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Studies on fractionated hyperthermia in experimental animal systems II. Response of murine tumors to two or more doses
036s3016/82/02022747SO3.00/0 Copyright 8 1982 Pcrgamon Press Ltd.
/III. J. Radiation Oncology Biol. Phys., Vol. 8. pp. 227-233 Printed in the U.S.A. All rights reserved
?? Original
Contribution
STUDIES ON FRACTIONATED HYPERTHERMIA IN EXPERIMENTAL ANIMAL SYSTEMS II. RESPONSE OF MURINE TUMORS TO TWO OR MORE DOSES MUNEYASU URANO, M.D., PH.D., LAURIE COCHRAN RICE, MS. AND VERNON MONTOYA, B. S. Department
of Radiation
Medicine,
Massachusetts General Boston. MA02114
Hospital,
Harvard
Medical School,
The effect of multiple hyperthermia and the kinetics of thermal resistance were studied in experimental murine tumors. A spontaneous C3Hf mouse mammary carcinoma, MCa and a chemically-induced fibrosarcoma, FSa-I, were used. Assay methods include determination of the TCD, i.e., the treatment time to yield a tumor control in half the treated animals and TG (tumor growth) time analysis, i.e., the time required for a tumor to reach a given size after first treatment. After equal dose fractions the TCDSO of MCa increased with increasing overall treatment time. This increase was most predominant when treatments were given with a time interval of one day. Isoeffect curves for the MCa were comparable to those for normal tissue damage (foot reaction), which was reported in the first part of this series of communications. The kinetics of thermal resistance in the MCa was compared with that in FSa-I since the fractionated hyperthermia for the FSa-I was reported to have resulted in an appreciable therapeutic gain. The magnitude of thermal resistance was far greater in the MCa than in the FSa-I, although the kinetics of thermal resistance was similar in both tumors; i.e., (a) the resistance reached a maximum in 24 hours after treatment and then decayed slowly, and (b) the development of thermal resistance increased with increasing initial dose. The thermal resistance in these tumors appeared to be greater than that in animal feet. Hyperthermia,
Thermal
resistance, Fractionated
hyperthermia,
INTRODUCTION
Mouse mammary carcinoma, Mouse fibrosarcoma.
possible repair of sublethal or potentially lethal thermal damage, progression of surviving cells through the cellcycle and alteration of hypoxic or acidic cell fraction in a tumor. The substantial development of thermal resistance, which is observed following a prior exposure to elevated temperature, has been demonstrated first in cultured mammalian cellP2 and recently in animal tissues. 1.13.16.17.23.31 Thermal resistance has appeared to be one of the most critical factors for the success of clinical hyperthermia. We have studied the kinetics of thermal resistance and fractionated hyperthermia regimen in murine tumors.
response to elevated temperature has been extensively studied in vitro and in vivo. A rationale for using hyperthermia in cancer therapy might be that hypoxic tumor cells, which are resistant to ionizing radiations, are at least as sensitive as aerobic tumor cells to elevated temperature.7 Recent studies revealed that the decrease in environmental pH is associated with an increase in thermal sensitivity of cultured mammalian cells.h.20~32Tumor tissue pH has been found to be lower as compared to blood or normal tissue pH, because of anerobic metabolism in hypoxic tumor cells that results in an accumulation of lactic acid.” In this situation, the hypoxic tumor cells should be more sensitive to heat than their well-oxygenated counterpart. This presumption has been confirmed by experimental animal studies.‘” Clinical hyperthermia will likely employ a fractionated regimen, which inevitably will be influenced by a variety of unresolved factors. We have studied fractionated hyperthermia in normal and tumor tissues in experimental animals.‘7.‘3.3’ Factors influencing the effect of multiple doses might include the kinetics of thermal resistance, Biological
METHODS
AND MATERIALS
Animals were 10-l 2 week old C3Hf/Sed mice derived from our defined flora colony. They were kept in an animal facility where defined flora conditions were maintained.24 Sterilized animal pellets* and acidified and vitamin K-fortified water were provided ad fibitum. Two tumors were used. One is a third generation isotransplant of a mammary carcinoma, MCa, which arose spontaneously in a female mouse. This tumor is weakly immu-
This work was supported in part by Grant Number CA26350 awarded by the National Cancer Institute, Department of Health, Education and Welfare.
Reprint requests to: M. Urano, M.D., Ph.D. Accepted for publication 7 October 198 I. *Wayne Lab Blox. 221
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Radiation Oncology 0 Biology 0 Physics
nogenic and has a volume doubling time of -2.5-3.0 days.25 The other is a fourth generation tumor of methycholanthlene-induced fibrosarcoma, FSa-I, which is moderately immunogenic and proliferates with a doubling time of 12.0 days.” Single cell suspensions of these tumor cells were prepared by trypsinization, which is described elsewhere.” Approximately lo5 viable tumor cells in 5 &! were transplanted into the right foot. Animals were randomly assigned into groups before treatment. Hyperthermia was given by immersing the animal foot into a water bath where a desired temperature +O. l°C was maintained by a constant temperature circulator.28 Tumor temperature was no less than 0.1% below water bath temperature. No anesthesia was given prior to treatment. Assay methods include TG (tumor growth) time analysis and determination of TCDso (50% tumor control dose). TCD,, is a treatment time at elevated temperature to yield a tumor control in half the treated animals. Tumors were treated when they reached an ‘average diameter of 4 mm since preliminary data indicated this sized tumors were controlled without loss of the foot. Following the treatment, animals were observed for a possible tumor recurrence once a week for 120 days; {he TCD,, was calculated on the basis of the recurrence-free rate in each dose group. Animals that died without recurrence before termination of the experiment were excluded from the assay. TG time is the time required for a tumor to reach a given volume after treatment. Three diameters of each tumor a, b and c were measured at least 3 times a week and the tumor volume was calculated by a formula of ?rabc/6. The TG time was obtained graphically for each tumor and the median value was calculated by logit analysis. RESULTS The MCa was treated with equal dose fractions at 43.5% with a Ti (time interval) between treatments of 1, 2 or 5 days; then TCD,, was determined. Figure 1 demonstrates TCD, values as a function of overall treatment time. The TCDSOincreased with increasing overall treatment time in each treatment regimen. The most predominant increase was seen in the treatment regimen with a Ti of one day, indicating substantial development of thermal resistance and/or possible repair of sublethal and potentially lethal thermal damage occurring in first 24 hours after each treatment. The TCDso values are listed in Table 1 together with the mouse foot RDso (~4.0) (treatment time to induce score 4.0 [loss of one toe] or greater reaction in half the treated animals) which were previously reported.3’ No difference can be found between TCDW and RD, in each regimen except 6 fractions given with a Ti of 2 days, which resulted in a larger therapeutic ratio (ratio of RD,, to TCD,,) as compared to a single dose, indicating that a therapeutic gain was rarely obtained for the equal dose fraction.
