Exploring the efficacy of tumor electric field therapy against glioblastoma: An in vivo and in vitro study

Abstract Aims Tumor electric fields therapy (TTFields) is emerging as a novel anti‐cancer physiotherapy. Despite recent breakthroughs of TTFields in glioma treatment, the average survival time for glioblastoma patients with TTFields is <2 years, even when used in conjugation with traditional anti‐cancer therapies. To optimize TTFields‐afforded efficacy against glioblastoma, we investigated the cancer cell‐killing effects of various TTFields paradigms using in vitro and in vivo models of glioblastoma. Methods For in vitro studies, the U251 glioma cell line or primary cell cultures prepared from 20 glioblastoma patients were treated with the tumor electric field treatment (TEFT) system. Cell number, volume, and proliferation were measured after TEFT at different frequencies (100, 150, 180, 200, or 220 kHz), durations (24, 48, or 72 h), field strengths (1.0, 1.5, or 2.2V/cm), and output modes (fixed or random sequence output). A transwell system was used to evaluate the influence of TEFT on the invasiveness of primary glioblastoma cells. For in vivo studies, the therapeutic effect and safety profiles of random sequence electric field therapy in glioblastoma‐transplanted rats were assessed by calculating tumor size and survival time and evaluating peripheral immunobiological and blood parameters, respectively. Results In the in vitro settings, TEFT was robustly effective in suppressing cell proliferation of both the U251 glioma cell line and primary glioblastoma cell cultures. The anti‐proliferation effects of TEFT were frequency‐ and “dose” (field strength and duration)‐dependent, and contingent on the field sequence output mode, with the random sequence mode (TEFT‐R) being more effective than the fixed sequence mode (TEFT‐F). Genetic tests were performed in 11 of 20 primary glioblastoma cultures, and 6 different genetic traits were identified them. However, TEFT exhibited comparable anti‐proliferation effects in all primary cultures regardless of their genetic traits. TEFT also inhibited the invasiveness of primary glioblastoma cells in transwell experiments. In the in vivo rat model of glioblastoma brain transplantation, treatment with TEFT‐F or TEFT‐R at frequency of 200 kHz and field strength of 2.2V/cm for 14 days significantly reduced tumor volume by 42.63% (TEFT‐F vs. control, p = 0.0002) and 63.60% (TEFT‐R vs. control, p < 0.0001), and prolonged animal survival time by 30.15% (TEFT‐F vs. control, p = 0.0415) and 69.85% (TEFT‐R vs. control, p = 0.0064), respectively. The tumor‐bearing rats appeared to be well tolerable to TEFT therapies, showing only moderate increases in blood levels of creatine and red blood cells. Adverse skin reactions were common for TEFT‐treated rats; however, skin reactions were curable by local treatment. Conclusion Tumor electric field treatment at optimal frequency, strength, and output mode markedly inhibits the cell viability, proliferation, and invasiveness of primary glioblastoma cells in vitro independent of different genetic traits of the cells. Moreover, a random sequence electric field output confers considerable anti‐cancer effects against glioblastoma in vivo. Thus, TTFields are a promising physiotherapy for glioblastoma and warrants further investigation.


