Biological activity of tumor-treating fields in preclinical glioma models

Glioblastoma is the most common and aggressive form of intrinsic brain tumor with a very poor prognosis. Thus, novel therapeutic approaches are urgently needed. Tumor-treating fields (TTFields) may represent such a novel treatment option. The aim of this study was to investigate the effects of TTFields on glioma cells, as well as the functional characterization of the underlying mechanisms. Here, we assessed the anti-glioma activity of TTFields in several preclinical models. Applying TTFields resulted in the induction of cell death in a frequency- and intensity-dependent manner in long-term glioma cell lines, as well as glioma-initiating cells. Cell death occurred in the absence of caspase activation, but involved autophagy and necroptosis. Severe alterations in cell cycle progression and aberrant mitotic features, such as poly- and micronucleation, preceded the induction of cell death. Furthermore, exposure to TTFields led to reduced migration and invasion, which are both biological hallmarks of glioma cells. The combination of TTFields with irradiation or the alkylating agent, temozolomide (TMZ), resulted in additive or synergistic effects, and the O6-methyl-guanine DNA methyltransferase status did not influence the efficacy of TTFields. Importantly, TMZ-resistant glioma cells were responsive to TTFields application, highlighting the clinical potential of this therapeutic approach. In summary, our results indicate that TTFields induce autophagy, as well as necroptosis and hamper the migration and invasiveness of glioma cells. These findings may allow for a more detailed clinical evaluation of TTFields beyond the clinical data available so far.

Glioblastomas are among the most lethal neoplasms. Despite multimodal therapy, including maximal safe surgical resection followed by radio-and chemotherapy, the median survival is limited to approximately 16 months in selected patient populations. 1 The highly invasive phenotype of these tumors precludes complete surgical resection and limits the efficacy of other local therapies. Moreover, defects in the apoptotic machinery of glioma cells account for their resistance to irradiation and chemotherapy. Therefore, novel therapeutic approaches are urgently needed.
Tumor-treating fields (TTFields) represent a novel treatment option for glioblastoma by creating alternating electric fields delivered through transducer arrays directly applied onto the scalp of patients. TTFields at intermediate frequencies between 10 kHz and 1 MHz circumvent the stimulation of excitable tissues such as nerves or muscles and do not induce tissue heating. 2 TTFields are supposed to exert antitumor effects by targeting dividing tumor cells while sparing other cells in the brain that are not undergoing division. 3,4 At the beginning of mitosis, the electric field in a cell is mostly uniform, thus oscillating electric forces yield minimal movement on charged molecules and dipoles. Thereby TTFields may prevent tubulin subunits to attain correct orientation to build the mitotic spindle apparatus, so that mitosis becomes arrested. In contrast, during cytokinesis the electric field is non-uniform with the highest field intensity at the furrow that is connecting the two forming daughter cells. TTFields may therefore disturb the internal cell structure by causing polarized molecules and organelles to move toward the furrow, finally resulting in cell death. 2,5 Moreover, preliminary data suggest that TTFields may also exert immune-modulating effects. 6 Thus, there are various hypotheses regarding the mechanisms that may contribute to the effectiveness of TTFields.
TTFields have been assessed in two phase III trials in patients with newly diagnosed, as well as recurrent glioblastoma. In patients with recurrent glioblastoma, TTFields had a comparable efficacy as that seen with a 'physician's best choice' chemotherapy regimen while having less toxicity. 7 The results of a randomized phase III trial in patients with newly diagnosed glioblastoma suggest that the addition of TTFields to maintenance temozolomide (TMZ) chemotherapy prolongs progression-free and overall survival. 8 Despite these encouraging data, TTFields are facing a lot of skepticism from patients and physicians, which, together with high treatment cost, has resulted in only limited use so far. 9,10 One of the major reasons causing low acceptance, has been the poor understanding of the underlying biology, which may explain the exact mechanism of action of alternating electric fields against tumors cells. The aim of the current project was to investigate the effects of TTFields on glioma cells including glioma-initiating cells (GICs), as well as the functional characterization of the underlying mechanisms. GIC ZH-161 or T-325 were exposed to increasing frequencies of TTFields to define the optimal parameters for penetrating the cell membrane. Most prominent effects on cell viability were observed at 100 kHz ( Figure 1a). This frequency was chosen for all further experiments. Next, LN-18, LN-229, T-325 or ZH-161 cells were exposed to increasing intensities of TTFields. Expectedly, TTFields induced cell death in an intensity-dependent manner, however, more extensively in LN-18 than in LN-229 cells, with a substantial proportion of annexin V-positive cells (Figure 1b Figure 1b). Importantly, the whole panel of glioma cell lines, composed of LTC and GIC, was sensitive to TTFields treatment to a variable degree in terms of cell death induction (Figure 1c).
