The Mechanisms of Action of Tumor Treating Fields

Tumor treating fields (TTFields) are a new, divergent treatment modality for cancer. Originally recognized for their antimitotic effects, it is now appreciated that TTFields exert a variety of effects (Fig. 1), making it a useful therapy for a variety of cancers. Here, we review the current understanding of its many biological effects and clinical data.

TTFields are 100 – 400 kHz alternating current (AC) electric fields transmitted transdermally to tumors using two orthogonal sets of transducer arrays. Transducer arrays are activated sequentially each second, effecting a direction change of the incident field on the target. The optimal AC frequency for different cancers has been determined experimentally and varies by tumor type, for example, 200 kHz for glioblastoma (GBM; ref. 1). TTFields’ effects are sensitive to directionality. Early hypotheses that TTFields’ mechanism of action involved polarizable intra-cellular structures and mitotic disruption were later confirmed empirically (1–5), which implies that the amplitude of TTFields’ effect seen by subcellular structures, and hence its efficacy, would depend on the direction of the imposed electric field. Thus, Kirson and colleagues predicted that TTFields’ efficacy would increase with a periodic change of the direction imposed on the cellular target, which was confirmed for direction-change intervals of 1 and 2 Hz (1, 2). The strongest TTFields effects occurred when the incident field was aligned with the cell axis during early mitosis, although a lesser, secondary effect was seen with an orthogonal field, indicating two mechanisms of action. No effect was seen when the field was oriented 45˚ to the cell axis, implying a lack of synergy between the two mechanisms of action. Later work showed that more frequent changes of direction, up to 20 Hz, improved TTFields efficacy as well (6).

Dose–response curves for multiple human cancer lines showed a threshold for significant efficacy of ∼1 V/cm. Since then, recommended TTFields’ field strength is often cited as 2 V/cm. However, the dose-response curves show that a field strength over 2 V/cm, for instance ∼2.45 V/cm for brain cancer (Fig. 2A), is predicted for 100% efficacy, ∼2.6 V/cm for lung cancer (Fig. 2B), nearly 3.5 V/cm for breast cancer (Fig. 2C), and 1.4 V/cm for melanoma (Fig. 2D). The curve-fits resemble those for linear-quadratic theory in radiation therapy. Assuming cells, organelles, and other structures, are randomly oriented in vivo, 2 orthogonal field direction changes will deliver high field strength to all structures in the 2-dimensional plane of field incidence. However, structures oriented orthogonal to that 2D plane may experience very little effective field strength (e.g., the cosine of 90° is 0). Thus, a third direction change may be necessary for full efficacy.

The electric field distribution of TTFields has been tested in various models. Studies using 3D shaped head models show that although electric field distribution is heterogeneous, large portions of the brain receive a dose greater than 1 V/cm (7). Computational models of the abdomen and thorax have also shown > 1 V/cm of electric field intensity with TTFields (8–10). In vivo studies have confirmed >1V/cm deliver of electric fields to the thorax (11).

Deregulated cellular proliferation is a hallmark of cancer (12, 13). Therefore, many anticancer agents have been developed to target dividing cells (14). Such agents induce the mitotic spindle assembly checkpoint (SAC), leading to a cell-cycle arrest, often followed by mitotic slippage, and subsequent cell death or senescence (15–17). SAC activation may also limit the efficacy of such drugs, by allowing time for spindle repair (18, 19). Indeed, it has been shown that GBM cells can be sensitized to antimitotic drugs by SAC inhibition (20).

Prolonged mitotic phase has been observed in TTField-treated cells, in addition to abnormal chromosome segregation and cellular multinucleation (2–5, 21, 22). In addition, GBM cells can be sensitized to TTFields by SAC inhibition (23). Blocking of MPS1, the key regulator kinase of the SAC, led to earlier and prolonged TTFields effects. The concomitant treatment significantly decreased cell proliferation and reduced the number of viable cells compared to the single treatments (23).

TTFields’ effect on telophase/cytokinesis is intensity- and frequency-dependent (24). Attachment of sister chromatids to spindle fibers (9) and stability of tubulin heterodimers are both modulated by TTFields field strength (25). The stability of microtubules is affected by electrical field-induced conformational changes that promote depolymerization (25). Hence, TTFields reduce the ratio between polymerized and total tubulin, preventing proper mitotic spindle assembly (4).