| INTRODUC TI ON
In 2004, KIRSON et al. 1 report for the first time in a variety of tumor cell lines and in animal models of malignant tumors that alternating electric fields of intermediate frequency (100-300 kHz) and low intensity (1-3V/cm) exert antimitotic effects selectively to dividing tumor cells, while have no effect on quiescent cells. Since then, the tumor electric fields or tumor-treating fields (TTFields) have been brought to the attention of more and more basic scientists and clinicians. [2][3][4][5][6] The TTFields interfere with the rapid division of tumor cells by acting on the spindle assembly in the middle and late stages of mitosis. In addition, uneven electric fields generated in mitotic tumor cells affect the organelles and macromolecules in the cells during the division period, resulting in abnormal chromosome segregation, multinucleated cells, and apoptosis. [7][8][9][10] Recently, breakthroughs have been made in the research on TTFields. Studies have found that it can enhance the sensitivity of tumor cells to DNA-damaging agents 11 and increase the permeability of tumor cell membranes. 12 In addition, low-frequency TTFields (100 kHz) can change the integrity of the blood-brain barrier (BBB). 13 The increased BBB permeability makes it possible to deliver drugs directly to the brain. 13 The tumor cell damages caused by TTFields promote the immune response. [14][15][16][17] Along with the advancement in preclinical research, clinical trials of TTFields have also been extensively carried out, including a phase II clinical trial of triple treatment with temozolomide + pembrolizumab, 18 the phase I study of a combined treatment with a personalized neoantigen vaccine in newly diagnosed glioblastoma (NCT03223103), and a clinical trial of combined treatment with radiotherapy alone or with radiotherapy and temozolomide for newly diagnosed patients (NCT03477110 and NCT03780569). With those advances in treating studies, patients with nervous tumor have potential to improve their prognosis. 19,20 In short, TTFields are a new type of treatment that utilizes lowintensity, medium-frequency, and alternating electric fields to exert biophysical forces on various charged and polarized molecules, resulting in a series of biological effects. 21 Its efficacy, reliability, and safety depend on specific parameters, including intermediate frequency (100~300 kHz) and low intensity (1~3V/cm). 22,23 Previous studies have shown that different tumor cell lines are sensitive to different electric field frequencies, 24 and the optimal frequency is inversely proportional to the size of the tumor cell. 1 The most suitable parameters for electric field therapy in glioma are frequency at 200 kHz and intensity at 1~2V/ cm. Additionally, the efficacy of TTFields relies on the relative direction of the mitotic axis and the field vector. In a single directional electric field, the damage to tumor cells increased 5 times when the mitotic axis was oriented the same as the electric field. 1 It is unknown whether the polyclonal primary tumor cells from different patients are all sensitive to the parameters above, and whether specific genetic components (such as O6-methylguanine-DNA methyltransferase (MGMT), isocitrate dehydrogenase IDH mutations, etc.) respond to TTFields. Less is known about the therapeutic effect of multi-directional random sequence electric field compared with bi-directional electric field.
Here, we adopted a domestic TEFT system to evaluate the treatment parameters in the glioma U251 cell line. We found that the domestic system can achieve comparable efficacy when the relative duration, field strength, frequency, and other parameters remain the same as the similar foreign products. We also used domestic systems to treat the cultured primary tumor cells collected from different glioma patients. The changes in cell volume, cell number, cell proliferation, and invasiveness were assessed. Finally, we improved the TEFT system by increasing the direction of the electric field from 1 to 3 random sequential directions. The therapeutic effect and safety of such random sequence electric field output were assessed in tumor-bearing rats by calculating tumor size and survival time and evaluating other immunobiological and blood parameters. Our results showed that all primary glioma cells with different genetic backgrounds responded to electric field treatment. They were sensitive to wide range of frequencies ranging from 100 to 220 kHz. Most of them had optimal frequencies at 200 kHz, and very few glioma cell types did not respond to 200 kHz TEFT. Three directional random sequence electric field output inhibited tumor growth and increased survival time compared with bi-directional fixed sequence electric field output. Mechanistically, both fixed and random sequence electric fields inhibited tumor cell proliferation, promoted cell apoptosis, and increased the infiltration of CD8 + T cells into the tumor mass. The random sequence electric field output showed better efficacy to inhibit proliferation and promote apoptosis.

| Tumor electric field therapy in cell lines
The tumor electric field treatment system (TEFTS, CL-301A) and the special electric field cell culture device were provided by Antai Kangcheng Biotechnology Co., Ltd. The working principles of the random sequence and fixed sequence output modes are shown in Figure 1, and the specific parameter settings are shown in Appendix 1.
A 20 mm diameter glass slide (Nest 801008) was placed in a ceramic petri dish. The tumor electric field intervention device (Antai Kangcheng Biotechnology Co., Ltd. CL-301A) was installed. 100-150 µL cell suspension (density 2 × 10 5 cells/mL) was applied evenly on a slide. After a 4-6 h incubation in a 37°C incubator, the medium was supplemented and cells were cultured overnight under the same conditions. The electric field treatment group was subjected to the TEFT with specific parameters (field strength and frequency), and the incubator temperature was set to ensure that the temperature in the petri dish was 37°C. The slides were taken out at specified times, and the cells were digested and counted. The inhibition rate was calculated as the relative number of cells % = (total number of cells in TEFT group/total number of cells in non-treated group)×100%. The smaller the relative number of cells, the better the inhibitory effect on tumor cells. The experiment was repeated 3 times independently.

