Abstract
Cancer cells feature a resting membrane potential (Vm) that is depolarized compared to normal cells, and express active ionic conductances, which factor directly in their pathophysiological behavior. Despite similarities to ‘excitable’ tissues, relatively little is known about cancer cell Vm dynamics. Here high-throughput, cellular-resolution Vm imaging reveals that Vm fluctuates dynamically in several breast cancer cell lines compared to non-cancerous MCF-10A cells. We characterize Vm fluctuations of hundreds of human triple-negative breast cancer MDA-MB-231 cells. By quantifying their Dynamic Electrical Signatures (DESs) through an unsupervised machine-learning protocol, we identify four classes ranging from “noisy” to “blinking/waving“. The Vm of MDA-MB-231 cells exhibits spontaneous, transient hyperpolarizations inhibited by the voltage-gated sodium channel blocker tetrodotoxin, and by calcium-activated potassium channel inhibitors apamin and iberiotoxin. The Vm of MCF-10A cells is comparatively static, but fluctuations increase following treatment with transforming growth factor-β1, a canonical inducer of the epithelial-to-mesenchymal transition. These data suggest that the ability to generate Vm fluctuations may be a property of hybrid epithelial-mesenchymal cells or those originated from luminal progenitors.
Introduction
All cells in the body exhibit a voltage difference (Vm) across the plasma membrane which regulates a wide range of functions such as gene expression, secretion, and whole-cell motility. Cellular Vm at rest varies both between and within cell types. Interestingly, whilst this is ca. −70 mV in mature ‘quiescent’ cells, including nerves and muscles, it is noticeably depolarized (Vm ca. −50 to −10 mV) in proliferating cells, including cancer cells and stem cells1,2.
Vm fluctuates dramatically, both spontaneously and in response to stimuli, in classically excitable tissues such as the heart, muscle, and nerve, which support the generation and conduction of action potentials. The resting Vm of several cell types has been shown to fluctuate3. These include cells with rhythmic activity, e.g. neurons controlling respiration4, arterial vasomotion5, biological ‘clocks’6, and sleep7,8. Oscillations of Vm also manifest in pathophysiological situations, such as epilepsy and neuronal degeneration, and can extend to network effects9,10.
In several carcinomas, functional expression of voltage-gated sodium channels (VGSCs) promotes the metastatic process11. Treating carcinoma cells in-vitro with VGSC blockers partially suppresses 3D invasion12,13. The most specific inhibitor of VGSCs is tetrodotoxin (TTX), which blocks the channel by binding to a site within the channel pore when the channel is in the open state14. TTX reduces invasion in carcinoma cells in-vitro, and this effect is abolished by siRNA silencing of the VGSC Nav1.5 in-vivo12,13,15,16. Gradek et al. recently demonstrated that silencing of SIK1 induces Nav1.5 expression, invasion, and the expression of the epithelial-to-mesenchymal transition (EMT)-associated transcription factor SNAl117. However, as noted above, the steady-state resting Vm of human breast cancer cells relative to normal epithelia is strongly depolarized18. In the case of the MDA-MB-231 cells, derived from a highly aggressive triple-negative breast cancer (TNBC), Vm rests between −40 and −20 mV15,19,20. The Vm-dependent inactivation of VGSCs means that the majority of channels should be permanently inactivated at such depolarized membrane potentials and therefore insensitive to TTX. Nevertheless, TTX has been shown repeatedly to inhibit the invasiveness of these cells and several other carcinomas11,15,19,21,22,23,24, potentially by blocking the persistent window current19,25.