TTFields also disrupt mitotic function (3) and confound cell transition from metaphase to anaphase by dielectrophoretic effects (5). TTFields interact with proteins possessing high dipole moments such as heterotrimeric Septin protein complex, a key protein in positioning the cytokinetic cleavage furrow (26, 27). At anaphase, TTFields inhibit Septin localization to the anaphase spindle midline and cytokinetic furrow. They also hinder Septin association with microtubules during cell attachment and spreading to fibronectin. Altogether, these lead to disordered membrane contraction, failed cytokinesis, mitotic catastrophe and p53-dependent apoptotic cell death after a G₀–G₁ cell cycle block (5). The result of aberrant mitotic division for cells that survive is aneuploid daughter cells due to abnormal and unbalanced chromosome segregation (2–5).

However, these effects are challenged by the biophysical state of the intracellular environment and tubulin properties. It is alternatively suggested that TTFields affect cell membrane potential and ion channels (28), leading to an influx of Ca²⁺ into the cell, reducing microtubule polymerization and thus the observed TTFields-effects during mitosis (29, 30).

TTFields exposure can stimulate autophagy in cancer cells. RNA analysis was performed on TTField-treated and control cells; TTFields resulted in >2 fold increase in multiple genes related to autophagy (31). Cellular changes consistent with autophagy have also been noted in TTFields-treated cells (31). In addition, electron microscopy of TTFields treated cells have also shown autophagy type changes such as increased vacuoles, autophagosomes, and mitochondria with swollen matrices and dilated endoplasmic reticulum (32–34). Using GFP tagged LC3 (LC3-GFP) cell lines, it was shown that the induction of autophagy by TTFields is due to replication stress (35). Treatment with TTFields reduced the number of mitotically active cells and increased the expression of LC3-GFP. In addition, daughter cells of TTFields-treated cells showed marked increase in LC3-GFP expression after completion of mitosis, suggesting that TTFields induced autophagy is due to abnormal mitosis of the treated parent cell, as well as the genomic instability in daughter cells.

Mechanistically, TTFields may induce autophagy via multiple pathways. AMPK, a known positive regulator of autophagy, was significantly upregulated in TTFields-treated cell lines, and silencing of AMPK with siRNA prevented the induction of autophagy in treated cells (35). Studies have also shown that TTFields treatment increases the interaction of Beclin1 with Vps34 or Atg14L, while reducing its interaction with Bcl2 (31). Treatment with 3-methyladenine (3MA) prevented autophagy in TTFields-treated cells. RNA analysis of TTFields-treated cells has shown reduced levels of AKT2, another known regulator of autophagy (31). Further studies revealed that this reduction may be due to the TTFields-induced expression of miR-29b, which acts to inhibit expression of AKT2.

It is currently unknown if autophagy is part of the cytotoxic effects of, or a resistance mechanism to TTFields. Knockdown of Beclin1 or ATG5 improve cell survival during treatment with TTFields (31). Treatment of cells with 3MA has also been shown to attenuate the cytotoxic effects of TTFields (32). However, other studies have shown that preventing autophagy using shATG-7 or chloroquine increased the cytotoxic effects of TTFields (35).

TTFields have been shown to improve efficacy for medications that cause DNA damage and replication stress. On the basis of this, it was evident that TTFields may play a mechanistic role in DNA damage. TTFields have been shown to decrease the expression of multiple Fanconi anemia pathway genes (36) in cancer cell lines. TTFields exposure also resulted in increased DNA damage over time and slowed DNA repair kinetics after ionizing radiation (IR). In addition, TTFields alone caused an increase in chromatid type aberrations and the number of γH2AX foci, as well as slowed the repair of IR-induced double-strand breaks. Amassing of γH2AX foci, a marker of DNA damage, has been seen following treatment with TTFields (22). It was proposed that the downregulation of the BRCA1/2 genes expression upon TTFields exposure caused conditional vulnerability, that is, BRCAness (37). It was shown that TTFields delay the repair of DNA damage caused by radiation or chemicals (38). More recently these effects were confirmed in in vivo models (39). Based upon IHC results, cisplatin combined with TTFields induced elevated γH2AX foci, suggesting TTFields amplified DNA damage induced by cisplatin (39). Elevated γH2AX foci is a marker of stalled replication forks and may be brought about by TTFields due to reduction in MCM6 and MCM10 (40, 41).