February 1982, Volume 8, Number 2
.c9 E
i
/
I -7
6 Trertment
9 Time;
6 t
4
3
i
2 t II
’ 0
1
2
3
4
5
6
7 Overall
IO” Days
Fig. 1. Isoeffect curves for TCDS, of C3Hf mouse mammary carcinoma. TCD,, (total dose) after equal dose treatments is plotted as a function of overall treatment time. Open circles, solid circles and open squares indicate treatment given with a Ti of 1, 2 and 5 days respectively. Vertical bars indicate 95% confidence limits.
Notably, the tumor control was exclusively associated with a foot reaction of score 5.0 (loss of partial foot or greater reaction) or with a concomitant, partial loss of the foot after fractionated treatments, although the tumor control after single dose was associated with less severe reaction, i.e. score 4.5. These results suggest that the true TCD,, values after fractionated treatments might have been greater than those obtained experimentally with a resultant decrease in the therapeutic ratio. An earlier report from our laboratory demonstrated that multiple treatments of FSa-I tumors significantly Table 1. TCDSo and RD,,, (~4.0) values after fractionated hyperthermia given at 43.YC Treatment
Ti (day) 0
1
2
5
V* 1 2 4 I 11 2 4 6 2 3
Schedule Overall treatment time (days) 0
1 3 6 10 2 6 10 5 10
TCDso (min) 146 (130-164)t 192 (155-238) 344 (300-395) 542 (449-656) 801 (718-892) 229 (188-279) 384 (330-446) 460(419-505) 175(143-215) 269 (221-328)
RDSI (min) 139 (128-152)t 202 (182-223) 331$ 592 (555-631) 888 (837-941) 256(245-267) 389 (361-420) 545(519-572) 209(193-226) 239 (212-270)
Note: The mammary carcinoma transplanted in the mouse right foot was treated when it reached an average diameter of 4 mm. RDS, (~4.0) values for mouse foot reactions were cited from our previous paper.” *Number of fractions. TNumbers in parenthesis indicate 95% confidence limit. *Estimated from the iso-effect curve.
Fractionated hyperthermia of mutine tumors 0 M. Table 2. Effect of daily hyperthermia on a normal tissue and FSa-I tumors. Hyperthermia was given at 43.5OC with a Ti of 22 hours26 Number of Fractions
i70, ..
E
83 142 229 367
I
2 5 10
173 265 530 915
229
4 50+
Therapeutic ratio?
RD,, (>5.0)*
TCD,,
URANO et al.
-
Mammary
2.08 1.87 2.31 2.49
Ca
30-
.. was used in this experiment instead (~4.0) which is used exclusively in the present study. tRD,, (rS.O)/TCD,,. *RDS,, (25.0)
of RDSo 20-
increased the therapeutic ratio.26 As cited in Table 2, the therapeutic ratios after single treatment and 10 daily treatments were 2.08 and 2.49 respectively, although the TCD,, values of the FSa-I were exclusively smaller than those of the MCa. The kinetics of thermal resistance was studied at 45.5% in order to investigate the different therapeutic ratio between MCa and FSa-I. The reasons for raising treatment temperature to 45.5V are: a) substantial development of thermal resistance required a prolongation of the treatment time to more than 5 hours at 43.5% with animal feet fixed in a water bath, b) the kinetics of thermal resistance in another murine tumor and a normal tissue has been studied at 45.5% and c) it is welldocumented for FSa-I and -11 that at temperatures above 42.5-43.0°C, a 1°C increase of treatment temperature halves the treatment time, which induces a given tumor response.2’.29 The effect of the Ti between D, (first dose) and D, (second dose) on the development of thermal resistance was investigated in the MCa. A D, of 10 min was
I
a80
2
I
I
I5 t
I
I
I
I
I
101
FSa.1
’ 0
I
I
1
I
LJ
1
2
3
4
5 Days
Time
Interval
between 2 Doses:
Fig. 3. The kinetics of thermal resistance in a C3Hf mouse mammary carcinoma and in a chemically induced fibrosarcoma, FSa-I. A D, of IO min was followed by D2 of 25 min with various Ti for mammary carcinoma, while D, of 7.5 min plus D, of 15 min were given to FSa-I with various Ti. Vertical bars show 95% confidence limits. (see text for detail.)
followed by a variable D, with various Ti, and the TG time was determined. The dose response curves after 2 doses demonstrated a less steep slope as compared to single dose response curve, indicating that substantial thermal resistance developed following the first exposure to heat (Fig. 2). Two doses with a Ti of 1 day resulted in a survival curve which showed the least steep slope; i.e., the thermal resistance developed rapidly in first 24 hours and decayed thereafter. Selected data from Figure 2 is shown
C3Hf/Sad, MCa
I
-I
I
t-
CJHVSed, MCa !pO” 0
._
E ._ c a +
30
5o
30 -
t 201
20 A
10’
0
6h
0
ld
??
Zd
0
16
0
3d
??
22.6
IO 20
40
60 Total Time at 45S’C;
80 min
Fig. 2. Development of thermal resistance in a C3Hf mouse mammary carcinoma. A D, of 10 min at 45.5% was followed by D, with various Ti. Each symbol indicates each Ti as shown in the figure. Vertical bars are 95% confidence limits.
’ 0
I
I 20
40 Total
I 60 Time at 45.5.C;
80 min
Fig. 4. Development of thermal resistance in a C3Hf mouse mammary carcinoma after a various D,. A D, was given 24 hours after D, . Different symbols show different sizes of D, as indicated in the figure. Vertical bars are 95% confidence limits.
230
Radiation Oncology 0 Biology 0 Physics
in Figure 3 where the TG time after a fixed D, (10min) plus a fixed D, (25 min) is plotted as a function of Ti between 2 doses. Figure 3 also shows the kinetics observed in FSa-I after a D, of 7.5 and D, of 15 min. It was necessary to use different treatment times in these 2 tumors because of the large difference in thermal sensitivity between them. The different treatment times made this comparison of these 2 tumors difficult since the kinetics of thermal resistance has been shown to depend on the size of D, as well as the Ti. Therefore, further studies were done. Effect of the size of D, on the development of thermal resistance was studied for MCa (Fig. 4) and FSa-I (Fig. 5) by TG time analysis. The Ti between doses was 24 hours. In both tumors the slope of the dose response curve after the second dose decreased with increasing D, indicating that an increase in D, was associated with increasing thermal resistance. The magnitude of thermal resistance appeared to be more dramatic in MCa than in FSa-I, in that a difference in slopes between single and 2 dose-response curves was far greater for MCa than for FSa-I. This was clearly seen when the thermal resistance ratio (TRR) was plotted as a function of D, (Fig. 6). The TRR is defined as: D (2 doses) - D, D, . . (1) D (single dose) - D, OrD (single dose) - D, ' .
