Murine sarcoma LPB cells, syngeneic to C57Bl/6 mice were used in the experiments. The cells were grown in Eagle minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS) (Sigma, Chemical Co., St. Louis, MO, USA). Cells were routinely subcultured twice per week and maintained in a humidified atmosphere with 5% CO₂ at 37°C. Inbred C57Bl/6 mice were purchased from the Institute of Pathology, University of Ljubljana (Slovenia). Mice were maintained at 21°C with natural day/night light cycle in a conventional animal colony. In the experiments mice of both sexes were used and distributed evenly between the groups. The average weight of mice subjected to treatment protocol was 22 g for female and 25 g for male mice.

Solid subcutaneous tumors were induced dorsolaterally by the injection of 1.3 × 10⁶ viable tumor cells in EMEM supplemented with 2% FCS prepared from cell culture in vitro. The tumors reached approximately 40 mm³ in volume in 10–12 days. Then, the mice were marked, divided randomly into experimental groups and subjected to specific experimental protocol. Treatment protocols were approved by the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia No. 323-02-237/01, and are in compliance with the standards required by the UKCCCR guidelines.

A stock solution (3 mg/ml) of BLM (Blenoxane, Bristol Myers Squibb Co., Princeton, NJ, USA) was prepared in phosphate buffered saline. Final concentration of BLM (0.28 μg/ml) was freshly prepared in EMEM before each experiment in vitro. In vivo experiments were performed using BLM at the dose of 0.5 mg/kg, which was daily prepared in 0.9% NaCl solution.

For irradiation an X-ray unit Darpac 2000 (Gulmay Medical Ltd, Shepperton, UK), operated at 220 kV, 10 mA, and with 0.55 mm Cu and 1.8 mm Al filtration was used. In experiments in vitro, cells (1 × 10⁶ cells/ml EMEM) were irradiated in low attachment plates at a dose rate 2 Gy/min with graded doses (2–8 Gy) and thereafter plated in Petri dishes for colony forming assay. In experiments in vivo, tumors were irradiated at a dose rate 2.1 Gy/min with single graded doses (5–50 Gy). Mice were put into a holder for 6 mice on the X-ray unit with the apertures for the irradiation of the tumors; the rest of the body of the mice was protected by lead block, and by the lead holder for the mice. In the holders, mice were restrained, but not anaesthetized during irradiation. To ensure uniform dose distribution through the tumor volume, the tumors were exposed to irradiation by two opposing treatment fields through each of which 50% of the dose was delivered 21.

For electroporation, an electric pulse generator Jouan GHT 1287 (St. Herblain, France) delivering 8 square wave electric pulses of amplitude over distance ratio of 1200 V/cm, duration 100 μs at 1 Hz was used. In experiments in vitro 90 μl of cell suspension (2.2 × 10⁷ cells/ml) was mixed with 10 μl of BLM at a concentration of 0.28 μg/ml, as described previously 20. Briefly, one half of the mixture was exposed to electric pulses and the other half served as a control for BLM treatment alone. Thereafter the cells were incubated for 5 min at room temperature in low attachment plates, diluted and plated on Petri dishes for colony forming assay. The survival of cells treated with ECT was normalized to electroporation treatment alone.

In in vivo experiments, ECT of tumors was performed as described previously 20. Briefly, 3 min after intravenous injection of BLM (injection volume 150 μl), electric pulses were applied to the tumors using plate electrodes with 8 mm distance between them. Electrodes were placed on the skin overlying the tumor at the opposing margins and electric pulses delivered in two sets of 4 pulses in opposed directions by rotating the electrodes for 90°.

To determine whether electroporation increases radiosensitising effect of BLM in vitro, LPB cells were exposed to electric pulses in the presence of BLM and placed in low attachment plates for 5 min. Thereafter, 1 ml of EMEM was added and after additional 5 min cells were irradiated and plated in Petri dishes (Figure 1A). The survival of cells treated with different treatment combinations that included irradiation, were normalized to the appropriate control group, in order to demonstrate the interaction between the treatments on cell's survival. Survival of cells after electroporation and irradiation was normalized to the effect of electroporation, survival of cells after combination of BLM and irradiation was normalized to the effect of BLM, whereas when the cells were irradiated after ECT, cell survival data were normalized to the effect of ECT. All the data were pooled from 3 independent experiments performed in triplicates. The effect of treatments was assessed by comparison of IC₉₀ values (drug concentration required to reduce cell survival for 90%). Enhancement factor (EF) was calculated on the basis IC₉₀ values.

