Therapeutic potential of tumor treating fields for malignant brain tumors

Abstract Background Malignant brain tumors are among the most threatening diseases of the central nervous system, and despite increasingly updated treatments, the prognosis has not been improved. Tumor treating fields (TTFields) are an emerging approach in cancer treatment using intermediate‐frequency and low‐intensity electric field and can lead to the development of novel therapeutic options. Recent Findings A series of biological processes induced by TTFields to exert anti‐cancer effects have been identified. Recent studies have shown that TTFields can alter the bioelectrical state of macromolecules and organelles involved in cancer biology. Massive alterations in cancer cell proteomics and transcriptomics caused by TTFields were related to cell biological processes as well as multiple organelle structures and activities. This review addresses the mechanisms of TTFields and recent advances in the application of TTFields therapy in malignant brain tumors, especially in glioblastoma (GBM). Conclusions As a novel therapeutic strategy, TTFields have shown promising results in many clinical trials, especially in GBM, and continue to evolve. A growing number of patients with malignant brain tumors are being enrolled in ongoing clinical studies demonstrating that TTFields‐based combination therapies can improve treatment outcomes.

by many domestic and international expert consensus and guidelines.
Afterward, breakthroughs have been made in the in vitro or animal studies on the biological mechanisms of TTFields, and the potential of TTField-based combination therapies continues to be explored.
Clinicians have carried out some significant trials of TTFields, including combined treatments or triple treatment, making TTFields a promising candidate in combination with other therapies. In this article, we will review the anti-tumor mechanism of TTFields and discuss the emerging framework of TTFields-based combination therapy for malignant brain tumors.

| ANTI-TUMOR MECHANISMS OF TTFIELDS
Tissues and cells possess endogenous EF that influence biological activities and cellular events. Bioelectrical signaling regulates many essential processes to cellular homeostasis, and the biological circuitry of cancer cells is modified. Such as the processes of tumor metastasis, which can be regulated by cellular ion channels. 14 When potassium ion channels are overexpressed in tumor cells, more negative charges will be carried inside the cells, and the imbalance of voltage will lead to an increase in tumor growth and metastasis. Manipulating the voltage characteristics of breast cancer cells can significantly reduce the number of metastatic sites in the lungs of mice by about 50%. 15 Exogenous EF has been long exploited for interference and/or stimulation of certain natural biological processes, such as depolarization of nerves, contraction of muscles, embryonic development and heat production of tissues. 16 Neither heat is generated nor action potentials are triggered, so the application of mid-frequency alternating EF is neglected. 17,18 Emerging studies have reported that TTFields can alter the bioelectrical state of macromolecules and organelles involved in cancer biology, thus showcasing the therapeutic potential against tumors. Moreover, the whole proteomic and transcriptomic analyzes proved the massive alteration of differentially expressed proteins, mRNAs, miRNAs, lncRNAs, and circRNAs by TTFields in GBM cells, which are related to cell mitosis-related events, varied cellular biological processes, and multiple organelle structures and activities. 19

| TTFields interfere with cancer cell mitosis
The inhibition of cell mitosis is the most commonly reported mechanism of TTFields and TTFields target cancer cells through unusual electrical polarity and rapid proliferative properties. Considering that all charged particles and dipoles in the cell will respond to EF/currents and will oscillate as EF forces alternate in opposite directions, 20 and dividing cells contain highly polar, spatially oriented microtubules and septins, TTFields are capable of interfering with cell mitosis and leading to the arrest of proliferation. 10 Specifically, during normal metaphase, tubulins are precisely choreographed and arranged to form microtubule spindles that extend into the genetic material lining the center of the cell and bind to chromosomes. 21,22 When exposed to TTFields, tubulin is forced to align along the direction of EF, resulting in the interference of tubulin polymerization and obstruction of microtubule spindle formation ( Figure 1A). 10 Another important basis for the effect of TTFields in dividing cells is the directional, hourglass-shaped cell morphology during the cytokinesis phase. 10 At the late stage of cell mitosis, the mitotic septin complex composes of septin 2, 6, and 7, which will be repositioned in alternating EF, leading to aberrant localization of the cytokinetic cleavage furrow (CCF), which results in improper cell division. 23 Septins cross-link to F-actin bundles within the submembrane actin cytoskeleton and must be sufficiently stiff to withstand the hydrostatic pressure generated within the cytoplasm by the invasion of CCF. 24,25 In cytokinesis, the applied EF intensity in the dividing cell shows an hourglass-like non-uniform field distribution, and all polar macromolecules and organelles will be pushed toward the CCF where the EF intensity is highest ( Figure 1B). [26][27][28] This so-called F I G U R E 1 Legend on next page.
"mesoelectrophoresis" prevents cells from dividing properly. Catastrophic mitotic errors that occur in late mitosis are impossibly rectified, and the cell membrane will rupture and blister. 29 Then cancer cells will undergo cell death or apoptosis. A systematic comparison of changes in cell number and clonogenic survival of 10 solid tumorderived cell lines after 72 h of TTFields exposure yielded that all cell lines exhibited a decrease in cell number and clonogenic survival. 27 However, the mechanism of apoptosis induced by TTfields has not been elucidated completely. Some studies revealed that TTFieldsinduced cell death occurs in a caspase 3-dependent manner, 30 while some argued that TTFields-induced apoptosis is not dependent on caspase 3. 31

