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Hyperthermia and radiation—A selective thermal effect on chronically hypoxic tumor cells in vivo
Inf. 1. Radiation
Oncology
Bid.
Phys.,
1!?77. Vol. 2, pp. 99-103.
Pergamon
Press.
Printed
in the U.S.A
??Original Contribution HYPERTHERMIA AND RADIATION-A SELECTIVE THERMAL EFFECT ON CHRONICALLY HYPOXIC TUMOR CELLS IN VIVOt WILLIAM C. DEWEY, Ph.D., DONALD E. THRALL, D.V.M., and EDWARD L. GILLETTE, D.V.M., Ph.D. Department
of Radiology and Radiation Biology, Colorado State University, CO 80523, U.S.A.
Ph.D.*
Fort Collins,
was evaluated by constructing survival curves for radiation doses required to control C3H mouse tumors (tumor control dose for 50% of the animals (TCD&) and for doses required for moist desquamatlon in 50% of the legs (DD&. A 15 min heat treatment at a water bath temperature of 44S”C was delivered under ambient conditions (mice breathing air, and the hind leg with the tumor not clamped) immediately after irradiation under ambient, hypoxfc, or hyperbaric 0, conditions. The analysis from survival curves suggested that thermal treatment after irradiation had a similar effect on oxygenated and acutely hypoxic skin and tumor cells. However, the chronically hypoxic tumor cells, i.e. those not oxygenated under hyperbaric 02, either were selectively killed or selectively radiosensitizedy the thermal treatment.
Thermal radiosensitization
Hyperthermia,
Hypoxia, Radiation
therapy.
INTRODUCTION Previous
to emphasize the selective effect of hyperthermia on chronically hypoxic tumor cells in vim, by analyzing previously reported in vim data*’ in terms of theoretical survival curves.
reports’.7,‘3.“,‘6,z’ suggest
that hypertreatment with Xthermic combined irradiation may lead to improvement in radiation therapy. There is an apparent increase in thermal sensitivity of chronically hypoxic tumor cells (i.e. those remaining hypoxic under hyperbaric 02) in relation to oxygenated and acutely hypoxic tumor cells;” therefore, hyperthermia may be most promising as an adjunct to radiation therapy when the tumor contains a large fraction of chronically hypoxic cells. This selective effect of hyperthermia on hypoxic cells, either by itself or in combination with X-irradiation, has been illustrated in in vitro systems5’9.10.17 and in the in viva bone marrow assay system.16 However, in these studies either low pH4,14 or poor nutritional states’ may have been an important factor in the selective thermal sensitization of the hypoxic cells. In this manuscript, we wish
METHODS
AND MATERIALS
Although details have been described elsewhere,” briefly, the hind leg of a C3H mouse bearing an 8 mm tumor was irradiated either: in air (ambient conditions); under three atmospheres absolute of hyperbaric 0,; or under hypoxia, by clamping the leg. For hyperthermic treatment, the legs were placed under ambient conditions into a 44S”C water bath for 15 min either immediately (within 5-15 min) before or after irradiation. The tumor and skin temperatures were 44.1”C and 44.4”C) respectively. Since Suit and Shwayder” and Westra and Dewey*“ indicate that a change of 1°C has a two-fold effect on thermal response, this difference of 0.3”C
tThis research was supported in part by American Cancer Society Grant No. ACS DT-9. $Present address: University of Georgia, School
of Veterinary Medicine, Department of Anatomy and Radiology, Athens, GA 30602, U.S.A. 99
100
Radiation Oncology 0 Biology 0 Physics
between tumor and skin may not be trivial. The end points were: DD50, the dose to produce moist desquamation in 50% of the legs, and TCD5,,, the dose to control the tumor as observed at 120 days in 50% of the animals.
January-February
1977, Volume
2, No. 1 and No. 2
:.. ‘.
:
X ONLY
‘. P.
