Determining Optimal Inhibitory Tumor Treating Field Frequency for Cancer Cells: A Non-invasive In Vitro Method to Assess the Effect of TTFields on Cancer Cell Viability

Tumor Treating Fields, or TTFields, use alternating electric fields to disrupt cancer cell proliferation. To study the effect of TTFields on cancerous cells in vitro, begin by assembling TTFields compatible dishes over a base plate. A pair of electrodes positioned on the dish walls helps deliver electric fields internally.

Place a glass coverslip inside the assembled dish. Seed a drop of cancer cell suspension onto the coverslip. Incubate to allow the cells to settle and adhere to the coverslip surface. Discard the spent media. Supplement it with a fresh growth media to support the growth and expansion of cancer cells.

Transfer the assembly to a refrigerated environment to compensate for dish heating during electric field application. Next, connect the assembly to a power supply. Subject the cells to an appropriate electric field for the desired duration.

Within rapidly dividing cells, the applied field causes charged molecules like tubulin dimers and septins to forcefully align in its direction. This mechanism interferes with key mitotic events like microtubule polymerization and septin fiber localization. The resulting mitotic arrest triggers apoptosis leading to tumor cell death.

To begin, install TTFields dishes with covers onto a base plate and prepare an additional 8 dishes that will be used for growing control cells. Place a sterile 2-millimeter coverslip on the bottom of each dish. Next, suspend 5 times 10 to the fourth U-87 MG cells in one milliliter of DMEM medium.

The appropriate number of cells plated in each dish depends on the properties of the cell line in use. It is crucial to calibrate it prior to the experiment in order to avoid cell overgrowth.

Pipet 200 microlitres of the cell suspension onto each coverslip such that a drop is formed on its surface and cover the dishes with their lids. Incubate all dishes at 37 degrees Celsius, 5% carbon dioxide overnight to allow the cells to adhere.

Once the cells are attached, aspirate the media from the coverslips using a 200 microliter pipette. Then, carefully pipet 2 milliliters of complete growth medium onto each dish. Gently tap with a sterile pipette tip on the coverslip edges to release any air bubbles caught under the slide.

Afterward, incubate the cells at 37 degrees Celsius, 5% carbon dioxide until TTFields treatment begins. To begin TTFields treatment, remove the base plate from the 37 degrees Celsius incubator and place it into a refrigerated carbon dioxide incubator.

The refrigerated incubator temperature will determine the electric field intensity. It is important to use the appropriate intensity, which is dependent on cell sensitivity to TTFields.

Then, plug a flat cable female connector into the base plate and turn the TTFields generator ON. Open the software dedicated for TTFields in vitro application system. After defining a new study by entering names of the experiment and experimenter, adjust the frequency and target temperature for each dish.

Click on the START button to initiate the TTField's treatment in the software and check if all dishes appear light blue on the monitor, which confirms their proper connection. To re-establish the connection, gently press the dish down and rotate it slowly back and forth until the dish appears light blue on the screen.

After 24 hours of the treatment, stop the experiment in the software by clicking PAUSE. Disconnect the flat cable connector from the base plate and then place the base plate in the laminar flow cabinet.

Then, remove the base plate with the dishes and the control cells grown in standard conditions from their incubators. Replace the medium in all dishes with fresh complete growth medium. Then, place the control cells in a 37 degree Celsius, 5% carbon dioxide incubator and return the base plate to the refrigerated incubator.

Reconnect the base plate to the generator. Continue the treatment by clicking the CONTINUE button. Once the treatment is completed, finish the experiment by clicking the END EXPERIMENT button in the software. Then, save the data uploaded from the system.

After turning the TTFields generator OFF, disconnect the flat cable from the base plate and remove the system from the incubator. Press down and turn the ceramic dish counterclockwise to detach it from the base plate.

Next, aseptically transfer the TTFields treated and control coverslips to sterile Petri dishes containing fresh medium for further evaluation.

• Tumor Treating Fields (TTFields) - An approach using alternating electric fields to disrupt the growth of cancer cells.
• Cancer cell viability - Assessing living, functional cells in a cancer population.
• Microtubule polymerization - The process of building cellular structures vital for cancer cell division.
• Apoptosis - The process of programmed cell death, heightened with TTFields.
• OVCAR3 doubling time - Time it takes for OVCAR3 cancer cell population to double, influenced by TTFields.

• Introduce Tumor Treating Fields (TTFields) - It applies electric fields to interfere with cancer cell growth (e.g., TTF therapy).
• Key Concepts - TTFields can disrupt cellular structures, hindering cancer cell propagation (e.g., microtubule polymerization).
• Underlying Mechanisms - Electric field causes molecules to align and interferes mitotic events triggering cell death (e.g., apoptosis).
• Connect to the Experiment - The investigation tests TTFields effectiveness in reducing OVCAR3 cell viability.

• What are Tumor Treating Fields and how do they affect cancer cell viability?
• What is the role of microtubule polymerization in TTFields?
• How does apoptosis occur as a result of TTFields application?

• Practical Applications - TTFields can be employed as therapeutics for managing cancer (e.g., TTF therapy).
• Industry Impact - Companies like Novocure, developing TTField-based therapies, impact the healthcare industry (e.g., Novocure).
• Societal Importance - TTFields provide a non-invasive, novel approach to disrupt tumor growth (e.g., TTFields).
• Link to Scientific Advancements - TTFields represent a promising avenue for future cancer research.