February 1982, Volume 8, Number 2 C3Hf/S~ed
0 ._
I
‘t;; K
I
I
I
-
15-
Mammary
Ca
--1
0
5
10
15
20 Dl ; min
Fig. 6. Thermal resistance ratio as a function of the size of D,. The biological endpoint used for calculation of the ratio was a treatment time to induce TG time of 30 days (T,,). Note a big difference between a C3Hf mouse mammary carcinoma (solid squares) and chemically-induced fibrosarcoma, FSa-I (open squares). The T,, for FSa-I treated with a D, of 3 and 7.5 min were obtained by extrapolating each dose response curve shown in Fig. 5. where D (single dose) or D (2 doses) is the treatment time to induce a given biological response after single treatment or 2 treatments, respectively.23 In Figure 6, the given biological response was a TG time of 30 days. The TRR increased with increasing D, and the magnitude of thermal resistance was much greater in MCa than in FSa-I. DISCUSSION
4f./ .
0
8 Single 0 a 0
q
... 15 37.5min
20 Total Time at 45.5’C;
40 min
Fig. 5. Development of thermal resistance in a chemically induced fibrosarcoma, FSa-I after a various D,.A D, was given 24 hours after D,.Different symbols show different sizes of D, as indicated in the figure. Vertical bars are 95% confidence limits.
Thermal resistance, or thermotolerance, has been demonstrated in animal tissues as well as in cultured mammalian cells.‘~” In general, it is a transient and non-heritable resistance to hyperthermia following a prior exposure to heat. The survival curve, of resistant cells is featured by an increased slope with or without a change in a shoulder. It has been shown both in vitro and in vivo that the magnitude of thermal resistance increases Some in vivo experiments with increasing D,.3y"*'6323 suggest rapid development of thermal resistance after a short D,.'6v23 Lower environmental pH has been found to be associated with less resistance.8*9.‘9 Our previous report discusses this topic in an animal tumor.” No consensus about the mechanism of thermal resistance has yet been obtained 1.4.13,14.18,22 Present
experiments
demonstrated
a substantial
differ-
Fractionated hyperthermia of murine tumors 0 M. URANOef al. Table 3. Effect of the size of D, on the development FSa-I
of thermal
resistance
231
in three murine tumors at 45S”C FSa-II
MCa
Foot reaction
(a) TX0 D, (min) 0 3 7.5 15 22.5 30 (b) Td (TG) D, (min) 0 3 7.5 15 22.5 30
T,0 (min) 17 31 40 28
TRR
Td (TG) 6 16 22 20
TRR
I .o 2.0 3.4 6.5
I .o 2.7 3.7 3.3
T,, (min) 19 46 74 82
TRR
Td (TG) 14 18 66 118 104
TRR 1.0 1.3 4.7 8.4 7.4
1.0 2.7 5.8 16.8
T,, (min) 40 53 80 135 115
TRR
Td (TG) 25 29 51 112 97
TRR 1.0 1.2 2.0 4.5 3.9
1.0 1.4 2.2 4.8 5.3
RD,, (min) 34 60 80 75 64 37
TRR
RD,, 34 60 80 75 64 37
TRR 1.0 1.9 3.0 3.9 5.6 9.3
1.0 1.9 3.0 3.9 5.6 9.3
Note: The second dose was given 24 hours after D,. Two different biological endpoints are used: (a) treatment time to induce TG time of 30 days or T3,, and (b) slope of the dose response curve, which is expressed as a treatment time required to double the TG time or Td (TG). Thermal resistance ratio or TRR is calculated by Eq. 1. For comparison, the TRR for mouse foot is tabulated from ref. thirty-one. The endpoint was RDS, (~4.0) i.e., treatment time to induce score 4.0 (loss of one toe) or greater reaction in half the treated mice.
in the magnitude of thermal resistance between 2 tumors. Although the possible development of thermal resistance in murine tumors was suggested by Crile,’ no systematic studies on thermal resistance in tumors have been reported to our knowledge, except one obtained in our laboratory in a spontaneous C3Hf/Sed mouse fibrosarcoma, FSa-II.” Table 3 summarizes all the data together with the data of a mouse normal tissue (foot response). As mentioned above, the magnitude of thermal resistance depends on the size of D,. The TRR may be altered by the biological endpoints of the level of damage assessed since the survival curve of thermal resistant cells may be associated with a change in the shoulder width as well as an increase in the slope. Therefore, the TRRs based on 2 different endpoints are shown in Table 3. The endpoints are (a) a treatment time to induce a TG time of 30 days and (b) a slope which is expressed as a treatment time to double the TG time in the exponential portion of the dose response curve. Common features are: thermal resistance is most substantial in MCa and least in FSa-II, and thermal resistance in the foot was only slightly greater than that in FSa-II, or less extensive than that in MCa and FSa-I. These results suggest that the therapeutic ratio is rarely greater than 1.0 after fractionated hyperthermia compared with a single treatment when the treatment intervals of 24-48 hours is used. No correlation could be found between the magnitude of thermal resistance and the thermal sensitivity of tumors. TCDSo at 43.5% of FSa-II was 170 (153-189*) min,*’ the greatest among three tumors, while the magnitude of thermal resistance was minimum. The TCD,, of ence
*95% confidence
limit.
MCa, which showed greatest resistance, was 146 (130164) min or intermediate among three tumors (see Tables I and 2). No correlation appeared between tumor immunogenecity and thermal resistance. The FSa-I, which was moderately immunogenic, yielded intermediate resistance, and the weakly immunogenic MCa and FSa-II demonstrated greatest and least resistance, respectively. Tumor volume doubling time was shortest in the FSaII (1.5-2.0 days) and longest in the MCa (2.5-3.0). Thus the longer doubling time appeared to be associated with greater thermal resistance. However, it is unknown whether this relation may exist in normal tissues. The RD,, (~4.0) might be the results from the skin or bone damage. It is also unknown if the cell cycle time of these cells may be shortened after the first hyperthermia treatment although cell cycle time of these tissue cells have been studied.‘5*34 Further studies in this relationship should be encouraged. The fractionated hyperthermia resulted in an increased therapeutic ratio for FSa-I, but not MCa. This increase for FSa-I most likely results from the small TCD,, value of this tumor rather than the less extensive thermal resistance. The TCD,, (single dose) at 43.5”C of FSa-I was 83 min while that of MCa was 146 min, which was comparable to RD,, (single dose) of 139 min (Tables 1 and 2). Consequently, each dose in fractionated treatments for FSa-I was smaller, thus less resistance was induced as compared to that for MCa or for animal feet, resulting in less increase in TCD,, for FSa-I than in TCD,, for MCa and in RD,,.
232
Radiation Oncology 0 Biology 0 Physics
Total
Radiation
Dose
HYPERTHERMIA
Total Treatment
Time
Fig. 7. Schematic demonstration of effectiveness of each dose in fractionated radiation doses (top panel) and in fractionated
hyperthermia (bottom panel). Thick dotted lines are single dose survival curves while thin dotted lines are survival curves after second dose. Solid line and solid squares are overall survival after multiple doses.
