The effect of hyperthermia on reoxygenation during the fractionated radiotherapy of two murine tumors, FSa-II and MCa

Int. J. Radiation Oncology Biol. Phys.. Vol. 29. No. I, pp. 141-148, 1994 Copyright 0 1994 Elwier Science Ltd Printed in the USA. All rights reserved 0360-3016/94 $6.00 + .OO

Pergamon

0 Hyperthermia Original Contribution THE EFFECT OF HYPERTHERMIA ON REOXYGENATION DURING THE FRACTIONATED RADIOTHERAPY OF TWO MURINE TUMORS, FSA-II AND MCA YASUMASA NISHIMURA,

M.D., PH.D. AND MUNEYASU URANO, M.D., PH.D.

Department of Radiation Medicine, University of Kentucky, Chandler Medical Center, 800 Rose St., Lexington, KY 40536-0084 Purpose: To investigate the effect of hyperthermia on the tumor reoxygenation during fractionated irradiations. It has been shown that hyperthermia increases the size of hypoxic cell fraction in some murine tumors and reoxygenation is critical for successful radiotherapy. Methods and Materials: Tumors were early generation isotransplants of spontaneous murine fibrosarcoma (FSaII) and mammary carcinoma (MCa) in C3Hf/Sed mice. Treatments were initiated when they reached an average diameter of 4 mm. A local heat treatment at 43S”C for 45 min was given in a constant temperature water bath 24 h before irradiation(s). This interval was selected to avoid heat-radiation interaction and to simply investigate the heat effect on the reoxygenation process. Tumors were irradiated under hypoxic conditions or in air and observed for recurrences for 120 days. The foot reaction of animals with controlled-tumors was scored on the last day of experiments. The TCDso (50% tumor control dose) and RDs (dose to induce partial foot atrophy in 50% of treated animals) were calculated. Results: The TCDSOs following a various number of fractions were obtained for FSa-II and MCa with or without hyperthermia. The difference between the TCDso (hypoxia) and TCDSO (in air) without hyperthetmia increased with an increasing number of fractions, suggesting that significant teoxygenation occuted during the fractionated irradiation. The TCD% (with heat, in air) were smaller than the TC&s (radiation alone, in air) following fractionated irradiations, indicating that hyperthermia did not affect tumor reoxygenation. The difference between these TCDSo values was greater for heat-sensitive MCa than for heat-resistant FSa-II, suggesting that this difference was due to additive heat cytotoxicity. An unexpected observation was that heat significantly enhanced the foot reaction with no resultant therapeutic gain for both MCa and FSa-II tumors. Conclusion: Hyperthetmia given independently prior to fractionated irradiation did not affect tumor teoxygenation, not was there a therapeutic gain for the two mutine tumors. These results suggest that selective tumor heating is essential in clinical thermoradiotbetapy. Hyperthetmia, diotherapy.

Fractionated radiotherapy, Reoxygenation,

FSa-II tumor, Mammary adenocarcinoma,

Thetmora-

Numerous experiments and clinical trials have suggested that hyperthermia given in combination with radiotherapy is advantageous (8). The rationale for this combined treatment is partly based on a radiosensitizing effect of hyperthermia. Moderate hyperthermia also has a selectively cytotoxic effect on acidic and nutritionally deprived tumor cells which are likely to be hypoxic and, therefore, radioresistant (8). However, various questions have been raised about the use of hyperthermia with fractionated radiotherapy. A critical finding for combined hyperthermia and radiotherapy may be that hyperthermia increases

the size of the hypoxic cell fraction (9, 17). This increase may be the result of decreased blood flow due to vascular damage in the heated tumor (3,9, 10,23). Reoxygenation occurring during fractionated radiotherapy is believed to favor subsequent irradiations (2), but it is unknown whether or not tumor cells can be reoxygenated in the heated tumor where the vasculature might have been damaged by the initial heat treatment. We have studied thermal effects on the reoxygenation process during the course of fractionated radiotherapy using two murine tumors. Hyperthermia was used as an independent agent, not a radiosensitizer (17). The late normal tissue response following a single dose or frac-

Reprint request to: Dr. Yasumasa Nishimura, Department of Radiology, Faculty of Medicine, Kyoto University, Sakyo-ku,

for her excellent technical and editorial assistance in this study and in the preparation of this manuscript. This study was par-

Kyoto 606, Japan. Acknowledgement:-We

tially supported by NIH CA 26350 and ACS PDT-444. Accepted for publication 17 December 1993.

