Synergism between hyperthermia, ultrasound and γ irradiation

Ultrasound in Med. & BioL Vol. 17, No. 6, pp. 607-612, 1991 0301-5629/91 $3.00 + .00 Printed in the U.S.A. © 1991 Pergamon Press plc

OOriginal Contribution

S Y N E R G I S M B E T W E E N H Y P E R T H E R M I A ,

U L T R A S O U N D A N D 3' I R R A D I A T I O N

PAM L O V E R O C K a n d G A I L TER H A A R Physics Department, Institute of Cancer Research, Sutton, Surrey, England

(Received 30 July 1990; in final Jbrm 31 December 1990)

Abstract--Hyperthermia is now established as a successful therapy for some turnouts. It has been found that, for some tumours, the combination of heat and ionizing radiation is more effective than either type of treatment on its own. Ultrasound is commonly used to produce hyperthermia. We have therefore studied the effect of combined heat, ultrasound and 6°Co 3" irradiation treatments on suspension cultures of V79 Chinese hamster lung fibroblasts. Cells were exposed to 43°C hyperthermia with or without simultaneous continuous wave ultrasound (2.6 MHz, 2.3 W cm -2) either before or after 6°Co 3" exposure (428 c Gy). It was found that when the two types of exposure followed one immediately after the other, then the ultrasound exposure increased the amount of cell killing produced by the combination of heat and ionizing radiation, whether the heat or the ionizing radiation came first. If an interval for repair of 72 minutes was left between the heat and ionizing radiation, then this enhancement was lost. The increase in cell killing produced by the ultrasound is shown to be caused by a mechanism that is nonthermal in origin.

Key Words: Hyperthermia, Ultrasound, 3' rays.

I N T R O D U C T I O N

It is now generally accepted that hyperthermia (up to 43-44°C) on its own is a useful therapy in only a few tumours . The main advantage of hyperthermia appears to lie in the synergistic effects that can be produced when it is used in conjunct ion either with ra- dio- or chemotherapy (Har -Kedar and Bleehen 1976). One technique for the induction of local heating uses ultrasound (Hand and ter Haar 1981). It is therefore of interest to discover whether there is any additional synergistic interaction between ultrasound and ionizing radiation that is nonthermal in origin.

There has been intermit tent interest in the exposure of cells and tissues to both ultrasonic and ionizing radiation. Spring (1969) studied the effect o f simultaneous 2 M H z ultrasound and 3' irradiation on the growth rate and germinative capacity of grass seeds. He found no effect on growth rate but a reduction in germinative capacity. Ult rasound on its own had no effect, but when the ultrasound and 3' exposures were simultaneous, the 3" dose required to reduce surviving fraction to 37% was reduced by a factor 2.6. No mechanism for this finding was hypothesized.

Address correspondence to: Gail ter Haar, Physics Depart- ment, Institute of Cancer Research, Sutton, Surrey, England.

Clarke and Hill (1970) looked for synergism between x-rays and ultrasound, when the ultrasound exposure was made either before, during or after x-ray exposure. They were unable to see any synergistic effect either in L5178Y lymphoma cells in suspension or in BICR/M 1 tumours implanted in the hind legs of rats. In all their experiments, the temperature was not allowed to rise above 37°C.

Todd and Schroy (1974) looked at the effect of ultrasound after x-irradiation in M3-1 Chinese hamster cells. Using a clonogenic assay, they found that ultrasound, while having no effect on its own, reduced by a factor 1.3 the x-ray dose needed to kill 99% of cells. Tempera ture increases within the cell suspensions are not quoted, but heating of the cells to 42°C for 10 minutes did not produce any enhancement of cell killing by x-irradiation.

Burr et al. (1982) looked for synergistic effects in producing chromosome damage in human lymphocytes exposed to therapeutic ultrasound (2 W cm -2) and 6°Co 3" irradiation. They found statistically significant differences between ultrasound exposures given simultaneously with the 3' rays and immediately afterwards. There was no significant difference if the ultrasound exposure was before the q/ exposure or if ultrasound was given more than two hours after 3, rays. Kuwabara et al. (1986) were unable

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to repeat these findings using diagnostic ultrasound exposures (spatial peak, temporal average intensity 46 mW cm-2).