February 1982, Volume 8, Number 2
Finally, it should be mentioned that each dose in the equal dose fractionation does not produce an equal effect. Figure 7 schematically compares the effect of fractionated hyperthermia on cell survival with that of fractionated radiotherapy. It is assumed that subsequent treatments were given when sublethal damage was fully repaired and the thermal resistance was fully developed. Each dose in fractionated radiotherapy has an equal effect throughout a treatment course, as has been frequently discussed by other investigators,2”3 while the effect of each dose in the fractionated hyperthermia is not equal because of the substantial development of thermal resistance. The first hyperthermic treatment modifies thermal sensitivity of tumor cells in such a way as to decrease sensitivity. Thermal resistance develops continuously after each treatment and an accumulation of fractionated doses might further increase the resistance. This means, as far as equal dose fractions are concerned, that the first dose exhibits the greatest effect and subsequent treatments are less effective. In 11 daily treatments, the largest fraction number tested (Table l), the daily dose was as large as 73 min, which was approximately half of the single dose TCDSO. This implies that equal dose fractionation given to thermally resistant cells eventually loses a fractionation effect that might increase the therapeutic ratio. Accordingly, as far as treatments are given to thermally resistant tumors, daily doses should be increased with a progress of treatments. Another choice may be the fractionation with a Ti which is long enough to eliminate thermal resistance. These regimen are now being studied.
REFERENCES 1.
2.
3.
4.
5. 6. 7.
8.
Crile, G., Jr.: The effects of heat and radiation on cancers implanted on the feet of mice. Cancer Res. 23: 372-380, 1963. Elkind, M.M., Sutton, H.: Radiation response of mammalian cells grown in culture I. Repair of X-ray damage in surviving Chinese Hamster Cells. Radiot. Res. 13: 556593.1960. Gerner, E.W., Boone, R., Connor, W.G., Hicks, J.A., Boone, Max L.M.: A transient thermotolerant survival response produced by single thermal doses in HeLa cells. Cancer Res. 36: 1035-1040, 1976. Gerner, E.W., Cress, A.E., Stickney, D.G., Holmes, D.K., Culver, P.S.: Factors regulating membrane permeability alter thermal resistance. In Thermal Characteristics of Tumors: Applications in Detection and Treatment, Vol. 335, R.K. Jain, P.M. Gullino (Eds.). New York, NY Acad. of Sciences. 1980, pp. 2 15-233. Gerner, EW., Schneider, M.J.: Induced thermal resistance in HeLa cells. Nature 256: 500-502, 1975. Gerweck, L.E.: Modification of cell lethality at elevated temperatures. The pH effect. Radial. Res. 70: 224-235, 1977. Gerweck, L.E., Gillette, E.L., Dewey, W.C.: Killing of Chinese hamster cells in vitro by heating under hypoxic or aerobic conditions. Eur. J. Cancer 10: 691-693, 1974. Gerweck, L.E., Jennings, M., Richards, B.: Influence of pH on the response of cells to single and split doses of hyperthermia. Cancer Res. 40: 4019-4024, 1980.
9. Goldin, E.M., Leeper, D.: The effect of low pH on thermotolerance induction using fractionated 45OC hyperthermia. Radiat. Res. 85: 472-479, 198 1. 10. Gullino, P.M., Grantham, F.H., Smith, S.H., Haggerty, A.C.: Modifications of the acid-base status of the internal milieu of tumors. J. N&l. Cancer Inst. 34: 857-869, 1965. 11. Harisiadis, L., Duk S., II, Hall, E.J.: Thermal tolerance and repair of thermal damage by cultured cells. Radiology 123:505-509,1977. 12. Henle, K.J., Karamuz, J.E., Leeper, D.B.: Induction of thermotolerance in Chinese hamster ovary cells by high (45O) or low (40°) hyperthermia. Cancer Res. 38: 570-574, 1978. 13. Hume, S.P., Marigold, J.C.L.: Transient, heat-induced thermal resistance in the small intestine of mouse. Radiat. Res. 82: 526536,198O. 14. Hume, S.P., Rogers, M.A., Field, S.B.: Heat-induced thermal resistance and its relationship to lysosomal response. tnt. J. Radiat. Biol. 34: 504-5 11, 1978. 15. Kember, N.F.: Cell survival and radiation damage in growth cartilage. Br. J. Radiol. 40: 496-505, 1967. 16. Law, M.P., Coultas, P.G., Field, S.B.: Induced thermal resistance in the mouse ear. Br. J. Radio). 52: 308-3 14, 1979. 17. Maher, J., Urano, M., Rice, L., Suit, H.D.: Kinetic studies of thermal resistance in spontaneous murine tumors. Br. J. Radiol. (In press). 18. Morgan, J.E., Honess, D.J., Bleehen, N.M.: The interac-
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tion of thermal tolerance with drug cytotoxicity in vitro. Br. J. Cancer 39: 422-428,1979. Nielsen, O.S., Overgaard, J.: Effect of extracellular pH on thermotolerance and recovery of hyperthermic damage in vitro. Cancer Res. 39: 2712-2118, 1919. Overgaard, J.: Influence of extracellular pH on the viability and morphology of tumor cells exposed to hyperthermia. J. National Cancer Inst. 56: 1243-l 250, 1976. Overgaard, J., Suit, H.D.: Time-temperature relationship in hyperthermic treatment of malignant and normal tissue in vivo. Cancer Res. 39: 3248-3253, 1979. Reeves, R.O.: Mechanisms of acquired resistance to acute heat shock in cultured mammalian cells. J. Cell Physio/. 79: 157-170,1972. Rice, L. Cochran, Urano, M., Maher, J.: The kinetics of thermotolerance in the mouse foot. Radiat. Res. (In press.). Sedlacek, R.S., Mason, K.A.: A simple and inexpensive method for maintaining a defined flora mouse colony. Part I. Lab. Animal Sri. 27: 667-670, 1977. Silobrcic, V., Suit, H.D.: Tumor-specific antigen(s) in a spontaneous mammary carcinoma of C3H mice. I. Quantitative cell transplants into mammary-tumor-agent-positive and -free mice. J. Natl. Cancer Inst. 39: I 113-I I 19, 1967. Suit, H.D.: Hyperthermic effects on animal tissues. Radiology 123: 483-487, 1977. Suit, H.D., Kastelan, A.: Immunologic status of host and response of a methylcholanthrene-induced sarcoma to local X-irradiation. Cancer 26: 232-238, 1970.