INTRODUCTION

are grateful

to Ms. Regina

Reynolds 141

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I. J. Radiation Oncology 0 Biology 0 Physics

tionated doses given alone or in combination with hyperthermia was also studied to investigate the therapeutic gain of this combined treatment. METHODS

AND MATERIALS

Eight to ten week-old C3Hf/Sed mice were used throughout this experiment. The mice were kept in the animal facility where microorganism-free conditions were maintained. They originated from the Department of Radiation Medicine, Massachusetts General Hospital, and were bred in the University of Kentucky Chandler Medical Center animal facility. Sterilized mouse pellets and acidified water were provided ad lib. The tumors were fourth generation isotransplants of a nonimmunogenic fibrosarcoma, FSa-II which arose spontaneously in a C3Hf/Sed mouse (15, 21) and of a spontaneous mammary carcinoma, MDAH MCa-IV ( 14, 17). The third generations of both tumors have been kept in a liquid nitrogen freezer to maintain their biological characters including radiosensitivities. They were transplanted in the mouse flank to make source tumors when needed. Cell suspensions were prepared by trypsinizing these source tumors as described elsewhere (19). These suspensions were transplanted into the mouse right foot in a 5 ~1 volume containing 2 X lo5 viable cells. The tumors were treated when they reached an average diameter of 4 mm (approximately 35 mm3). Hyperthermia was given by immersing the animal foot (up to 1 cm above the ankle) in a water bath where a temperature of 43.5 f 0.05”C was maintained by a constant temperature circulator.] The tumor temperature was equilibrated within 90 s and was no less than 0.1 “C below the water bath temperature. Further details, including tumor temperature measurement, are given elsewhere ( 15). Tumors were irradiated with a one-portal 4000 Ci ‘37Cs irradiator,2 specifically designed for mouse irradiation. Animals were placed inside a lucite chamber and appropriately shielded by a lead and tungsten collimator. Tumors were irradiated at the center of a 3 X 20 cm field. Animals were turned over in the middle of each irradiation to give uniform doses in the tumor. The dose rate at the tumor center was 5.5-5.6 Gy/min during the experiments. A constant air flow of 2 l/min was maintained in the chamber during irradiation. Tumors were irradiated under hypoxic conditions or in air at room temperature. Tumor hypoxia was obtained by applying a heavy brass clamp above the ankle for at least two minutes before and during each irradiation. Animals were anesthetized with sodium pentobarbital (60 mg/Kg) only before irradiation. No anesthesia was given before hyperthermia. A single dose or fractionated regimens with 2, 5, 10, and 20 fractions were given alone or in combination with hyperthermia. Hyperthermia of 43.5”C for 45 min was

’Lauda model MS, Lauda, Germany.

Volume 29, Number I. 1994

administered 24 h before the first radiation dose. Fractionation regimens were equal graded daily doses with a constant interfraction interval of 24 + 1 h. For the 20 fraction experiments, twice-a-day (bid) irradiations were also performed to investigate tumor cell proliferation during the course of fractionated doses. The bid doses were given with intervals of 6 and 18 h, alternately. Following the completion of treatments, irradiated areas were palpated once a week for 120 days for possible tumor recurrences. A tumor that regrew more than 8 mm in average diameter was scored as a recurrence. Animals dying without a tumor within 120 days were excluded from the assay. For the various assays, 5- 15% of the mice died of metastatic tumors. The TCDso was calculated by logit analysis based on the tumor control frequency within 120 days following the final treatment. Approximately 40-50 animals were used in an experiment and at least two experiments were performed for each assay. A total of 1600 mice were used in this study. The hypoxic cell fraction (HCF) was calculated on the basis of the vertical displacement of the dose-response curves according to the following equation (18) using a Do value of 4.7 Gy for FSa-II tumors (16): In HCF = [TCDsO(in air) - TCD,,,(hypoxia)]/Do

