Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.

Cancer Technologies

Cancer Technologies - Poster Session E

Poster S18 - Electrophysiologic Monitoring of Cell Death Mechanisms in Liver Cancer

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Cellular functions, identity, and responses to various conditions are characterized by a cell’s electrophysiological properties and how these properties can change during cellular processes or disease. Dielectrophoresis (DEP) is a technique that derives a cell’s electrophysiological properties from a cell’s distinctive pattern of movement when exposed to various electric frequencies, such as: cytoplasmic conductivity, membrane conductance, and membrane capacitance.
This label-free cell characterization has many applications. Most relevant to this project is the ability of DEP to distinguish small differences in cells’ response over time or as a result of drug treatment. Presented in this abstract is novel research that tracks the electrophoretic response of HEPG2 human liver cancer cells to cisplatin, a cancer drug that is known to induce apoptosis, and RSL3, which induces ferroptosis. By correlating dielectric property changes with both cell death mechanisms, this research aims to both study and quantify the rate of cell death through apoptosis and ferroptosis to understand mechanistic differences and potentially new predictive markers for cancer cell death.

Materials and

Methods:  Cell Treatment and Preparation:
The liver cancer line HEPG2 were cultured in RPMI under standard conditions (ATCC). Prior to 3DEP experiments, individual populations of HEPG2 were grown to confluence. Cells were plated with 2 million cells per flask prior to treatment. Cells were treated with 5uM of cisplatin and 2.5uM of RSL3. Treatments were incubated for 24 hrs.

Cells were trypsinized and centrifuged at 250 g for 5 minutes. Cells were washed twice and resuspended at 106 cells per ml in isosmotic DEP 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 and with NaOH for 7 pH. Cell viability, radius, and conductivity were measured.

DEP Analysis:
We measure DEP response using the 3DEP platform. Approximately 80 μL of cells, suspended in DEP media, were loaded into the 20-well 3DEP chip and loaded into the 3DEP reader. The 3DEP program was run, energizing cells in each well with a unique frequency within a set range (1kHz- 20MHz) for 30 seconds. This produces a spectra from the dielectrophoretic responses across all 20 wells. Five technical replicates were produced for each treatment group.

Data Extraction:
The 3DEP system fits each dataset to a model of the Clausius-Mossoti factor and determines values for cytoplasmic conductivity (σcyt), specific membrane conductance (Geff) , and specific membrane capacitance (Ceff). One-way ANOVA tests were used to analyze property changes between control and treatment groups across experimental timepoints.

Results, Conclusions, and Discussions: The mean values of each treatment’s cytoplasmic conductivity (σcyt), specific membrane conductance (Geff) , and specific membrane capacitance (Ceff) indicated general trends, with minimal significant differences found between our control (non-treated) cells and the apoptosis and ferroptosis treatments (Figure 1). Membrane conductance (GEFF) indicative of ionic transport across the membrane and its surface increased with both treatments. Similar to other cell death investigations using DEP, we also found that cytoplasm conductivity (σcyt) indicative of free ionic concentration within the cytoplasm decreased with treatment.

From the results, we found that with both treatments while Geff increases, σcyt decreases with treatment. We believe this is due to what similar DEP studies investigating apoptosis have observed, where ions begin to leak out of the cell with induced cell death. While we did not find any significant difference between the cisplatin and RSL3 treatments in the DEP parameters, we did find a 40.4% percent difference between the cisplatin and RSL3 treatment’s Ceff of the cells. Ceff is dependent on cell morphology. This decrease is in agreement with previous DEP studies that monitored the morphological changes of Jurkat T-cells as they progressed through chemically induced apoptosis. These cells also showed decreases in membrane capacitance, reflecting a smoothing of the membrane through loss of microvilli.

Future work will investigate different concentrations of cisplatin and RSL3 to better determine if further DEP-based differences can be found between the two different cell death mechanisms. We will investigate other electrical parameters specific to the cell membrane, such as surface zeta potential, and membrane potential. We will also conduct SEM imaging of the treatment groups to better characterize physical changes to the cell membrane.

This work demonstrated DEP’s ability to monitor cancer cells undergoing different induced cell death mechanisms (ferroptosis and apoptosis). Membrane capacitance, indicative of a cell’s morphology, showed the largest difference with treatment giving promise to this technique as a way to monitor varying cell death pathways in cancer treatments.

Acknowledgements (Optional):  This project was supported in part by the eREU KEEN grant in the Department of Biomedical Engineering at Wake Forest University School of Medicine. HEPG cells were provided by Prof. Bierbach from the WFU department of chemistry.