Although the depolarization of resting Vm and the enriched VGSC expression in aggressive cancer cell lines are established (reviewed by Yang and Brackenbury2 and Pardo and Stühmer26), unlike classical excitable tissues (e.g., heart, muscle, nerve), comparatively little is known about cancer’s Vm dynamics. Studies utilizing multi-electrode arrays detected Vm fluctuations but could not attribute them to individual cells27,28. Here, in contrast, we captured cellular-resolution, spatially resolved Vm dynamics in human breast cancer cells with a fast, electrochromic voltage-sensitive dye, enabling optical monitoring of Vm changes in hundreds of cells simultaneously. Through an unsupervised machine learning protocol, we classified and characterized the dynamic electrical signatures (DESs) of the cellular Vm time series obtained with high-throughput imaging. A subset of MDA-MB-231 breast cancer cells exhibited hyperpolarizing “blinks” and “waves”, in contrast with the quiescent, static Vm of non-tumorigenic MCF-10A cells. Application of TTX suppressed the Vm fluctuations in MDA-MB-231 cells whilst treatment of MCF-10A cells with transforming growth factor-β1 (TGF-β), which stimulates EMT, induced Vm fluctuations in these cells. Taken together, these data suggest that the ability to generate Vm fluctuations is acquired during the EMT and may participate in cancer progression.
Results
Di-4-AN(F)EP(F)PTEA fluorescence ratio linearly reports change in V m
We imaged the membrane potential of cultured cell monolayers with extracellularly applied di-4-AN(F)EP(F)PTEA, a dye that inserts into the outer membrane, shifting its absorption and emission spectra as a function of membrane potential with sub-microsecond temporal fidelity29. We sequentially excited the dye with blue and green light-emitting diodes (LEDs, Figs. 1a, b and S1), taking the ratio of fluorescence excited by each color at each point in time and dividing by the baseline ratio (ΔR/R0). A change in Vm causes the fluorescence excited by each color to change in opposite directions (Fig. 1c), amplifying the corresponding change in the ratio. The ratiometric imaging scheme also partially mitigates the confounds of uneven dye labeling, photobleaching decay, and mechanical motion30. This approach enabled us to image the dynamics of hundreds of human breast cancer cells simultaneously with cellular resolution.
We first verified that the fluorescence ratio (ΔR/R0) linearly reported changes in Vm. By imaging Di-4-AN(F)EP(F)PTEA fluorescence, while stepping Vm through a range of values in whole-cell voltage clamp of MDA-MB-231 cells, we observed that the ratio of blue-to-green Di-4-AN(F)EP(F)PTEA fluorescence varied linearly with changes in Vm over a physiological range of Vm from −60 to +30 mV. The slope of ΔR/R0 vs. Vm showed an average sensitivity of 5.1 ± 0.43% per 100 mV (mean ± standard error of the mean (s.e.m.); n = 12 cells; Fig. 1d–g). Furthermore, as expected, global depolarization of all cells on the coverslip by washing in a high-potassium (100 mM) extracellular solution also increased ΔR/R0 (Fig. S2). These results show that the ratio ΔR/R0 faithfully reports Vm changes in subsequent recordings of spontaneous Vm fluctuations (Fig. 1h).
Membrane voltage fluctuates in human breast cancer cells
We compared the frequency of optically detected, transient, negative-going events (“−VEs”) in non-tumorigenic MCF-10A cells to that of eight human breast cancer cell lines: MDA-MB-231, MDA-MB-468, Cal-51, SUM-159, Hs578T, MDA-MB-453 and BT-474, and T-47D. Figure 2 displays the rate of −VEs for each field of view imaged. All of the cancer cell lines exhibit a −VE rate significantly greater than that of the benign MCF-10A breast epithelial line: MDA-MB-231 (p = 1.4 × 10−8), MDA-MB-468 (p = 1.1 × 10−8), Cal-51 (p = 1.1 × 10−5), SUM-159 (p = 3.4 × 10−3), Hs578T (p = 5.7 × 10−6), MDA-MB-453 (p = 5.2 × 10−5), BT-474 (p = 2.2 × 10−11), and T-47D (p = 0.01). The p-values are for the comparison of the average per field-of-view negative event “−VE” rate for each cancer cell line compared to the non-tumorigenic MCF-10A line using a Mann–Whitney U test with Benjamini–Hochberg correction for multiple comparisons (false discovery rate = 0.05). Strikingly, the extent of activity correlated strongly with the line sub-type. Cancer lines previously classified as Luminal B (BT-474, MDA-MB-453) or Basal A (MDA-MB-468) were the most active31 (Fig. 2). But while Luminal A and Basal B lines were less active than Luminal B or Basal A, they were nonetheless markedly more active than non-transformed cells….
