Cancer Therapy Vol 2, 533-548, 2004

Abscopal regression of subcutaneously implanted N29 rat glioma after treatment of the contra-lateral tumours with pulsed electric fields (PEF) or radiation therapy (RT) and their combinations (PEF+RT)

Research Article

Bertil R.R. Persson^1,3,*,Catrin Baureus Koch^1,2,3, Gustav Grafström^1,3, Crister Ceberg^1,3 and Leif Salford^2,3

¹Dept. of Medical Radiation physics

²Dept. of Neurosurgery

³Rausing Laboratory, Biomedical Centre, Lund University, 221 85 LUND, Sweden

__________________________________________________________________________________

*Correspondence: Bertil R.R. Persson, PhD, MD h.c., professor, Dept. of Medical Radiation Physics, University Hospital in Lund, Klinikgatan 7, S-221 85 LUND, Sweden; Tel: +4646173110; Fax: +4646134249; e-mail: bertil.persson@radfys.lu.se

Key words: Abscopal effect, Fischer rat, glioma, N29 tumour, electric pulses, PEF, radiation therapy, ⁶⁰Co-g radiation

Abbreviations: Abscopal enhancement ratio, (AER); control, (Ctrl); intraperitoneally, (i.p.); Pulsed Electric Field, (PEF); radiation therapy, (RT); source-skin distance, (SSD); Specific abscopal effect ratio, (SAER); Specific abscopal effect, (SAE); Specific therapeutic effect, (STE); Therapeutic enhancement ratio, (TER); tumor volume, (TV); tumour volume growth rate, (TGR); unexposed controls, (UC); unexposed tumour, (UE)

Received: 8 November 2004; Accepted: 26 November 2004; electronically published: January 2005

Summary

The aim of the present work is to study the Abscopal regression of subcutaneously implanted contra-laterally tumors treated with pulsed electric fields and/or radiation therapy. The study was performed on rats of the Fischer-344 strain with N29 rat glioma tumors implanted subcutaneously on both the hind legs. Only the tumours on the right hind legs were treated with pulsed electric field "PEF" (16 exponentially decaying pulses with a maximum electric field strength of 1300 V/cm and t_l/e= 1 ms) or/and radiation therapy "RT" (⁶⁰Co-g radiation given in daily fractions of 5 Gy, to a total absorbed dose of 20 Gy). The animals were arranged into one group of untreated controls and 3 groups of various treatments: PEF only, RT only, and in combination PEF + RT. The tumour growth rate of the right-lateral treated tumours was significantly decreased for independent treatment with pulsed electric fields (PEF, p<0.005) or radiation therapy (RT, p<0.001) as well as combined treatments (PEF+RT, p<0.005). For the left-lateral untreated tumours the growth rate was significantly decreased in rats with right-lateral tumours treated with RT (p<0.00 1) and the combination PEF+RT (p<0.00l). In rats treated with PEF only, there was no significant decrease in tumour growth rate of the left-lateral tumour. The specific therapeutic effect "STE" of the right-lateral treated tumours were all significantly different from zero (p<0.00l) STE = 0.28 ± 0.02 for PEF; 0.44 ± 0.02, for RT and 0.3 ± 0.10 for the combined treatment PEF+RT. Corresponding quantity for the left-lateral unexposed tumours is named the specific abscopal effect "SAE", which is highly significantly different from zero p<0.00l for animals treated on the opposite side with RT, SAE = 0.22 ± 0.02, or with the combined treatment PEF+RT 0.20 ± 0 03. In rats treated with PEF on the opposite side the SAE= 0.01 ± 0.08 that is not significantly different from zero (p=0.8). Therapeutic enhancement ratio (TER) and Abscopal enhancement ratio (AER) at combined treatment with PEF and RT is 0.43 ± 0.05 and 1.1 ±1 0.1 respectively. It is interesting to note that the AER is about twice the Therapeutic enhancement ratio (TER). Thus by combining radiation therapy with pulsed electric fields and there is an enhancement therapeutic effect of the treated tumour as well as an effect on the distant non-treated tumours.