(1)

The late foot reaction was scored in animals which developed no recurrence within 120 days. Our numerical score system is shown in Table 1 (20). The radiation dose inducing a foot reaction of 5.0 (partial foot atrophy) in 50% of the treated animals within 120 days after the treatment, that is, RDsO, was calculated by logit analysis. The thermal enhancement ratio (TER) was calculated as the ratio of the TCDSo (radiation alone) to TCDSO(test

Table 1. Foot reaction scores at 120 days after local irradiation Score 0 0.5

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 6.0

with or without

hyperthermia

Signs Normal foot Epilation in less than 50% of the foot Epilation in approximately 50% of the foot Epilation in more than 50% of the foot Complete epilation in the foot or dry desquamation in less than 50% of the foot Complete fusion of toes, mild edema, and/or desquamation in more than 50% of the foot Moderate edema, moist desquamation, or skin necrosis in less than 50% of the foot Severe edema, moist desquamation, or skin necrosis in more than 50% of the foot Atrophy of one toe or less than five segments Atrophy of two or more toes Partial atrophy of the foot Complete atrophy of the foot

’ J. R. Shepherd,

San Fernando,

CA.

Heat effect on reoxygenation 0 Y.

alone) to RDso (test treatment) for tumor response or late foot reaction, respectively. The therapeutic gain factor (TGF) was the ratio of TER (tumor) to TER (late foot reaction). Statistical analysis was performed using Student’s t-test and p -c 0.05 was regarded as significant.

treatment)

or as the ratio of RDso (radiation

RESULTS

The TCDsos of the FSa-II tumors were investigated following l-20 radiation doses given alone or following a heat dose of 43.5”C for 45 min (Table 2). The relationships between the tumor control rate and total radiation dose are shown for single, 5-, lo-, and 20(U)-fraction experiments in Figure 1. The TCDso (in air) of 67.1 Gy following heat plus a single radiation dose was not significantly different from that of 66.2 Gy following radiation (in air) alone. The TCDsos (in air) following combined treatments were approximately 10 Gy smaller than those following radiation alone when the number of fraction was increased to five and 10 doses (Fig. 1 and Table 2). This reduction was significant compared to the TCDsos (in air) for five and 10 doses given without heat (p < 0.02 and p < 0.01, respectively). However, the TCDsos following heat plus 20 radiation doses given either daily or twice-a-day was not significantly lower than those following radiation

143

NISHIMURA AND M. URANO

alone. The hypoxic cell fraction in the untreated 4 mm FSa-II tumor was 8.7% (95% confidence limit; 4.6-16.2), using Eq. 1. The RDso values obtained from animals with controlled tumors are also listed in Table 2 with 95% confidence limits. Figure 2 shows the relationship between the TCD50 values and the number of fractions for the FSa-II tumor. The TCDso (hypoxia) substantially increased from single to two fractions and then linearly from two to 20 daily fractions. The TCDso (hypoxia) following 20 daily doses (given in 19 days) was significantly larger than that following 20 bid irradiations (given in 9 days). The TCDW (in air) with or without hyperthermia also increased with an increasing number of fractions, but this increase was less substantial compared to the increase in the TCDW (hypoxia) (Fig. 2). The TCDSOs (in air) following 20 bid doses were not significantly different from those following 10 daily doses for radiation given either alone or in combination with hyperthermia. The TCDso (in air) of MCa tumors was investigated for a single dose, five daily doses, and 20 bid doses given alone or following hyperthermia (Table 3 and Fig. 3). In Figure 3, the TCDSOs (hypoxia) were redrawn from the data published by Suit et al. (14) because the same MCa tumors were used in both studies. As shown for the FSaII tumor, the difference between the TCDSO(hypoxia) and

Table 2. Effects of hvnerthermia on fractionated radiotheranv in the FSa-II tumors and foot reaction Tumor response Foot reaction Treatment

condition

Single dose In air H + R (in air) Hypoxia 2 fractions (Ti = 24 hr) In air H + R (in air) Hypoxia 5 fractions (Ti = 24 hr) In air H + R (in air) Hypoxia 10 fractions (T, = 24 hr) In air H + R (in air) Hypoxia 20 fractions (Ti = 24 hr) In air H + R (in air) Hypoxia 20 fractions (Ti = 6 and 18 h, bid) In air H + R (in air) Hypoxia

Fraction size (Gy) X no. of fraction

TCD5,, (Gy) (95% C.L.)