RESULTS We have attempted to analyze the results for skin vs tumor in terms of theoretical survival curves. In particular, we investigated the nature of the large effect of heat when delivered following X-irradiation under hyperbaric O2 conditions. As reported** and illustrated (compare Figs. l-3), heat following irradiation under hyperbaric O2 conditions had a rad equivalent of 5500-2700 = 2800 rad, much greater than any value seen for tumor or skin under any other condition. For the construction of theoretical survival curves, we assumed that the tumor cells and the skin cells had the same intrinsic radiosensitivity, i.e. an extrapolation number (n) of 10 and a D,- of 128 rad for oxygenated cells.20~25~26 Thus, with the observed oxygen enhancement ratio for skin of 2.5 (Fig. l), a Do” of 320 rad with n = 10 was obtained for hypoxic skin and tumor cells. We assume that the differences observed in viva (Fig. 1) between tumor and skin irradiated under ambient or hyperbaric 0, conditions resulted primarily from variations in the nutrients, temperature, or O2 tension at the time of irradiation.22.23 Because of these complications, which were particularly apparent when heat preceded irradiation, survival curves were constructed only for conditions when heat followed irradiation (X + A), i.e. the condition where the greatest differential response was observed for tumor relative to skin. We assumed that the heat treatment acted primarily like a dose of radiation in that it killed some of the cells and greatly reduced or eliminated the shoulder of the radiation survival curve. As defined and illustrated previously,* this is called an additive type of interaction between heat and radiation. Only a small synergistic effect, which was seen as a 2-4% increase in slope was assumed. This assumption of additive interaction is in reasonable agreement with in vitro data6 for Chinese hamster ovary (CHO) cells irradiated
L-TCD
50
\ 1000
2000
3000 4000 DOSE-RAD
\
‘,\\
5000
6000
I
7000
Fig. 1. Survival curves for radiation only were drawn through values (with 95% confidence limits) observed for DD,, and TCD,, when irradiation was delivered under different conditions of oxygenation. The value of n, the extrapolation number, is determined by extrapolating the straight-line portion of the survival curve to D = 0. The Do- is the increment of dose on this straight-line portion corresponding to an incremental reduction of survival to 37% (i.e. l/e) of an initial value. in air within 5-15 min either before or after a 45.5”C heat treatment. Incidentally, the time interval between irradiation and heat treatment is important because the sublethal radiation damage which interacted with subsequent heat damage was completely repaired by 1 hr.6 The effects of irradiation only are shown in Fig. 1. The actual data points (with 95% confidence limits) were plotted on curves having the assumed values for Do- and II in order to establish the levels of survival (L) required for the observed DDso and TCD,, doses. We assumed that these same levels of survival always applied for any condition of tissue oxygenation or combinations of heat and radiation; i.e. the DDs, and TCD,, values, although different than those shown in Fig. 1, were still those required to reduce the survival values to those shown in Fig. 1. If a value for the Do- under oxygenated conditions was assumed to differ from 128 rad by as much as lO-20%, the survival levels for the DD,, and
Hyperthermia
and radiation 0 W. C. DEWEY et al.
TCD5, values would be changed, but the comparisons between the curves and conclusions which follow would be essentially the same (curves not shown). As shown by the extrapolations of the hyperbaric O2 and ambient curves for tumor, about 60% of the tumor cells were oxygenated under ambient conditions, with 40% being hypoxic; but as shown for irradiation under hyperbaric 02, about 10% of the cells in the tumor were hypoxic. These cells that were hypoxic under hyperbaric 0, conditions, i.e. unable to be oxygenated under three atmospheres of oxygen, are called chronically hypoxic tumor cellsI in relation to the acutely hypoxic tumor cells that become oxygenated under hyperbaric OZ. As shown for skin that was irradiated under ambient conditions, the skin cells appeared to be partially hypoxic as reported by others.‘8,2” The results for heat following irradiation under hypoxic conditions were plotted in Fig. 2 for both skin and tumor. Note that the rad equivalents were approximately equal for skin and tumor, and the curve constructed through
IO-‘L-TCD50 lo-‘-
I 1000
I
I 2000
I
I I I 3000 4000 DOSE-MD
I
I 5000
! 6000
I
I 7DDO
Fig. 2. Survival curves were drawn for heat treatments after irradiation under hypoxia. The values for DDSo and TCD,, were plotted at the same survival levels shown in Fig. 1. For comparison, the 95% confidence limits for heat before irradiation were: 1867-2346 rad for skin and 4946-5568 rad for tumor.