| TTFields disrupt genomic integrity
During interphase, TTFields possess the function of disrupting the integrity of the genome, leading to efficient cytocidal actions against tumor cells. The phosphorylation level of γ-H2AX, an indicator of DNA damage, was higher when combining TTFields with radiation treatment (RT) than RT alone. 32 RT-induced cytotoxicity depends on the extent of DNA double-strand breaks (DSBs) repair, most of which are repaired within 24 h after RT. In TTFields-treated cells after RT, more than 40% of DSB failed to be repaired, indicating the efficacy of TTFields to enhance RT-damaged DNA may be through blocking the homologous recombination repair pathway. 33 The expression of BRCA1 pathway genes was found significantly down-regulated during TTFields treatment. 34 The BRCA1 pathway not only plays a vital role in homologous recombinant DNA repair, 35 but maintains the stability of DNA replication forks in association with the Fanconi anemia proteins and promotes alternative endjoining DNA repair. 36 Therefore, DNA replication stress is increased by TTFields, including the reduction of replication fork speed and the increase in R-loop formation, resulting in the disruption of DNA integrity. 37 In turn, TTFields not only slow DNA damage repair kinetics but also induce replication stress in cancer cells, resulting in cell death ( Figure 1C).

| TTFields inhibit cell migration and invasion
TTFields have been reported with the capacity to inhibit the metastatic spread of solid tumors. 38 Tumor metastasis is a multi-step process, including tumor cell invasion of basement membranes and movement to surrounding tissues, intravasation into blood vessels, and spreading to other organ sites. It's well known that microtubules in TTFields-treated cells tend to align with the EF. Alterations of microtubules lead to the mediation of the GEF-H1/RhoA/ROCK signaling pathway and the consequent formation of focal adhesions and induction of peripheral actin bundling, thereby hindering the motility of cancer cells ( Figure 1D). 23 TTField also exert the suppression of ciliogenesis in GBM cell lines, which is related to the development of tumor and resistance to therapy. 39 Epithelial-mesenchymal transition (EMT) programs promote the acquisition of aggressive properties by enhancing the motility of cancer cells, damaging the intercellular junctions, and remodeling the extracellular matrix (ECM). 40 EMT-related biomarkers in GBM cells were found significantly affected by TTFields involving a series of potential mechanisms.
Remarkably, mesenchymal markers (e.g., vimentin, smooth muscle actin) were down-regulated, while epithelial markers (e.g., the adherens junction protein E-cadherin) were up-regulated, and its loss serves as the core role in the loss of epithelial differentiation. 41 ECM is the first tissue barrier to prevent tumor invasion peripherally, which can be degraded by matrix metalloproteinases (MMPs). TTFields can inhibit the degradation of ECM by suppressing the expression of nuclear factor kappa-B (NF-κB), a transcription factor that regulates the expression of MMPs. 41,42 Besides, the migration and invasion of cancers require an adequate supply of oxygen and nutrients, hence neovascularization is a decisive factor in cancer progression. 43 The levels of hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor (VEGF) were decreased in TTFields-treated cells, leading to reduced angiogenesis. 41 Furthermore, a significant time-dependent inhibition in PI3K/AKT and MAPK signaling was observed in the TTFields-treated cells, resulting in a reduction in cell migration and invasion by decreasing EMT-and ECM-related marker expression and reducing angiogenesis ( Figure 1D). 41