| Glioma tissue specimen
Human glioma tissues were collected from the Department of Neurosurgery, General Hospital of the Chinese People's Liberation Army. Twenty cases of glioma were surgically resected and pathologically diagnosed as glioma grade III and IV (glioblastoma) according to the WHO pathological classification. The patient was informed F I G U R E 1 Schematic diagram of the orderly (fixed) and random sequence output modes generated by the tumor electric field treatment system for in vitro experiments. (A) When the output of electric field was in an orderly sequence, the cell suspensions were evenly inoculated into a quadrilateral petri dish (a') and electrodes were placed on the four sides of the quadrilateral petri dish. The electric field output of fixed frequency and strength was applied at the directions as illustrated (a, b), and the two directions alternated every second (c). (B) When the output of electric field was in a random sequence, the cell suspensions were inoculated into a hexagonal petri dish (a') and electrodes were placed on the six sides of the hexagonal petri dish. The electric field output was applied at the directions as illustrated (a, b, c), and the three directions were switched randomly every second (d) [Colour figure can be viewed at wileyonlinelibrary.com] of the purpose of this study before the operation. The patient knew the content of the study and signed an informed consent. The study was approved by the ethics review committee of the PLA General Hospital (batch number: S2020-200-02). The clinical data of the patients are shown in Table 1.

| Isolation and culture of primary glioblastoma cells
Tumor specimens were subjected to primary cell isolation immediately after resection. The tissues were washed with PBS containing penicillin-streptomycin to remove fat and connective tissue, cut into small pieces, and then incubated at 37°C for 0.5-1 h with DMEM medium containing appropriate amount of trypsin. After the digestion was terminated, the cell suspension was filtered and centrifuged. Cell pellet was resuspended and transferred to a 25 cm 2 cell culture flask. The primary cells <4 passages were used for the experiment.
Primary cells were identified using GFAP immunofluorescence staining. Cells were fixed, blocked, permeabilized, and incubated with anti-GFAP diluted with 1% BSA (abcam ab68428, 1:250) at 4°C overnight. Isotype IgG was used as a negative control. After wash, cells were stained with rabbit anti-IgG H&L (Alexa Fluor 488 ® , abcam ab150077, 1:1000) at room temperature for 1 h. Cell nuclei were stained with Hoechst. Cells were mounted and then imaged using the PerkinElmer Vectra 3.

| Transwell experiment
The treated and control cells were collected to prepare a single

| Animals
Male Wistar rats (6-8-week-old, 150 ± 20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. All rats were kept and fed in the laboratory animal center of the Institute of Biophysics, Chinese Academy of Sciences in Beijing. Cages, bedding, and food were sterilized and changed regularly. Protocols for animal handling, experimentations, and post-surgery care were approved by the Institutional Animal Care and Use Committee of the PLA General Hospital and performed in strict accordance with the National Institute of Health ethical guidelines for animal care and use. All animal data reporting followed the ARRIVE 2.0 guidelines. 25 Rats were shaved under isoflurane inhalation to ensure that the head top and jaw where the electrode attached were hairless before surgery. The electrodes were installed on the 3rd day after in situ inoculation of tumor cell suspension. The experiment was divided into three groups: the random sequence output group, fixed sequence output group, and control group. Blood and tissues were collected at specific times. A timeline of the experimental design is shown in Figure 5A. Rats that had severe neurological dysfunction were euthanized and excluded.

