cell Proteolytic assays to investigate clonal heterogeneity and cell dynamics using efficient microfluidic cell loading

Proteolytic degradation of extracellular matrix is critical in cancer invasion. In this work, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, allowing the characterization of proteolytic activities for a small sample (10-100 cells). Combined with a clonal sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activities and also investigated the differences among clones. Furthermore, the platform allows monitoring single-cell dynamics in proteolytic activity. The presented tool facilitates single-cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.