TTFields have also been shown to induce the formation of DNA R-loops, a marker of replication stress (42, 43). This replication stress may contribute to the anticancer effects of TTFields. However, it could also lead to resistance via genomic instability. Prolonged treatment with TTFields has been reported to induce activating mutations in mTOR and/or deep loss of CDKN2A raising the possibility of resistance to TTFields treatment (44).

Because of the absence of DNA replication in noncancerous cells, TTFields have little effect on DNA in normal tissue. This is supported by the lack of toxicity seen in the many clinical trials of TTFields, reviewed below.

TTFields may stimulate antitumor immune effects. This was first suggested by a leporine model of abdominally implanted VX -2 tumors. TTFields treatment of abdominal tumors, with limited exposure to the lungs, reduced burden of subsequent metastatic lung disease as compared with controls (45). Metastatic tumors from treated animals also exhibited a higher number of total CD45⁺ cells and increased CD4⁺ and CD8⁺ populations. In addition, the histologic distribution of infiltrating lymphocytes differed between treated and control tumors (45) and was reminiscent of immunologically “hot” (TTField-treated) versus “cold” (control) tumors (46).

TTFields may induce antitumor immunity via multiple mechanisms. TTFields treatment has been shown to increase expression of calreticulin, and the secretion of ATP and high mobility group protein 1 (HMGB1; ref. 33); markers of immunogenic cell death (ICD; ref. 47). One possible driver of this ICD mitotic catastrophe that results in aneuploidy (2, 5, 32). Aneuploid cells exhibit the hallmarks of ICD including the ability to vaccinate mice against tumor formation in syngeneic tumor models (48). ICD can also be stimulated by DNA damage, cell stress responses, and increased reactive oxygen species (47), which have been documented in response to TTFields exposure in cell culture (34, 36, 49). Autophagy may also play a significant role in improving immune recognition and elimination of TTFields-treated tumors (35). Importantly, none of these effects on cells necessarily excluded the other's ability to produce cellular stress that may culminate in stimulating antitumor immunity.

In vivo studies showed that TTFields-treated Lewis lung carcinoma cells activated bone marrow-derived DCs, which exhibited stimulated expression of MCH IIB, CD80, CD40, and increased phagocytosis of the TTFields-treated cells, indicative of DC maturation (33). Similarly, coculturing of murine bone marrow-derived macrophages (BMDM) with previously exposed TTFields-treated CT26 cells resulted in increased expression of proinflammatory activation markers (50). TTFields treatment of in vitro M1 and M2 macrophages has also resulted in increased production of proinflammatory cytokines (CXCL1, IL18, IL23, IL12p70, TNFα, IL12p40, CCL22, G-CSF, CCL17, and IL1β) as well as skewing of M2 macrophages to M1 phenotype (51). Together, these data suggest the intriguing possibility that TTFields-damaged cells stimulate immune responses and possibly promote adaptive immune response against tumor neoantigens.

TTFields-exposed T cells retain interferon gamma secretion, cytotoxic degranulation, and antigen-directed cell cytotoxicity (52); however, exposure to TTFields reduces viability of activated but not nonproliferating, naïve T-cell cultures. Interestingly, there were significant increases in the numbers of infiltrating lymphocytes with signs of activation in gliomas from newly diagnosed patients following TTFields + standard chemoradiation (52).

A recent study revealed that TTFields-induced mitotic catastrophe results in the formation of micronuclei that possess weak nuclear envelopes, resulting in DNA release into the cytoplasm thereby activating the cGAS/STING and AIM2 DNA detection pathways (53). TTFields were shown to drive the expression of inflammatory mediators including IL1, IL6, IL8, type I interferons, and CXLC10, but this effect was absent when both STING and AIM2 were knocked down. Importantly, when TTFields-treated murine glioma cells were orthotopically implanted into syngeneic mice they provoked antitumor immunity that was also STING/AIM2-dependent. This immunity further protected mice from re-challenge with the same cell line. Tumor immune phenotyping revealed shifts in leukocyte phenotypes toward activated states in both the lymphoid and myeloid compartments including the production of durable central memory T cells. Analysis of glioma patient PBMCs also showed increased T-cell numbers and clonal expansion as well as increases in memory T cells and activation of macrophages and dendritic cells following TTFields treatment, similar to the previous analysis (52). However, T cells also showed increased PD-1 expression, suggesting that TTFields stimulation may also drive T-cell exhaustion, and possible need for combination with checkpoint inhibitor therapy.