There are many references in the literature showing, for different endpoints, that when ultrasound is used to induce a hyperthermic temperature rise, then the radiation dose required to give an effect may be reduced. (See, for example, Baker et al. 1982, Corry et al. 1982, Gavrilov et al. 1975, Kremkau and Wit- cofski 1977, Lehmann and Krusen 1955, Shuba et al. 1976 and Woeber and Stein 1963).

In the work reported here we describe a synergistic effect between ultrasound and 3' irradiation found in mammalian cells exposed in vitro under conditions for which the ultrasound produces no measurable temperature rise. This effect disappears if an interval of 72 minutes is left between exposures.

MATERIALS AND METHODS

Cell culture Chinese hamster lung fibroblasts V79-379A,

maintained in suspension culture, were used for this work. Cells were kept in log phase at concentrations of 105-106 cells per ml. The doubling time of 10-12 hours meant that the cultures were diluted daily (Stratford and Adams 1977).

Ultrasound exposure All ultrasound treatments were carried out using

the exposure system described in detail elsewhere (ter Haar et al. 1980, 1988; Loverock et al. 1990). The 2.6-MHz 2.5-cm diameter plane transducer was mounted at the bottom of a thermostatically con- trolled water bath (Grant TA Immersion thermostat, temperature stability _+0.005°C), at an angle of 45 ° to the horizontal, tilted upward. The water bath temperature was controllable to within _+0.05°C. The cylin- drical stainless steel sample holders, which had acous- tically transparent windows (2.5-cm diameter, 50-urn thick) and contained 8 ml of cell suspension, were mounted at the same 45 o angle to the horizontal, at a distance of 25 cm from the transducer front face. This is the position of the last axial maximum. With this arrangement, the ultrasonic beam passes through the sample chamber and hits the water surface where it is reflected through 90°; thus, standing waves are avoided. The total water path from transducer to surface is approximately 45 cm. Identical chambers, con- taining control suspensions, were also placed in the bath but away from the ultrasound beam.

Spatially averaged ultrasound intensities were measured using a tethered float radiometer (Shotton 1980). It is these intensities that are quoted in this article. All exposures were continuous.

Acoustic pressure profiles across the beam were measured using a calibrated PVDF membrane hydro- phone (GEC Marconi). These have been published elsewhere (Loverock et al. 1990). The maximum pressure within the sample holder was 0.6 MPa, and the beam profile, which was approximately Gaussian, had a full width at half maximum of 10 mm. The spatial average intensity within the chamber was 2.3 W cm -2.

During ultrasound exposures, the temperature of the cell suspension did not rise by more than 0.1 °C, as measured by a fine thermocouple (ter Haar et al. 1980; Loverock et al. 1990). The contents of the chamber reached thermal equilibrium within two minutes. This was true for both ultrasonically exposed and sham-exposed chambers.

6°Co 3" irradiation Cells were irradiated in the stainless steel cham-

bers, at room temperature, using a 6°Co 3, source, for 70 seconds. A mean dose of 428 c Gy was used. This was measured by LiF thermoluminescence dosime- try. This dose was chosen as it gave a convenient base- line surviving fraction of about 30%. Treatment re- gimes for specific chambers were allocated in a ran- dom fashion.

Cell clonogenic assay After treatment, cells were removed from the

chambers using a Pasteur pipette, washed by centrifu- gation, resuspended, counted and then serially diluted. They were then plated into petri dishes contain- ing M-MEM + 15% fetal calf serum (fcs) and incubated in a 95% air/5% Co/atmosphere for seven days. Colonies of more than 30 cells were scored as survi- vors, and the surviving fraction was calculated as the number of colonies scored divided by the initial number of cells plated.

The plating efficiency (or surviving fraction of control cells maintained at 37°C) was routinely greater than 85%. Survival curves showing the loga- rithm of surviving fraction as a function of time of exposure to 37°C or 43°C were plotted.