I. Introduction

A. Radiation therapy and the Abscopal effect

Radiation therapy is beside surgery still the most widely used therapy modality for cancer treatment and in developed countries it is given to one out of two patients with cancer (Knöös 1991, SBU 2003). Although a lot of progress is made in development of new methods and techniques for radiotherapy about half of the curatively intended treatments fail as a result of either distal or local recurrences (DeVita 983). Improvements of local radiotherapy are focused on fractionation and higher absorbed dose to the clinical target volume without exceeding the tolerance of surrounding normal tissue (Suit and Miralbell 1989; Suit et al, 1988, 2002). The therapeutic effect of radiation therapy refers to the direct exposed tumours and effects on tumours outside the treated target area are mostly not considered in radiation therapy.

Effects of radiation therapy on cancer tumors outside of the radiation field have, however, been reported in many malignancies (Nobler et al, 1969; Ehlers et al, 1973; Kingsley 1975; Antoniades et al, 1977; Rees et al, 1981; Rees and Ross 1983; Sham et al, 1995; Obba et al, 1998). This phenomenon was originally described as abscopal effect by R. J. Mole in 1953. The definition of abscopal effect comes from the Latin ab (position away from) and scopus (mark or target). The abscopal mechanism of action remains still unexplained, although a variety of under-lying biologic events can be hypothesized, including a possible role for the immune system (Uchida et al, 1989; Macklis et al, 1992). The Abscopal effect studied in mice with 67NR tumor after RT with 2 or 6 Gy, was recently proven to be an immune mediated effect (DeMaria 2004). The abscopal effect is, however, not often observed clinically, possibly because many tumor-bearing hosts develop immune suppression (Kusmartsev and Gabrilovich 2002).

Nagasawa and Little (1992) observed that cells hit by a-particles and neighbouring cells without hit both exhibit same type of damage. The phenomenon was called “bystander effect” borrowed from the gene therapy field. Since then several reports and reviews have appeared dealing with this kind of nonlinear dose-response relationship that is called both bystander effect and abscopal effect (Mothersill and Seymour 2001, Azzam et al 2004).

B. The effect of pulsed electric fields

Pulsed Electric Field (PEF) treatment is the application of short, intense electric pulses of high field strength that cause transient permeabilization of the cell membrane, thus allowing extra cellular administered compounds, e.g. DNA plasmids, cytotoxic drugs or other pharmaceuticals to reach the interior of the cell. This method has been employed in vivo to introduce anticancer agents into tumors (Engström et al, 1998, 2001a; Jaroszeski et al, 1997a, b, 1999; Salford et al, 1993) and recently also DNA-plasmids and proteins into various tissues (Heller et al; 1996a, 2001; Nishi et al, 1996; Aihara 1998; Mir et al, 1998; Rols et al 1998). A series of clinical trials of this treatment modality has been performed and with encouraging results in cancer therapy (Heller 1996b, 1997, 1998, 1999; Mir et al, 1998; Sersa et al, 1998, 1999, 2000).

Pulsed Electric Field (PEF) treatment can by itself create cellular and sub cellular lesions by inducing lipid per-oxidation associated with the membrane area being permeabilized, and appears to be correlated to cell survival (Maccarrone et al, 1995a, b; Danfelter et al, 1998). In biological tissue peroxide can form hydroxyl radicals OH·, which enable single strand breaks in DNA (Mello-Filho and Meneghini, 1984). DNA damage from pulsed electric fields has thus been reported, where the number of DNA strand breaks was correlated to the field strength and duration of the electric pulses (Meaking, et al, 1995; Meldruni et al, 1999). Pulsed electric fields have also been found to induce apoptosis and activation of caspases (Pinero et al, 1997; Hofmann et al, 1999).

C. Effect of pulsed electric fields in combination with radiation therapy

Pulsed Electric fields used in combination with radiation therapy has recently been found to increase the differential response between tumour and normal tissue (Engström et al, 2001b; Persson et al, 2003). The therapeutic effect of pulsed electric fields combined with radiation therapy was first studied in rats of the Fischer-344 strain with glioma tumors implanted subcutaneously on the thigh (Engström et al, 2001b). Exponentially decaying pulsed electric fields of 1400 V/cm and t_l/e= 1ms were applied to the tumors with 16 pulses in the 4 consecutive days combined with radiation therapy of total absorbed radiation dose of 20 Gy, in 4 fractions of 5 Gy each day in four consecutive days. The combined treatment with pulsed electric fields was found to have a sensitizing effect on radiation therapy and resulted in a high fraction of complete remissions (6/9) and increased survival compared to the controls (60% at 100 days) (Engström et al, 200lb).