TTField-induced cell death occurs in a caspaseindependent manner. Next, we aimed at assessing the mode of cell death upon TTFields treatment in more detail. Increased caspase activity, as determined by DEVD-amc cleavage, was detected in cells treated with staurosporine, a prototypic inducer of canonical, caspase-dependent apoptosis, but not upon exposure to TTFields. Accordingly, the broad spectrum caspase inhibitor, zVAD-fmk, inhibited caspase activity induced by staurosporine, but had no effect in TTField-treated cells (Figure 2a). Furthermore, caspase-3 processing was not detected upon exposure to TTFields, indicating that cell death induced by TTField occurs in a caspase-independent manner. Interestingly, when LN-18, LN-229 or ZH-161 cells were exposed to TTFields or staurosporine, increased LC3A/B-II levels, an indicator for the induction of autophagy, were observed (Figure 2b, Supplementary Figure 2c). We therefore went on to examine the morphology of TTField-induced cell death using transmission electron microscopy. TTField-exposed LN-18 or ZH-161 cells showed typical signs of autophagy such as a markedly increased frequency of autophagosomes, mitochondria with swollen matrices or a dilated endoplasmatic reticulum. In contrast, staurosporine, as a classical inducer of apoptosis, led to membrane blebbing or DNA condensation along the nucleus and dark cytoplasm (Figure 2c). To clarify whether autophagy in the context of TTFields acts as a survival or cell death signal, glioma cells were cultured in the absence or presence of the autophagy inhibitor 3-methyladenine. Blocking autophagy reduced the amount of dead cells upon TTFields exposure, suggesting that autophagy mediates TTField-induced cell death (Figure 2d, Supplementary  Figure 2a). In addition, when cells were co-treated with the receptor-interacting protein-1 inhibitor necrostatin-1, which blocks necroptosis, cell death induced by TTFields was partly attenuated, suggesting that necroptosis also contributes to cell death upon TTFields administration (Figure 2e, Supplementary Figure 2B).
TTFields interfere with cell cycle progression. Next, we performed cell cycle analyses, which demonstrated severe alterations that may ultimately result in cell death. In fact, we observed an increased number of cells in the G2/M phase after 24 h, followed by an accumulation of cells in the sub-G1 phase after 48 h of treatment with TTFields ( Figure 3a). In line with these findings, confocal microscopy revealed an increased number of abnormal mitotic events in TTFieldtreated cells such as poly-or micronucleation. In addition, many cells exhibited aberrant changes in cellular shape and accumulations of actin in the nucleus, which are further indicators of cellular stress (Figure 3b).
TTFields reduce migration and invasion of glioma cells. As glioma cells are characterized by their ability to migrate and invade into the surrounding healthy tissue, we asked whether TTFields modulate their migration and invasiveness. We observed impaired migration and invasiveness of LN-18 and LN-229 cells upon exposure to TTFields using transwell migration and Matrigel invasion assays (Figures 4a and b). Importantly, the effects on migration and invasion exceeded those predicted from the loss of viable cells at the same time (Supplementary Figure 3a). These data were further corroborated using a scratch wound-healing assay, which showed reduced migration and growth toward the center of the gap of TTField-exposed LN-18 or LN-229 cells (Figure 4c). Importantly, TTFields also interfered with the migration of the GIC lines T-325 and ZH-161, which are thought to be of particular importance for tumorigenesis and tumor recurrence ( Figure 4d). Again, the effects on migration of GIC were more prominent than those expected from loss of viability (Supplementary Figure 3b).
TTFields act in an additive or synergistic manner with irradiation or TMZ. As radiation therapy and alkylating chemotherapy with TMZ are part of the standard treatment for glioblastoma patients, we assessed the activity of TTFields in combination with these treatment modalities. In acute cytotoxicity assays, mostly additive effects were observed in LN-18 cells when TTFields were combined with TMZ or irradiation, whereas synergistic effects were seen in LN-229, T-325 or ZH-161 cells by either combination (Figure 5a). Analyses of clonogenic survival in limiting dilution assays revealed synergistic effects of TTFields in combination with irradiation in LN-18, as well as T-325 cells, and with TMZ in LN-229 and ZH-161 cells (Figure 5b). Hence, depending on the cell line, the combination approach and the read-out, the effects may be additive or synergistic, however, never antagonistic in the models studied here.