| Establishment of glioma orthotopic transplantation model in rats
Animals were anesthetized with isoflurane inhalation and fixed on a stereotactic frame. After sterilization, an incision was made approximately 1 cm from the sagittal line (avoiding the attached electrodes).
A small burr hole (diameter of 1 mm) was drilled using a dental drill on the right frontal bone. A 26-G Hamilton syringe was used to inject a 5 μL C6 glioma cell suspension (containing 5 × 10 5 cells) into the caudate nucleus according to the following coordinates: 1 mm anterior, 3 mm lateral to the bregma, and 6 mm below the skull (with a 1 mm withdrawal later). The cell suspension was slowly injected at a rate of 1 μL/min. The needle was maintained for 5 min after injection and retracted at 2 mm/min. The burr hole was sealed with sterilized medical bone wax, and the skin was sutured.

| Electrode installation
Rats were anesthetized with isoflurane inhalation. The electrodes were fixed as shown ( Figure S2Ca  sinusoid. The voltage for 5-6 directions was 27Vpp. Frequency was 200 kHz, sinusoid. There were three random direction outputs.
Switching time was 1 s.

| Measurement of electric field intensity
Rats were anesthetized with isoflurane inhalation. Two small burr holes, 0.73 cm apart, were drilled on the left and right parietal bones.
Two insulated probes were inserted 0.8 cm below the skull. One end (1 mm long) of the probe was unwrapped and was used to detect electric signals. The other end of the probe was connected to an oscilloscope and used to detect the voltage and wave curve of the signal ( Figure S2Ba-b). Similarly, probes were placed 0.5 cm below the temporal parietal bone as described above. The distance between two probes was 0.41 cm. One end of the probe was used to detect electric signals. The other end of the probe was connected to an oscilloscope and used to detect the voltage and wave curve of the signal ( Figure S2Bc-d).

| Magnetic resonance imaging (MRI) and tumor volume measurement
Rats were anesthetized and positioned on an animal cradle with a stereotaxic head holder. Gadolinium diamine (0.6 mmol/kg) was injected through the tail vein. T1-weighted images of the tumor were acquired by a Ingenia 3.0T-PHILIPS MRI (parameters in Table 2).
The length and width of the tumor were measured using the Syngo fastview DICOM imaging device. Tumor volume was calculated with the following formula: Tumor Volume=length × width 2 /2 (mm 3 ). 26

| Dermatological side effects
The dermatological side effects were evaluated according to the criteria below 27 ( Figure S3D): Animals without any dermatological side effects were scored as 0.
Grade I side effect was scored 1. The symptoms were shown as contact dermatitis, including local erythema, edema, and small hemorrhagic spots.
Grade II side effect was scored 2. The symptoms were shown as secondary local and invasive skin erosion with clear tissue exudate.
Grade III side effect was scored 3. The symptoms were shown as larger areas of skin ulceration. The base of the ulcer was clean, necrotic, granulated, or with small bleeding.
Grade IV side effect was scored 4. The symptoms were shown as Grade III skin side effect with concurrent skin or soft tissue infection. Yellow purulent discharge was observed around skin erosion.

| Immunohistochemistry
4% PFA-fixed tissues were paraffin embedded and sectioned into serial slices of 5 μm thickness. After permeabilization, antigen retrieval, and blocking, the brain slices were incubated with primary antibod-

| Hematoxylin and eosin (H&E) staining
The slides with cells were fixed with 4% paraformaldehyde for 20 min and washed with PBS. Cells were then stained with hematoxylin for 15 min, followed by eosin staining for 5 min. Cells were mounted on the slide using neutral balsam. Four fields in each slide were imaged using a microscope (Nikon-Eclipse Ti) under a 20X lens. ImageJ V1.8.0 was used to count the cells and calculate the average cell surface area. The experiment was repeated 3 times independently.

| Tumor electric field treatment for primary glioma cells
Primary cells in the logarithmic growth phase and within 3 passages were used for electric field treatment. The electric field frequency setting is displayed in Appendix 1. Cells were digested with 0.25% trypsin, centrifuged, and resuspended to prepare a single cell suspension. Cells were counted using a cell counter (Nexcelom Cellometer Mini). Cell diameter was measured.

