The human ovarian endometrioid adenocarcinoma cell lines A2780 and A2780cis were obtained from the European Collection of Cell Cultures and from AddexBio, respectively. The human ovarian high grade ovarian serous adenocarcinoma cell line OVCAR-3 was obtained from the American Type Culture Collection (ATCC). Human cell lines were grown in RPMI media supplemented with 10% (v/v) fetal bovine serum (FBS), 1 mM sodium pyruvate, 12 mM HEPES and penicillin/streptomycin (50 µg/ml) in a 37°C humidified incubator supplied with 5% CO₂. The media of A2780cis cells was additionally supplemented with 1 µM cisplatin (Sigma, C2210000) in every passage to maintain platinum resistance. Media and supplements were purchased from Sartorius Israel Ltd. (Biological Industries Ltd., Beit HaEmek). Murine ID8 cells were previously transduced by the Laboratory of Molecular Virology and Gene Therapy in the Leuven Viral Vector Core of KU Leuven, using a lentiviral vector (pCHMWS_CMV-fluc-I-PuroR) to create the stable luciferase producing cell line, ID8-fLuc (25). These ID8-fluc cells were cultured at 37°C with 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FCS, 100 U/ml penicillin/streptomycin, 2mM glutamine, 2.5µg/ml amphotericin B and 10 mg/ml gemcitabine, which were obtained from Gibco.

Cells were seeded on coverslip (22 mm diameter; 20×10³ cells/coverslip for A2780 and A2780cis; 40×10³ cells/coverslip for OVCAR-3). After overnight incubation, the coverslips were transferred into inovitro^TM dishes containing 2 ml of media. TTFields at a frequency of 200 kHz (and intensity of 1 V/cm RMS) were applied to the cells for 72 hr using the inovitro™ system (Novocure, Haifa, Israel), as previously described (26).

For efficacy outcomes (cell count, overall effect, and apoptosis), various concentrations of carboplatin (MCE MedChemExpress, HY-17393), olaparib (Cayman Chemical, 10621), or niraparib (Cayman Chemical, 20842) were applied, with or without TTFields.

For DNA damage and cell cycle examination, the following drug concentrations were selected: For A2780 – 6 µM carboplatin, 1 µM olaparib, and 0.5 µM niraparib; For OVCAR-3 – 16 µM carboplatin, 0.5 µM olaparib, and 0.8 µM niraparib; For A2780cis – 36 µM carboplatin, 10 µM olaparib, and 1.5 µM niraparib.

Cell count was examined following treatment using Cytek Northern Lights flow cytometer (Cytek Biosciences, USA). Results are presented as percentage relative to control.

Treated cells were harvested, re-plated in 6-well plates (500 cells/well for A2780 and A2780cis; 1000 cells/well for OVCAR-3), and grown for 7 (A2780 and A2780cis) or 21 (OVCAR-3) days. Colonies were stained with 0.5% crystal violet, quantified with ImageJ, and expressed as percentages relative to control. Overall effect was calculated by multiplying colony formation with the corresponding cell count.

The surviving fraction predicted for an additive effect between TTFields and drug was calculated (per the various drug concentrations) by multiplying the actual measured surviving fractions for the individual treatments one by the other (SF_{calculated additive} = SF_TTFields × SF_drug; where SF are expressed as probability) (27–29). Based on the calculated values, a trendline was determined. Additivity, synergy, or antagonism was defined when the calculated additive trendline overlapped, was above, or was below the actual measured line for TTFields+drug, respectively.

For quantifying the magnitude of TTFields-drug interaction, interaction index (I_i) values were calculated by the Bliss independence method using mortality values (M_x = 1 – SF_x) (27–29). Per the various drug concentrations, mortality predicted for an additive effect between TTFields and drug (M_{calculated additive} = M_TTFields + M_drug – M_TTFields × M_drug) was divided by the actual measured mortality for TTFields+drug. Additivity was determined when the 95% confidence interval (CI) overlapped 1, synergy when 95% CI < 1, and antagonism when 95% CI > 1. Lower I_i values were considered indicative of higher synergy levels.

Treated cells were stained with FITC-conjugated Annexin V (AnnV) and 7-Aminoactinomycin D (7-AAD) using a commercial kit (BioLegend, San Diego, CA, USA), according to the manufacturer’s instruction. Data acquisition and analysis were done on the Cytek Northern Lights flow cytometer.

Extracts were prepared from treated cells and subjected to western blot analysis (25 μg protein/sample) as previously described. Primary antibodies are outlined in Table 1 . Horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, Cambridge, UK; cat #ab97023 or #ab6721, 1:10,000) and a chemiluminescent substrate (Immobilon Forte, Millipore, Burlington, MA, USA) were used for visualization. Bands were recorded on GeneGnome XRQ gel imager (AlphMetrix Bitech, Rödermark, Germany). Densitometric readings were normalized to GAPDH with FIJI software and expressed as fold change relative to control.

