Tumor Treating Fields (TTFields) are an effective anti-tumor treatment modality delivered via the continuous, noninvasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application to cell lines using a TTFields in vitro application system allows for the determination of the optimal frequency that leads to the highest reduction in cell counts.
Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), alternating electric fields in the frequency range of several hundred kHz. The study of TTFields in tissue culture is carried out using the TTFields in vitro application system, which allows for the application of electric fields of varying frequencies and intensities to ceramic Petri dishes with a high dielectric constant (Ɛ > 5,000). Cancerous cell lines plated on coverslips at the bottom of the ceramic Petri dishes are subjected to TTFields delivered in two orthogonal directions at various frequencies to facilitate treatment outcome tests, such as cell counts and clonogenic assays. The results presented in this report demonstrate that the optimal frequency of the TTFields with respect to both cell counts and clonogenic assays is 200 kHz for both ovarian and glioma cells.
Tumor Treating Fields (TTFields) are an anti-mitotic modality for the treatment of glioblastoma multiforme and potentially other cancer types. The fields are delivered via the continuous application of low-intensity (1-3 V/cm), intermediate-frequency (100-500 kHz), alternating electric fields to the region of the tumor1,2. TTFields application in vitro and in vivo was shown to inhibit both the growth of various cancerous cell lines and the progression of the tumors in several animal tumor models1,2,3,4,5,6,7. Pilot clinical trials and larger randomized studies in patients with solid tumors, including glioblastoma and non-small cell lung cancer, have demonstrated the safety and efficacy of continuous TTFields application8,9,10. The efficacy of the TTFields was found to be: (1) frequency-dependent, with specific optimal frequencies leading to the highest reduction in the cell counts of cell lines from different origins1,2,4,5,6,7; (2) electric field intensity-dependent, with a minimal threshold for activity at around 1 V/cm and more potent higher intensities1,2,7,11; (3) enhanced when the treatment duration was longer5; and (4) higher when 2 directional TTFields were applied perpendicularly to each other, as compared to electric fields applied from a single direction1. Based on the above findings, TTFields can be applied to patients for long durations using 2 sets of transducer arrays localized on the patients' skin to maximize the electric field intensities in the tumor bed12,13.
Studying the effects of TTFields on cancerous cells in vitro currently provides the only way to determine the optimal frequency to apply to a specific tumor type. Testing for the optimal frequency requires a device that allows for the application of different frequencies in the range of 100-500 kHz and at intensities of up to 3 V/cm root mean square (RMS) to the cell culture. As TTFields application produces heat, the application system requires the ability to dissipate excessive heat while maintaining tight control over the temperature.
Several devices were developed throughout the years to allow for TTFields application to cell cultures1,2,5,14,15,16. In all of these devices, the electrodes used were insulated in order to avoid the caveats involved with the use of conductive electrodes, such as electron exchange at the electrode surface and the release of toxic metal ions into the medium1. The main difference between the various TTFields application systems tested is the type of electrode insulation used, with either electrodes made of metal wires insulated with a thin film of insulator2,14,15,16 or with a high dielectric-constant material (e.g., lead magnesium niobate-lead titanate (PMN-PT))6. While the insulated-wire electrodes offer a relatively simple and cost-effective solution for TTFields application, they are often limited by the high voltage required to achieve effective electric field intensities above the 1 V/cm threshold and by the surface available for cell plating, as the distance between the electrodes is relatively small. Systems based on electrodes insulated using a high dielectric-constant material require special design and manufacturing capabilities, yet they do not require high voltage and can offer a larger area for cell growth between the electrodes.