TTFields may also affect the tumor microenvironment (TME) through metabolic changes. Analysis of glioma cell lines following treatment with TTFields demonstrated decreased uptake of [18F]DASA-23, a probe that detects the presence of PKM2, an isoform of pyruvate kinase that promotes anaerobic glycolysis and lactate production (54). Western blot analysis showed a decrease in PKM2 expression (55). This may have direct and indirect effects on the tumor, as dimeric PKM2 is important in driving anaerobic glycolysis required for tumor growth (56) as well as driving the production of lactic acid, which is immunosuppressive in the TME (57). In addition, dimeric PKM2 also acts as a HIF1α coactivator and controls the expression of genes that play cell autonomous roles in immune evasion and tumor progression (58).

Dexamethasone is a commonly used supportive medication in patients with GBM. Retrospective analysis of patients in the phase III EF-11 trial testing TTFields in recurrent GBM revealed that a strong predictor of response to TTFields was low dexamethasone usage (59). Further analysis revealed a cutoff of less than 4.1 mg dexamethasone/day, which correlated with better outcomes in TTFields-treated patients (60). Interestingly, the effects of high dexamethasone were also seen in the control arm (60). While dexamethasone usage and dose could be related to disease severity, when dexamethasone dose was aggressively reduced to below 4.1 mg/day, mOS was similar to that found in the low dexamethasone population (60). Corticosteroids such as dexamethasone are potent anti-inflammatory agents that have been shown to reduce antitumor immunity in patients with glioma (61–63). Therefore, it is likely that the inferior outcomes associated with dexamethasone use are due to its immunosuppressive effects.

Cell migration and invasion are processes central to the metastatic spread of solid tumors. These aspects require dynamic changes in biophysical properties and are targetable by TTFields. TTFields have been shown to suppress the migration and invasion of LN-18 glioma cells in Boyden chamber and wound-healing assays (22, 32, 64). Confocal microscopy showed abnormal changes in cellular shape and accumulation of actin within the nucleus of TTFields-treated cells, indicating cell stress (32). Similar findings of reduced migration and invasion have been seen in vitro (32). TTFields treatment also led to an upregulation of the epithelial marker E-cadherin and downregulation of vimentin expression, a mesenchymal marker, suggesting reversal of epithelial–mesenchymal transition, a known marker of malignant transformation. TTFields also showed inhibition of p38, ERK, JNK, and AKT phosphorylation. It was later hypothesized that this inhibition of phosphorylation could indicate a mechanism of how cell migration and invasion are inhibited (64).

To date there have been limited published studies evaluating TTFields on cell motility in animal models (32, 45, 64). Two studies in the lungs of mice and New Zealand White rabbits revealed that TTFields-treated animals had a reduced number and size of lung melanoma and VX2 pancreatic cancer metastases, respectively, compared with control animals (45, 65), which could be the result of reduced cell migration.

Impacted cell migration and invasion properties can also be shown by reduced cell dispersal and reduced cell velocity in vitro. TTFields-treated human GBM cell lines showed decreased cell dispersal compared with control cells (65). TTFields also led to a reduction in cell migration velocity compared with control cells (65), because slower cell motility can impact the intensity of cell invasion.

The majority of studies of TTFields at the cellular level have focused on microtubules, however TTFields likely have similar effects on filamentous actin. TTFields have been shown to reduce tunneling nanotubules formation, ultrafine thread-like protrusions of F-actin that connect cells and mediate transfer of intracellular content, shown in Supplementary Fig. S1A and S1B, in MPM cells. This effect is independent of cell proliferation (66). Further studies are necessary to understand this topic of ell communication and invasion properties.

One of the most recently discovered mechanisms of action of TTFields is its permeabilization of cancer cell membranes (67). It has been demonstrated that the application of TTFields to cancer and noncancer cell lines facilitated the ingress of reagents up to 20 kDa in size, by increasing the size and number of fenestrae in the cancer cell membrane (67). This effect was not observed in human fibroblast cells exposed to the same TTFields conditions. In a separate series of experiments, a 24-hour cessation period during which TTFields exposure was halted demonstrated a reversal of the increased size and number of membrane pores (67). 5-aminolevulinic acid (5-ALA) was also studied in human GBM cells after they were exposed to TTFields for 6 or 24 hours, and in both cases, there was increased fluorescence in the TTFields-exposed cells (67). However, no increased fluorescence was seen in cocultured noncancer fibroblasts. These findings suggest that TTFields could be used to help increase penetration of diagnostic (e.g., 5-ALA used in the operating room during neurosurgical resection of brain cancers) and therapeutic (e.g., chemotherapy) agents into cancer cells.