| TTFields intervene autophagy process
Abnormal mitotic events can invite autophagy to occur ( Figure 1E). 44 When exposed to TTFields, the expression of autophagosome marker LC-II/LC-I was increased and the cells exhibited typical signs of autophagy. 31 Akt2/mTOR/p70S6K axis (a negative regulator of autophagy) is a vital regulator of autophagy by TTFields. Moreover, many miRNAs can be induced by TTFields, especially miR-29b which directly targets Akt2 to trigger autophagy. 45 In most cases, autophagy can maintain the organization and stability of the centrosome to protect cells, 46 while in the presence of the autophagy inhibitor 3-methyladenine, the F I G U R E 1 Diagram of the anti-cancer mechanism of TTFields. (A) During metaphase mitosis, tubulin will align in the direction of the EF, resulting in interference with its polymerization and thus impaired chromosome segregation. (B) During cell division, the cell morphology under TTFields (hourglass-like shape) produces an inhomogeneous intracellular EF, and all polar macromolecules and organelles will be pushed toward the CCF. In addition to interfering with mitosis, TTFields have multiple biological mechanisms, including (C) inhibiting DSB repair, enhancing DNA replication pressure, (D) inhibiting tumor cell migration and metastasis, (E) promoting autophagy, and (F) inflammatory responses to kill tumor cells, (G) increasing cell membrane and (H) BBB permeability to facilitate the uptake of agents.
number of dead GBM cells treated with TTFields was diminished, indicating that autophagy-mediated TTFields-induced cell death. 31 Such called "autophagic programmed death" may be an alternative to programmed cell death. 47 However, inconsistent findings have been reported. Some studies proposed that autophagic flux might be interrupted by TTFields. 19 Autophagy may be a protective mechanism of cells against TTFields in some cases. Phosphorylated AMP-dependent kinase (AMPK) can inhibit mTORC1, thereby suppressing its ability to negatively regulate autophagy. Depletion of AMPK inhibits the up-regulation of autophagy in response to TTFields and sensitizes GBM cells to treatment. 48 It is unknown whether autophagy exerts a protective or killing effect on TTFields-treated cancer cells and may be relevant to the genetic traits of cancer cells, which remains to be further investigated.

| TTFields induce an anti-tumor immune response
Systemic anti-tumor immune responses can be activated in TTFieldstreated tumor-bearing animals ( Figure 1F). 38 High doses of dexamethasone (a steroid used to relieve inflammation) could interfere with the therapeutic effect of TTFields on rGBM. 49 In TTFields-treated cells, damage-related molecular patterns including high-mobility group box 1 (HMGB1) and ATP were released, and calreticulin was exposed on the cell surface leading to increased infiltration of antigen-presenting cells into the tumor site. Endoplasmic reticulum stress induced by TTFields may be the trigger that drives exposure of calreticulin to the cell surface. 50 The dying cells release ATP, which serves as a "find me" signal for apoptotic cells to increase the recruitment of lymphocytes to induce immunogenic cell death. 51 Furthermore, cell death caused by TTFields can promote the maturation and phagocytosis of dendritic cells. 50 Emerging evidence suggested that TTFields fostered the activation of RAW 264.7 macrophages and its output of NO and ROS.

When co-cultured with 4TI cells (a breast cancer cell line) under
TTFields, macrophages secreted elevated levels of pro-inflammatory cytokines, like IL-1β, TNF-α, and IL-6, and the viability of 4TI cells was diminished. Besides, the phosphorylation levels of IκB-α, NF-κB p65 subunit, and p38 MAPK were observed higher in TTFields-treated RAW 264.7 cells than in control, indicating that TTFields induced the p38 MAPK/NF-κB pathway of macrophages to exert inflammatory effects. 52 Recently, TTFields were reported to cause local disruption of the nuclear envelope of cancer cells during interphase, resulting in the release of large cytoplasmic clusters of naked micronuclei, which recruited and activated two major DNA sensors (cGAS and AIM2).
Subsequently, the activated cGAS/STING inflammasome tended to up-regulate pro-inflammatory cytokines, type 1 interferons (T1IFNs) and T1IFN-responsive genes, thereby activating the peripheral immune system and creating a potential intrinsic immune platform for cancers 53 ( Figure 1C).