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M., 28. Urano, M., Gerweck, L.E., Epstein, R., Cunningham, Suit, H.D.: Response of a spontaneous murine tumor to hyperthermia: Factors which modify the thermal response in vivo. Radiat. Res. 83: 3 12-322, 1980. M., Maher, J., Rice, L. Cochran, Kahn, J.: 29. Urano, Response of spontaneous murine tumors to hyperthermia: temperature dependence in 2 different sized tumors. J. Natl. Cancer Inst. Monograph 60, (In press). 30. Urano, M., Nesumi, N., Ando, K., Koike, S., Ohnuma, N.: Repair of potentially lethal radiation damage in acute and chronically hypoxic tumor cells in vivo. Radiology 118: 441-451, 1976. 31 Urano, M., Rice, L. Cochran, Kahn, J., Sedlacek, R.S.: Studies on fractionated hyperthermia in experimental animal systems. I. The foot reaction after equal doses: Heat resistance and repopulation. Int. J. Radiat. Oncol. Biol. Phys. 6: 1519-1523, 1980. 32. Von Ardenne, M., Reitnauer, P.G.: Supplementary information on pH, Hyperthermia, and cell survival: Tumor hyperacidification and multistep cancer therapy. J. Natl. Cancer Inst. 61: 3-4, 1978. 33. Withers, H.R.: The four R’s of radiotherapy. In Advances in Radiation Biology, Vol. 5, J.T. Lett, H. Adler, (Eds.). New York, Academic Press. 1975, pp 241-27 1. 34. Yamaguchi, T., Tabachnik, J.: Cell kinetics of epidermal repopulation and persistent hyperplaria in locally /3irradiated guinea pig skin. Radial. Res. 50: 158-180, 1972.
/III. J. Radiation Oncology Biol. Phys., Vol. 8. pp. 227-233 Printed in the U.S.A. All rights reserved
?? Original
Contribution
STUDIES ON FRACTIONATED HYPERTHERMIA IN EXPERIMENTAL ANIMAL SYSTEMS II. RESPONSE OF MURINE TUMORS TO TWO OR MORE DOSES MUNEYASU URANO, M.D., PH.D., LAURIE COCHRAN RICE, MS. AND VERNON MONTOYA, B. S. Department
of Radiation
Medicine,
Massachusetts General Boston. MA02114
Hospital,
Harvard
Medical School,
The effect of multiple hyperthermia and the kinetics of thermal resistance were studied in experimental murine tumors. A spontaneous C3Hf mouse mammary carcinoma, MCa and a chemically-induced fibrosarcoma, FSa-I, were used. Assay methods include determination of the TCD, i.e., the treatment time to yield a tumor control in half the treated animals and TG (tumor growth) time analysis, i.e., the time required for a tumor to reach a given size after first treatment. After equal dose fractions the TCDSO of MCa increased with increasing overall treatment time. This increase was most predominant when treatments were given with a time interval of one day. Isoeffect curves for the MCa were comparable to those for normal tissue damage (foot reaction), which was reported in the first part of this series of communications. The kinetics of thermal resistance in the MCa was compared with that in FSa-I since the fractionated hyperthermia for the FSa-I was reported to have resulted in an appreciable therapeutic gain. The magnitude of thermal resistance was far greater in the MCa than in the FSa-I, although the kinetics of thermal resistance was similar in both tumors; i.e., (a) the resistance reached a maximum in 24 hours after treatment and then decayed slowly, and (b) the development of thermal resistance increased with increasing initial dose. The thermal resistance in these tumors appeared to be greater than that in animal feet. Hyperthermia,
Thermal
resistance, Fractionated
hyperthermia,
INTRODUCTION
Mouse mammary carcinoma, Mouse fibrosarcoma.
possible repair of sublethal or potentially lethal thermal damage, progression of surviving cells through the cellcycle and alteration of hypoxic or acidic cell fraction in a tumor. The substantial development of thermal resistance, which is observed following a prior exposure to elevated temperature, has been demonstrated first in cultured mammalian cellP2 and recently in animal tissues. 1.13.16.17.23.31 Thermal resistance has appeared to be one of the most critical factors for the success of clinical hyperthermia. We have studied the kinetics of thermal resistance and fractionated hyperthermia regimen in murine tumors.
response to elevated temperature has been extensively studied in vitro and in vivo. A rationale for using hyperthermia in cancer therapy might be that hypoxic tumor cells, which are resistant to ionizing radiations, are at least as sensitive as aerobic tumor cells to elevated temperature.7 Recent studies revealed that the decrease in environmental pH is associated with an increase in thermal sensitivity of cultured mammalian cells.h.20~32Tumor tissue pH has been found to be lower as compared to blood or normal tissue pH, because of anerobic metabolism in hypoxic tumor cells that results in an accumulation of lactic acid.” In this situation, the hypoxic tumor cells should be more sensitive to heat than their well-oxygenated counterpart. This presumption has been confirmed by experimental animal studies.‘” Clinical hyperthermia will likely employ a fractionated regimen, which inevitably will be influenced by a variety of unresolved factors. We have studied fractionated hyperthermia in normal and tumor tissues in experimental animals.‘7.‘3.3’ Factors influencing the effect of multiple doses might include the kinetics of thermal resistance, Biological
METHODS
AND MATERIALS
Animals were 10-l 2 week old C3Hf/Sed mice derived from our defined flora colony. They were kept in an animal facility where defined flora conditions were maintained.24 Sterilized animal pellets* and acidified and vitamin K-fortified water were provided ad fibitum. Two tumors were used. One is a third generation isotransplant of a mammary carcinoma, MCa, which arose spontaneously in a female mouse. This tumor is weakly immu-
This work was supported in part by Grant Number CA26350 awarded by the National Cancer Institute, Department of Health, Education and Welfare.
Reprint requests to: M. Urano, M.D., Ph.D. Accepted for publication 7 October 198 I. *Wayne Lab Blox. 221
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nogenic and has a volume doubling time of -2.5-3.0 days.25 The other is a fourth generation tumor of methycholanthlene-induced fibrosarcoma, FSa-I, which is moderately immunogenic and proliferates with a doubling time of 12.0 days.” Single cell suspensions of these tumor cells were prepared by trypsinization, which is described elsewhere.” Approximately lo5 viable tumor cells in 5 &! were transplanted into the right foot. Animals were randomly assigned into groups before treatment. Hyperthermia was given by immersing the animal foot into a water bath where a desired temperature +O. l°C was maintained by a constant temperature circulator.28 Tumor temperature was no less than 0.1% below water bath temperature. No anesthesia was given prior to treatment. Assay methods include TG (tumor growth) time analysis and determination of TCDso (50% tumor control dose). TCD,, is a treatment time at elevated temperature to yield a tumor control in half the treated animals. Tumors were treated when they reached an ‘average diameter of 4 mm since preliminary data indicated this sized tumors were controlled without loss of the foot. Following the treatment, animals were observed for a possible tumor recurrence once a week for 120 days; {he TCD,, was calculated on the basis of the recurrence-free rate in each dose group. Animals that died without recurrence before termination of the experiment were excluded from the assay. TG time is the time required for a tumor to reach a given volume after treatment. Three diameters of each tumor a, b and c were measured at least 3 times a week and the tumor volume was calculated by a formula of ?rabc/6. The TG time was obtained graphically for each tumor and the median value was calculated by logit analysis. RESULTS The MCa was treated with equal dose fractions at 43.5% with a Ti (time interval) between treatments of 1, 2 or 5 days; then TCD,, was determined. Figure 1 demonstrates TCD, values as a function of overall treatment time. The TCDSOincreased with increasing overall treatment time in each treatment regimen. The most predominant increase was seen in the treatment regimen with a Ti of one day, indicating substantial development of thermal resistance and/or possible repair of sublethal and potentially lethal thermal damage occurring in first 24 hours after each treatment. The TCDso values are listed in Table 1 together with the mouse foot RDso (~4.0) (treatment time to induce score 4.0 [loss of one toe] or greater reaction in half the treated animals) which were previously reported.3’ No difference can be found between TCDW and RD, in each regimen except 6 fractions given with a Ti of 2 days, which resulted in a larger therapeutic ratio (ratio of RD,, to TCD,,) as compared to a single dose, indicating that a therapeutic gain was rarely obtained for the equal dose fraction.