Fitting the data obtained from consecutive measurements of tumor volume (TV) of each individual tumor to an exponential model estimated the tumor growth rate (TGR %per day) after the first day of treatment (t=0). The TGR of N32 tumors treated with the combination of 4PEF+4RT are significantly decreased compared to the controls p<0.0001) compared to RT alone p<0.0001) and compared to PEF alone p<0.001). The combined treatment of N32 gives significant effect on the tumor growth rate after 2, 3 and 4 treatment session while RT alone seems to be most efficient after one treatment of 5 Gy and PEF alone is most efficient after 2 treatments at 2 consecutive days (Persson et al, 2003).

The specific therapeutic effect STE is defined as the difference between the average tumor growth rates of controls and exposed tumors divided by the average tumor growth rate of the controls. With 4 PEF treatments alone the average STE value was 0.32 for N32 tumors and 0 for N29; for 4RT alone the STE values were 0.29 and 0.42 respectively, and for combined treatments 4PEF+4RT 0.67 and 0.17 respectively. For the N29 tumors treated with 2PEF+4RT the STE value was 0.53. The values of the therapeutic enhancement ratio, , increase with the number of treatment sessions and the TER of the combined treatments is above 1 in two of the N32 series, which indicates a synergistic effect of 4PEF+4RT (Persson et al, 2003).

So far, there are no studies performed of the abscopal effect of pulsed electric fields or its combination with radiation therapy. In the present study we investigate the abscopal effect in Fischer rats with a N29 tumor inoculated on both the right leg and on the left leg. The right tumor was treated at about 30 days after inoculation with either RT or PEF and their combination (PEF+RT) while the left tumor was untreated. The size of the tumors on the flank was measured daily and growth of tumor was evaluated in terms of the change in tumor growth rate of exposed tumor relative to the control.

II. Materials and methods

A. Experimental animals and tumour inoculation

1. Animals

Rats of the Fischer 344 strain were used. The strain was maintained by continuous, single line, brother/sister mating in our laboratory.

2. Tumour cell cultures

All tumour cells were cultivated in antibiotic-free RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 10 mM Hepes, 0.5 mM pyruvate, and 0.096% NaHCO₃. Cell cultures were maintained in culture flasks Nunc, Denmark) and harvested with trypsin-EDTA.

3. Subcutaneous tumours

The rat glioma N29 was induced in our laboratory by subcutaneous administration in the hind legs. 200 000 cells were inoculated into the right leg, whilst 50 000 cells were inoculated into the left leg in order to simulate a secondary smaller tumour. Tumour volume is estimated as an ellipsoid by length, width and thickness measured with a calliper. When the tumours reached a volume of 9 cm³, the animal was sacrificed of ethical reasons.

B. Procedure of tumour treatment

The effect of pulsed electric fields and radiation therapy and their combination was investigated in male rats of the Fischer-344 strain with rat glioma N29 tumours implanted subcutaneously on the flank or on the thigh of the hind leg. The animals were arranged into five groups, which received electric pulses followed by radiation (PEF+RT) radiation treatment followed by electric pulses (RT+ PEF) radiation treatment only (RT) electric pulses only (PEF) or no treatment (Ctrl). The delay between electro pulsation and radiation treatment was kept as short as possible (4-5 minutes). The animals were arranged into groups of various treatments as shown in Table 1.

1. Radiation treatment

Animals were given fractionated radiation treatment using a ⁶⁰Co radiotherapy unit (Siemens Gammatron S) with a source-skin distance (SSD) of 80 cm and the maximum absorbed dose rate 0.65-0.70 Gy/min. A 0.5 cm thick, tissue-equivalent bolus (Super Flab, Mike Radio-nuclear instruments inc. NY, USA) was placed over the tumor to achieve full dose buildup and a more homogeneous dose distribution in the tumor. The radiation field size was collimated to cover the tumor area with a margin of at least 1 cm (Figure 1).

The absorbed dose to the exposed right-lateral tumour was 5 Gy/day x 4 (consecutive) days, in all 20 Gy, which in preparatory experiments showed to be a suitable, suboptimal and non-curative dose. While the absorbed dose to the left-lateral tumour was negligible.