It is well known that the DNA repair protein O 6 -methylguanine DNA methyltransferase (MGMT) influences the antitumor activity of the alkylating agent, TMZ, in vivo and in vitro. 11,12 Thus, we determined a possible impact of the cells' MGMT status on their sensitivity to TTFields. To this end, we used genetically engineered cells with modulated MGMT expression. LN-18 cells with a silenced MGMT gene or LN-229 cells overexpressing MGMT displayed similar sensitivity to TTFields as their wild-type counterparts. Thus, TTFieldinduced cell death seems to be independent from the MGMT status ( Figure 6a, Supplementary Figure 3c). Virtually all patients relapse or progress during or after TMZ therapy, which encouraged us to analyze whether TTFields exert antitumor activity against TMZ-resistant glioma cells, which had been generated by repeated exposure to TMZ. 13

Discussion
Glioblastoma is a highly lethal brain tumor with a median overall survival of approximately 16  trials. Since 2005, the standard of care for newly diagnosed glioblastoma has been maximal safe tumor resection, followed by irradiation with concomitant and maintenance chemotherapy with TMZ. However, the survival benefit conferred by TMZ is largely restricted to patients with tumors that harbor a methylation of the MGMT gene promoter. 12,14 TTFields represent a novel treatment approach that has already been approved by FDA for newly diagnosed, as well as recurrent glioblastoma based on the results of two phase III trials. 7,8 However, there is a mismatch between the encouraging clinical data and the biological effects of TTFields on tumor cells, which have only been poorly understood.
Here we demonstrate that TTFields potently induce cell death in all glioma cell lines. Importantly, TTFields treatment displayed strong activity against glioma cells with stem-like properties, too, which is of particular interest because of the potential contribution of this cell population to tumor recurrence and therapy resistance ( Figure 1). 15,16 Glioblastomas are characterized by extensive heterogeneity at a cellular and molecular level. 17,18 Owing to advances in single-cell technology it became clear that each tumor contains multiple distinct populations of tumor cells with variation in the expression of a range of transcriptional programs, including oncogenic signaling, proliferation, immune response and hypoxia. 19 Hence, inter-and intra-tumoral heterogeneity may contribute to the failure of targeted therapeutic treatment strategies, which greatly depend on the activity of certain molecular pathways. 20 As TTFields are a physical rather than chemical treatment modality, they may be effective over a wider range of tumors with heterogeneous characteristics. 5 The antitumor activity of TTFields may depend on parameters like the cell size that may determine the optimal frequency to induce cell death or the cell's doubling time as TTFields is believed to act mainly during cellular division. 5,21 Accordingly, GIC may be less responsive to TTFields than other tumor cells because of slower proliferation and higher heterogeneity in cell size.
In contrast to a report suggesting that HCT-116 colon cancer cells undergo apoptosis upon TTFields treatment, 22 we did not observe caspase activation, a hallmark of apoptosis, in the cell lines tested in this study. However, our experiments revealed an important role for autophagy and necroptosis in TTFieldinduced cell death (Figure 2). Electron microscopy further supported the induction of autophagy, whereas no cellular changes typically associated with apoptosis were detected. Thus, the type of cell death induced upon TTFields treatment may differ between tumor entities and cell lines. In line with other studies, we observed an increased number of nuclear aberrations in TTField-treated cells that finally result in cell death ( Figure 3). 22,23 Moreover, we noticed an accumulation of nuclear actin filaments, which has been attributed a consequence of cellular stress and may have a protective role. 24 Glioblastoma is characterized by highly infiltrative growth, which precludes complete surgical resection and thus contributes to its aggressive phenotype. 25 Upon exposure to TTFields, we found aberrant changes in the shape of glioma cells (Figure 3), which may indicate altered cell motility. 26 Accordingly, we observed reduced migration and invasion of TTField-treated glioma cells (Figure 4). In line with these data, reduced metastatic spread of solid tumors to the lungs by TTFields treatment has been reported and may be a result of impaired formation of microtubule-based processes in migrating cells. 6,26,27 The combination of TTFields with irradiation or TMZ showed additive or synergistic effects supporting the clinical combination of these treatment modalities as done for TMZ in one trial ( Figure 5). 8 Similarly, it has been reported that combining TTFields with drugs, such as paclitaxel or doxorubicin, may result in reduced tumor cell proliferation and viability in preclinical models. 28,29 In our experiments, altering MGMT expression did not influence the efficacy of TTFields, and TMZ-resistant glioma cells remained responsive to TTFields treatment suggesting that the mechanisms of resistance toward TMZ, which may involve MGMT but also other mediators 13 do not overlap with a putative resistance to TTFields ( Figure 6). Accordingly, TTFields may be particularly attractive for the majority of glioblastoma patients with tumors that are unlikely to benefit from TMZ treatment because of the unmethylated MGMT promoter. These findings are in line with data demonstrating that TTFields are also active against mitoxantrone-or doxorubicin-resistant breast cancer cells that overexpress ABC transporters. Moreover, it was shown that TTFields re-sensitizes drug-resistant tumor cells to chemotherapeutic agents, a finding that was interpreted as TTField-induced changes on the integrity of the cytoskeleton and microtubules, as well as mitochondria distribution. 29 In summary, TTFields represent a novel therapeutic approach that has entered late-stage clinical development in glioblastoma patients. Our data set reveals that TTFields induce autophagy and necroptosis and interfere with the migration and invasion of glioma cells in vitro. The additive or synergistic effects of TTFields in combination with irradiation or TMZ, as well as their ability to induce cell death in TMZresistant glioma cells may represent a biological rationale for the further clinical evaluation of TTFields in glioma patients.      Migration and invasion. A cell suspension containing 40 000 or 80 000 viable cells pretreated with TTFields or not for 24 h before migration or invasion, respectively, was added to the upper well of transwell migration inserts or to BD BioCoatTM MatrigelTM invasion chambers (pore size: 8 μm, BD Biosciences, Franklin Lakes, NJ, USA). In the lower well, 700 μl of NIH-3T3 cell-derived conditioned medium were used as a chemoattractant. After 16 h at 37°C and 5% CO 2 , the cells were fixed in ice-cold methanol for 10 min and stained with Mayer's alum hematoxylin for 60 min. Inserts were mounted in glass slides and nine fields per sample were counted on a microscope.