To determine whether application of electric pulses increases radiosensitising effect of BLM, ECT was combined with local tumor irradiation with 20 min interval between the treatments (Figure 1B). Antitumor effectiveness of ECT combined with irradiation was evaluated by comparing the effects to single treatments or to the other treatment combinations: control untreated tumors, tumors treated with BLM, electroporation or irradiation only, tumors treated by ECT, and tumors treated with combination of BLM or electroporation and irradiation. Tumors were irradiated with single doses (5, 10, 15, 20, 25, 30, 35, 40, 45, 50 Gy). Each experimental group consisted of 3–7 mice and the data were pooled from 3 independent experiments.

The tumor volume was determined by measuring three orthogonal tumor diameters (e₁, e₂ and e₃) with Vernier calliper. Tumor volume was calculated by the formula V = π × e₁ × e₂ × e₃/6. Tumor regression and regrowth was followed until the tumors grew up to 350 mm³, and then the animals were sacrificed. If the tumors regressed after therapy, animals were checked for the presence of the tumor in the irradiation field at 4–5 day intervals up to 100 days. The animals were considered cured if they were tumor free at day 100. Radiation dose needed to control 50% of irradiated tumors (TCD₅₀) was used to determine response of tumors (included were 9–21 mice per irradiation dose). Dose response curves were computed by the logit method of analysis 24. Enhancement factor (EF) was calculated based on TCD₅₀ values.

EPR oximetry was used to measure partial oxygen pressure (pO₂) in normal muscle, subcutaneous tissue, untreated LPB tumors as well as in the tumors subjected to electric pulses, as described previously 25. Briefly, EPR oximetry is based on the fact that molecular oxygen is paramagnetic with two unpaired electrons and has a very rapid relaxation rate. It provides an effective relaxation mechanism to other paramagnetic species via Heisenberg spin exchange interaction. Consequently, the presence of oxygen influences both, spin-spin and spin-lattice relaxation times of the paramagnetic probe. Therefore, an increase in oxygen concentration increases the EPR spectra line-width, decreases the resolution of hyperfine structure, and decreases the microwave power at which the saturation of the EPR absorption lines occurs. All these parameters can be measured by EPR. Therefore, 3 small crystals of the oxygen sensitive paramagnetic probe lithium phtalocyanine (LiPc, 15 – 40 μm in diameter) were inserted into tumor (one in centre and one in periphery) and one in selected normal tissue (subcutaneous tissue or skeletal muscle) one day before the treatment. Immediately after the treatment, the EPR spectra were recorded continuously for 60 min and thereafter at each selected time point for 15 min. The mice were anaesthetized by intraperitoneal injection of a mixture of Domitor (1.0 mg/kg body weight; Pfizer GmbH, Karlsruhe, Germany) and 10% ketamine (75.0 mg/kg body weight; Veyx-Pharma GmbH, Schwarzenborn, Germany). During the anesthesia, warm air was used to keep the body temperature as close as possible to 37°C with variations of up to 0.5°C during single measurement. The measurements were performed on Varian E-9 EPR spectrometer, with a custom-made low frequency microwave bridge operating at 1.1 GHz and an extended loop resonator (11 mm in diameter), both designed by Professor T. Walczak (Darmouth Medical School, Hanover, NH). Typical spectrometer settings were: modulation frequency, 100 kHz; modulation amplitude not more than one-third of the peak-to-peak line-width, and scan range, 2 mT. The line width of the EPR spectra reflects the pO₂, on the site of the paramagnetic probe and was determined from the calibration curve presented elsewhere, as the changes in the EPR spectrum can be calibrated with known concentration of oxygen 25.