RESULTS

Ultrasound and 6°Co 3" exposure at 37°C Figure 1 shows survival curves for cells exposed

to ultrasound (2.6 MHz, 2.3 W cm -2) for different times while being maintained at 37 °C, either immediately before (u/s --~ 3", Fig. 1 a) or immediately after (7 --~ u/s, Fig. lb) 3, exposure. It can be seen that ultrasound has no effect on the level of survival resulting from the 3" exposure.

Ultrasound and 3' irradiation • P. LOVEROCK and G. TER HAAR 609

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Fig. 1. Graphs showing the survival of cells exposed to 6°Co 3" irradiation and continuous wave ultrasound (2.6 MHz, 2.3 W cm -2) at 37°C. (A) Ultrasound immediately before 3" exposure; (B) ultrasound immediately after 3, exposure; • survival of cells exposed only to I, rays; © survival of cells

exposed to both ultrasound and 3' rays.

Ultrasound exposure at 43°C Figure 2 shows the survival curves obtained

when cell suspensions are maintained at 43°C for different lengths of time, with or without simultaneous ultrasound exposure (2.6 MHz, 2.3 W cm-2). At this intensity a small synergistic effect is seen, but this is not statistically significant.

43°C with or without ultrasound followed by 6°Co 3' exposure

Figure 3 shows the survival curves for cells exposed to 43°C, with or without simultaneous ultrasound exposure (2.6 MHz, 2.3 W cm-2), for different lengths of time immediately before 3" irradiation (43°C _+ u/s --* 3"). The graph shows the means and standard errors of the mean for 10 experiments. A linear regression line has been fitted to the data. It can be seen that the number of cells surviving treatment is reduced for any given heating time, when the cells have been exposed to ultrasound as well as 43°C. The ratio of the slopes of the linear portion of the curve is 1.34. These lines have been shown statistically to be significantly different (p < .001).

43°C with or without ultrasound immediately after 6°Co 3, exposure

Figure 4 shows the survival curves for cells exposed to hyperthermia, with or without simultaneous

ultrasound (2.6 MHz, 2.3 W cm -2) immediately after 3, irradiation (3, --* 43°C + u/s). Again, ultrasound is found to enhance the amount of cell killing produced by the combination of heat and 3' irradiation. The graph shows the means and standard errors of the mean. The ratio of the linear regression slopes is 1.22. These have been shown statistically to be significantly different (p < .01).

The effect of allowing an interval for repair between treatments

Figures 5 and 6 show survival curves for cells that have been exposed to 3, irradiation either followed by, or preceded by, 43°C (with or without simultaneous ultrasound, 2.6 MHz, 2.3 W cm -2) for different times, but with an interval of 72 minutes between treatments, during which the cells were held at 37°C.

In one sequence (Fig. 5), the cells were heated to 43 °C (in some cases exposed simultaneously to ultrasound), incubated at 37°C for 72 minutes and then exposed to 6°Co 3, irradiation (43°C + u/s --* 37°C,

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o ib 2b 3'0 s.0 do 7b 8b Fig. 2. Graph showing survival of cells exposed to simultaneous 43°C and ultrasound (2.6 MHz, 2.3 W cm -2) for different lengths of time. • Cells exposed to 43°C; © cells

exposed simultaneously to 43°C and ultrasound.


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lb 2b 3'0 K0 5b 6b 7'0 Fig. 3. Graph showing survival of cells exposed to simultaneous 430C and ultrasound (2.6 MHz, 2.3 W cm -2) for different times immediately before 3" exposure. • 43°C --~

3'; 0 43°C + u/s -,- 7.

721 ~ "y). In the other (Fig. 6), the cells were exposed to 7 rays, incubated at 37°C for 72 minutes and then exposed to heat, with or without simultaneous ultrasound for different times (-y --~ 37°C, 72 t --~ 43°C + u/s). The graphs show the means and the standard errors of the means and the linear regression curves fitted to the data. There is no statistical difference between the lines for cells exposed to ultrasound and for those not exposed to ultrasound for either sequence of treatments.

DI SCUSSION

We have shown earlier that ultrasound exposures (3 W cm -2) of single cells in suspension may enhance the cytotoxicity of 43 °C hyperthermia (ter Haar et al. 1980). At the intensity used in the study reported here (2.3 W cm-2), there is a small increase in cell killing. Ultrasound on its own at these intensity levels has no effect on cell survival at 37°C, nor does it affect the

level of survival of cells exposed to 6°Co 3, irradiation at this temperature.