| TEFT inhibits the proliferation of glioblastoma cells in cultures
A tumor electric field treatment system was built for the current study. For in vitro experiments, two different sets of electric field outputs were applied to cell cultures, respectively, including the fixed sequence mode ( Figure 1A) and the random sequence mode ( Figure 1B). In the fixed sequence mode, the electric field is applied in two different directions, which alternate every second; in the random sequence mode, the electric field is applied in three different directions that are switched randomly every second.

| TEFT suppresses the proliferation of patientderived primary glioblastoma cells in vitro
In the next set of experiments, we determined whether TEFT could suppress cell proliferation of primary glioblastoma cells. To this end, we collected resected specimens from a total of 20 patients who underwent surgical treatment of glioma in the Department of Neurosurgery at the PLA General Hospital (  Figure 3A). TEFT-treated cells (frequency 180 kHz or 220 kHz) appeared to be enlarged compared with the non-treated cells ( Figure 3B). Quantitative analysis confirmed that TEFT-treated cells showed overall increases in cell diameters. Figure 3C illustrates the relative cell distribution (% of total cell counts) at each of the cell diameter scales after control treatment or TEFT at 180 kHz or 220 kHz. Figure 3D shows the relative cell numbers (% of total cell counts) with small diameters

| TEFT effectively inhibits the proliferation of primary glioblastoma cells at different optimal frequencies
Primary glioblastoma cells from different patients showed different sensitivity to electric field frequencies ( Figure 4A). Figure S1 shows the results of all 20 primary cell preparations in responses to TEFT at a range of frequencies from 100 kHz to 220 kHz. The optimal frequency (to achieve the maximal proliferation-suppression effect) was different among all primary cell preparations: 150 kHz for 3 cases (15% of all cases), 180 kHz for 6 cases (30% of all cases), 200 kHz for 5 cases (25% of all cases), and 220 kHz for 6 cases (30% of all cases). In 2 cases (T6 and T10), TEFT at frequency of 200 kHz failed to suppress cell proliferation ( Figure S1). For the 15 cases in which the optimal frequency was not 200 kHz, we compared the effect at their respective optimal frequency with that of 200 kHz ( Figure 4B) and those that showed no statistical difference were reclassified into the 200 kHz group. The proportion of the 200 kHz group increased to 55% after this reclassification ( Figure 4C).

| TEFT effectively inhibits the proliferation of primary glioblastoma cells regardless of different genetic traits
Eleven of the 20 specimens collected in this study underwent 6 tumor genetic tests (Table 1). Taking all experimental results together, we found that primary glioblastoma cells with MGMT methylation, and IDH, TERT, or BRAF mutations, or 1p/19q co-deletions were all sensitive to TEFT and showed no resistance compared to cells without any of the above 6 genetic traits. Each of the primary cell preparations with a specific genetic trait remains sensitive to one or more frequencies within the range of 100-220 kHz ( Figure S1).

| TEFT inhibits the invasiveness of primary glioblastoma cells
We further assessed the invasive phenotype of primary glioblastoma cells after TEFT using the transwell setting. Two representative primary cell preparations (T8 and T9) were treated with their optimal frequencies (180 and 220 kHz, respectively) of electric fields for 72 h. The results revealed that TEFT significantly suppressed the invasiveness of both primary cell preparations compared with their control groups (T8 vs. control, p = 0.0001; T9 vs. control, p = 0.0004, Figure 4D).

| TEFT suppresses tumor growth in an in vivo rat model of glioblastoma implantation
To determine the in vivo effect of TEFT, we established a rat model of glioblastoma by inoculating C6 glioma cells (cell number, 5 × 10 5 ) into the caudate nucleus. The TEFT was set up for treatment in rats as illustrated ( Figure S2), which allows either the TEFT-F or TEFT-R mode.

