(697f) High-Throughput, Single Cell Analysis of Peptide Uptake and Deubiquitinating Enzyme Activity Using a Microfluidic Droplet Trapping Array

Recent advances in molecularly-targeted therapeutics, such as the proteasome inhibitors Bortezomib and Carfilzomib, have shown significant promise in the treatment of multiple myeloma. However, challenges in drug efficacy frequently occur due to tumor heterogeneity, drug resistance, and personalized responses. In the case of multiple myeloma, recent efforts have demonstrated that increased activity of deubiquitinating enzymes (DUBs) facilitate a developed resistance to proteasome-based therapeutics. As such, attention has shifted towards personalized medicine consisting of multiple agent therapies. This has created a need for high-throughput biochemical assays capable of quantifying enzyme activity in intact single cells to examine the potential effectiveness of multiplexed therapeutics. The goal of this project was to develop a microfluidic droplet trapping array capable of dynamic, high-throughput, single cell analysis of DUB activity in intact single cells. The microfluidic platform can generate aqueous droplets at a rate of 200 Hz which are immediately trapped in a downstream trapping array with ~99% trapping efficiency. Single cells were encapsulated in ~40-50% of the trapped droplets. As a proof-of-concept, the droplet trapping array was first used to assess cell penetrating peptide (CPP) uptake across a population of cancer cells. These experiments demonstrated a significant heterogeneity in CPP uptake with ~20% of the population of cells exhibited no observable uptake. Next, the microfluidic platform was used to quantify DUB activity in a population of myeloma-derived cell lines. Here, the microfluidic platform was combined with a novel cell permeable, long lived, fluorescent, DUB specific, peptide-based reporter developed by our group. Preliminary findings demonstrate significant heterogeneity in DUB activity across the population of cells in addition to a variable response to the commercially available DUB inhibitor PR-619. Finally, a statistical analysis was performed across cells challenged with the DUB inhibitor to identify drug resistant subpopulations. The work presented here serves a technology first step for ex vivo diagnostics to help in developing treatment protocols using molecularly-targeted therapeutics towards DUBs.