February 1982, Volume 8, Number 2
.c9 E
i
/
I -7
6 Trertment
9 Time;
6 t
4
3
i
2 t II
’ 0
1
2
3
4
5
6
7 Overall
IO” Days
Fig. 1. Isoeffect curves for TCDS, of C3Hf mouse mammary carcinoma. TCD,, (total dose) after equal dose treatments is plotted as a function of overall treatment time. Open circles, solid circles and open squares indicate treatment given with a Ti of 1, 2 and 5 days respectively. Vertical bars indicate 95% confidence limits.
Notably, the tumor control was exclusively associated with a foot reaction of score 5.0 (loss of partial foot or greater reaction) or with a concomitant, partial loss of the foot after fractionated treatments, although the tumor control after single dose was associated with less severe reaction, i.e. score 4.5. These results suggest that the true TCD,, values after fractionated treatments might have been greater than those obtained experimentally with a resultant decrease in the therapeutic ratio. An earlier report from our laboratory demonstrated that multiple treatments of FSa-I tumors significantly Table 1. TCDSo and RD,,, (~4.0) values after fractionated hyperthermia given at 43.YC Treatment
Ti (day) 0
1
2
5
V* 1 2 4 I 11 2 4 6 2 3
Schedule Overall treatment time (days) 0
1 3 6 10 2 6 10 5 10
TCDso (min) 146 (130-164)t 192 (155-238) 344 (300-395) 542 (449-656) 801 (718-892) 229 (188-279) 384 (330-446) 460(419-505) 175(143-215) 269 (221-328)
RDSI (min) 139 (128-152)t 202 (182-223) 331$ 592 (555-631) 888 (837-941) 256(245-267) 389 (361-420) 545(519-572) 209(193-226) 239 (212-270)
Note: The mammary carcinoma transplanted in the mouse right foot was treated when it reached an average diameter of 4 mm. RDS, (~4.0) values for mouse foot reactions were cited from our previous paper.” *Number of fractions. TNumbers in parenthesis indicate 95% confidence limit. *Estimated from the iso-effect curve.
Fractionated hyperthermia of mutine tumors 0 M. Table 2. Effect of daily hyperthermia on a normal tissue and FSa-I tumors. Hyperthermia was given at 43.5OC with a Ti of 22 hours26 Number of Fractions
i70, ..
E
83 142 229 367
I
2 5 10
173 265 530 915
229
4 50+
Therapeutic ratio?
RD,, (>5.0)*
TCD,,
URANO et al.
-
Mammary
2.08 1.87 2.31 2.49
Ca
30-
.. was used in this experiment instead (~4.0) which is used exclusively in the present study. tRD,, (rS.O)/TCD,,. *RDS,, (25.0)
of RDSo 20-
increased the therapeutic ratio.26 As cited in Table 2, the therapeutic ratios after single treatment and 10 daily treatments were 2.08 and 2.49 respectively, although the TCD,, values of the FSa-I were exclusively smaller than those of the MCa. The kinetics of thermal resistance was studied at 45.5% in order to investigate the different therapeutic ratio between MCa and FSa-I. The reasons for raising treatment temperature to 45.5V are: a) substantial development of thermal resistance required a prolongation of the treatment time to more than 5 hours at 43.5% with animal feet fixed in a water bath, b) the kinetics of thermal resistance in another murine tumor and a normal tissue has been studied at 45.5% and c) it is welldocumented for FSa-I and -11 that at temperatures above 42.5-43.0°C, a 1°C increase of treatment temperature halves the treatment time, which induces a given tumor response.2’.29 The effect of the Ti between D, (first dose) and D, (second dose) on the development of thermal resistance was investigated in the MCa. A D, of 10 min was
I
a80
2
I
I
I5 t
I
I
I
I
I
101
FSa.1
’ 0
I
I
1
I
LJ
1
2
3
4
5 Days
Time
Interval
between 2 Doses:
Fig. 3. The kinetics of thermal resistance in a C3Hf mouse mammary carcinoma and in a chemically induced fibrosarcoma, FSa-I. A D, of IO min was followed by D2 of 25 min with various Ti for mammary carcinoma, while D, of 7.5 min plus D, of 15 min were given to FSa-I with various Ti. Vertical bars show 95% confidence limits. (see text for detail.)
followed by a variable D, with various Ti, and the TG time was determined. The dose response curves after 2 doses demonstrated a less steep slope as compared to single dose response curve, indicating that substantial thermal resistance developed following the first exposure to heat (Fig. 2). Two doses with a Ti of 1 day resulted in a survival curve which showed the least steep slope; i.e., the thermal resistance developed rapidly in first 24 hours and decayed thereafter. Selected data from Figure 2 is shown
C3Hf/Sad, MCa
I
-I
I
t-
CJHVSed, MCa !pO” 0
._
E ._ c a +
30
5o
30 -
t 201
20 A
10’
0
6h
0
ld
??
Zd
0
16
0
3d
??
22.6
IO 20
40
60 Total Time at 45S’C;
80 min
Fig. 2. Development of thermal resistance in a C3Hf mouse mammary carcinoma. A D, of 10 min at 45.5% was followed by D, with various Ti. Each symbol indicates each Ti as shown in the figure. Vertical bars are 95% confidence limits.
’ 0
I
I 20
40 Total
I 60 Time at 45.5.C;
80 min
Fig. 4. Development of thermal resistance in a C3Hf mouse mammary carcinoma after a various D,. A D, was given 24 hours after D, . Different symbols show different sizes of D, as indicated in the figure. Vertical bars are 95% confidence limits.
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in Figure 3 where the TG time after a fixed D, (10min) plus a fixed D, (25 min) is plotted as a function of Ti between 2 doses. Figure 3 also shows the kinetics observed in FSa-I after a D, of 7.5 and D, of 15 min. It was necessary to use different treatment times in these 2 tumors because of the large difference in thermal sensitivity between them. The different treatment times made this comparison of these 2 tumors difficult since the kinetics of thermal resistance has been shown to depend on the size of D, as well as the Ti. Therefore, further studies were done. Effect of the size of D, on the development of thermal resistance was studied for MCa (Fig. 4) and FSa-I (Fig. 5) by TG time analysis. The Ti between doses was 24 hours. In both tumors the slope of the dose response curve after the second dose decreased with increasing D, indicating that an increase in D, was associated with increasing thermal resistance. The magnitude of thermal resistance appeared to be more dramatic in MCa than in FSa-I, in that a difference in slopes between single and 2 dose-response curves was far greater for MCa than for FSa-I. This was clearly seen when the thermal resistance ratio (TRR) was plotted as a function of D, (Fig. 6). The TRR is defined as: D (2 doses) - D, D, . . (1) D (single dose) - D, OrD (single dose) - D, ' .