All data were tested for normality of distribution. The statistical differences between the treatment groups were assessed by a t-test after one-way ANOVA was performed and fulfilled. SigmaStat statistical software (SPSS inc.) was used for statistical analysis. P levels of less than 0.05 were taken as significant.

In order to determine the radiosensitising effect of BLM, LPB cells exposed to BLM alone or ECT were irradiated with graded X-ray doses up to 8 Gy (Figure 2). Exposure of cells either to BLM, electroporation or ECT statistically significantly increased radiation response of LPB cells. Increase in radiation response was the lowest when the cells were exposed to BLM (EF = 1.19), probably due to low BLM concentration and a very short exposure time. On the other hand, the radiosensitivity of LPB cells was greatly enhanced when the cells were pretreated with ECT (EF = 1.53). When cells were exposed to electric pulses prior to irradiation the enhancement factor was 1.25. Therefore, radiosensitising effect of ECT might not be due to just increased BLM delivery into the cells by electroporation, but also to radiosensitisation by electroporation of cells (Figure 2). Surviving fraction of LPB cells after pertinent control treatments BLM alone, electric pulses alone and ECT is presented in Table 1.

Radiosensitising effect of ECT was evaluated also on subcutaneous LPB tumors in mice. As endpoints for evaluation of antitumor effectiveness were used tumor growth delay and local tumor control (TCD₅₀ assay). Neither treatment of animals with BLM alone nor application of electric pulses to the tumors prior to irradiation of tumors had any effect on local tumor control, as observed by TCD₅₀ values (Table 2, Figure 3). However, ECT of tumors statistically significantly increased radiation response of the tumors (Table 2, Figure 3). TCD₅₀ value was reduced from 23.1 Gy in mice that were treated with irradiation only, to 12.4 Gy in mice that were treated with ECT prior to irradiation. Since the enhancement factor was 1.9, it is evident that electroporation of tumors significantly contributed to radiosensitisation of tumors with BLM, specifically because combined treatment of ECT with irradiation was statistically significantly more effective than treatment of tumors that were irradiated in combination with electroporation or BLM.

Antitumor effectiveness of pertinent control groups BLM alone, electric pulses alone and ECT is shown in Table 1. ECT of tumors had good antitumor effect and some of the tumors were cured (8.7%). Treatment with BLM only or application of electric pulses to the tumors had minimal effect on tumor growth and no tumor cures were obtained after either of the treatments.

Side effects associated with application of electric pulses were instantaneous contractions of muscles located beneath the site of treatment, which disappeared immediately after each pulse. Irradiation alone or combined with BLM, electric pulses and ECT resulted in hair loss, but none of the treatments induced skin desquamation.

In order to determine whether electroporation of tumors can induce radiobiologically relevant hypoxia, that could reduce radiosensitising effect of ECT, pO₂ was measured in the tumors after exposure to electric pulses. To determine whether reduction in tumor oxygenation is confined to the electroporated area (tumor), pO₂ was measured in the same animal also in the normal tissues (skeletal muscle, subcutaneous tissue) that were not exposed to electric pulses. Application of electric pulses to the tumors statistically significantly reduced pO₂ in the tumors (Figure 4). Five min after application of electric pulses to the tumors pO₂ was lowered in the centre of the tumors down by 75% and in the periphery of the tumors down by 50%. Immediately thereafter, tumors started to reoxygenate; however 6 h after electroporation the oxygen level was still at 70% of pretreatment level and even after 24 h was not completely restored (Figure 4). Specifically, at 20 min after electroporation of the tumors, i.e. at the time of tumor irradiation, in the tumor centre as well as in the tumor periphery there was still reduced tumor oxygenation, at the level of radiobiologically relevant hypoxia (4.6 ± 0.6, 6.1 ± 0.3, respectively). In normal tissues; skeletal muscle and subcutaneous tissue pO₂ was not affected by application of electric pulses to the tumors (Figure 4). In addition, pO₂ was measured in untreated LPB tumors at the same time points to evaluate the whether tumor growth can affect tumor oxygenation. As shown on Figure 4, pO₂ in untreated tumors did not change throughout the 24 h measurement.