RD5,, (Gy) (95% CL.)

66.2 (64.1-68.3) 67.1 (64.2-70.1) 77.7 (75.2-80.3)

72.6 (69.9-75.4) 62.5 (57.0-68.6) 91.2 (84.1-98.9)

40.8 x 2 39.6 x 2 47.65 x 2

81.6 (78.2-85.2) 79.2 (75.0-86.3) 95.3 (88.8-102.2)

90.5 (83.3-98.2) 76.2 (71.6-81.2) > 110*

19.98 x 5 18.14 x 5 23.46 x 5

99.9 (95.6-104.4) 90.7 (85.7-95.8) 117.3 (110.4-124.5)

112.4 (105.3-120.0) 80.9 (70.4-93.0) 162.7 (121.7-217.5)

10.66 x 10 9.39 x 10 15.15 x 10

106.6 (100.5-l 13.0) 93.9 (87.3-101.1) 151.5 (141.3-162.4)

122.4 (114.5-130.9) 89.6 (79.6-101.0) 167.5 (155.5-180.5)

5.955 x 20 6.015 x 20 11.34 x 20

119.1 (110.8-128.1) 120.3 (111.6-129.8) 226.8 (207.6-247.9)

147.3 (137.8-157.4) < 110* - 220*

5.425 x 20 5.140 x 20 8.395 X 20

108.5 (102.1-l 15.3) 102.8 (94.0-l 12.4) 167.9 (161.2-174.9)

128.5 (121.2-136.3) 97.8 (86.8-l 10.2)

167.9 (155.2-181.6)

Note: Hyperthermia (43.5”C, 45 min) was given 24 h before the first radiation dose. Late foot reaction was scored in animals which developed no recurrence within 120 days after completion of the treatment, and the radiation dose which induced a reaction of score 5.0 or greater in 50% of the treated feet (RD,,) was calculated. * RDso could not be calculated because the dose range examined did not cover the RDso dose or number of mice was too small.

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Volume 29, Number I. 1994

'0 ?ilOO-

QlW _.._.

0

0

hair

____.

0

In air

W 0;

i

80

I I I

40

: I

i 0:

/

20 :

40

60

80

100

120

140

160

180

200

Total

Dose

0

50

100

150

20

Fr(bid), FSa-II

200

2;o

(Gy)

Fig. 1. Tumor control probability of 4 mm FSa-II tumors treated with a single, 5, 10, and 20 (&&fractionated radiation doses. Each figure has three dose response curves: Radiation given in air (O), radiation given under hypoxic conditions (0), and hyperthermia plus radiation given in air (A). Hyperthernna (43.5”C for 45 min) was administered 24 h before the first radiation dose.

the TCDSO (in air) without hyperthermia increased with an increasing number of fractions. Unlike the FSa-II, the TCDsos (in air) following heat plus l-20 radiation doses were significantly smaller than the TCDsos (in air) following radiation alone. This indicated that heating at 435°C for 45 min was more cytotoxic to the MCa than the FSaII tumors; namely, MCa tumors were more heat-sensitive than FSa-II tumors. To evaluate the late normal tissue reaction, the foot reaction of score 5.0 (partial foot atrophy) was selected because the RDso values for a score 5.0 were relatively close to the TCDsos of FSa-II tumors. These RDso values are shown in Tables 2 and 3 together with the TCDso values. An unpredicted observation was that the TERs for the late foot reaction were significantly greater than those for the TCDSO of heat-resistant FSa-II tumors regardless of the number of fractions with the exception of 20 bid doses (Table 4). As a result, all TGFs for the treatment of FSa-II tumors were < 1.0, that is, a single treatment of moderate hyperthermia given 24 h before frac-

tionated irradiations led to therapeutic loss. None of the TGFs for MCa were significantly different from 1.O (Table 4) that is, no significant therapeutic gain was obtained for either heat-sensitive MCa or heat-resistant FSa-II by adding hyperthermia independently 24 h before fractionated irradiations.

