Human Breast Cancer Cells Demonstrate Electrical Excitability

Abstract

Breast cancer is one of the most prevalent types of cancers worldwide and yet, its pathophysiology is poorly understood. Single-cell electrophysiological studies have provided evidence that membrane depolarization is implicated in the proliferation and metastasis of breast cancer. However, metastatic breast cancer cells are highly dynamic microscopic systems with complexities beyond a single-cell level. There is an urgent need for electrophysiological studies and technologies capable of decoding the intercellular signaling pathways and networks that control proliferation and metastasis, particularly at a population level. Hence, we present for the first time non-invasive in vitro electrical recordings of strongly metastatic MDA-MB-231 and weakly/non-metastatic MCF-7 breast cancer cell lines. To accomplish this, we fabricated an ultra-low noise sensor that exploits large-area electrodes, of 2 mm², which maximizes the double-layer capacitance and concomitant detection sensitivity. We show that the current recorded after adherence of the cells is dominated by the opening of voltage-gated sodium channels (VGSCs), confirmed by application of the highly specific inhibitor, tetrodotoxin (TTX). The electrical activity of MDA-MB-231 cells surpasses that of the MCF-7 cells, suggesting a link between the cells' bioelectricity and invasiveness. We also recorded an activity pattern with characteristics similar to that of Random Telegraph Signal (RTS) noise. RTS patterns were less frequent than the asynchronous VGSC signals. The RTS noise power spectral density showed a Lorentzian shape, which revealed the presence of a low-frequency signal across MDA-MB-231 cell populations with propagation speeds of the same order as those reported for intercellular Ca²⁺ waves. Our recording platform paves the way for real-time investigations of the bioelectricity of cancer cells, their ionic/pharmacological properties and relationship to metastatic potential.

Keywords: bioelectronics; breast cancer; electrophysiology; metastasis; multi-electrode arrays; sensors; voltage-gated sodium channels.

FIGURE 1

(A) Representative diagram of the MEA device with a layer of adherent cells (depicted in red). (B) MC7 cells forming a confluent layer on electrode. The bright part denotes cells on top of the Au electrode; the darker region indicates the cells outside the Au electrode. (C) MDA-MB-231 cells attached to the electrode. (D) Box plot of cell viability studies indicating no significant statistical difference between viability of cells on the MEA vs. culture dish (“X” represents P = 0.98).

FIGURE 2

Electrical activity of MDA-MB-231 cells recorded over a period of up to 3 days. (A) Baseline current measured with only medium over the electrodes’ surface, showing a current of 0.5 pA. (B) Typical asynchronous spiking activity. (C) An expanded trace showing a fast-asynchronous spike. (D) Detailed section of fast spiking activity. (E) Detailed section of square shaped pulses. (F,G) Results from spike characterization over a 12 h period of asynchronous spiking activity of MDA-MB-231 cells, showing the distribution of amplitudes with bin intervals of 20 pA each (F) and pulse widths with bin intervals of 5 ms each (G) Bars represent average values of all 3 repeats and error bars represent standard deviation. (H) Experiment results comparing the spike counts obtained from 24 h measurements on strongly metastatic MDA-MB-231 cells vs. weakly metastatic MCF-7 cells, showing a significantly smaller spike count for the latter.

FIGURE 3

VGSC activity of MDA-MB-231 cells. VGSC activity was blocked using TTX (20 μM). (A) Current trace showing electrical activity before, during and after application of TTX. (B) Quantification of the spikes recorded in (A). Number of spikes were measured in time bins of 6 min. Bars represent average values of all 3 repeats and error bars represent SDs.

FIGURE 4

Time up/down characterization for square pulses seen in MDA-MB-231 current recordings. (A) Distribution of time “up” values with the inset showing the boundaries for this measurement. (B) Distribution of time “down” values with the inset showing the boundaries for this measurement. (C) Current PSD of the square pulses extracted from one of the experiments (black scatter plot) and a calculated PSD using the time constants τ_UP = 1.1 s and τ_DOWN = 2.5 s. A good agreement between theory (curve) and experimental data (points) is apparent. (D) Amplitude histogram of the current magnitudes recorded with a resolution of 5 pA, agglomerating 1260 RTS events which occurred during 3 different experiments. In red, a Gaussian fit guides the eye, to 3 distinct peaks.

FIGURE 5

An overview of a long-term recording of a population of MDA-MB231 cells, at different experimental stages (A–D). (A) RTS and asynchronous bursting prior to adding the chelating agent EGTA. (B) Effect of EGTA on the RTS activity during the 10 min treatment. (C) RTS quiescent region, where some occasional, low amplitude asynchronous spikes and some RTS activity resumes. (D) Recovery of RTS activity seen 8 h following EGTA wash. (E–G) Time traces of the different regions prior, during and following EGTA treatment. (H–J) Show the morphological changes to MDA-MB-231 cells throughout EGTA experiments, with tight junctions being clearly visible in (H), decreased in (I) and forming again in (J).