| Assessment for the field strength of TEFT in rat brain
The effective field strength for TEFT was determined through a probe inserted into the brain. For the TEFT-F mode (1-2 and 3-4 direction, Figure S2

| TEFT-R effectively inhibits tumor growth in tumor-bearing rats
The timelines for the TEFT efficacy studies are illustrated ( Figure 5A).
TEFT significantly prolonged the overall survival time compared with control rats ( Figure 5B): TEFT-F vs. control group, p = 0.0415 and TEFT-R vs. control group, p = 0.0064. TEFT-R appeared to be more effective than TEFT-F in prolonging the survival time of tumorbearing rats (p = 0.0471, Figure 5B).
The effect of TEFT on tumor growth was assessed by measuring tumor volume by MRI. MRI was performed at 9, 11, 13, 15, and 17 days after C6 glioma inoculation ( Figure 5C). There was no significant difference in tumor volume among the three experimental groups at 9 days after glioma inoculation (p > 0.05). Both TEFT-F and TEFT-R markedly reduced tumor volume at 11, 13, 15, and 17 days after glioma inoculation ( Figure 5D,E). Notably, TEFT-R showed greater effects in reducing tumor volume than TEFT-F ( Figure 5D,E).

| Side effects of TEFT
The impacts of TEFT on liver and kidney functions were evaluated.
Pathological examinations revealed no gross changes in vital organs of TEFT-treated rats, including the liver, kidney, and brain ( Figure S2B).
Skin reaction is a common adverse reaction observed after TEFT.
We observed local contact dermatitis and skin erosion in animals treated with TEFT-F (occurring in 40% rats) or TEFT-R (occurring in 80% rats) at 11 days after glioma inoculation ( Figure S2E). Grade III skin reactions were observed in 10% of TEFT-R treated rats and in 20% of TEFT-F-treated rats at 13 days after glioma inoculation ( Figure S2E). Skin side effects peaked at 15 days after glioma

| DISCUSS ION
The Inovitro™ system (Novocure) 24    Our in vivo experiments show that TEFT-R has better tumorsuppressive effect than TEFT-F. However, is TEFT-R better than TEFT-F in all aspects? We find that the body weight of TEFT-Rtreated rats is lower than the control at multiple time points, although the difference is not statistically significant. Additionally, the skin side effects are more severe in the TEFT-R group than TEFT-F group, which is manifested as more rats having grade II or higher skin reactions. Finally, blood tests reveal that TEFT-R increases serum creatinine, red blood cell count, and hemoglobin levels. The serum urea level in all groups is within normal range but at its upper limit, probably due to reduced water intake. Therefore, TEFT-R has more adverse impacts in rats, including body weight loss, skin reaction, and abnormal blood tests. Parameters of TEFT-R need to be further optimized to ensure long-term usage and better therapeutic effects.
There are several limitations in this study, especially the in vivo animal work. First, the current rat model of glioblastoma was produced by inoculation of the C6 cell line, which does not reflect the intra-and inter-tumoral heterogeneity on molecular characteristics and sensitivities to therapies. 49 While it has been a considerable challenge in the field to inoculate primary glioblastoma cells into rodent brain, we will need to establish such models in the future to identify the optimal treatment paradigms for TEFT. Second, the side effects of TEFT were only partially studied in the current study, with a focus on local skin reactions. However, other side effects could occur, for example, would TEFT aggravate peritumoral cerebral edema? The latter is a major contributor to neurological impairment and mortality in clinical glioblastoma. 50 Future studies should be performed to examine the impact of different TEFT paradigms on brain edema that are associated with inoculated glioblastoma. Third, biomarkers for TEFT are not explored in this study; however, non-invasive biomarkers are essential for testing a novel therapy in clinical trials. Several non-invasive brain imaging technologies have been merging for such purposes, including novel MRI approaches that measure neurometabolic alterations 51 or visualize the tumor microenvironment with specific parameters on oxygen metabolism and neovascularization. 52,53

| CON CLUS IONS
The fixed sequence output TEFT effectively inhibits the proliferation and invasiveness of glioma cells with common genetic mutations. The TEFT random sequence output mode further improves the therapeutic effect on glioblastoma. Importantly, different primary glioblastoma cells are sensitive to specific frequencies of electric field.
The precision treatments are strongly advocated recently.
Optimal treatment regimen should be designed according to the specific conditions of patients to achieve the best curative effect and minimize adverse reactions. For electric field therapy, optimizing treatment parameters according to the conditions of individual patients and improving the potability of the equipment are critical to further improve its overall efficacy.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.