February 1982, Volume 8, Number 2 C3Hf/S~ed
0 ._
I
‘t;; K
I
I
I
-
15-
Mammary
Ca
--1
0
5
10
15
20 Dl ; min
Fig. 6. Thermal resistance ratio as a function of the size of D,. The biological endpoint used for calculation of the ratio was a treatment time to induce TG time of 30 days (T,,). Note a big difference between a C3Hf mouse mammary carcinoma (solid squares) and chemically-induced fibrosarcoma, FSa-I (open squares). The T,, for FSa-I treated with a D, of 3 and 7.5 min were obtained by extrapolating each dose response curve shown in Fig. 5. where D (single dose) or D (2 doses) is the treatment time to induce a given biological response after single treatment or 2 treatments, respectively.23 In Figure 6, the given biological response was a TG time of 30 days. The TRR increased with increasing D, and the magnitude of thermal resistance was much greater in MCa than in FSa-I. DISCUSSION
4f./ .
0
8 Single 0 a 0
q
... 15 37.5min
20 Total Time at 45.5’C;
40 min
Fig. 5. Development of thermal resistance in a chemically induced fibrosarcoma, FSa-I after a various D,.A D, was given 24 hours after D,.Different symbols show different sizes of D, as indicated in the figure. Vertical bars are 95% confidence limits.
Thermal resistance, or thermotolerance, has been demonstrated in animal tissues as well as in cultured mammalian cells.‘~” In general, it is a transient and non-heritable resistance to hyperthermia following a prior exposure to heat. The survival curve, of resistant cells is featured by an increased slope with or without a change in a shoulder. It has been shown both in vitro and in vivo that the magnitude of thermal resistance increases Some in vivo experiments with increasing D,.3y"*'6323 suggest rapid development of thermal resistance after a short D,.'6v23 Lower environmental pH has been found to be associated with less resistance.8*9.‘9 Our previous report discusses this topic in an animal tumor.” No consensus about the mechanism of thermal resistance has yet been obtained 1.4.13,14.18,22 Present
experiments
demonstrated
a substantial
differ-
Fractionated hyperthermia of murine tumors 0 M. URANOef al. Table 3. Effect of the size of D, on the development FSa-I
of thermal
resistance
231
in three murine tumors at 45S”C FSa-II
MCa
Foot reaction
(a) TX0 D, (min) 0 3 7.5 15 22.5 30 (b) Td (TG) D, (min) 0 3 7.5 15 22.5 30
T,0 (min) 17 31 40 28
TRR
Td (TG) 6 16 22 20
TRR
I .o 2.0 3.4 6.5
I .o 2.7 3.7 3.3
T,, (min) 19 46 74 82
TRR
Td (TG) 14 18 66 118 104
TRR 1.0 1.3 4.7 8.4 7.4
1.0 2.7 5.8 16.8
T,, (min) 40 53 80 135 115
TRR
Td (TG) 25 29 51 112 97
TRR 1.0 1.2 2.0 4.5 3.9
1.0 1.4 2.2 4.8 5.3
RD,, (min) 34 60 80 75 64 37
TRR
RD,, 34 60 80 75 64 37
TRR 1.0 1.9 3.0 3.9 5.6 9.3
1.0 1.9 3.0 3.9 5.6 9.3
Note: The second dose was given 24 hours after D,. Two different biological endpoints are used: (a) treatment time to induce TG time of 30 days or T3,, and (b) slope of the dose response curve, which is expressed as a treatment time required to double the TG time or Td (TG). Thermal resistance ratio or TRR is calculated by Eq. 1. For comparison, the TRR for mouse foot is tabulated from ref. thirty-one. The endpoint was RDS, (~4.0) i.e., treatment time to induce score 4.0 (loss of one toe) or greater reaction in half the treated mice.
in the magnitude of thermal resistance between 2 tumors. Although the possible development of thermal resistance in murine tumors was suggested by Crile,’ no systematic studies on thermal resistance in tumors have been reported to our knowledge, except one obtained in our laboratory in a spontaneous C3Hf/Sed mouse fibrosarcoma, FSa-II.” Table 3 summarizes all the data together with the data of a mouse normal tissue (foot response). As mentioned above, the magnitude of thermal resistance depends on the size of D,. The TRR may be altered by the biological endpoints of the level of damage assessed since the survival curve of thermal resistant cells may be associated with a change in the shoulder width as well as an increase in the slope. Therefore, the TRRs based on 2 different endpoints are shown in Table 3. The endpoints are (a) a treatment time to induce a TG time of 30 days and (b) a slope which is expressed as a treatment time to double the TG time in the exponential portion of the dose response curve. Common features are: thermal resistance is most substantial in MCa and least in FSa-II, and thermal resistance in the foot was only slightly greater than that in FSa-II, or less extensive than that in MCa and FSa-I. These results suggest that the therapeutic ratio is rarely greater than 1.0 after fractionated hyperthermia compared with a single treatment when the treatment intervals of 24-48 hours is used. No correlation could be found between the magnitude of thermal resistance and the thermal sensitivity of tumors. TCDSo at 43.5% of FSa-II was 170 (153-189*) min,*’ the greatest among three tumors, while the magnitude of thermal resistance was minimum. The TCD,, of ence
*95% confidence
limit.
MCa, which showed greatest resistance, was 146 (130164) min or intermediate among three tumors (see Tables I and 2). No correlation appeared between tumor immunogenecity and thermal resistance. The FSa-I, which was moderately immunogenic, yielded intermediate resistance, and the weakly immunogenic MCa and FSa-II demonstrated greatest and least resistance, respectively. Tumor volume doubling time was shortest in the FSaII (1.5-2.0 days) and longest in the MCa (2.5-3.0). Thus the longer doubling time appeared to be associated with greater thermal resistance. However, it is unknown whether this relation may exist in normal tissues. The RD,, (~4.0) might be the results from the skin or bone damage. It is also unknown if the cell cycle time of these cells may be shortened after the first hyperthermia treatment although cell cycle time of these tissue cells have been studied.‘5*34 Further studies in this relationship should be encouraged. The fractionated hyperthermia resulted in an increased therapeutic ratio for FSa-I, but not MCa. This increase for FSa-I most likely results from the small TCD,, value of this tumor rather than the less extensive thermal resistance. The TCD,, (single dose) at 43.5”C of FSa-I was 83 min while that of MCa was 146 min, which was comparable to RD,, (single dose) of 139 min (Tables 1 and 2). Consequently, each dose in fractionated treatments for FSa-I was smaller, thus less resistance was induced as compared to that for MCa or for animal feet, resulting in less increase in TCD,, for FSa-I than in TCD,, for MCa and in RD,,.