The TTFields in vitro application system used in this work belongs to the latter class of systems, with the core unit being a Petri dish (TTFields dish, see Figure 1) composed of high dielectric-constant ceramic (i.e., PMN-PT). Two pairs of electrodes are printed perpendicularly on the outer walls of a TTFields dish to allow for the application of electric fields from 2 directions. The electrodes are connected to a sinusoidal waveform generator and an amplifier, which allow for TTFields application in the frequency range of 50-500 kHz. In order to dissipate the excessive heat, the TTFields dishes are kept inside a refrigerated incubator, with the medium temperature-control performed using constant monitoring of the dish temperature and adjustments to the voltage applied by the system. In practice, setting the incubator to a lower temperature would lead to higher electric field intensities, as the system increases the voltage until the target temperature within the dish is achieved. The difference between the temperature within the dish and the incubator temperature may lead to some evaporation, depending on the temperature gradients; hence, the culture medium needs to be replaced every 24 h to maintain adequate growth conditions.
The protocol below describes the experimental procedure to optimize the application of TTFields frequencies to cancerous cells so that a maximum reduction in cell count and a reduction in the potential of the surviving cells to form colonies are achieved.
1. TTFields In Vitro Application System — Base Plate and Dish Maintenance
2. Experiment Setup
NOTE: All equipment and materials used in this protocol are described in the Materials List. All steps below should be performed inside a laminar flow cabinet while sterile conditions are maintained.
3. TTFields Application
4. Control Samples
Note: Grow control cells in similar conditions to TTFields-treated cells, excluding TTFields application.
5. Experiment End
6. Evaluation of the Effect of TTFields
NOTE: TTFields' effects can be determined in several ways. Use one or more of the following methods to compare treated cells to untreated control cells:
Outcomes after TTFields application when scanning different frequencies can be quantified based on different assays, such as cell counts, colorimetric assays, clonogenic assays, and examinations on the changes in the number of invading cells using a Boyden chamber placed inside a special high-wall TTFields cell culture dish. Careful experimental planning based on predetermined cell doubling times will allow the cells to reach the maximal number of mitotic events during the treatment duration, thus maximizing treatment outcomes.
Figure 2 illustrates a typical frequency scan outcome for an average cell count (i.e., number of cells) and clonogenic assay of A2780 (i.e., human ovarian cancer cells; Figure 2A)7 and F-98 (i.e., rat glioma cells; Figure 2B)1 treated with two directional TTFields (frequency range: 100-500 kHz, RMS: 1.7 V/cm, and incubator temperature: 18 °C). The results demonstrate a significant reduction in the cell counts at all applied frequencies, with the maximal reduction at 200 kHz for all cell lines tested (one-way ANOVA with multiple comparison). The optimal frequency leading to the highest reduction in the clonogenic potential was the same for both A2780 and F98 cells (Figures 2B and C). The Pearson correlation coefficient between the cell number and the clonogenic effect at each frequency was 0.967 (p = 0.002) and 0.755 (p = 0.083) for A2780 and F98, respectively.
Figure 3 demonstrates a frequency scan outcome for the average cell count for OVCAR-3 (i.e., human ovarian cancer cells; Figure 3A) and U-87 MG (i.e., human glioma cells; Figure 3B) cells treated with two directions of TTFields at different intensities (RMS: 1.7, 1.3, and 1.0 V/cm, respectively and incubator temperature: 18 °C, 24 °C, and 28 °C, respectively). The results demonstrate that while there is still a significant reduction in OVCAR-3 cell count after treatment with 1.3-V/cm TTFields (incubator temperature: 24 °C), the effect is relatively small compared to the results obtained when the cells were treated with 1.7 V/cm (incubator temperature: 18 °C). U-87 MG cells treated with 1.0-V/cm TTFields (incubator temperature: 28 °C) also demonstrate a similar trend in the reduction of cell number as when treated with 1.7 V/cm, yet the effect was not significant at lower intensities.
Figure 1. TTFields dishes were installed onto a base plate unit and connected to the flat TTFields control cable. Please click here to view a larger version of this figure.