DISCUSSION Our previous study demonstrated that the hypoxic cell fraction of the FSa-II and MCa tumors increased immediately after hyperthermia (43.5”C for 45 min) and remained higher, at least for the next 3 days, than that in the nontreated tumors (17). A similar temporary increase in the hypoxic cell fraction following hyperthermia was also reported by Song et al. (11). Heat-induced vascular damage is considered a cause of this increase in the hypoxic cell fraction (3, 9, 11). The tumor vasculature in rodent transplantable tumors is quite fragile to heat, and

Heat effect on reoxygenation 0 Y.

TCD-50,

NKHIMURA

AND

145

M. URANO

FSa-II f--

20 Fr(qd)

Fr(bid)

~20

<-

20 Fr(bid)

I

I

10

100

No. of Fractions Fig. 2. Relationship between the TCDSo values and the number of fractions for FSa-II tumors. Tumors were treated with radiation given in air (Cl), under hypoxic conditions (0), or with hyperthermia plus radiation given in air (A). Hyperthermia (43.5’C for 45 min) was administered 24 h before the first radiation dose. For 20 fractions, two TCDSO values are shown: Upper and lower points are from assays using once-a-day (qd) and twice-a-day (bid) irradiation, respectively. Bars indicate 95% confidence limits.

hyperthermia at 43S”C for 45 min can cause the nearly complete destruction of the tumor vasculature (3, 4, 9, 23). Although secondary cell death due to ischemia may occur in tumors, some hypoxic tumor cells may remain clonogenic and could cause treatment failure. Present study also showed that the TCDsos (in air) of the FSa-II tumors treated with single or two doses following hyperthermia were not significantly different from the TCD+ of tumors treated with radiation alone. Accordingly, our major question was how this heat-increased hypoxic cell fraction modifies the tumor response to subsequent fractionated radiation doses. The present study demonstrated an increase in the difference between the TCDSO (hypoxia) and TCDso (in air)

Table 3. Effects of hyperthermia

on fractionated

with an increasing number of fractions (Fig. 2). This suggested that the hypoxic tumor cells were reoxygenated during fractionated irradiations. Although hyperthermia caused slight radioresistance following a single dose or two doses, the TCDsO (in air, heat) was smaller than the TCDSO (in air, without heat) in five and ten fractionated doses. This indicated that tumor reoxygenation was not inhibited by hyperthermia. Heating at 43.5”C for 45 min can permanently destroy a significant portion of the vasculature in experimental tumors (3, 4, 9, 23). However, tumor vessels in the tumor periphery are more resistant to heat than those in the tumor center (3,23) and angiogenesis of tumor vessels occur from the tumor periphery within a few days after hyperthermia (3). This suggests

radiotherapy Tumor

in the MCa tumor

and foot reaction

response Foot reaction

Treatment

Fraction size (Gy) X no. of fraction

condition

Single dose In air H + R (in air) 5 fractions (Ti = 24 hr) In air H + R (in air) 20 fractions (Ti = 6 and 18 h, bid) In air H + R (in air) Note: RD,,s

are taken from Table

1.

TCDs,, (Gy) (95% C.L.)

RDSo (Gy) (95% C.L.)