Three biophysical models of cancer cells were reviewed to explain the observed phenomenon of TTFields-induced cancer cell membrane permeabilization (68). The possibility of TTFields influencing ion channel activation was considered (68). However, the size of voltage-gated calcium channels is much smaller than the fenestrate visualized on scanning electron microscopy. Second, the bioelectrorheological model (69) of the cell unites the relationships between TTFields, cell membrane destabilization, and shape deformations, which could ultimately lead to the third proposed model, electroporation (68). Electroporation has long been used in the biomedical sciences for cell transfection (70). Although aqueous pore formation causes increased plasma membrane permeability, structural and chemical alterations in the lipid and protein components of the membrane itself also contribute to the permeabilization effect (71).

The restrictiveness of the blood–brain barrier (BBB) remains to be the greatest challenge for drug delivery into the brain. Majorly composed of endothelial cells, with developmental and functional support from astrocytes, pericytes, and neurons, the BBB acts as a gateway sealed together by junctional proteins (72–74).

As stated above, the ability of TTFields to increase cancer cell membrane permeability was recently identified. Healthy human fibroblasts, however, did not demonstrate similar changes, pointing to cancer-specificity of the phenomenon (67). Although the effects of TTFields on tumor cells such as human skin, brain, lung, prostate, breast (2) and ovarian (9), as well as hamster pancreatic (21), rat brain, mouse skin (2), and small lung (75) cancer have been demonstrated, little is known about their effects on normal cells. Preliminary experiments with murine cerebellar microvascular endothelial cells cerebEND (76), a cell line used for drug transport studies as well as an in vitro model of stroke and the senescent BBB (77–81), revealed the ability of TTFields to induce alterations in the cellular morphology.

Using this cell line, it was shown that the administration of frequency-, intensity-, as well as duration- dependent TTFields results in the delocalization of its major tight junction protein claudin-5, as well as the cytoplasmic accessory protein zonula occludens-1 (ZO-1), from the cell membrane boundaries into the cytoplasm (82). Allowing the cells to recover up to 96h post-TTFields led to the restoration of the original cellular morphology, pointing to the reversibility of the process. Furthermore, the morphological alteration was accompanied by a compromised BBB integrity demonstrated by decreased transendothelial electrical resistance and significant increase in the permeation of fluorescein-conjugated dextran. A similar occurrence was observed in vivo, in rats, whereby treatment with TTFields resulted in increased permeability for immunoglobulin (Ig)-G in the microvessel, as well as for dyes and contrast agents in the brain. Combined treatment of brain-tumor bearing rats with TTFields and paclitaxel, a drug not able to cross the BBB, led to significantly reduced tumor proliferation and volume, indicating enhanced drug permeation and efficacy (83). These findings implicate the potential of TTFields as an innovative method of drug delivery for treatment of brain tumors and other related diseases of the CNS. Hence, further validation of these results in clinical studies would be favorable.

TTFields were first tested clinically in a phase I trial in patients with advanced solid tumors, Table 1 (84). Six patients with refractory cancer were included and treated with TTFields at 100–200 kHz based on tumor type. TTFields were applied via electrodes placed over shaved skin and held in place with a layer of adhesive hydrogel and strips. Of the 6 patients treated, a partial response was noted in one patient with a cutaneous metastasis from breast cancer and stable disease was noted in 3 others.

TTFields have been most extensively studied in patients with GBM. Patients with recurrent GBM were randomized to receive TTFields at 200 kHz or standard salvage chemotherapy (85). 237 patients were randomized with 120 patients receiving TTFields and 117 receiving standard chemotherapy. Although a trend towards improved outcomes with TTFields was seen, overall response rate (ORR) of 14% versus 9.6% and median overall survival (mOS) 6.6 versus 6 months, these were not statistically significant. Post hoc noninferiority analysis of the study showed TTFields to be equivalent to chemotherapy. Grade 1/2 contact dermatitis was seen in 16% of patients treated with TTFields.

