A Microfluidic Platform for Single Cell Fluorometric Granzyme B Profiling

Abstract

Granzyme B (GrB) is an essential cytotoxic effector in cancer immunotherapy as it can be a potential biomarker to predict the efficacy of immunotherapies including checkpoint inhibitors. Monitoring the Granzyme B activity in cells would help determine a patient's clinical response to treatment and lead to better treatment strategies by preventing administration of ineffective therapies and avoid adverse events resulting in a delay in subsequent treatment.Methods: A microfluidic device with hydrodynamic traps and pneumatic valving system was fabricated using photo and soft lithography. Single cell Granzyme B (GrB) activity was detected and measured fluorometrically using a commercial assay kit with a peptide substrate containing GrB recognition sequence (Ac-IEPD-AFC) and AFC (7-Amino-4-trifluoromethylcoumarin) label. Fluorescence was observed and measured using a confocal microscope with CSU-W1 scanner unit and CCD camera as well as an inverted microscope with photodetector. Model cells (NK-92, GrB-transduced Jurkat, and THP1 cells) and human PBMCs from healthy donor and lung cancer patients including an anti-PD-1 antibody treated patient were profiled of its GrB activity as proof of concept.Results: GrB expression from the model cells was found to be markedly different. NK-92 cells were found to have higher GrB activity than the GrB-transduced Jurkat cells. THP-1 was found to have relatively no significant activity. A marked increase in GrB expression was also observed in anti-PD-1 treated lung cancer patient sample in comparison to PBMC from a healthy donor. TCR+ Ig-G4+ PBMC cells were found to have high activity which signifies a clear response to PD-1 blockade.Conclusion: As proof of concept, we have shown the capability of a microfluidic platform to measure GrB production through a single cell enzymatic activity assay. Our platform might be a promising tool for evaluating the sensitivity of immunotherapies and identifying specific T cell subset responsible for the anti-tumor response.

A design and optimization of a high throughput valve based microfluidic device for single cell compartmentalization and analysis

“…The previous version of the fabricated device, reported elsewhere 20 , had 1080 traps and corresponding chambers. In order to have a higher throughput and better representation of the sample population, the microfluidic chip was expanded up to 5000 traps/chambers.…”

“…Initially, the fabricated microfluidic chip following the simulation results made use of a 10:1 PDMS ratio for the flow layer and 5:1 ratio for the control layer. The said ratios were used in congruence with the device used for single cell granzyme B profiling of model cells and PBMC samples reported elsewhere 20 . However, the said ratio for PDMS-PDMS bonding is not the optimized ratio to yield a stronger bonding for multi-layer softlithography.…”

Design and Optimizations of a High-throughput Valve-based Microfluidic Device for Single Cell Compartmentalization and Analysis

“…The previous version of the fabricated device, reported elsewhere 22 , had 1080 traps and corresponding chambers. In order to have a higher throughput and better representation of the sample population, the microfluidic chip was expanded up to 5000 traps/chambers.…”

The Present and Future Role of Microfluidics for Protein and Peptide-Based Therapeutics and Diagnostics

“…Complex MF structures can be printed with high resolution to allow for precise control and monitoring of the environment within the MF chip. Recent enzyme studies using MFs [73] incorporate hydrodynamic traps within the MF chip to monitor an enzyme's activity, like Granzyme B, to determine an individual's response to therapeutic treatment and improve prognosis [74]. This type of assessment offers a high standard for determining clinical response as it focuses on single-cell activity, using a life (rather than fixed) cell [73].…”

“…Recent enzyme studies using MFs [73] incorporate hydrodynamic traps within the MF chip to monitor an enzyme's activity, like Granzyme B, to determine an individual's response to therapeutic treatment and improve prognosis [74]. This type of assessment offers a high standard for determining clinical response as it focuses on single-cell activity, using a life (rather than fixed) cell [73]. By exploiting this fact, it is possible to measure a cell's response over a prolonged duration of time, giving a clearer image of a patient's response to therapy.…”