Table 1. Number of animals in each group of various treatments.

Group	Treatment	Number of rats
Ctrl	Controls with no treatment	40
RT	Radiation therapy 4x5 Gy	15
PEF	Pulsed Electric Fields	25
PEF+RT	Pulsed Electric Fields + Radiation Therapy	15
Total		95

2. PEF treatment

Two rectangular flat electrodes were mounted on a slide calliper and connected to an exponential pulse generator (Figure 1). In the first series of experiment we used a BTX6OO (Genetronics, San Diego, USA) and since year 2000 a CytorExp 2000 (Aditus Medical AB, Lund Sweden) was used.

The pulsed delivered was monitored by an oscilloscope and the load was adjusted so that the time constant of the exponential pulse was 1 ms.

The hair over the tumor was shaved off and the skin was carefully covered with electrocardial paste to ensure good electrical contact between electrodes and skin. The paste also moistened the skin, reducing the transdermal impedance and limited the risk of skin necrosis from the pulse treatment. The tumours were gently fixed in position between the two electrodes, and the voltage was adjusted to the distance between the electrodes to deliver pulses of identical field strength to all tumours. Sixteen pulses of approximately 1400 V/cm with a time constant of 1 ms were delivered transdermally to the whole tumor during approx. 20 seconds. This treatment was repeated daily for four days.

C. Model for tumour growth analysis and synergistic enhancement

The tumours are treated with PEF radiation or a combination of these two. The question is how to express the enhancement of the therapeutic effect of the combined treatment with the single treatment and a hypothetical independent combination of the two single treatments.

1. Tumour growth rate

The tumour volume measurements of each tumour fitted to a model of exponential growth made the tumour volume growth rate (TGR) according to the following equation.

where

TV Tumour volume

TGR Tumour growth rate constant day^-1

TV₀ Tumour volume at the time of treatment

The therapeutic effect is defined as the ratio of the tumour volume between the treated tumour and the control group.

2. Specific therapeutic effect (STE)

The ratio of the tumour volume of the exposed tumour and corresponding control is a measure of surviving fraction, SF, of the cells in the treated tumour:

The therapeutic effect, TE, is a measure of the number of lethal events that has occurred in the cells of the treated tumour volume and thus defined as:

In order to get a therapeutic effect measure independent of time a quantity named "specific therapeutic effect" STE is defined. That is the tumour growth rate difference between the control and exposed tumour divided by tumour growth rate of the controls.

where

The average of the individual tumour growth rate constant (day^-1) in the group of N exposed rats.

The average of the individual tumour growth rate constant (day^-1) in the group of unexposed control rats.

The STE is equal to 0 when the average of tumour growth rate constant of the exposed group, is equal to the average of the tumour growth rate constant of the control

The STE is equal to 1 when the average tumour growth rate constant of the exposed group, is equal to 0, which means arrested tumour growth.

The STE is larger than 1 when the average tumour growth rate constant of the exposed group, is less than 0, which means a declining tumour volume.

3. Specific abscopal effect (SAE)

The "specific abscopal effect" SAE is defined as the tumour growth rate difference between the left-lateral unexposed tumour and corresponding control divided by tumour growth rate of the controls.

where

The average of the individual tumour growth rate constant of the unexposed (UE) left-lateral tumours in the group of N exposed rats.

The average of the individual tumour growth rate constant in the left-lateral tumours in the group of unexposed controls (UC).

4. The therapeutic enhancement ratio and abscopal effect enhancement ratio

The enhancement effect the combined treatments (PEF+RT) is the ratio of the effect of the experimental combination of PEF and RT and the therapeutic effect the hypothetical independent combination of the two agents.

The therapeutic enhancement ratio of the exposed turnours is thus defined as:

and the abscopal enhancement ratio of the left-lateral unexposed tumour is defined as:

where the hypothetical effect by independent (additive) action of ionizing RT and PEF is given by

STE_Independent= STE_RT + STE_PEF

for the exposed tumours

SAE_Independent = STE_RT + STE_PEF

for the left-lateral unexposed tumours

The enhancement ratios are measures of any synergistic or diminishing effect obtained in the combination of the two agents. It may be due to interaction of sub lethal lesions induced by both agents to produce lethal events that cause the enhancement ratio> 1. If the individual therapies are highly aggressive by themselves there might, however, also be an "over killing" effect that reduce the effect compared to the additive action, so that enhancement ratios <1. It is thus important to investigate the effect of combined treatments at various dose levels to find the maximum value of enhancement ratios.