Figure 2. Frequency scans for the determination of the optimal TTFields inhibitory frequency for (A) A2780 and (B) F98 cells. Cells were treated for 72 h with TTFields of different frequencies (1.7 V/cm and 100-500 kHz). The effect of TTFields treatment was estimated using cell count and clonogenic assays. The arrow indicates the optimal frequency. The results represent averages ± SD based on at least 6 replicates for each frequency tested. (C) Representative images of the clonogenic survival of A2780 cells following TTFields treatment at various frequencies. Please click here to view a larger version of this figure.
Figure 3. Frequency scans for the determination of the optimal TTFields inhibitory frequency for (A) OVCAR-3 and (B) U-87 MG cells. Cells were treated for 72 h with TTFields of different frequencies (100-500 kHz) and intensities (OVCAR-3: 1.3 and 1.7 V/cm; U-87 MG: 1.0 and 1.7 V/cm). The effect of TTFields treatment was estimated using cell counts. The arrow indicates the optimal frequency. The results represent averages ± SD based on at least 6 replicates in each frequency tested. Please click here to view a larger version of this figure.
Incubator ambient temperature | Expected TTFields intensities |
(°C) | (V/cm RMS) |
18 | 1.62 |
19 | 1.55 |
20 | 1.48 |
21 | 1.41 |
22 | 1.33 |
23 | 1.26 |
24 | 1.19 |
25 | 1.12 |
26 | 1.04 |
27 | 0.97 |
28 | 0.9 |
29 | 0.83 |
30 | 0.76 |
Table 1. Incubator ambient temperature and expected TTFields intensities inside the TTFields dish.
TTFields are an emerging anti-tumor modality based on the continuous application of properly tuned alternating electric fields1,2,8,9,10,17. Maximizing anti-tumor efficacy is a desirable outcome for all treatment modalities. Thus, "fighting" for every additional percent of cancerous cell growth inhibition may have a significant effect on the long-term clinical outcome for patients. This is because of the required continuous nature of TTFields application and the resulting cumulative effect. Maximizing TTFields application can be achieved in several ways: (1) increasing the electric field intensity1,7, (2) lengthening treatment duration5, (3) finding the most effective combination with other treatment modalities18,19, and (4) defining the optimal frequency1,2,4,6,7. Maximizing the electric field intensity at the site of the tumor is achieved by optimizing the location of the arrays on the patient skin; this allows for the delivery of the maximal field intensity to the tumor based on the individual anatomy of the patient20. Lengthening treatment duration mostly relies on patient compliance with treatment (for at least 18 h per day)17. Finding the right combination with other therapies and determining the optimal frequency relies heavily on in vitro results, as no validated markers for TTFields treatment outcomes are currently available. In this work, we have outlined the experimental procedures required to determine the optimal TTFields frequency for cancerous cell lines using the TTFields in vitro application system. The methods described here can potentially be used to screen the combination of other cancer treatment modalities (e.g., chemotherapy agents or irradiation) with TTFields and to determine the optimal frequency for TTFields administration for each specific combined treatment.
In line with previous publications, the results shown here demonstrate that the optimal frequency for the treatment of both glioma cells and ovarian cancer cells is 200 kHz1,7. In this work, we demonstrated for the first time that the optimal TTFields frequency to reduce the clonogenic potential is associated with the frequency that leads to the maximal cytotoxic effect. The methods used in this work to quantify the effects of TTFields (i.e., cytotoxic and clonogenic) are only two of many possible standard endpoint assays to evaluate treatment outcomes. Additional treatment outcome tests include: (1) fixing, staining, and mounting the coverslips on which the cells are plated over a microscope for the visualization of intracellular structures; (2) performing assays of protein and RNA extracts, either from the TTFields dishes themselves or after transferring the coverslip to a new disposable dish; and (3) trypsinizing cells stained for flow cytometry analysis.