101
the points for X + A gave a Do- of 308 rad, which was 96% of the Do- for X-irradiation only. Furthermore, this curve intersected the ordinate at a survival value of 0.07. This value of 0.07 representing thermal killing of hypoxic skin and tumor cells agrees reasonably well with the value of 0.20 estimated from the data for cultured CHO cells.24 We wish to emphasize that this comparison has been made under hypoxic conditions where there should be no difference in the state of oxygenation for the tumor and skin cells, and thus the basic cellular response to heat and radiation, we assume, would be the same for skin and tumor cells. Actually, Fig. 3 shows that the acutely hypoxic tumor cells (about 90% of the hypoxic cells) effectively determined the response of the tumor to X + A because the chronically hypoxic cells either were radiosensitized or selectively killed by the heat treatment; however, if these chronically hypoxic cells are considered, the curve in Fig. 2 for hypoxic tumor cells should start at 7 x 10m2x 0.9 = 6.3 x lo-’ instead of 7 x lo-‘, and the DC would change only from 308 to 310 rad. Curves were constructed through the observed DDso and TCDso values (Fig. 3) in order to better compare the effects of heat on skin and tumor irradiated under various conditions preceding heat treatment. For irradiation under hyperbaric 02, the curve for tumor was drawn two ways. First (curve labeled No. l), it was assumed that the chronically hypoxic tumor cells were killed by heat to the same extent as oxygenated and acutely hypoxic cells. Thus, the curve for chronically hypoxic cells would begin at point B, i.e. 10% of the value of the intercept for the whole population since about 10% of the tumor cells were chronically hypoxic. However, as shown for this assumption, the chronically hypoxic tumor cells would be (Do- of about more sensitive to irradiation 223 rad) than the acutely hypoxic tumor cells (Do of 308 rad). The second assumption (curve labeled No. 2) for irradiation under hyperbaric OZ was that the chronically hypoxic tumor cells were killed by heat (point C) much more readily than the other cells (survival fraction of 3 X lO~“/lO~“/lO~’ = 0.003 in contrast to 0.07 for hypoxic cells illustrated in Fig. 2). Then, as
102
Radiation Oncology 0 Biology 0 Physics
January-February
1977, Volume 2, No. 1 and No. 2
mined the response of the tumor. As the curves show, these chronically hypoxic tumor cells resulted in a much lower TCD, for irradiation under hyperbaric O2 conditions when compared to irradiation under ambient or hypoxic conditions. In other words, under ambient or hypoxic conditions, heat sensitization of the relatively few chronically hypoxic cells was of little consequence because the tumor response was determined primarily by the large number of acutely hypoxic cells. UMOR AND SKIN
DISCUSSION
1000
2000
3000
4000
5oOD
6000
7DDo
DOSE-RAD
Fig. 3. Survival curves were plotted for skin and tumor when irradiated under different conditions of oxygenation and then heated. Point A at 4 x lo-* shows the extrapolation of the curve for tumor irradiated under ambient conditions. Points B and C illustrate the survival values of chronically hypoxic tumor cells resulting from heat only; as discussed in the text, heat either killed a large number of chronically hypoxic cells (point C at 3 x 1O-4 and curve 2 with a D,- of 308 rad) or radiosensitized them (point B and curve 1 with a D,- of 223 rad). The curve for ambient skin has a Doe of 230 rad. For comparison, the 95% confidence limits when heat preceded irradiation were: for tumor, 4257-4375 rad under ambient conditions and 3651-4310 rad under hyperbaric 0, conditions, and for skin 591-999 rad under ambient conditions and 497-770 rad under hyperbaric O2 conditions.