III. Results

A. TGR

The tumour growth of each individual tumour was measured during the entire lifetime of all the animal in the following experimental groups (Table 2). The female Fischer-344 rats had N29 glioma tumours implanted on both thighs and only the tumours on the right side were treated.

In the series B the right-lateral tumours were treated with four fractions of exponential PEF, RT and their combination (PEF+RT). The PEF treatment was performed at days 42, 43, 45 and 46 after inoculation of the tumours by applying 16 exponential pulses at electric field strength of 1400 V/cm, and 1.0 ms time constant with plate electrodes over the tumours. The radiation therapy was performed at days 29, 30, 32 and 33 after inoculation with four daily fractions of 5 Gy (total 20 Gy). The combined treatment was performed with less than 1 h between the PEF and RT treatments performed as above at days 30, 31, 33 and 34 after the day of inoculation.

The tumour volume was estimated by more or less daily measurements. At those occasions the rats were also observed for symptoms from the tumour growth. The Figures 2 display the average tumour volume at each time of measurement of tumours in the animals of the series-B. At about 30 days after inoculation and thereafter the tumour growth data fit well to an exponential growth model. The fitted curves for all tumours in each group are displayed in the Figure 2 as solid lines.

The time the tumour reached a volume of about 9 cm³, when the animal was sacrificed, varied within the group and at the end only few or a single rat survived more than 50 or 60 days in the control groups. It is, thus, not quite correct to draw quantitative conclusions directly out of the growth curves of the averaged data. In order to perform a more correct analysis of the results, the tumour growth rate is estimated from the volume measurements of each individual tumour. Thus the tumour volume data after the time of treatment of each individual tumour were fitted to a model of exponential growth . Where "TV" is tumour volume, ⁹⁹TGR" is tumour growth rate constant (day^-1) and time "t" is the number of days after the first treatment.

The results thus obtained from the growth rate of right lateral tumours tumours in all series-A, -B, -C, and -D with average of treated with either PEF or RT and their combinations (PEF+RT) are displayed in Figure 3 in blue columns. The tumour growth rate results of the corresponding untreated left-lateral tumours are displayed in red. The p-values of significant difference to the controls are displayed in the columns.

Series-A include controls only and series-C controls and tumours treated with pulse electric fields (PEF) only. In the series-D the right-lateral tumours were treated with two fractions of exponential pulsed electric fields (PEF) and 4 fractions of radiation therapy (RT) and their combination (PEF+RT). The PEF treatment was performed on days 40 and 44 after inoculation of the tumours by applying 16 exponential pulses at electric field strength of 1400 V/cm, and 1.0 ms time constant. The radiation therapy was performed at days 33, 34, 36 and 37 after inoculation with four daily fraction of 5 Gy (total 20 Gy). The combined treatment was performed with less than 1 h between the PEF and RT treatments performed as above at days 33, and 36, while at days 34 and 37 were given RT only.

Table 2. Number of animals in each experimental series and groups of treatment.

Exp. series	Controls	PEF	RT	PEF+RT
A	8
B	17	9	8	7
C	8	9
D	7	7	7	8
All	40	25	15	15

B. STE and SAE

The therapeutic effect is equivalent to the difference in tumour growth rate between the controls and the exposed tumours calculated from the day of treatment. The "specific therapeutic effect" STE is obtained by normalizing the difference in tumour growth rate to the tumour growth rate of each individual exposed tumour (F) of the average of its controls at the same period of time.

This quantity is independent of time and individual variations in the tumour growth characteristics of each experiment.

The corresponding quantity "specific abscopal effect" SAE is obtained by normalizing the difference in tumour growth rate to the tumour growth rate of each individual unexposed tumour (UE) of the average of its corresponding UC at the same period of time.

Since the STE and SAE values are normalized to the controls of each experimental series they can be pooled and treated as one population. The results the pooled data of series B, C, D are displayed in Figure 5.