| TTFields increase cell membrane permeability
When applying high-pulsed EF to the cells, depending on the field intensity, irreversible electroporation, induction of necrotic cell death, or reversible electroporation may occur. 54 In TTFields-treated glioblastoma cells, the number and size of membrane pores were reversibly increased, allowing greater permeability to substances as large as 20 kDa (e.g., 5-aminolevulinic acid) but not exceeding 50 kDa ( Figure 1G). 55 Interestingly, the effect was tumor-specific and did not appear on normal cells. EF-induced transient increase in plasma membrane permeability, with contributions from structural rearrangement of lipids and protein changes, causing fatigue of the membrane structure. 54 Since the membrane composition of cancer cells is altered and more deformable than normal cells that may help in explaining the inconsistent response to alternating EF. 54 The cell membrane permeability of tumor cells increases in response to TTFields, allowing transmembrane transport of chemical agents that explain the increased efficacy of other drugs to some extent. The cell-membrane permeabilization also contributes to the release of innate tumor antigens and activates the immune system. 56 Ion channels in the cell membrane can be affected by alternating EF, and the L-type Ca 2+ channels CACNA1C (Cav1.2) were identified as TTFields target recently, 57 thereby influencing cell cycle progression, cell migration, and clonogenic survival of GBM cells. 58 Cell membrane potential is a key factor controlling the switching on and off of ion channels in cell membranes, and has been identified as a target for TTFields. Employing the Schwan equation (used to calculate transmembrane potential); it was found that in tumor cells, TTFieldsinduced changes in cell membrane potential can be much higher than 10% of the resting cell membrane potential, thus affecting intracellular ion homeostasis. 59 TTFields were also reported to disrupt tight junction proteins (e.g., claudin 5 and ZO-1) of the BBB to increase the permeability of the brain ( Figure 1H). 60 Till now, the presence of BBB has posed limitations for the treatment of brain malignancies and the cerebral application of some agents. Recently, TTFields have been proven to temporarily increase the permeability of the BBB in a human in vitro model. 61 TTField-induced opening of BBB is similarly reversible and expands the range of intracranial drug applications. when compared with chemotherapy as an option in the maintenance phase. Of note, most of the recruited patients were in the advanced stages of the disease (failure of more than 2 chemotherapy agents), and there was heterogeneity in the patient population. In the OptimalTTF-1 trial (NCT02893137), the combination of cranial remodeling surgery with TTFields and BPC in patients with rGBM yielded an OS of 15.5 months and PFS of 4.6 months, 69 much higher than that of TTFields arm in EF-11, and was well tolerated by patients. 11 The removal of a standard craniotomy bone flap increased the EF intensity at the tumor site and was more effective with multiple smaller burr holes than with a single craniectomy. 69 Since the new multimodal approach has shown a preliminary survival benefit, a large-scale randomized, controlled trial, phase 2 trial (OptimalTTF-2, NCT0422399) was initiated 2 years ago to valid the novel combination therapy. 77

| New diagnosed glioblastoma
The SOC for ndGBM is maximal surgical resection, 6 weeks of postoperative RT and TMZ, and 6 months of TMZ maintenance therapy. 78  TTFields are a promising candidate radiosensitizer which induced an abnormal increase in mitotic catastrophe and DNA damage of cells. 32 A prospective, single-arm study that recruited 10 patients with ndGBM was conducted to assess the feasibility and safety of combined RT and TTFields therapy with maintenance TMZ and TTFields.
The mean PFS of this trial was 8.9 months and limited toxicity was reported. 73 When TTFields were given concurrently with RT which was delivered through the TTFields arrays and with maintenance TMZ, the PFS of 9.3 months was illustrated. 72 It follows that the feasibility of TTFields both as maintenance therapy and alongside chemoradiotherapy is endorsed by prior clinical practice, and a more large-scale clinical trial, EF-32(NCT04471844), is underway.

| Other gliomas
The experience with the application of TTFields to other gliomas is Ganglioglioma is classified as grade I, and although grows slowly, some patients will experience recurrence or malignant progression.
Transformation of ganglioglioma to high-grade glioma is rare but usually with a poor prognosis. 80

| Metastatic tumors
The majority of brain metastases result from lung cancer, while in women the most common primary histology is breast cancer. 82 The development of brain metastases complicates many solid tumors and is attributed to the death of patients with advanced cancer. 83  and is ongoing currently. 84