Given these results, a randomized trial of TTFields in combination with maintenance temozolomide for patients with newly diagnosed GBM who had previously been treated with surgery, when feasible, and chemoradiation was initiated. Patients were randomized 2:1 to maintenance TTFields and temozolomide or temozolomide alone. Uninterrupted treatment with TTFields was recommended. In an interim analysis of 315 patients in the intention to treat population, an improvement in mOS was seen for TTField-treated patients (19.6 vs. 16.6 months, P = 0.03), resulting in FDA approval (86). An improvement in the 2-year survival rate was also noted (43% vs. 29%, P = 0.006). Final analysis with a minimum follow up of 24 months confirmed benefit for maintenance TTFields (87). Median progression-free survival (mPFS) and mOS were significantly longer for patients who received TTFields (6.7 vs. 4.0 months, P ≤ 0.001 and 20.9 vs. 16.0 mo, P ≤ 0.001). In patients who received TTFields, increased use correlated with improved outcomes (88). Although patients only received TTFields therapy for up to 24 months, a persistent survival benefit was noted with an increase in the 5-year OS rate of 13% versus 5%.

Patients who had progression on maintenance temozolomide with TTFields and were treated with 2^nd line investigators choice therapy could continue TTFields along with second- line therapy. In a post hoc analysis, OS was compared between those who received second-line treatment with and without continuation of TTFields. OS for those who continued on TTFields was longer (11.8 vs. 9.2 mo, P = 0.049; ref. 89). As the majority of patients were treated with a bevacizumab-based regimen, OS was also compared for only patients treated with bevacizumab based therapies; the benefit with TTFields in this setting persisted (11.8 vs. 9.0 mo, P = 0.043).

TTFields was FDA approved for the treatment of mesothelioma based on the single-arm phase II STELLAR trial. Patients with malignant mesothelioma who were not candidates for surgical resection were treated with platinum agent in combination with pemetrexed and TTFields at 150 kHz. mOS, compared with the reported mOS, 12.1 months, in prior phase III trial of platinum agent with pemetrexed (90), was the primary endpoint. Eighty patients were enrolled and included in the final analysis. An overall response rate of 40% was seen with an mOS of 18.2 months with mPFS being 7.6 months, meeting its primary endpoint (91).

TTFields has also been evaluated, and is currently undergoing evaluation, in other tumor types as well, Table 1. In a small phase II trial of TTFields in combination with gemcitabine or gemcitabine and nab-paclitaxel in patients with pancreatic cancer, no increased safety concerns were noted with the addition of TTFields (92). In addition, there were signals of improved survival for treated patients, particularly in those with locally advanced disease. The phase III PANOVA-3 trial (NCT03377491) is currently underway and may confirm these findings. A small single-arm phase II safety study of TTFields in combination with paclitaxel for patients with recurrent ovarian cancer also showed no concerning safety signals (93). PFS and OS survival of this study was longer than that reported from meta-analyses of other trials in this line of therapy. The phase III INNOVATE-3 study (NCT03940196) is currently underway and may confirm these results. Safety of TTFields in combination with chemotherapy for patients with non–small cell lung cancer has also been confirmed in a phase I/II study, and is currently under evaluation in phase III trial (NCT02973789; ref. 94). TTFields is currently being studied in many other tumor types as well including hepatocellular carcinoma, small cell lung cancer, gastric cancer, astrocytoma, ependymoma, brain metastases, and metastatic cancer to the liver, Table 1.

Overall, clinical trials of TTFields have shown them to be safe with little side effects. In the initial phase I study grade 1 skin rash was seen in 3/6 patients; no other adverse events were seen. Skin rash resolved in all patients with repositioning of the electrodes and topical steroid ointments. Other than dermatitis, no other significant safety signals have been noted in subsequent clinical trials. Despite being well tolerated, TTFields do have limitations, mainly patch application and time requirement of use. 2 orthogonal sets of patches can be difficult to place on certain areas of the body, such as the extremities, which add difficulty to studying this therapy in these areas. Additionally, it is generally recommended that TTFIelds be used 18 hours a day, as previous studies show increased use correlate with improved survival (87). Use of TTFields requires a patient to carry around a power supply; however, newer devices models have smaller easier to carry devices and batteries that mitigate this burden.