C. Specific abscopal effect ratio (SAER)

The Specific Abscopal Effect Ratio of various types of treatments is evaluated by comparing the specific therapeutic effect growth of the exposed tumour to the corresponding untreated left- lateral tumours in the same group of treatment.

Where SAE_Left is the average of the specific abscopal effect evaluated for the untreated left tumours and SAE_Right is the average of the specific therapeutic effect of the treated right tumours.

The "Specific abscopal effect ratio" SAER of tumours in all series with animals treated with PEF, RT and their combinations (PEF+RT) are given in Figure 6, which includes data from the experimental series, B, C, D.

D. Therapeutic enhancement ratio (TER) and AER

The therapeutic enhancement ratio of the combined treatment is defined as the ratio of the specific therapeutic effect of the combined treatment and the sum of the independent treatments.

The abscopal enhancement ratio of the combined treatment is defined as the ratio of the specific therapeutic effect of the untreated tumours on rats with combined treatment of the left-lateral tumour and the sum of the corresponding independent treatments.

TER and AER at combined treatment with PEF and RT are given in Figure 7 with data from the experimental series; B, and D to perform a statistical analysis the results the tumour growth rate of each individual tumour is estimated from the tumour volume measurements.

In the experimental series-B the tumour on the left side was implanted 8 days after the tumour was implanted on the right side, in order to simulate the occurrence of a distant smaller secondary tumour. The right tumour was considered as the main tumour and was treated with PEF, RT and their combination. The tumour growth rate of the treated tumour was significantly decreased for independent treatments with PEF (p<0.05) and RT (p<0.001). But there was no significantly decrease (p=0.2) in the tumour growth rate with combined treatment (PEF+RT). Although there is a large effect in some tumours as seen in Figure 3, the variation is large between individual tumours.

The tumour growth rate of the untreated left-lateral tumour, however, is most significantly reduced (p<0.0005) in the group of rats with right-lateral tumours treated with the combination PEF+RT. In the groups with independent treatments of the right-lateral tumour, the tumour growth rate was significantly reduced for RT (p<0.01) but no significant effect (p=0.08) with PEF treatment.

In the experimental series D the tumour on the left side was simultaneously inoculated with the tumour on the right side, but with 1/4 fewer cells to get a smaller tumour at the time of treatment. The right lateral tumours were treated with PEF, RT and their combination PEF+RT. The tumour growth rate of the right-lateral treated tumour in this series was significantly decreased for independent treatments with PEF (p<005) and RT (p<0.005) and with combined treatment PEF+RT (p<0.005).

The tumour growth rate of the left-lateral untreated tumour is most significantly reduced in the group of rats independently treated with RT on the right-lateral tumour. In the groups with independent treatments with PEF and with combined treatments PEF+RT of the right-lateral

tumour, there was no significant reduction of tumour growth rate on the left-lateral tumour.

By combining the data from all comparable experimental series (A, B, C, D) the tumour growth rate of the right-lateral treated tumour was significantly decreased for independent treatments with PEF (p<0.005) in 25 tumours and RT (p<0.001) in 15 tumours and with combined treatment PEF+RT (p<0.005) in 15 tumours.

The tumour growth rate of the left-lateral untreated tumour, however, is significantly reduced in the group of rats treated with RT (p<0.000l) in 15 tumours and the combination PEF + RT (p<0.000l) in 15 tumours. In the groups with independent treatment of right-lateral tumours with PEF, there was no significant reduction of the tumour growth rate of the left-lateral tumours.

IV. Discussion

A. Tumour growth rate (TGR)

It is quite difficult to draw any quantitative conclusions out of the average growth curves because of the variations in the growth of the individual tumour and the time of death. In order to perform a statistical analysis the results the tumour growth rate of each individual tumour is estimated from the tumour volume measurements of each tumour. The results of the tumour growth rate thus obtained are summarized in Table 3.

B. Specific therapeutic effect (STE) and specific abscopal effect (SAE)

The therapeutic effect is the difference in tumour growth rate between the controls and the exposed tumours. The “specific therapeutic effect” STE is obtained by normalizing the difference in tumour growth rate to the tumour growth rate of the controls. This quantity is independent of time and the tumour growth characteristics of each experiment.

As a measure of the Abscopal effect the “specific abscopal effect” SAE was evaluated as the difference in tumour growth rate between the controls and the untreated left tumour. The results of the “specific therapeutic effect” STE are summarized in Table 4.

