(265e) High-Throughput Single Cell Analysis of Deubiquitinating Enzyme Activity in Intact Cells

Molecularly-targeted therapeutics have shown significant promise in the treatment of several different cancers. In the treatment of multiple myeloma, drugs designed to inhibit members of the ubiquitin-proteasome system (UPS) have increased median patient survival rates. However, challenges in drug efficacy frequently occur due to tumor heterogeneity, drug resistance, and personalized responses. In the case of proteasome inhibition, recent studies have found that tandem therapies targeting both the proteasome and deubiquitinating enzymes (DUBs) have allowed cells to overcome drug resistance. DUBs are responsible for removing the polyubiquitin chain that targets proteins for proteasomal degradation. This work will demonstrate a new approach to directly quantify DUB activity and DUB inhibitor efficacy at the single cell level across a heterogeneous population. This is accomplished using a previously developed long-lived, cell permeable, fluorescent, DUB-specific, peptide-based reporter capable of delivering a visual readout of DUB activity in intact cells using fluorescent microscopy. Here, the peptide reporter is incorporated into a droplet microfluidic trapping array capable of encapsulating single cancer cells in aqueous droplets and trapping them in an overhead, circular array to facilitate direct imaging. The microfluidic platform is supplemented with a Python algorithm for rapid image analysis and hierarchical cluster analysis for single cell quantification of DUB activity and identification of distinct subpopulations with heterogeneous DUB activity. To assess difference in drug efficacy, the system was used to evaluate DUB activity in three different model myeloma cell lines challenged with different doses and combinations of DUB and proteasome inhibitors. This platform is a first of its kind to perform high-throughput single cell analysis of DUB activity and offers the potential for new studies in personalized diagnostics and drug development.