Treated cells were fixed with 4% paraformaldehyde for 10 min, permeabilized for 20 min with 0.5% Triton X-100 in PBS, and blocked with donkey serum (PBS with 0.3% Triton X-100 and donkey serum 1:100). Cells were incubated at 4°C overnight with anti-ɣH2AX antibody (Cell Signaling, Danvers, MA, USA; #9718, 1:400), followed by incubation at room temperature for 1 hr with Alexa Flour 488-conjugated secondary antibody (Jackson Immunoresearch, Cambridge, UK; #711–545-152, 1:500) and 0.2 μg/ml 4’,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, Rehovot, Israel). LSM 700 laser scanning confocal system (Zeiss, Gottingen, Germany) was utilized to obtain images, and the mean number of foci per nucleus was determined using the FIJI software with the BioVoxxel plugin.

Treated cells were fixed with 70% ice-cold ethanol for 30 min, pelleted, washed, and stained for 30 min at 37°C in phosphate buffered saline (PBS) containing 1% FBS 5 µg/ml 7-AAD (BioLegend), 200 µg/ml RNase, 1 mM EDTA and 0.1% Triton X-100. Data acquisition (at 665/30 nm) and analysis were done on the Cytek Northern Lights flow cytometer and the FlowJo 10.8.1 software (BD Biosciences), respectively.

Murine experiments were approved by the KU Leuven ethical committee (P082/2021). NIH guidelines for the Care and Use of Laboratory Animals were followed along with the 2010/62/EU directive and the ARRIVE (Animal Research: Reporting of In Vivo Research: Reporting of In Vivo Experiments) guidelines. Syngeneic ID8-fluc cells were harvested using 0.05% Trypsin-EDTA and inoculated (5x10⁶ cells in 100µL DPBS) intraperitoneally in female C57BL/6 mice (six- to eight-week-old, obtained from Envigo (Horst, The Netherlands)), leading to the development of a stage III-IV ovarian cancer model (25).

Seven days post inoculation, treatment was initiated. Mice were divided into four groups receiving either: sham-heat and vehicle (n=8), sham-heat and olaparib (n=8), TTFields and vehicle (n=7) or TTFields and olaparib (n=11). TTFields treatment (200 kHz) was administered continuously using the inovivo^TM system (Novocure, Israel) by applying arrays to the shaven abdomen of the mice as previously described (30). Sham-heat used analogous non-therapeutic arrays. Olaparib (MedKoo Biosciences, USA) was dissolved in a vehicle of 10% DMSO, 50% PEG300 and 40% DPBS and administered at a concentration of 50 mg/kg/day through daily oral gavage. Overall, therapeutic interventions lasted for four weeks, given in four cycles of five consecutive treatment days, followed by two days without treatment.

Tumor load was observed through bioluminescent imaging analysis before (day 5) and after (day 35) treatment administration. All mice were anaesthetized using isoflurane gas (2 L/min) and received 126 mg/kg of D-luciferin through subcutaneous injection. Ten minutes post injection, photon flux was measured using the IVIS-spectrum preclinical In Vivo Imaging System (Perkin-Elmer, USA). Normalized photon flux was calculated by subtracting the photon flux before treatment from the paired photon flux after treatment per mouse.

Mice were followed up and weighed daily once ascites development started as indicated by the appearance of abdominal distention. Mice were sacrificed when their body weight reached ≥32 grams as a surrogate endpoint for survival.

In vitro experiments were repeated at least three times, and data are presented as mean ± standard error of the mean (SEM), and analyzed with ANOVA or student’s t-test as appropriate. To determine the in vivo sample size, a statistical power analysis was performed to reach a power of at least 0.80. Photon-flux measured through BLI was summarized with means and standard deviations and visualized using bar charts. These results were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Kaplan-Meier curves were compared using the log-rank test. Multiple comparison adjustment was performed using the Benjamini-Hochberg procedure. Statistical analyses were performed using GraphPad Prism 10.1 software (La Jolla) and differences considered significant at (adjusted) p-values of: *p < 0.05, **p < 0.01, and ***p < 0.001.

We examined carboplatin dose response curves, based on cell count measurements, in three different human ovarian cancer cell lines: A2780 (HRP cells), OVCAR-3 (HRD cells), and A2780cis (platinum-resistant cells, commercially available generated by repeated exposures of the A2780 cell line to cisplatin). The OVCAR-3 cells demonstrated highest sensitivity to carboplatin, while the A2780cis cells demonstrated highest resistance, as would be expected by the HRD phenotype of the former and the acquired resistance of the latter ( Supplementary Figure 1 ).

TTFields significantly amplified (i.e. lower cell count) the effect induced by carboplatin alone in all examined cell lines ( Figures 1A–C ; p<0.0001 for all cell lines). Similarly, the overall effect (cell count × colony formation) induced by carboplatin was significantly elevated after addition of TTFields ( Figures 1D–F ; p<0.0001 for all cell lines). Apoptosis analysis demonstrated increases in the fraction of apoptotic cells when TTFields were applied with carboplatin, suggesting a cytotoxic effect ( Figures 1G–I ).