the curve (No. 2) shows, these chronically hypoxic cells would have the same sensitivity to irradiation as the acutely hypoxic cells, i.e. from a Do- of 308 rad. The main conclusion the curves in Fig. 3 is that tumor and skin cells which were irradiated under hyperbaric O2 conditions and then heated, responded to irradiation so that the lower doses of irradiation (below 1000 rad) must have been killing oxygenated tumor cells to about the same extent as oxygenated skin cells. However, as the dose was increased, the oxygenated tumor cells were eliminated, and the chronically hypoxic tumor cells which remained deter-
The general conclusion derived by comparing Figs. l-3 is that heat (44.1”C for 15 min) following irradiation had a selective effect on chronically hypoxic tumor cells. Thus, the hypothesis that heat should be considered as a means of eliminating the radioresistance of chronically hypoxic tumor cells appears to be valid, especially in single dose X-ray regimens. However, the failure to observe a selective thermal effect on acutely hypoxic tumor or skin cells relative to oxygenated cells (Do- reduced from 320 rad to 308 rad vs from 128 rad to 119 rad) may imply that the most important factor in increasing thermal radiosensitization is either low pH4.14 or nutritional deficiency;8 both of these have been shown to increase thermal sensitization and probably exist in chronically hypoxic tumor cell populations.3,‘2 Thus, when possible, future studies should be designed to differentiate thermal effects that are related to oxygen tension from thermal effects that are related to pH and nutritional states. If indeed, heat sensitization is more related to low pH and poor nutrition than to low oxygen tension, thermal treatment should be most effective when combined with radiation therapy delivered under hyperbaric OZ. Finally, much information is needed for a variety of tumors and normal tissues concerning optimal thermal treatments (i.e. temperature and duration in relation to time of irradiation), when hyperthermia is delivered with water baths, microwaves, 27 or ultrasound.”
Hyperthermia and radiation 0 W. C. DEWEY et al.
103
REFERENCES 1. Crile, Jr., G.: The effects of heat and radiation 15. Robinson, J.E., Wizenberg, M.J., McCready, on cancers implanted on the feet of mice. W.A.: Combined hyperthermia and radiation Cancer Res. 23: 372-380, 1963. suggest an alternative to heavy particle therapy for reduced oxygen enhancement ratios. Nature 2. Dewey, W.C., Stone, L.E., Miller, H.H., Radiosensitization with SGiblak, R.E.: 251: 521-522, 1974. bromodeoxyuridine of Chinese hamster cells 16. Robinson, J.E., Wizenberg, M.J., McCready, X-irradiated during different phases of the cell W.A.: Radiation and hyperthermal response of cycle. Radiat. Res. 47: 672-688, 1971. normal tissue in situ. Radiology 113: 195-198, 3. Eden, M., Haines, B., Kahler, H.: The pH of rat 1974. tumors measured in vivo. J. Nat1 Cancer Inst. 17. Schulman, N., Hall, E.J.: Hyperthermia: Its 16: 541-556, 1955. effect on proliferative and plateau phase cell 4. Freeman, M.L., Dewey, W.C., Hopwood, L.E.: cultures. Radiology 113: 207-209, 1974. Effect of pH on hyperthermic cell survival. J. 18. Suit, H.D., Howard, T.C.: Normal tissue Nat1 Cancer Inst. to be published. damage and tumor cure probability in hybrid 5. Gerweck, L.E., Gillette, E.L., Dewey, W.C.: mice. Radiology 89: 720-726, 1967. Killing of Chinese hamster cells in vitro by 19. Suit, H., Urano, M.: Repair of sublethal heating under hypoxic or aerobic conditions. radiation injury in hypoxic cells of a C,H Europ. J. Cancer 10: 691-693, 1974. mouse mammary carcinoma. Radiat. Res. 37: 6. Gerweck, L.E., Gillette, E.L., Dewey, W.C.: 423434, 1969. Effect of heat and radiation on synchronous 20. Suit, H.D., Shalek, R.J., Wette, R.: Radiation Chinese hamster cells: Killing and repair. response of C,H mouse mammary adenocarRadiat. Res. 64: 611-623, 