52.2 (49.2-55.4)* 40.4 (35.4-46.0)

72.6 (69.9-75.4) 62.5 (57.0-68.6)

14.42 X 5 11.26 X 5

= =

72.1 (66.8-77.9) 56.3 (51.5-61.7)

112.4 (105.3-120.0) 80.9 (70.4-93.0)

4.235 X 20 3.310 x 20

= =

84.7 (75.9-94.5) 66.2 (60.8-72.2)

128.5 (121.2-136.3) 97.8 (86.8-l 10.2)

146

1. J. Radiation Oncology 0 Biology 0 Physics

TCD-50, 160

Volume 29, Number 1, 1994

MCa

Hypoxia(data from Suit et al.) -I)-

R (in air)

--t_

H + R (in air) .-

.-

POFr(bid)

60 60

100

No. of Fractions Fig. 3. Relationship between the TCD50 and the number of fractions for MCa tumors. Radiation was administered in air with (A) or without hyperthermia (0). Hyperthermia (43S”C for 45 min) was administered 24 h before the first radiation dose. The TCD=,,, values of hypoxic irradiations (A) were redrawn from the data published by Suit et al. (14). Bars indicate 95% confidence limits.

that this new formation of blood vessels may contribute to the reoxygenation which occurs during fractionated irradiations. The additive effect of hyperthermia was observed, especially for the MCa, in fractionated irradiations (Fig. 3). Because hyperthermia was given 24 h before fractionated irradiations, the thermal effect was mainly due to the direct heat-cytotoxicity as shown in a previous study ( 17). This heat-cytotoxicity corresponded to approximately 10 Gy for the FSa-II tumors treated with five or ten radiation doses, and 16- 18 Gy for the MCa treated with five daily or 20 bid doses. No significant heat-effect was observed, however, for the FSa-II tumors treated with either 20 daily or bid doses. This may simply reflect a lesser degree of heat sensitivity of the FSa-II tumors. It might be possible Table 4. The effect of preheating

No. of fraction FSa-II tumors Single dose 2 (Ti = 24 h) 5 (Ti = 24 h) IO (Ti = 24 h) 20 bid (Ti = 6 and 18 h) 20 qd (Ti = 24 h) MCa tumors Single dose 5 (Ti = 24 h) 20 bid (Ti = 6 and 18 h) Note: Hyperthermia limit.

that tumor cell repopulation which occurred during the prolonged overall treatment time (22) counteracted heatcytotoxicity for the FSa-II tumors. An unknown factor may be a potential damage on the tumors and normal tissues of repeated clamping which was applied for hypoxic irradiations. In the present study, only one hyperthermia was given before the initiation of irradiations, although the heat treatment has been given once- or twice-a-week in the clinic. It is likely that more additive heat effect could have been obtained if more than one heat dose was given in this study. Two reasons to have limited to one heat dose were: (a) the aim of this study was to investigate the heat effect on the reoxygenation process; and (b) two weekly heat doses given with fractionated irradiations induced

on the thermal enahancement ratio (TER) and therapeutic MCa tumors treated with fractionated irradiations

TER. tumor

0.99 1.03 1.10 1.14 1.06 0.99

(A)

(0.94-I .04) (0.96-1.10) (1.02-1.18) (1.04-1.24) (0.95- 1.17) (0.89-I .09)

1.29 (1.11-1.49) 1.28 (1.14-1.44) 1.28 (1.11-1.47)

gain factor (TGF) for the FSa-II and

TER, foot reaction (BI

1.16 1.19 1.39 1.37 1.19

TGF (A/B)

(1.05-1.28) (1.07-1.13) (1.19-1.62) (1.19-1.55) (1.03-1.37) > 1.34

0.85 0.87 0.79 0.83 0.89

1.16 (1.05-1.28) 1.39 (1.19-1.62) 1.31 (1.15-1.49)

(43.5”C, 45 min) was given 24 hr before single or fractionated

irradiation.