In the experimental series-B the tumour on the left side was implanted 8 days after the tumour was implanted on the right side, in order to simulate the occurrence of a distant smaller secondary tumour. The right tumour was considered as the main tumour and was treated with PEF, RT and their combination. The tumour growth rate of the treated tumour was significantly decreased for independent treatments with PEF (p<0.05) and RT (p<0.001). But there was no significantly decrease (p=0.2) in the tumour growth rate with combined treatment (PEF+RT). Although there is a large effect in some tumours as seen in Figure 3, the variation is large between individual tumours.

The tumour growth rate of the untreated left lateral tumour, however, is most significantly reduced (p<0.0005) in the group of rats with right-lateral tumours treated with the combination PEF+RT.

Table 3. Average mean tumour growth-rate (% per day) of all experiments

Series of Experiment	Type of treatment	TGR Right	SE	N	t-test vs Ctrl Right	TGR Left	SE	N	t-test vs Ctrl left
All	Controls	8.4	± 0.3	40	1.0	9.1	± 0.3	40	1.0
A	Controls	9.4	± 1.1	8	1.0	10.6	± 0.6	8	1.0
B	Controls	7.7	± 0.3	17	1.0	8.7	± 0.3	17	1.0
C	Controls	9.1	± 0.7	8	1.0	9.6	± 0.6	8	1.0
D	Controls	8.2	± 0.8	7	1.0	7.8	± 0.7	7	1.0

Series of Experiment	Type of treatment	TGR Right	SE	N	t-test vs Ctrl Right	TGR Left	SE	N	t-test vs Ctrl left
All	PEF	5.9	± 0.6	25	0.003	8.1	± 0.4	25	NS
A	PEF
B	PEF	5.1	± 0.9	9	0.03	7.2	± 0.7	9	NS
C	PEF	6.7	± 1.4	9	NS	9.9	± 0.6	9	NS
D	PEF	5.7	± 0.6	7	0.03	7.1	± 0.6	7	NS

Series of Experiment	Type of treatment	TGR Right	SE	N	t-test vs Ctrl Right	TGR Left	SE	N	t-test vs Ctrl left
All	RT	4.5	± 0.3	15	< 0.0001	6.1	± 0.4	15	< 0.0001
A	RT
B	RT	4.4	± 0.5	8	< 0.0001	6.6	± 0.6	8	0.01
C	RT
D	RT	4.5	± 0.3	7	0.002	5.6	± 0.3	7	0.02

Series of Experiment	Type of treatment	TGR Right	SE	N	t-test vs Ctrl Right	TGR Left	SE	N	t-test vs Ctrl left
All	PEF+RT	5.4	± 0.7	15	0.003	6.69	± 0.18	15	< 0.0001
A	PEF+RT
B	PEF+RT	6.0	± 1.3	7	NS	6.71	± 0.32	7	0.0004
C	PEF+RT
D	PEF+RT	4.9	± 0.4	8	0.004	6.67	± 0.21	8	NS

Table 4. Average Specific Therapeutic effects STE and Specific Abscopal effect SAE of all experiments

Series of Experiment	Type of treatment	STE Right	SE	N	t-test STE > 0	SAE Left	SE	N	t-test SAE > 0
All	PEF	0.28	± 0.02	27	0.0005	0.01	± 0.08	27	NS
B	PEF	0.33	± 0.1	9		0.17	± 0.1	9
C	PEF	0.26	± 0.2	9		-0.03	± 0.1	9
D	PEF	0.26	± 0.1	9		-0.10	± 0.1	9

Series of Experiment	Type of treatment	STE Right	SE	N	t-test STE > 0	SAE Left	SE	N	t-test SAE > 0
All	RT	0.44	± 0.02	15	< 0.0001	0.22	± 0.02	15	0.0003
B	RT	0.42	± 0.06	8		0.24	± 0.07	8
C	RT
D	RT	0.47	± 0.06	7		0.20	± 0.07	7

Series of Experiment	Type of treatment	STE Right	SE	N	t-test STE > 0	SAE Left	SE	N	t-test SAE > 0
All	PEF+RT	0.32	± 0.10	15	0.003	0.20	± 0.03	15	< 0.0001
B	PEF+RT	0.22	± 0.17	7		0.23	± 0.04	7
C	PEF+RT
D	PEF+RT	0.41	± 0.05	8		0.17	± 0.03	8

specific abscopal effect was as low as 0.17.

