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. 2022 Aug 31;11(17):2712.
doi: 10.3390/cells11172712.

Real-Time Monitoring of the Effect of Tumour-Treating Fields on Cell Division Using Live-Cell Imaging

Hoa T Le  1 , Michael Staelens  2 , Davide Lazzari  3 , Gordon Chan  4 , Jack A Tuszyński  2   4
Affiliations

Real-Time Monitoring of the Effect of Tumour-Treating Fields on Cell Division Using Live-Cell Imaging

Hoa T Le et al. Cells. .

Abstract

The effects of electric fields (EFs) on various cell types have been thoroughly studied, and exhibit a well-known regulatory effect on cell processes, implicating their usage in several medical applications. While the specific effect exerted on cells is highly parameter-dependent, the majority of past research has focused primarily on low-frequency alternating fields (<1 kHz) and high-frequency fields (in the order of MHz). However, in recent years, low-intensity (1-3 V/cm) alternating EFs with intermediate frequencies (100-500 kHz) have been of topical interest as clinical treatments for cancerous tumours through their disruption of cell division and the mitotic spindle, which can lead to cell death. These aptly named tumour-treating fields (TTFields) have been approved by the FDA as a treatment modality for several cancers, such as malignant pleural mesothelioma and glioblastoma multiforme, demonstrating remarkable efficacy and a high safety profile. In this work, we report the results of in vitro experiments with HeLa and MCF-10A cells exposed to TTFields for 18 h, imaged in real time using live-cell imaging. Both studied cell lines were exposed to 100 kHz TTFields with a 1-1 duty cycle, which resulted in significant mitotic and cytokinetic arrest. In the experiments with HeLa cells, the effects of the TTFields' frequency (100 kHz vs. 200 kHz) and duty cycle (1-1 vs. 1-0) were also investigated. Notably, the anti-mitotic effect was stronger in the HeLa cells treated with 100 kHz TTFields. Additionally, it was found that single and two-directional TTFields (oriented orthogonally) exhibit a similar inhibitory effect on HeLa cell division. These results provide real-time evidence of the profound ability of TTFields to hinder the process of cell division by significantly delaying both the mitosis and cytokinesis phases of the cell cycle.

Keywords: electromagnetic fields; human breast epithelial cells; human cervical carcinoma cells; non-invasive therapies; oncology; spinning disk microscopy; thymidine block; time-lapse microscopy.

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Conflict of interest statement

This study was funded, in part, by Novocure Ltd. The funder had limited involvement with the study; they supplied us with the inovitro™ live system, and gave a brief introduction on its usage and functionality. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
A digital photograph of the Novocure inovitro™ live system, with all four main components shown.
Figure 2
Figure 2
Representative graphs obtained from data logged every 3 s by the inovitro™ live system throughout the exposure of HeLa cells to TTFields with the following parameters: a frequency of 100 kHz, the target medium temperature set to 37 °C, a duty cycle of 1-1, and a duration of ~18 h. The ambient temperature was set to 23 °C. (A) The measured temperature (T) as a function of time. (B) The measured resistance (R) as a function of time. (C) The measured current (I) as a function of time.
Figure 3
Figure 3
A schematic of the full procedure used in our live-cell imaging experiments with TTFields-exposed cells. The microscope images of exposed HeLa cells shown here were obtained in our experiments with 100 kHz TTFields with a 1-1 duty cycle.
Figure 4
Figure 4
A total of 3 × 104 HeLa cells were seeded on a coverslip of a 35 mm ibidi dish and allowed to adhere overnight. Cells were synchronized in the G1/S phase by a single thymidine block and then released for 6 h before imaging. Cells were maintained in an atmosphere of 5% CO2 with an ambient temperature of 37 °C for the control cells and 23 °C for the TTFields-treated cells. (A) Representative images of unexposed HeLa cells acquired every 10 min throughout cell division. (B) Representative images of TTFields-treated HeLa cells (100 kHz, 1-1 duty cycle) acquired every 10 min throughout cell division.
Figure 5
Figure 5
A total of 3 × 104 MCF-10A cells were seeded on a coverslip of a 35 mm ibidi dish and allowed to adhere overnight. Cells were synchronized in the G1/S phase by a single thymidine block and then released for 6 h before imaging. Cells were maintained in an atmosphere of 5% CO2 with an ambient temperature of 37 °C for the control cells and 23 °C for the TTFields-treated cells. (A) Representative images of unexposed MCF-10A cells acquired every 10 min throughout cell division. (B) Representative images of TTFields-treated MCF-10A cells (100 kHz, 1-1 duty cycle) acquired every 10 min throughout cell division.
Figure 6
Figure 6
The durations of mitosis and cytokinesis obtained from three independent experiments with HeLa cells in a variety of scenarios: control, 100 kHz (1-1 duty cycle), 100 kHz (1-0), and 200 kHz (1-1). The p-value overlaid on each plot corresponds to the results obtained from two-sample t-tests comparing the displayed data for HeLa cells in each TTFields-exposed group to those of the control cells. (A) Boxplots representing the duration of mitosis, calculated from nuclear envelope breakdown (NEBD) to anaphase. (B) Boxplots representing the duration of cytokinesis, calculated from telophase to the formation of two separated daughter cells. Data are displayed as the median duration flanked by upper and lower quartiles, with outliers shown as red circles.
Figure 7
Figure 7
The HeLa cells from each set of experiments were grouped based on the length of mitosis (tm ≤ 60 min, 60 < tm ≤ 120 min, or tm > 120 min) and the length of cytokinesis (tc ≤ 120 min, 120 < tc ≤ 240 min, or tc > 240 min). Graphs represent the percentage of the cells in each category, reported as the mean ± SD from three independent experiments. (A) Results obtained by grouping the control and TTFields-exposed HeLa cells by their duration of mitosis. (B) Results obtained by grouping the control and TTFields-exposed HeLa cells by their duration of cytokinesis.
Figure 8
Figure 8
The durations of mitosis and cytokinesis obtained from our experiments with MCF-10A cells. The p-value overlaid on each plot corresponds to the results obtained from each two-sample t-test comparing the displayed data for MCF-10A cells in the TTFields-exposed group to those of the control cells. (A) Boxplots representing the duration of mitosis, calculated from nuclear envelope breakdown (NEBD) to anaphase. (B) Boxplots representing the duration of cytokinesis, calculated from telophase to the formation of two separated daughter cells. Data are displayed as the median duration flanked by upper and lower quartiles, with outliers shown as red circles.
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This research was funded by Novocure Ltd. and the National Sciences and Engineering Research Council of Canada (NSERC), grant number RGPIN-2018-03837.

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