While the concomitant application of TTFields with carboplatin, led to enhanced treatment efficacy relative to each treatment alone, we further sought to elucidate the nature of interaction between the two modalities. We calculated the expected dose curve for an additive effect ( Figures 1A–F , dashed lines) and the interaction index (I_i, Figure 1J ). An antagonistic interaction was demonstrated in OVCAR-3 cells, as the actual measured curves were above the calculated additive curves for co-treatment and the 95% confidence intervals (CI) for I_i were larger than 1. On the other hand, for A2780 and A2780cis cells synergy was determined, as the actual measured curves were below the calculated additive curves and the 95% CI for I_i were smaller than 1. The lower I_i determined for A2780cis relative to A2780 cells suggested higher levels of TTFields plus carboplatin synergy in A2780cis cells.

We next measured cell count dose response curves of olaparib and niraparib in the three different human ovarian cancer cell lines. As per the case with carboplatin, OVCAR-3 demonstrated highest sensitivity to PARPi while A2780cis demonstrated highest resistance ( Supplementary Figure 2 ). This trait of A2780cis cells suggests that their acquired platinum resistance was also conferring some resistance to PARPi.

TTFields significantly augmented the effect induced by olaparib and niraparib alone in all examined cell lines, as seen based on cell count ( Figures 2A–C , 3A–C , respectively; p<0.0001 for all cell lines with both drugs) and on the overall effect ( Figures 2D–F , 3D–F , respectively). Apoptosis analysis demonstrated elevation in the apoptotic cell fraction when TTFields were co-applied with olaparib or niraparib, suggesting a cytotoxic effect ( Figures 2G–I , 3G–I , respectively p<0.0001 for all cell lines with both drugs).

Additivity was determined for TTFields with PARPi in OVCAR-3 cells, as the actual measured curves overlapped the calculated additive curves for the co-treatment (and 95% CI for I_i spanned 1) ( Figures 2A–F , 3A–F , dashed lines; Figures 2J , 3J ). Different levels of synergy were determined in A2780 and A2780cis cells, with the lower I_i determined for the former indicating higher levels of TTFields plus PARPi synergy in A2780 cells. Interestingly, the levels of synergy for TTFields plus PARPi in the A2780 cells was higher than that demonstrated with carboplatin in either A2780 and A2780cis cells.

We next examined accumulation of DNA damage in treated cells, by fluorescence microscopy detection of γH2AX foci in cell nuclei ( Figures 4A–I ). For these experiments, per each cell line, drugs were used at concentrations that induce 70 to 80 percent reduction in cell count when co-applied with TTFields. Under the selected conditions olaparib and niraparib alone, induced only a mild elevation in the levels of γH2AX in all cell lines. Carboplatin facilitated a more pronounced effect that was especially dramatic in the OVCAR-3 cells. Application of TTFields alone to the cells induced low or no effect on the level of γH2AX foci formation relative to control. However, co-application of TTFields together with either of the three drugs elevated the foci levels significantly relative to control and to TTFields or drug monotherapy.

To further understand how TTFields were involved in elevating DNA damage, we examined possible changes in expression levels of the cyclin-dependent kinase inhibitor p21 ( Figures 4J, K ), a key mediator of DNA damage-induced cell cycle arrest (31, 32), and of proteins from the FA-BRCA pathway for DNA damage repair ( Figures 4L–N ). TTFields application to the various cell lines elevated expression levels of p21 in A2780 and A2780cis cells (we could not detect p21 in the OVCAR-3 cells, as previously reported (33)), and decreased the expression of FANCB, FANCD2, FANCJ and BRCA2 relative to control cells in all three cell lines.

We next tested whether the various treatments and their consequent DNA damage formation and p21 elevation could induce cell cycle arrest ( Figures 5A–I ; Supplementary Figure 3 ). Carboplatin, olaparib, and niraparib alone all induced G2/M arrest, while co-application of TTFields with each of the drugs significantly elevated the fraction of cells in G2/M.

We measured the effect of TTFields application together with olaparib in mice bearing orthotopic ID8-fLuc (HRP cells) ovarian cancer tumors. Experimental timeline and a schematic illustration of the TTFields/sham arrays attached to the mouse torso are depicted in Figures 6A, B , respectively. The photon flux measured before treatment showed 100% tumor engraftment, and no significant difference in tumor volume between treatment groups at the start timepoint ( Supplementary Figure 4 ). After the 4 weeks treatment period, significantly smaller tumor volumes were seen for mice treated with TTFields plus olaparib, which were lower by about 80% compared to controls (p=0.0183) and to olaparib only treated mice (p=0.0066) ( Figure 6C ). Additionally, about 70% reduction in tumor growth was observed in mice treated with TTFields alone compared to olaparib monotherapy (p=0.0288) and to control (p=0.0658).

The Kaplan-Meier curve showed median overall survival of 62 days for control, 64 days for TTFields alone, 64.5 days for olaparib alone, and 70 days for TTFields plus olaparib ( Figure 6D ). Survival was significantly prolonged in the mice co-treated with TTFields plus olaparib compared to control mice (p=0.0003), olaparib monotherapy (p=0.0003), and TTFields monotherapy (p=0.0130). Notably, survival benefit was also observed in olaparib only treated mice compared to control mice (p=0.0401).