We have shown here that, if cells are exposed to ultrasound, the cytotoxic effect of a combined heat and 3' exposure is increased, whether the heat treatment is immediately before, or immediately after, the ionizing irradiation. The effect is greater if the heat treatment is given first. However, i fa resting period is left between the two treatments, during which repair can take place, then the enhancing effect of ultrasound is lost.

It is well known that the synergistic effect of heat and ionizing radiation is lost if an interval is left between treatments in which damage may be repaired (Field 1978; Stewart and Denekamp 1977). Heat treatments given prior to ionizing radiation exposure are effective for longer in their ability to potentiate ionizing radiation exposures than are those given after. This is also our finding here. In the absence of ultrasound, the ratio of the slopes of the linear por-

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1'0 20 3'0 t,,.b 50 6'0 7b Fig. 4. Graph showing survival of cells exposed to simultaneous 43°C and ultrasound (2.6 MHz, 2.3 W cm -2) for different times immediately after 3' exposure. • 3" --~ 43°C;

O 3' ~ 43°C + u/s.

Ultrasound and 3' irradiation • P. LOVEROCK and G. TER HAAR 61 1

tions of the survival curves is 1.06 for the two treatment sequences 43°C ~ 3' and 43°C --~ 37°C, 72 min --* 3' and 1.54 for the t rea tment sequences 3' --* 43°C and 3' --* 37°C, 72 min --* 43°C. Although there is still an enhancement in cell killing over that seen by either t rea tment alone when the 3' exposure is made first, it is not as great.

More work is required to determine the mechanism for the synergistic effect demonstra ted here. We have shown that there is no synergism between ultrasound and 3" irradiation at 37°C. Therefore, the effect is one that occurs only when the cell is heated, but it is nonthermal in origin as the ultrasound does not induce any additional temperature rise in the suspension. We have shown that a s imultaneous t reatment of 43°C and ultrasound for an hour can put cells into a block in the G1 phase of the cell cycle (Ormerod et al. 1990). After four hours, the cells start to cycle again, but normal cycling is not achieved until approximately 24 hours after t reatment. The ultrasound ef-

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10 20 30 t~0 50 6'0 70 Fig. 5. Graphs showing the survival of cells exposed to simultaneous 43°C and ultrasound for different times 72 minutes before 3' exposure. • 43°C --~ 72 min ~ 3"; © 43°C + u/s

72 min ~ 3'.

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lb 2b 3b sb 6b 7'0 Fig. 6. Graphs showing the survival of cells exposed to 3' irradiation 72 minutes before simultaneous 43°C and ultrasound exposure for different times. • 3' ~ 72 min --~ 43°C;

© 3' "-~ 72 min ~ 43°C + u/s.

fect seen in the present study has caused damage that appears to have been largely repaired after 72 minutes.

There is no evidence that ultrasound, at the intensity levels used for this work, can produce direct effects on DNA, but it is possible that the synergism seen here may be caused by an indirect effect on the cell nucleus. Further work is planned to study effects on cell cycling and to look for effects on nuclear pro- tein content. It is also possible that the effects seen here are membrane mediated. It is well known that heat causes changes in extracellular membranes and that ultrasound can affect ionic transport across membranes . Further studies are planned to elucidate the mechanism involved in producing the effects reported here.

REFERENCES

Baker, D. C.; Sager, H. T.; Elk©n, D.; Constable, W.; Rinehart, L.; Wills, M.; Savory, J.; Lacher, D. The response of kidney to ion-


izing radiation combined with hyperthermia induced by ultrasound. Radiology 145:515-519; 1982.

Burr, J. G.; Wald, N.; Pan, S.; Preston, K. The synergistic effect of ultrasound and ionizing radiation on human lymphocytes. In: Evans, H. J.; Lloyd, D. C., eds. Mutagen induced chromosome damage in man. New Haven, CT: Yale University Press; 1982:120-128.

Clarke, P. L. R.; Hill, C. R. Synergism between ultrasound and x-rays in tumour therapy. Brit. J. Radiol. 43:97-99; 1970.