Scratch wound-healing assay. Cells were seeded on Thermanox coverslips (Thermo Scientific). At confluency, two straight lines in perpendicular direction were scratched into the monolayer using a 1 ml pipette tip. Cells were washed twice to remove detached cells and then grown in serum-containing medium for additional 24 h in the absence or presence of TTFields. The gap distance was imaged on a microscope.
Immunoblot analysis. Cells were lysed in radioimmunoprecipitation assay buffer (10 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with 1x complete inhibitor mix (Roche Diagnostics, Grenzach-Wyhlen, Germany) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich) and protein levels were analyzed by immunoblot using 30 μg of protein per lane mixed with Laemmli buffer containing β-mercaptoethanol, using the respective antibodies. Protein bands were visualized using horseradish peroxidase-coupled secondary antibodies (Sigma-Aldrich) and enhanced chemiluminescence (Perbio, Bonn, Germany). 32 Viability assay. Cells, treated as indicated, were detached with accutase and resuspended in PBS. The number of viable cells was assessed by counting the cells with the Trypan blue dye exclusion method using a Vi-CELL Cell Viability Analyzer (Beckman Coulter, Nyon, Switzerland).
Annexin V/propidium iodide (PI) flow cytometry. For cell death analysis, cells were treated as indicated, followed by resuspension in annexin buffer (10 mM HEPES, 140 nM NaCl, 2.5 mM CaCl2, pH 7.4) and staining with Pacific bluelabeled annexin V from Biolegend (San Diego, CA, USA) and PI (Sigma-Aldrich) for 15 min at room temperature in the dark. Samples were analyzed by flow cytometry. Annexin V-or PI-positive cells were counted as dead cells (either apoptotic or necrotic), and the remaining cells were designated the surviving cell fraction.
Cell cycle analysis. Cells were fixed in ice-cold 70% ethanol for 1 h on ice, subsequently washed, stained with a solution containing 0.5 mg/ml PI, 1 mg/ml RNase A (Sigma-Aldrich) and 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 30 min at 4°C, washed and then analyzed by flow cytometry.
Caspase activity assay. Glioma cells, exposed to TTFields or not, were grown in phenol red-free medium. Subsequently, the cells were lysed and exposed to 12.5 μM of the fluorescent substrate DEVD-amc (Bachem AG, Bubendorf, Switzerland) for 1 h. Caspase activity was assessed using a Mithras microplate reader (Berthold Technologies, Bad Wildbad, Germany).
Transmission electron microscopy. The cells including supernatant were fixed in a 2x fixation solution (2.5% glutaraldehyde and 1.6% formalaldehyde in a 100 mM sodium cacodylate buffer, pH 7.4, final concentration), dehydrated in a graded ethanol series and embedded into Epon. Sections of 60 nm were imaged with a Tecnai Spirit transmission electron microscope (FEI, Hillsborough, OR, USA).

Conflict of Interest
MW has received research grants from Actelion, Alpinia Institute, Bayer, Isarna, MSD, Merck Serono, Piqur and Roche and honoraria for lectures or advisory board participation from Celldex, Isarna, Magforce, MSD, Merck Serono, Pfizer, Roche and Teva. RS is the principal investigator for the pivotal clinical trials on TTFields in newly diagnosed and recurrent glioblastoma. RS served as an advisor to Novocure (non-remunerated), and has received travel assistance for trial associated activities, data review and data presentation at scientific meetings. PR has received honoraria for advisory boards and lectures from BMS, Roche, MSD, Novartis and Molecular Partners. MS declares no conflict of interest.