1975. cinema evaluated in terms of cellular radiation 7. Hahn, E.W., Alfieri, A.A., Kim, J.H.: Increased sensitivity. In Cellular Radiation Biology. cures using fractionated exposures of XBaltimore, Maryland, Williams & Wilkins, irradiation and hyperthermia in the local 1965, pp. 514-530. treatment of the Ridgway osteogenic sarcoma 21. Suit, H.D., Shwayder, M.: Hyperthermia: in mice. Radiology 113: 119-202, 1974. Potential as an antitumor agent. Cancer 34: 8. Hahn, G.M.: Metabolic aspects of the role of 122-129, 1974. hyperthermia in mammalian cell inactivation 22. Thrall, D.E., Gillette, E.L., Dewey, W.C.: and their possible relevance to cancer treatEffect of heat and ionizing radiation on normal ment. Cancer Res. 34: 3117-3123, 1974. and neoplastic tissue of the C,H mouse. Radial. 9. Harisiadis, L., Hall, E.J., Kraljevic, U., Borek, Res. 63: 363-377, 1975. C.: Hyperthermia: Biological studies at the 23. Thrall, D.E., Gerweck, L.E., Gillette, E.L., cellular level. Radiology 117: 447452, 1975. Dewey, W.C.: Effects of hyperthermia on the 10. Kim, S.H., Kim, J.H., Hahn, E.W.: Enhanced X-ray response of cells and tissues in vivo and killing of hypoxic tumor cells by hyperthermia. in vitro. In Advances in Radiation Biology, Vol. Br. J. Radiology 48: 872-874, 1975. 6, ed. by Lett, J., Adler, H. New York, 11. Lehman, J.F.: Ultrasound therapy. In Academic Press, 1976, pp. 211-227. Therapeutic Heat and Cold, 2nd Edn, ed. by 24. Westra, A., Dewey, W.,: Variation in sensitivLicht, S. Baltimore, Maryland, Waverley Press, ity to heat shock during the cell cycle of 1972, pp. 321-386. Chinese hamster cells in vitro. Int. J. Radiat. Biol. 19: 467-477, 1971. 12. Meyer, K.A., Kammerling, E.M., Amtman, L., Koller, M., Hoffman, S.J.: pH studies of 25. Withers, H.R.: Recovery and repopulation in malignant tissues in human beings. Cancer Res. vivo by mouse skin epithelial cells during 8: 513-518, 1948. fractionated irradiation. Radiat. Res. 32: 22713. Overgaard, K., Overgaard, J.: Investigations on 239, 1967. the possibility of a thermic tumor therapy-II. 26. Withers, H.R.: The effect of oxygen and Action of combined heat-roentgen treatment on anesthesia on radiosensitivity in vivo of a transplanted mouse mammary carcinoma. epithelial cells of mouse skin. Br. J. Radiol. 40: Europ. J. Cancer 8: 573-575, 1972. 14. Overgaard, J., Overgaard, K.: Effect
335-343,
of environmental acidity on the hyperthermic treatment of tumour cells. IRCS Med. Sci.: Biochem. Cancer, Cell and Membrane 386. 1975.
Biol. 3:
1967.
27. Zimmer, R.P., Ecker, H.A., Popovic, V.P.: Selective electromagnetic heating of tumors in animals in deep hypothermia. IEEE Trans. Microwave Theory Techniq. 19: 238-244,197l.
Oncology
Bid.
Phys.,
1!?77. Vol. 2, pp. 99-103.
Pergamon
Press.
Printed
in the U.S.A
??Original Contribution HYPERTHERMIA AND RADIATION-A SELECTIVE THERMAL EFFECT ON CHRONICALLY HYPOXIC TUMOR CELLS IN VIVOt WILLIAM C. DEWEY, Ph.D., DONALD E. THRALL, D.V.M., and EDWARD L. GILLETTE, D.V.M., Ph.D. Department
of Radiology and Radiation Biology, Colorado State University, CO 80523, U.S.A.
Ph.D.*
Fort Collins,
was evaluated by constructing survival curves for radiation doses required to control C3H mouse tumors (tumor control dose for 50% of the animals (TCD&) and for doses required for moist desquamatlon in 50% of the legs (DD&. A 15 min heat treatment at a water bath temperature of 44S”C was delivered under ambient conditions (mice breathing air, and the hind leg with the tumor not clamped) immediately after irradiation under ambient, hypoxfc, or hyperbaric 0, conditions. The analysis from survival curves suggested that thermal treatment after irradiation had a similar effect on oxygenated and acutely hypoxic skin and tumor cells. However, the chronically hypoxic tumor cells, i.e. those not oxygenated under hyperbaric 02, either were selectively killed or selectively radiosensitizedy the thermal treatment.