Corry, P. M.; Spanos, W. J.; Tilchen, E. J.; Barlogie, B.; Barkley, H. T.; Armour, E. P. Combined ultrasound and radiation therapy treatment of human superficial tumors. Radiology 145:165-169; 1982.

Field, S. B. Response of normal tissues to hyperthermia alone or in combination with x-rays. In: Streffer et al., eds. Cancer therapy by hyperthermia and radiation. Munich, Germany: Urban & Schwarzenberg; 1978:37-48.

Gavrilov, L. R.; Kalendo~ G. S.; Ryabukhin, W.; Shaginyan, K. A.; Yarmonenko, S. P. Ultrasonic enhancement of the gamma radiation of malignant tumours. Sov. Phys. Acoust. 21 : 119-121; 1975.

ter Haar, G. R.; Stratford, I. J.; Hill, C. R. Ultrasonic irradiation of mammalian cells in vitro at hyperthermic temperatures. Brit. J. Radiol. 53:784-789; 1980.

ter Haar, G. R.; Walling, J.; Loverock, P.; Townsend, S. The effect of combined heat and ultrasound on multicellular tumour spheroids. Int. J. Radiat. Biol. 53:813-827; 1988.

Hand, J. W.; ter Haar, G. R. Heating techniques in hyperthermia. Brit. J. Radiol. 54:443-466; 1981.

Har-Kedar, I.; Bleehen, N. M. Experimental and clinical aspects of hyperthermia applied to the treatment of cancer with special reference to the role of ultrasonic and microwave heating. Adv. Rad. Biol. 6:229-266; 1976.

Kremkau, E. W.; Witcofski, R. L. Ultrasonic enhancement of cancer radiotherapy. J. Ac. Soc. Am. 61(Suppl. 58); 1977.

Kuwabara, Y.; Matsubara, S.; Yoshimatsv, S.; Suzuki, S. Com- bined effects of ultrasound and ionizing radiation on lympho- cyte chromosomes. Acta Radiologica Oncology 25:291-294; 1986.

Lehmann, J. F.; Krusen, F. H. Biophysical effects of ultrasonic en- ergy on carcinoma and their possible significance. Arch. Phys. Med. Rehab. 36:452-459; 1955.

Loverock, P.; ter Haar, G. R.; Ormerod, M. G.; lmrie, P. R. The effect of ultrasound on the cytotoxicity of adriamycin. Brit. J. Radiol. 63:542-546; 1990.

Ormerod, M. G.; Loverock, P.; ter Haar, G.; lmrie, P. R. Ultra- sound enhances the effect of hyperthermia on the cell cycle of chinese hamster V79 cells. Inst. Phys. Conf. Ser. 98:817-820; 1990.

Shotton, R. C. A tethered float radiometer for measuring the output power from ultrasonic therapy equipment. Ultrasound Med. Biol. 6:131-133; 1980.

Shuba, E. P.; Balitsky, K. P.; Panfilova, T. K.; Baran, L. A. Com- bined action of x-ray radiation and ultrasound on the growth of experimental tumours. Meditsinskaya Radiol. (Mosk) 21:42- 47; 1976.

Spring, E. Increased radiosensitivity following simultaneous ultrasonic and gamma ray irradiation. Radiology 93:175-176; 1969.

Stewart, F.; Denekamp, J. Sensitization of mouse skin to x-irradiation by moderate heating. Radiology 123:195; 1977.

Stratford, 1. J.; Adams, G. E. Effect of hyperthermia on the differen- tial cytotoxieity ofa hypoxic cell radiosensitizer Ro-07-0582, on mammahan cells in vitro. Brit. J. Cancer 35:307-313; 1977.

Todd, P.; Schroy, C. B. X-ray inactivation of cultured mammalian cells: Enhancement by ultrasound. Radiology 113:445-447; 1974.

Woeber, K.; Stein, G. Ergebnisse bei kombinierter Rt~ntgen-und Ultraschallbehandlung bi~sartiger Haut tumoren. Strahlenther 122:285-289; 1963.

Synergism between hyperthermia, ultrasound and γ irradiation

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