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.

癌细胞的MMP9活性在肿瘤的发展中起着至关重要的作用。在细胞-细胞和细胞-基质相互作用过程中，肿瘤细胞处于粘附状态。然而，对于当前平台（例如，典型的液滴系统）来说，在粘附概况中测量单个细胞分泌的MMP9并检索感兴趣的细胞用于下游分析是具有挑战性的。本文中，我们报告了一种基于“虚拟液滴”的单细胞蛋白酶传感器（SCPS），该传感器可以满足上述两个要求。SCPS可以在一分钟内形成一个液滴一个液滴的轮廓，从而在一分钟内形成648个虚拟液滴，该液滴提供了一个隔离的空间，带有支持细胞粘附的基质，并可以在两个小时内对数百个单个粘附的细胞进行MMP9测量。除了MMP9产生中癌细胞的高度异质性外，我们还发现粘附的MDA-MB-231细胞比悬浮细胞产生的MMP9相对更多。此外，回收产生高MMP9和低MMP9的细胞，并对其进行mRNA序列。结果表明，在高MMP9细胞中，丰富了与细胞迁移和侵袭相关的KEGG途径，并且上调了一些相关基因（例如FYN，MAPK3，RAF）。这种SCPS系统为测量单细胞的蛋白酶活性提供了一种有前途的策略。RAF）在高MMP9细胞中上调。这种SCPS系统为测量单细胞的蛋白酶活性提供了一种有前途的策略。RAF）在高MMP9细胞中上调。这种SCPS系统为测量单细胞的蛋白酶活性提供了一种有前途的策略。