Thermal radiosensitization
Hyperthermia,
Hypoxia, Radiation
therapy.
INTRODUCTION Previous
to emphasize the selective effect of hyperthermia on chronically hypoxic tumor cells in vim, by analyzing previously reported in vim data*’ in terms of theoretical survival curves.
reports’.7,‘3.“,‘6,z’ suggest
that hypertreatment with Xthermic combined irradiation may lead to improvement in radiation therapy. There is an apparent increase in thermal sensitivity of chronically hypoxic tumor cells (i.e. those remaining hypoxic under hyperbaric 02) in relation to oxygenated and acutely hypoxic tumor cells;” therefore, hyperthermia may be most promising as an adjunct to radiation therapy when the tumor contains a large fraction of chronically hypoxic cells. This selective effect of hyperthermia on hypoxic cells, either by itself or in combination with X-irradiation, has been illustrated in in vitro systems5’9.10.17 and in the in viva bone marrow assay system.16 However, in these studies either low pH4,14 or poor nutritional states’ may have been an important factor in the selective thermal sensitization of the hypoxic cells. In this manuscript, we wish
METHODS
AND MATERIALS
Although details have been described elsewhere,” briefly, the hind leg of a C3H mouse bearing an 8 mm tumor was irradiated either: in air (ambient conditions); under three atmospheres absolute of hyperbaric 0,; or under hypoxia, by clamping the leg. For hyperthermic treatment, the legs were placed under ambient conditions into a 44S”C water bath for 15 min either immediately (within 5-15 min) before or after irradiation. The tumor and skin temperatures were 44.1”C and 44.4”C) respectively. Since Suit and Shwayder” and Westra and Dewey*“ indicate that a change of 1°C has a two-fold effect on thermal response, this difference of 0.3”C
tThis research was supported in part by American Cancer Society Grant No. ACS DT-9. $Present address: University of Georgia, School
of Veterinary Medicine, Department of Anatomy and Radiology, Athens, GA 30602, U.S.A. 99
100
Radiation Oncology 0 Biology 0 Physics
between tumor and skin may not be trivial. The end points were: DD50, the dose to produce moist desquamation in 50% of the legs, and TCD5,,, the dose to control the tumor as observed at 120 days in 50% of the animals.
January-February
1977, Volume
2, No. 1 and No. 2
:.. ‘.
:
X ONLY
‘. P.
RESULTS We have attempted to analyze the results for skin vs tumor in terms of theoretical survival curves. In particular, we investigated the nature of the large effect of heat when delivered following X-irradiation under hyperbaric O2 conditions. As reported** and illustrated (compare Figs. l-3), heat following irradiation under hyperbaric O2 conditions had a rad equivalent of 5500-2700 = 2800 rad, much greater than any value seen for tumor or skin under any other condition. For the construction of theoretical survival curves, we assumed that the tumor cells and the skin cells had the same intrinsic radiosensitivity, i.e. an extrapolation number (n) of 10 and a D,- of 128 rad for oxygenated cells.20~25~26 Thus, with the observed oxygen enhancement ratio for skin of 2.5 (Fig. l), a Do” of 320 rad with n = 10 was obtained for hypoxic skin and tumor cells. We assume that the differences observed in viva (Fig. 1) between tumor and skin irradiated under ambient or hyperbaric 0, conditions resulted primarily from variations in the nutrients, temperature, or O2 tension at the time of irradiation.22.23 Because of these complications, which were particularly apparent when heat preceded irradiation, survival curves were constructed only for conditions when heat followed irradiation (X + A), i.e. the condition where the greatest differential response was observed for tumor relative to skin. We assumed that the heat treatment acted primarily like a dose of radiation in that it killed some of the cells and greatly reduced or eliminated the shoulder of the radiation survival curve. As defined and illustrated previously,* this is called an additive type of interaction between heat and radiation. Only a small synergistic effect, which was seen as a 2-4% increase in slope was assumed. This assumption of additive interaction is in reasonable agreement with in vitro data6 for Chinese hamster ovary (CHO) cells irradiated
L-TCD
50
\ 1000
2000
3000 4000 DOSE-RAD
\
‘,\\
5000
6000
I
7000