Careful experimental planning will impact the treatment outcomes after the delivery of TTFields. The key steps include ensuring that the cell proliferation throughout the experiment does not lead to overgrowth and using the appropriate electric field intensity, as intensities that are too high when applied to sensitive cell lines will result in too-few cells for the required assays to determine the optimal frequency. Conversely, TTFields applied at very low intensities on less sensitive cell lines will result in small effects that may be masked by inherent variation. The treatment logs should be examined for valuable information regarding temperature stability, electrical currents, and resistance for each and every dish throughout the experiment. Replacing faulty dishes at treatment start and excluding data from a dish that did not meet the desirable treatment parameters will minimize variability between replicates.
In summary, TTFields are an emerging anticancer treatment modality that has already demonstrated efficacy and safety in clinical settings8,9,10. Testing TTFields in an in vitro setting using the protocols described here may allow for the optimization of TTFields treatment parameters in the clinical setting and may broaden our understanding of the underlying mechanism of action.
The authors have nothing to disclose.
Authors have no acknowledgements.
inovitro system and software | Novocure | ITG1000 and IBP1000 | Each unit contains 1 TTFields generator, 1 base plate, 8 TTFields dishes with covers and 1 flat cable. |
Sterilization bags | Westfield medical | 24882 | |
Plastic cover slides | Thermo Scientific (NUNC) | 174977 | Pre treated and sterilized |
Glass cover slides | Thermo Scientific (Menzel-Gläser) | CB00220RA1 | Sterilize if necessary |
Dulbecco’s modified Eagle’s medium | Biological Industries (Israel) | 01-055-1A | Warm in 37 °C water bath before use |
RPMI 1640 | Gibco | 21875-034 | Warm in 37 °C water bath before use |
Fetal Bovine Serum (FBS) | Biological Industries (Israel) | 04-007-1A | Warm in 37 °C water bath before use |
L-Glutamine 200mM (100X) | Gibco | 25030-029 | |
Pen/Strep (10000 U/mL Penicillin, 10000 µg/mL Streptomycin) | Gibco | 15140-122 | |
Sodium Pyruvate solution 100 mM | Biological Industries (Israel) | 03-042-1B | |
Hepes buffer 1M | Biological Industries (Israel) | 03-025-1B | |
Insuline solution from bovine pancreas | Sigma-Aldrich | 10516-5ML | |
0.25% Trypsin/EDTA | Biological Industries (Israel) | 03-050-1B | Warm in 37 °C water bath before use |
Methanol | Merck | 1.06009.2511 | Cool to -20 °C in the freezer before use |
Crystal violet | Sigma-Aldrich | 120M1445 | Harmful. Prepare 0.1% w/v crystal violet solution in 25% Methanol 75% water. |
Light detergent | Alcononx | 242985 | Prepare 5% solution in water, or according to manufacurer's instrutions. |
PBS | Biological Industries (Israel) | 02-023-1A | Without calcium and magnesium |
A2780 | ECACC | 93112519 | Grow in RPMI 1640 supplemented with FBS (10%), pen/strep (100 U/mL / 100 µg/Ml), sodium pyruvate (1 mM) and Hepes buffer (12mM). |
F98 | ATCC | CRL-2397 | Grow in Dulbecco’s modified Eagle’s medium supplemented with FBS (10%), pen/strep (100 U/mL / 100 µg/Ml), sodium pyruvate(1 mM) and glutamine (2mM). |
Ovcar-3 | ATCC | HTB-161 | Grow in RPMI 1640 supplemented with FBS (20%), pen/strep (100 U/mL / 100 µg/Ml), sodium pyruvate (1 mM), Hepes buffer (12 mM) and insuline (10 µg/mL). |
U-87 MG | ATCC | HTB-14 | Grow in Dulbecco’s modified Eagle’s medium supplemented with FBS (10%), pen/strep (100 U/mL / 100 µg/Ml), sodium pyruvate(1 mM) and glutamine (2mM). |
refrigirated CO2 incubator | CARON | 7404-10-3 | |
Laminar flow cabinet | ADS Laminair | Bio12 and VSM12 |