TTFields are a new cancer treatment modality that is currently FDA-approved for two different tumor types. Preclinical studies have shown multiple downstream effects of TTFields on cancer cells, as outlined above. Combinational studies using drugs that target mitosis, ER stress, autophagy, and DNA replication stress in combination with TTFields may be beneficial in this setting. For tumors where drug penetration is a concern, studies combining standard therapies with TTFields should be considered given its effects on the blood brain barrier and cell membrane permeability. It is well known that intratumoral immune responses may spread to nontreated tumors. Therefore, studies of TTFields targeting large volume areas of disease, or areas with known low response rates such as the liver, in combination with anti–PD-1 and other emerging immunotherapies should be considered as well. Translational studies accompanying clinical trials are crucial for deeper understanding of TTFields mechanism of action and potential use. Given the many mechanisms of action, TTFields have potential to be effectively combined with many types of anticancer therapy for many tumor types.

J. Moser reports other support from Novocure during the conduct of the study; personal fees from BMS, Amunix, Thirona Bio, Adagene, and Imaging Endpoints; other support from Novocure, Genentech, Alpine Immune Sciences, Amgen, Trishula Therapeutics, BioEclipse Therapeutics, FujiFilm, ImmunSensor, Simcha, Repertiore Immune Sciences, Nektar Therapeutics, Synthorx Inc., from Istari Oncology, Ideaya Biosciences, Rubius Therapeutics, University of Arziona, and Senwha Biosciences; personal fees from Caris Life Sciences, Daiichi-Sankyo, TGen, and Immunocore outside the submitted work. E. Salvador reports grants from Novocure Ltd. during the conduct of the study. K.D. Swanson reports grants and other support from Novocure Inc. during the conduct of the study and personal fees from Tegus consulting outside the submitted work. J. Tusynski reports grants from Novocure during the conduct of the study. K.W. Carlson reports personal fees from Novocure Ltd. during the conduct of the study. N. Karanam reports grants from American Association of Cancer Research and grants and personal fees from Novocure during the conduct of the study; in addition, N. Karanam has a patent for Treating tumors using Tumor Treating Fields combined with a PARP inhibitor. (Patent No.: US 10,953,209 B2) issued, licensed, and with royalties paid from Novocure, a patent for Combination of Tumor Treating Fields together with CDK4/6 inhibitor and E2F inhibitor for tumor treatment (provisional patent application US 62/993,603) pending, licensed, and with royalties paid from Novocure, a patent for Combination of therapeutics using Tumor Treating Fields (TTFields) (provisional patent application 17/208,107) pending, and a patent for Methods for applying TTFields combined with personalized ultra-fractionated stereotactic adaptive radiotherapy (provisional patent application filed in 2022 April) pending. C.B. Patel reports grants, personal fees, nonfinancial support, and other support from Novocure Ltd. outside the submitted work; in addition, C.B. Patel has a patent for US11103698B2 Using alternating electric fields to increase cell membrane permeability issued to Novocure GmbH Leland Stanford Junior University and a patent for US20210199640A1 Methods of Normalizing Aberrant Glycolytic Metabolism in Cancer Cells pending to Leland Stanford Junior University. M.D. Story reports grants from Novocure during the conduct of the study; personal fees from Novocure outside the submitted work; in addition, M.D. Story has a patent for 10953209 B2 issued, licensed, and with royalties paid from Novocure, a patent for USP 62/993,603 pending, licensed, and with royalties paid from Novocure, and a patent for USP 63/160,692 pending. E. Lou reports nonfinancial support and other support from Novocure and grants from AACR-Novocure Tumor-Treating Fields Research grant during the conduct of the study; and research funding from a Research Scholar Grant RSG-22-022-01-CDP from the American Cancer Society 2022-26; research funding from the Minnesota Ovarian Cancer Alliance in 2019, 2021, and 2022; honorarium and travel expenses for a research talk at GlaxoSmithKline in 2016; honoraria and travel expenses for lab-based research talks and equipment for laboratory-based research, Novocure, Ltd, 2018 to 2021; honorarium for panel discussion organized by Antidote Education for a CME module on diagnostics and treatment of HER2+ gastric and colorectal cancers, funded by Daiichi-Sankyo, 2021 (honorarium donated to lab); consultant, Nomocan Pharmaceuticals (unpaid); scientific advisory board member, Minnetronix, LLC, 2018 to present (unpaid); consultant and speaker honorarium, Boston Scientific US, 2019. Institutional Principal Investigator for clinical trials sponsored by Celgene, Novocure, Intima Biosciences, and the National Cancer Institute, and University of Minnesota membership in the Caris Life Sciences Precision Oncology Alliance (unpaid). No disclosures were reported by the other authors.

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