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Nano and Micro Technologies

Nano and Micro Technologies - Poster Session B

Poster J17 - Dynamic Analysis of Deubiquitinating Enzymes Activity in Single Multiple Myeloma Cells Using a Droplet Microfluidic Approach

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  In recent years, there has been increased interest in studying molecular-targeting therapeutics, specifically targeting the ubiquitin-proteasome system (UPS), the primary cellular pathway responsible for degrading and managing misfolded proteins. Protein ubiquitination is facilitated by a cascade of enzymes to conjugate a polyubiquitin chain to a protein to signal the proteasome to degrade the protein. This can be reversed by deubiquitinating enzymes (DUBs), which cleaves the polyubiquitin chain from the target protein, saving it from degradation. Increased DUB and proteasome activity have been correlated with several diseases, especially cancer, that require it to maintain increased protein levels making both enzymes favorable clinical targets. Clinically approved proteasome inhibitors (e.g., Bortezomib, BTZ) and pre-clinical DUB inhibitors (e.g., PR-619) have been developed as potential therapeutics. These therapeutics have shown success in multiple myeloma (MM). MM cells are proliferative, meaning they overproduce defective proteins that need to be degraded by the proteasome; however, a subset of patients either relapse or do not respond to the therapeutics due to the heterogeneity of MM cells. Currently the underlying mechanism(s) of drug resistance are unknown due to the heterogeneous nature of cancer with single cells all behaving differently. In this project, we aimed to quantify DUB activity in a model MM cell line under both basal and drugged conditions to identify distinct subpopulations. We will accomplish this by leveraging a long lived, cell-permeable, fluorescent, DUB specific, peptide-based reporter and a droplet microfluidic trapping array to perform high-throughput screening (HTS) of DUB activity.

Materials and

Methods:  We have previously developed the DUB reporter (Fig. 1A) which combines a β-hairpin sequence (RWRWR) with a DUB recognition sequence (LRGG). Enzyme quantification occurs by DUB-mediated cleavage of a C-terminal fluorophore (AFC, 7- amino-4-trifluoromethylcoumarin, Fig. 1A) by DUBs causing the fluorophore to emit a bright green signal that can be measured using fluorescence microscopy with a higher the fluorescence signal correlating to enhanced DUB activity. A droplet microfluidic device allows for the co-encapsulation of single MM cells and the DUB reporter in aqueous droplets in a continuous oil phase that are isolated in a downstream trapping array (Fig. 1B) allowing for direct measurement of DUB activity in intact single cells (Fig.1C). We quantified DUB activity at the single cell level using a model MM cell line with enhanced DUB activity (OPM2) under basal conditions and in the presence of either a DUB inhibitor (PR-619) or proteasome inhibitor (BTZ). Cells were imaged under either basal or drugged conditions for 6 h on a 37°C temperature-controlled fluorescence microscope (Lecia Dmi8) fitted with an automated image acquisition platform to generate a rich data set that was analyzed using an automated imaging algorithm (Figure 2) that then identifies the fluorescent cells and then subtracts the background to provide numerical values correlating to DUB activity. Cluster analysis was performed on this large dataset using K-means segmentation across all experimental conditions.

Results, Conclusions, and Discussions: We hypothesized that significant heterogeneity exists in DUB activity across a population of MM cells that can ultimately lead to drug resistance. We anticipated that cells treated with PR-619 will exhibit diminished DUB activity whereas cells treated with only BTZ may have enhanced DUB activity to induce a resistance to proteasome inhibitors which has been shown in the literature. We also suspect that cells under both basal and drugged conditions exhibit a wide range of DUB activity that varies both temporally and across the population of cells suggesting that distinct subpopulations exist with enhanced enzyme activity and low susceptibility to drugs that drive drug resistance. To reach the goal of quantifying DUB activity, we optimized the microfluidic device fabrication workflow to overcome challenges with channel clogging with debris causing the device not to perform properly. A multi-step process was devised and implemented including multiple cutting, flushing, and cleaning steps to minimize debris presence. After the device fabrication process was optimized, different cellular seeding densities were explored to maximize the number of droplets with single cells to assist with image acquisition. We found that a density of 6 million cells per mL resulted in the highest number of individual droplets with single cells to minimize multiple cells confounding image acquisition and analysis. Work is underway using the device to collect the time-dependent fluorescence microscopy images to measure the fluorescent signals correlating to DUB activity under basal and drugged conditions. The large datasets obtained from this approach will be clustering using K means segmentation to identify subpopulations and drug responsiveness that is needed to better characterize MM that is currently not possible using existing bioanalytical techniques.

Acknowledgements (Optional):