(J-385) Investigating Oxidative Stress in Cancer Using Electrophysiology

Introduction::

Oxidative stress (OS) is one of the leading causes of cytotoxicity and is linked to many human physio-pathological conditions. There are critical gaps in the study of cellular oxidative stress (OS) including mechanistic pathways, OS agent-specific biophysical effects, as well as the assessment of therapeutic agents. Recent examples include myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), T-cell dysregulation in inflammatory/infectious diseases, anti-cancer therapies, neurodegenerative diseases, and blood-related diseases. 

The sensitivity of electrophysiology has been shown to discriminate subtle differences between cells- differences that can occur prior to any changes in morphology or labeling. Some attention has been given to electrophysiology as a marker for OS and in examining anti-oxidant agents. Dielectrophoersis (DEP) is a technique in which non-uniform alternating (AC) electric fields induce cellular motion, dependent on both AC field frequency and unique, cellular electrical properties.  3DEP (Deptech, UK) measures the mean response of approximately 20,000 cells yielding real-time electrophysiological parameters including effective membrane conductance - indicative of ionic transport across the membrane and on its surface, effective membrane capacitance- indicative of membrane morphology, and cytoplasmic conductivity- indicative of  ionic concentration within the cytoplasm.

Cancer-associated redox imbalance suggests that redox-modulating treatments could provide an effective strategy to induce cancer-specific cell death. Differences in cellular susceptibility to redox imbalance can serve as a basis for the development of promising anticancer therapies. The objective of this work was to establish a dose response of the 3DEP assay to characterize OS in Jurkat cells, and distinguish the severity of OS-related cellular damage.

Materials and Methods:: Cell Maintenance
Jurkat cells were suspended in RPMI culture media supplemented with 1% glucose, 1% Pen-Strep, and 10% FBS (Millipore Sigma, US) and proliferated in an incubator at 37℃ at 5% oxygen. At confluence, cells were passed and prepared for experiments.

Cell preparation
A stock solution of 98mM of hydrogen peroxide (Fisher Scientific, US) in RPMI complete media was used for all treatments media for a final treatment concentration of 10 µM for two hours drug exposure. Once the drug incubation was complete, cells were imaged, counted, and centrifuged to remove the drug treatment. Pellets are resuspended in isosmotic 3DEP media, consisting of 8.5% (w/v) sucrose, 0.5% (w/v) dextrose, 100 µM CaCl2 and 250 µM MgCl2 and adjusted with PBS to 10 mS*m^-1 conductivity.

3DEP Analysis:
Approximately 80 mL Jurkat cells, suspended in 3DEP media, are loaded into 20-well DEP chip. The chip is covered with a glass cover slip and loaded into the 3DEP chip holder. The DEP program is run, energizing cells with a range of frequencies for 30 seconds. The program produces a spectra from the dielectrophoretic responses across all 20 wells. Approximately 5 technical replicates of 3DEP spectra are produced for each treatment group.
Data Extraction:
The 3DEP system fits each dataset to a mathematical model of the Clausius-Mossoti factor and derives values for cytoplasmic conductivity and membrane conductivity and capacitance. Two-way ANOVA tests are used to identify significance in dielectrophoretic property changes between control and Jurkat treatment groups across experimental timepoints.

Results, Conclusions, and Discussions:: Data were analyzed in Prism 8 (Graphpad) with ANOVA analysis. There was no statistical variance (p >0.01) observed between the three control groups for membrane conductance (GEFF), membrane capacitance (CEFF), or cytoplasmic conductivity (σCYT). There was a slight significant difference between (*) between trial 1 and trial 2 treatment groups for membrane capacitance (CEFF). A significant drop in membrane conductance and membrane capacitance was observed in the ROS-treated cells when compared to our control groups. This is in agreement with previous studies of induced cell death and apoptosis in Jurkats that saw reduced membrane capacitance and calcium flux across the membrane.

Though more experiments need to be performed to increase the robustness of our statistical results, we have demonstrated that DEP can be used to assess OS effects in Jurkats providing functional (membrane capacitance and conductance) measures of the cell electrophysiology. Further investigations will examine different mechanisms of actions in various OS-inducing agents to determine whether DEP can differentiate between different OS processes.

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