Fig. 1. Survival curves for radiation only were drawn through values (with 95% confidence limits) observed for DD,, and TCD,, when irradiation was delivered under different conditions of oxygenation. The value of n, the extrapolation number, is determined by extrapolating the straight-line portion of the survival curve to D = 0. The Do- is the increment of dose on this straight-line portion corresponding to an incremental reduction of survival to 37% (i.e. l/e) of an initial value. in air within 5-15 min either before or after a 45.5”C heat treatment. Incidentally, the time interval between irradiation and heat treatment is important because the sublethal radiation damage which interacted with subsequent heat damage was completely repaired by 1 hr.6 The effects of irradiation only are shown in Fig. 1. The actual data points (with 95% confidence limits) were plotted on curves having the assumed values for Do- and II in order to establish the levels of survival (L) required for the observed DDso and TCD,, doses. We assumed that these same levels of survival always applied for any condition of tissue oxygenation or combinations of heat and radiation; i.e. the DDs, and TCD,, values, although different than those shown in Fig. 1, were still those required to reduce the survival values to those shown in Fig. 1. If a value for the Do- under oxygenated conditions was assumed to differ from 128 rad by as much as lO-20%, the survival levels for the DD,, and
Hyperthermia
and radiation 0 W. C. DEWEY et al.
TCD5, values would be changed, but the comparisons between the curves and conclusions which follow would be essentially the same (curves not shown). As shown by the extrapolations of the hyperbaric O2 and ambient curves for tumor, about 60% of the tumor cells were oxygenated under ambient conditions, with 40% being hypoxic; but as shown for irradiation under hyperbaric 02, about 10% of the cells in the tumor were hypoxic. These cells that were hypoxic under hyperbaric 0, conditions, i.e. unable to be oxygenated under three atmospheres of oxygen, are called chronically hypoxic tumor cellsI in relation to the acutely hypoxic tumor cells that become oxygenated under hyperbaric OZ. As shown for skin that was irradiated under ambient conditions, the skin cells appeared to be partially hypoxic as reported by others.‘8,2” The results for heat following irradiation under hypoxic conditions were plotted in Fig. 2 for both skin and tumor. Note that the rad equivalents were approximately equal for skin and tumor, and the curve constructed through
IO-‘L-TCD50 lo-‘-
I 1000
I
I 2000
I
I I I 3000 4000 DOSE-MD
I
I 5000
! 6000
I
I 7DDO
Fig. 2. Survival curves were drawn for heat treatments after irradiation under hypoxia. The values for DDSo and TCD,, were plotted at the same survival levels shown in Fig. 1. For comparison, the 95% confidence limits for heat before irradiation were: 1867-2346 rad for skin and 4946-5568 rad for tumor.
101
the points for X + A gave a Do- of 308 rad, which was 96% of the Do- for X-irradiation only. Furthermore, this curve intersected the ordinate at a survival value of 0.07. This value of 0.07 representing thermal killing of hypoxic skin and tumor cells agrees reasonably well with the value of 0.20 estimated from the data for cultured CHO cells.24 We wish to emphasize that this comparison has been made under hypoxic conditions where there should be no difference in the state of oxygenation for the tumor and skin cells, and thus the basic cellular response to heat and radiation, we assume, would be the same for skin and tumor cells. Actually, Fig. 3 shows that the acutely hypoxic tumor cells (about 90% of the hypoxic cells) effectively determined the response of the tumor to X + A because the chronically hypoxic cells either were radiosensitized or selectively killed by the heat treatment; however, if these chronically hypoxic cells are considered, the curve in Fig. 2 for hypoxic tumor cells should start at 7 x 10m2x 0.9 = 6.3 x lo-’ instead of 7 x lo-‘, and the DC would change only from 308 to 310 rad. Curves were constructed through the observed DDso and TCDso values (Fig. 3) in order to better compare the effects of heat on skin and tumor irradiated under various conditions preceding heat treatment. For irradiation under hyperbaric 02, the curve for tumor was drawn two ways. First (curve labeled No. l), it was assumed that the chronically hypoxic tumor cells were killed by heat to the same extent