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Total
Radiation
Dose
HYPERTHERMIA
Total Treatment
Time
Fig. 7. Schematic demonstration of effectiveness of each dose in fractionated radiation doses (top panel) and in fractionated
hyperthermia (bottom panel). Thick dotted lines are single dose survival curves while thin dotted lines are survival curves after second dose. Solid line and solid squares are overall survival after multiple doses.
February 1982, Volume 8, Number 2
Finally, it should be mentioned that each dose in the equal dose fractionation does not produce an equal effect. Figure 7 schematically compares the effect of fractionated hyperthermia on cell survival with that of fractionated radiotherapy. It is assumed that subsequent treatments were given when sublethal damage was fully repaired and the thermal resistance was fully developed. Each dose in fractionated radiotherapy has an equal effect throughout a treatment course, as has been frequently discussed by other investigators,2”3 while the effect of each dose in the fractionated hyperthermia is not equal because of the substantial development of thermal resistance. The first hyperthermic treatment modifies thermal sensitivity of tumor cells in such a way as to decrease sensitivity. Thermal resistance develops continuously after each treatment and an accumulation of fractionated doses might further increase the resistance. This means, as far as equal dose fractions are concerned, that the first dose exhibits the greatest effect and subsequent treatments are less effective. In 11 daily treatments, the largest fraction number tested (Table l), the daily dose was as large as 73 min, which was approximately half of the single dose TCDSO. This implies that equal dose fractionation given to thermally resistant cells eventually loses a fractionation effect that might increase the therapeutic ratio. Accordingly, as far as treatments are given to thermally resistant tumors, daily doses should be increased with a progress of treatments. Another choice may be the fractionation with a Ti which is long enough to eliminate thermal resistance. These regimen are now being studied.
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2.
3.
4.
5. 6. 7.
8.
Crile, G., Jr.: The effects of heat and radiation on cancers implanted on the feet of mice. Cancer Res. 23: 372-380, 1963. Elkind, M.M., Sutton, H.: Radiation response of mammalian cells grown in culture I. Repair of X-ray damage in surviving Chinese Hamster Cells. Radiot. Res. 13: 556593.1960. Gerner, E.W., Boone, R., Connor, W.G., Hicks, J.A., Boone, Max L.M.: A transient thermotolerant survival response produced by single thermal doses in HeLa cells. Cancer Res. 36: 1035-1040, 1976. Gerner, E.W., Cress, A.E., Stickney, D.G., Holmes, D.K., Culver, P.S.: Factors regulating membrane permeability alter thermal resistance. In Thermal Characteristics of Tumors: Applications in Detection and Treatment, Vol. 335, R.K. Jain, P.M. Gullino (Eds.). New York, NY Acad. of Sciences. 1980, pp. 2 15-233. Gerner, EW., Schneider, M.J.: Induced thermal resistance in HeLa cells. Nature 256: 500-502, 1975. Gerweck, L.E.: Modification of cell lethality at elevated temperatures. The pH effect. Radial. Res. 70: 224-235, 1977. Gerweck, L.E., Gillette, E.L., Dewey, W.C.: Killing of Chinese hamster cells in vitro by heating under hypoxic or aerobic conditions. Eur. J. Cancer 10: 691-693, 1974. Gerweck, L.E., Jennings, M., Richards, B.: Influence of pH on the response of cells to single and split doses of hyperthermia. Cancer Res. 40: 4019-4024, 1980.
9. Goldin, E.M., Leeper, D.: The effect of low pH on thermotolerance induction using fractionated 45OC hyperthermia. Radiat. Res. 85: 472-479, 198 1. 10. Gullino, P.M., Grantham, F.H., Smith, S.H., Haggerty, A.C.: Modifications of the acid-base status of the internal milieu of tumors. J. N&l. Cancer Inst. 34: 857-869, 1965. 11. Harisiadis, L., Duk S., II, Hall, E.J.: Thermal tolerance and repair of thermal damage by cultured cells. Radiology 123:505-509,1977. 12. Henle, K.J., Karamuz, J.E., Leeper, D.B.: Induction of thermotolerance in Chinese hamster ovary cells by high (45O) or low (40°) hyperthermia. Cancer Res. 38: 570-574, 1978. 13. Hume, S.P., Marigold, J.C.L.: Transient, heat-induced thermal resistance in the small intestine of mouse. Radiat. Res. 82: 526536,198O. 14. Hume, S.P., Rogers, M.A., Field, S.B.: Heat-induced thermal resistance and its relationship to lysosomal response. tnt. J. Radiat. Biol. 34: 504-5 11, 1978. 15. Kember, N.F.: Cell survival and radiation damage in growth cartilage. Br. J. Radiol. 40: 496-505, 1967. 16. Law, M.P., Coultas, P.G., Field, S.B.: Induced thermal resistance in the mouse ear. Br. J. Radio). 52: 308-3 14, 1979. 17. Maher, J., Urano, M., Rice, L., Suit, H.D.: Kinetic studies of thermal resistance in spontaneous murine tumors. Br. J. Radiol. (In press). 18. Morgan, J.E., Honess, D.J., Bleehen, N.M.: The interac-
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M., 28. Urano, M., Gerweck, L.E., Epstein, R., Cunningham, Suit, H.D.: Response of a spontaneous murine tumor to hyperthermia: Factors which modify the thermal response in vivo. Radiat. Res. 83: 3 12-322, 1980. M., Maher, J., Rice, L. Cochran, Kahn, J.: 29. Urano, Response of spontaneous murine tumors to hyperthermia: temperature dependence in 2 different sized tumors. J. Natl. Cancer Inst. Monograph 60, (In press). 30. Urano, M., Nesumi, N., Ando, K., Koike, S., Ohnuma, N.: Repair of potentially lethal radiation damage in acute and chronically hypoxic tumor cells in vivo. Radiology 118: 441-451, 1976. 31 Urano, M., Rice, L. Cochran, Kahn, J., Sedlacek, R.S.: Studies on fractionated hyperthermia in experimental animal systems. I. The foot reaction after equal doses: Heat resistance and repopulation. Int. J. Radiat. Oncol. Biol. Phys. 6: 1519-1523, 1980. 32. Von Ardenne, M., Reitnauer, P.G.: Supplementary information on pH, Hyperthermia, and cell survival: Tumor hyperacidification and multistep cancer therapy. J. Natl. Cancer Inst. 61: 3-4, 1978. 33. Withers, H.R.: The four R’s of radiotherapy. In Advances in Radiation Biology, Vol. 5, J.T. Lett, H. Adler, (Eds.). New York, Academic Press. 1975, pp 241-27 1. 34. Yamaguchi, T., Tabachnik, J.: Cell kinetics of epidermal repopulation and persistent hyperplaria in locally /3irradiated guinea pig skin. Radial. Res. 50: 158-180, 1972.