In a previous investigation it was shown that the therapeutic response of the combined treatment increased with the number of PEF treatments (Persson et al 2003). In the series-D only two PEF treatments were given in comparison to 4 in series-B. Thus the difference in the number of PEF treatments in the two series might be the reason for the difference in therapeutic response.

C. Therapeutic (TER) and abscopal enhancement ratio (AER)

The therapeutic enhancement ratio of the combined treatment is defined as the ratio of the specific therapeutic effect of the combined treatment and the sum of the independent treatments.

The abscopal enhancement ratio of the combined treatment is defined as the ratio of the specific therapeutic effect of the left-lateral untreated tumours on rats at combined treatment of the right-lateral tumour and the sum of the corresponding independent treatments.

Therapeutic enhancement ratio (TER) and Abscopal enhancement ratio (AER) at combined treatment with PEF + RT are 0.29 ± 0.07 and 0.55 ± 0.05(SE) respectively in series-B, and 0.57 ± 0.08 and 1.7± 0.2 respectively in series-D. The average of the enhancement ratios of the two experimental series-B, -D are TER =0.43 ± 0.07 and AER=1.1 ± 0.1 respectively. It is interesting to note that the values Abscopal enhancement ratio (AER) are about twice the Therapeutic enhancement ratio (TER) in both experimental series. In a previous study of the therapeutic effect of PEF+RT on rat glioma the TER values were >1 in a few cases (Persson, 2003). Thus by combining tumour treatment with pulsed electric fields and radiation therapy there is an enhancement effect of the treated tumour as well as on the distant non-treated tumours that is more than additive. The average of the abscopal enhancement value AER>1 might indicate that the combination PEF+RT has a synergistic effect.

After PEF- treatment only there is no abscopal effect, while it is significantly increased after RT and PEF+RT treatment. This might indicate that RT produces specific factors responsible for the abscopal effect. Emerit et al, (1995) observed radiation-induced clastrogenic factors in plasma samples from RT - patients. It has also been observed that cells exposed with ⁶⁰Co g-radiation produced a factor that mediates cell death in cells never exposed to radiation (Mothersill 2001). Other studies suggest that ionizing radiation induces the release of cytokines which mediate a systemic anti-tumour effect by activation of immune activity (Uchida 1989). The existence of radiation induced factors in vivo is now well accepted and they are likely to be tissue and patient specific (Mothersill 2002).

The mechanisms explaining this phenomenon might be either a molecular or an immunological effect which will be further considered in future investigations.

V. Conclusions

The tumour growth rate of the right-lateral treated tumours was significantly decreased for independent treatment with pulsed electric fields (PEF, p<0.005) or radiation therapy (RT, p<0.001) as well as combined treatments (PEF+RT, p<0.001).

The tumour growth rate of the left-lateral untreated tumour was significantly reduced in the group of rats independently treated with RT (p<0.001) and the combination PEF+RT (p<0.001) of the right-lateral tumours. In the groups with independent treatments with PEF of the right-lateral tumour, there was no significant reduction of tumour growth rate of the left-lateral tumours.

The specific therapeutic effect “STE” of the right-lateral treated tumours were significant different from zero (p<0.001) for all type of treatments. Corresponding quantity for the left-lateral unexposed tumours, the specific abscopal effect “SAE”, is highly significantly different from zero (p<0.001) for animals treated on the opposite side with RT, or with the combined treatment PEF+RT. But in rats treated with PEF on the right-lateral side the SAE is not significantly different from zero.

Abscopal enhancement ratio (AER) at combined treatment with PEF + RT is about twice the Therapeutic enhancement ratio (TER). Thus by combining tumour treatment with pulsed electric fields and radiation therapy there is an enhanced therapeutic effect of the treated tumour as well as on the distant non-treated tumours.

Acknowledgements

We thank Susanne Strömblad and Catarina Blennow for excellent technical assistance. Financial support from Swedish Cancer Society, Hedvig Foundation, John and Augusta Persson Foundation for Medical Research, Lund University Hospital's donation funds and Faculty of Medicine at Lund University is gratefully acknowledged.

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