All cell culture media, serum and media supplements were obtained from Biological Industries, Beth Haemek, Israel. All drugs and chemical agents were obtained from Sigma.
The following cell lines and their drug resistant derivatives were used: A clonal derivative (C11) of parental Chinese hamster ovary AA8 cells and their emetine-resistant sub-lines EmtR1 cells having ATP dependent MDR1 type drug resistance , a kind gift from Prof. G. Eytan Dept. of Biology, Technion, Haifa, Israel; Human breast cancer wild type MCF-7 cells, obtained from ATCC and their mitoxantrone-resistant sub-lines MCF-7/Mx having ABCG2 transporter , a kind gift from Prof. M. Liscovitch, Dept. of Biological Regulation Weizmann Institute of Science, Rehovot, Israel; Human breast cancer wild type MDA-MB-231 cells obtained from ATCC and from which doxorubicin resistant MDA-MB-231/Dox cells were developed in our laboratory using a stepwise increase in drug concentration protocol. This procedure is identical with that developed for these cells in other laboratories  for inducing MDR1 type of ABC transporters. The AA8/EmtR1 cell lines were maintained as a monolayer in -minimal essential medium containing 5% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin G, and 100 μg/ml streptomycin sulphate. The EmtR1 cell medium also included 1 μM of emetine. The MCF-7/MCF-7MX and MDA-MB-231/MDA-MB-231Dox cell lines were maintained under monolayer conditions in DMEM containing 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin G, and 100 μg/ml streptomycin sulphate. The MCF-7/Mx cell medium also included 250 nM of mitoxantrone and the MDA-MB-231/Dox cells medium also included 0.1 μM of doxorubicin.
All cells were kept in a 5% CO2 incubator at 37°C. Exponentially growing cells were passaged twice a week using a standard trypsinization procedure.
The level of resistance to doxorubicin and paclitaxel was determined by means of the XTT assay as previously described [8, 9]. Briefly, 2 × 104 cells/well were plated in 24-well plate (NUNC), incubated without drugs for 24 h and then the initial number of cells, OD0, was determined following incubation of with the XTT reagent using ELISA Reader (TECAN Sunrise, USA). The medium was then exchanged with ones containing different drug concentrations, 4 wells for each drug concentration (doxorubicin: 0.001-100 μM; paclitaxel: 0.0001-100 μM). After 72 h, the culture media was discharged, XTT reagent was added and the final cell number, OD72 h, was determined. Data obtained from 3 - 5 experiments were collected and the mean values and standard deviations (SEM) of OD72 h, representing final number of viable cells, were calculated for each drug concentration. Cell survival was presented as percentage of viable cells as compared to the corresponding viable cell number in no - drug controls. Drug concentrations inhibiting cell growth by 50% (IC50) were calculated from relative survival curves using the median-effect principle .
Exposure to TTFields
As previously described [9, 11], two pairs of electrodes, insulated by a ceramic having a very high dielectric constant (NovoCure Ltd, Haifa, Israel), were positioned at 90° with respect to each other in both treatment and control Petri dishes. The distance between the electrodes in each pair was 20 mm. Each pair of electrodes was alternatively connected for 250 ms to a sinusoidal waveform generator (NovoTTF, NovoCure Ltd. Haifa, Israel) that produced 1.75 V/cm, 150 kHz fields in the medium . The 150 kHz frequency of TTFields was found to be effective for treatment of all cells studied.
Four different sets of conditions in each experiment were conducted for each cell line in conjunction with each chemotherapeutic agent: untreated control cells, cells treated by the chemotherapeutic agent alone, cells exposed to TTFields, and cells having a combined TTFields - Chemo exposure (8 Petri dishes for each condition). After 72 h, the culture media was discharged, XTT reagent was added and the final number of viable cells, OD72 h, was determined. Data obtained from 3 - 5 experiments were collected and the mean values and standard deviations (SEM) of OD72 h, representing final viable cell numbers were calculated for each set of conditions. Cell survival was presented as percentage of viable cells out of the corresponding viable cell number in untreated controls. Student t-test was applied to asses the significance of the differences between results obtained for each of the four conditions tested. In order to assess the extent of possible chemotherapeutic dose reduction when applied in combination with TTFields, dose reduction indexes (DRI) for each TTFields/drug combination were calculated according to .
The DRI for the same level of effect (DRIm) was calculated as the ratio of the concentration of drug alone to that of the combined drug-TTFields treatment:
DRIm = Dm(drug alone)/Dm(combined treatment). The DRIs determine the magnitude of dose reduction allowed for each drug when given in combination with TTFields, as compared with the agent dose that achieves the same level of effect. DRI values larger than 1 indicate increased sensitivity to the drug.
Intracellular Doxorubicin Accumulation
The intracellular accumulation of doxorubicin was determined for both wild type and drug resistant sub-lines. Cells were grown in total 16 Petri dishes (35 mm, NUNC) as monolayers for 24 h in drug-free medium and then incubated for 1 h in the absence or presence of doxorubicin with or without exposure to TTFields (1.75 V/cm, 150 kHz) (4 Petri dishes for each treatment condition). The cells were washed with ice cold PBS three times and solubilised with 100 μl of 2% SDS. The solutions were then transferred to black 96-well plates (NUNC) and doxorubicin fluorescence was measured by spectrofluorometry (ELISA Reader TECAN F-200) at λem 600 nm and λex 450 nm. Data obtained from 2 - 4 experiments were collected and the mean values and standard deviations (SEM) of doxorubicin fluorescence were calculated for each condition. Student t-test was applied to asses the significance of the differences between results obtained for each of the three cell pairs.