as oxygenated and acutely hypoxic cells. Thus, the curve for chronically hypoxic cells would begin at point B, i.e. 10% of the value of the intercept for the whole population since about 10% of the tumor cells were chronically hypoxic. However, as shown for this assumption, the chronically hypoxic tumor cells would be (Do- of about more sensitive to irradiation 223 rad) than the acutely hypoxic tumor cells (Do of 308 rad). The second assumption (curve labeled No. 2) for irradiation under hyperbaric OZ was that the chronically hypoxic tumor cells were killed by heat (point C) much more readily than the other cells (survival fraction of 3 X lO~“/lO~“/lO~’ = 0.003 in contrast to 0.07 for hypoxic cells illustrated in Fig. 2). Then, as
102
Radiation Oncology 0 Biology 0 Physics
January-February
1977, Volume 2, No. 1 and No. 2
mined the response of the tumor. As the curves show, these chronically hypoxic tumor cells resulted in a much lower TCD, for irradiation under hyperbaric O2 conditions when compared to irradiation under ambient or hypoxic conditions. In other words, under ambient or hypoxic conditions, heat sensitization of the relatively few chronically hypoxic cells was of little consequence because the tumor response was determined primarily by the large number of acutely hypoxic cells. UMOR AND SKIN
DISCUSSION
1000
2000
3000
4000
5oOD
6000
7DDo
DOSE-RAD
Fig. 3. Survival curves were plotted for skin and tumor when irradiated under different conditions of oxygenation and then heated. Point A at 4 x lo-* shows the extrapolation of the curve for tumor irradiated under ambient conditions. Points B and C illustrate the survival values of chronically hypoxic tumor cells resulting from heat only; as discussed in the text, heat either killed a large number of chronically hypoxic cells (point C at 3 x 1O-4 and curve 2 with a D,- of 308 rad) or radiosensitized them (point B and curve 1 with a D,- of 223 rad). The curve for ambient skin has a Doe of 230 rad. For comparison, the 95% confidence limits when heat preceded irradiation were: for tumor, 4257-4375 rad under ambient conditions and 3651-4310 rad under hyperbaric 0, conditions, and for skin 591-999 rad under ambient conditions and 497-770 rad under hyperbaric O2 conditions.
the curve (No. 2) shows, these chronically hypoxic cells would have the same sensitivity to irradiation as the acutely hypoxic cells, i.e. from a Do- of 308 rad. The main conclusion the curves in Fig. 3 is that tumor and skin cells which were irradiated under hyperbaric O2 conditions and then heated, responded to irradiation so that the lower doses of irradiation (below 1000 rad) must have been killing oxygenated tumor cells to about the same extent as oxygenated skin cells. However, as the dose was increased, the oxygenated tumor cells were eliminated, and the chronically hypoxic tumor cells which remained deter-
The general conclusion derived by comparing Figs. l-3 is that heat (44.1”C for 15 min) following irradiation had a selective effect on chronically hypoxic tumor cells. Thus, the hypothesis that heat should be considered as a means of eliminating the radioresistance of chronically hypoxic tumor cells appears to be valid, especially in single dose X-ray regimens. However, the failure to observe a selective thermal effect on acutely hypoxic tumor or skin cells relative to oxygenated cells (Do- reduced from 320 rad to 308 rad vs from 128 rad to 119 rad) may imply that the most important factor in increasing thermal radiosensitization is either low pH4.14 or nutritional deficiency;8 both of these have been shown to increase thermal sensitization and probably exist in chronically hypoxic tumor cell populations.3,‘2 Thus, when possible, future studies should be designed to differentiate thermal effects that are related to oxygen tension from thermal effects that are related to pH and nutritional states. If indeed, heat sensitization is more related to low pH and poor nutrition than to low oxygen tension, thermal treatment should be most effective when combined with radiation therapy delivered under hyperbaric OZ. Finally, much information is needed for a variety of tumors and normal tissues concerning optimal thermal treatments (i.e. temperature and duration in relation to time of irradiation), when hyperthermia is delivered with water baths, microwaves, 27 or ultrasound.”
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