| PRECLINICAL STUDIES OF TTFIELDS IN MALIGNANT BRAIN TUMORS
With an optimal output pattern, TTFields could significantly inhibit the viability, proliferation, and invasiveness of different cell lines, irrespective of their genetic traits. 85 Several studies have shown synergistic effects of TTFields and targeted agents, of which BEV is widely applied. 68,86 Sorafenib is a multi-kinase inhibitor and a first-line agent for the treatment of high-grade gliomas as well, but it has failed to improve the outcomes of patients when combined with TMZ. 87 While combined with TTFields, sorafenib significantly inhibited motility, invasiveness, and angiogenesis of GBM cells. 88 Evolutionary conserved protein kinase monopolar spindle 1 (MPS1) inhibition also has an impact on the mitotic process, resulting in more than an additive effect when combined with TTFields to GBM cells, and the antiproliferative benefit of the combination therapy begins to work earlier than mono-therapy. 89 Some inhibitors of MPS1 have been developed and can potentially serve as a bridge for TTFields therapy interruption.
Likewise, hyperthermia has been reported to increase the efficacy of other approaches against cancers. Combining heat therapy with TTFields has been reported to enhance each other's therapeutic effects and inhibit the metastasis of GBM cells. 90 The combination of two physical therapy may be easily tolerated by patients. Since Ca 2+ channels contribute to cellular stress response to TTFields, combining TTFields with Ca 2+ antagonists (e.g., benidipine) may augment the efficacy and outcome of TTFields. 57 It provides the possibility of combining TTFields with Ca 2+ antagonists, which are already applied in clinical. In brief, TTFields hold great promise to address the challenge across the spectrum of the management of patients with high-grade gliomas by optimizing other treatment strategies.
Given the special immune environment of intracranial tumors, brain malignancies suppress immune cell activity and anti-cancer function. Revitalizing the central immune system has become the emerging for malignant brain tumors and experienced tremendous growth. 5 Considering the transformation of tumors under TTFields exposure to a state more favorable for an anti-tumor immune response, 53  increased mean diffusivity and decreased fractional anisotropy, maximum relative cerebral blood volume, and reduced choline/creatine were noted at 2 months follow-up periods. 95 The MRI results verified Positron emission tomography (PET) scanning provides more biological information than just anatomical information in a non-invasive way. 96 As the most widely clinically applied tracer, 18 F-FDG( 18 F-2-fluoro-2-deoxy-D-glucose) can be highly taken up by normal brain tissue, thus limiting the application in patients with malignant brain tumors. 97 The Response Assessment in Neuro-Oncology group has recommended more wide-scale diagnostic access to amino acidsbased PET for the management of patients with malignant brain tumors, 98 due to the low uptake by normal brain tissue. 99  This research proved that TTFields can cause a shift in GBM metabolism from glycolysis to oxidative phosphorylation for the first time.
Even in the presence of sufficient oxygen, cancer cells prefer to use the inefficient process of glycolysis for energy production, 105 the socalled Warburg effect, which produces lactic acid that is beneficial for tumor growth and metastasis. 106 113 which can be applied between chemoradiotherapy or concurrently with RT. 114 Furthermore, TTFields are inherently nonspecific to tumor types, may cause therapeutic effects for a wide range of solid tumors, and are being investigated for application to extracranial tumors. 50,63,115 Unfortunately, the mechanism of TTFields is far less clear and suffers from skepticism than other therapies. 116 Pharmacological profiling may help reveal the synergistic effects of TTFields in combination with other therapeutic agents. Continued enhancement of research and understanding of the molecular mechanisms will facilitate the adoption of this novel therapy for integration into existing or new treatment strategies.
Although already become an established treatment for ndGBM or rGBM in adults and has been proven effective in combination with SOC of GBM, TTFields were not recommended to be added to SOC of GBM by the leading experts. 114 High cost is the biggest obstacle to broadening the application of TTFields, which needs to be addressed immediately, and additional cost-effectiveness studies are recommended. 117,118 Therefore, improving the efficiency of TTFields will greatly benefit cancer treatment and reduce global medical costs. The phase II clinical trial EF-33 (NCT04492163) for rGBM is being carried out, which applies higher field strength to the tumor by increasing the number of electric field arrays under the condition of safety assurance to achieve a better therapeutic effect. To improve the convenience of use, further refinement of the TTFields system is required to improve the ease of use and device performance through physical and other means (e.g., changing the size and weight of the instrument, implanting electrodes around the tumor, and increasing battery capacity).
Conducting an outpatient clinic for TTFields therapy consultation is suggested, which may lead to a great promotion of motivation and compliance rate, and ensures the clinical efficacy of TTFields. 119 Besides, it's still a mission for researchers to figure out the biomarkers to select the suitable patients who might be responsive to TTFields, and the markers or technique predicting optimal frequency or response in different cancer types or individuals. The best combination formula based on TTFields for special populations should also be defined. The framework of TTFields therapy, from patient selection and treatment to the follow-up efficacy assessment system, needs to be refined in detail. As an innovative treatment with great potential for tumor therapy, we firmly believe that the TTFields treatment system will be a boon to patients with cancer.

ACKNOWLEDGMENTS
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CONFLICT OF INTEREST STATEMENT
The authors have stated explicitly that there are no conflicts of interest in connection with this article

DATA AVAILABILITY STATEMENT
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ETHICS STATEMENT
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