(0.76-0.95) (0.76-0.98) (0.66-0.93) (0.70-0.96) (0.74-I .05) < 0.74

1.11 (0.92-1.32) 0.92 (0.75-l. 11) 0.98 (0.80-l. 18) Parenthesis

in Table: 95% confidence

Heat effect on reoxygenation 0 Y. NISHIMURAAND M. URANO

severe foot reactions that did not allow further treatments in a pilot study. Another question about hyperthermia is its effect on rapid repopulation observed during the radiotherapy of various animal and human tumors ( 14, 22). It has been observed that tumors treated with hyperthermia regress more rapidly than tumors treated with radiation alone. This rapid regression might further accelerate tumor cell repopulation. In the present study, the TCD,O (hypoxia) increased with an increasing number of fractions. This increase is most likely the result of the repair of the sublethal and potentially lethal radiation damages during each intertreatment interval ( 14). A significant observation was that the TCDSO (hypoxia) of the FSa-II tumors treated with 20 daily doses was significantly larger than that of tumors treated with 20 bid doses. The difference between these two TCD+ was 59 Gy, which might have been due to repopulation of clonogenic tumor cells. This means that approximately 6 Gy per day was required to compensate for the tumor cell repopulation when all doses were given under hypoxic conditions. Although the TCDSOs (in air) of the tumors treated with heat plus radiation and those of tumors treated with radiation alone also increased more significantly in 20 daily irradiations compared to 20 bid irradiations, the differences in TCDsO values between daily and bid irradiations were not as large as the differences in the TCDsos (hypoxia). These results suggest that hypoxic tumor cells effectively reoxygenated during fractionated irradiations when all irradiations were given in air with or without hyperthermia. This reoxygenation apparently compensated for the rapid repopulation of tumor cells. Since it is unknown how hyperthermia modifies the response of normal tissues to fractionated irradiations, the present study investigated this response and found that the thermal effect on the foot reaction was significantly greater than that on the response of heat-resistant FSa-II tumors. This suggests that hyperthermia given 24 h before fractionated irradiations significantly enhanced the late foot reaction (Tables 2 and 3). Similar normal tissue sensitization was observed by Overgaard in the mouse skin using a 24-h interval between heat and fractionated irradiations (7). However, he observed no thermal enhancement of the skin reaction to radiation if the treatment interval was prolonged to 72 h. Two possible explanations can be given: One is that the repair halftime of sublethal heat damage may be considerably longer in the mouse normal tissues than in the tumors and this damage may interact with radiation (1). Another is that the blood flow in the skin increases substantially after

147

heating at 43.5”C. Song et al. (10) reported that the blood flow measured 24 and 72 h after heating at 43.5”C for 1 h was more than doubled compared to nonheated skin. This result suggests a possibility that preheating increases the oxygenated cell fraction in the skin and makes the skin more sensitive to subsequent irradiations. Accordingly, this differential heat effect on the tumor and normal tissues may result from the differential vulnerability to heat between blood vessels in the tumor and normal tissues. It should be noted that the human skin has been cooled with appropriate devices in the clinic to avoid therapeutic loss. Although a therapeutic gain has been demonstrated in many studies for hyperthermia combined with a single dose irradiation (6, 12, 13) no significant therapeutic gain was obtained in the present study for either FSa-11or MCa tumors treated with fractionated doses. Stewart (13) reported no therapeutic gain for hyperthermia combined with two or five fractionated doses, although they demonstrated a therapeutic gain for a single radiation dose. On the other hand, Overgaard demonstrated a TGF of 1.4 when hyperthermia was combined sequentially with the last (fifth) radiation dose using his heat-sensitive MCa (7). Our previous study investigated the effect of hyperthermia on five or ten fractionated doses using the FSaII tumor (5). In this study, one heat treatment at 43.5”C for 45 min was combined simultaneously or independently (24-h interval) with the first or last dose. Although greater thermal enhancement was observed for both the tumor response and foot reaction when heat was combined simultaneously than when heat was given independently, no significant therapeutic gain was obtained. Regarding the combined heat and fractionated radiotherapy where a single heat treatment is given prior to radiotherapy, it can be concluded that: (a) on the tumor response, the thermal effect is largely due to direct heatcytotoxicity, whereas, (b) on the normal tissue response, thermal radiosensitization continues for more than 24 h perhaps because of increased blood flow in the normal tissue and, as a result, (c) hyperthermia given prior to radiation sensitizes the normal tissue response to radiation regardless of whether radiation is given in a single dose or fractionated doses, with no significant thermal radiosensitization in the tumor tissue. In this situation, it is difficult to obtain a significant therapeutic gain if the tumor and normal tissues are equally heated or the tumor cells are not strongly heat-sensitive. These results suggest that selective tumor heating or normal tissue cooling is essential to obtain a favorable therapeutic gain in the combined heat and fractionated radiotherapy.

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