??Original Contribution

TIME-TEMPERATURE RELATIONSHIP FOR HYPERTHERMIA INDUCED STOPPAGE OF THE MICROCIRCULATION IN TUMORS

A. E. VAN DEN BERG-BLOK, B.A.’ AND H. S. REINHOLD, M.D.. PH.D.’

‘Radiobiological Institute TNO, Rijswijk and 2Erasmus Unlversit). Rotterdam. The Netherlands

The time-temperature dependence for microcirculation stoppage Has investigated for the Rhabdomyosarcoma BAI I1 2 growing in observation chambers (“sandwich chambers”). The tumor microcirculation could be observed continuously during the treatment, and the condition of the microcirculation was recorded every 15 minutes as “flowing” or “stoppage” By using large numbers of tumors, the SOW stoppage time (STSO) could be derived for the four temperatures investigated: 42”, 42.9, 43’ and 435°C. The respective ST50 values were 226, 152, 101 and 70 minutes. The results can be expressed as a log-linear relationship with a slope value of 0.4551 2 0.03 (SD) per degree centigrade. This value probably does not differ significantly from the “t% for every degree c” rule that has been found for the thermal response of many biological systems.

Tumor microcirculation, Hyperthermia, Time-temperature relationship.

INTRODUCTION

Hyperthermia is presently considered to be a comple- mentary treatment to radiation therapy.5.6.‘2 This is partly because tumors have an inferior microenvironment with an acidic PH~.‘~,” and hypoxia. While this hypoxic and acidic milieu protects the tumor cells against radia- tion ‘I”.~~ there is ample evidence that cells in such an environment are very sensitive to hyperthermia.‘3-‘6 However, not only the tumor cells are affected by heat; microcirculation in tumors also exhibits a thermal sen- sitivity. R-Il.27.2R.30.31.34

A hyperthermic treatment consists of a certain tem- perature applied over a given period of time. For hyper- thermic cell killing. treatment time can be shortened when the treatment temperature is increased. Roughly, in the range of about 43 to 46°C. treatment time can be ap- proximately halved for every degree increase in temper- ature.5,‘R.23.24 The same holds true for experimental tu- mors26 and some normal tissues.‘9.22.24 Determinations of the response of tumor microcirculation to hyperthermia are often performed by physiological methods, which may be the reason why no data are available on the time- temperature relationship for the response of the tumor microcirculation to hyperthermia. The experiments de- scribed here were performed in view of increased interest in the possible contribution of tumor microcirculation damage to the overall treatment effect of hyperthermia. The results indicate that the time-temperature relationship

for this endpoint appears rather similar to that of other systems.

METHODS AND MATERIALS

The undifferentiated Rhabdomyosarcoma BA I 112. a tumor which is isogeneous in the WAG/Rij strain of rats. was used in this study. The “sandwich” tumor is grown in a thin layer of subcutls between a mica base plate and a glass coverslip. In this system, the tumor grows in a sheet-like fashion and its thickness is limited to about 200 microns.” allowing transillumination with obser- vation of the microcirculation by use of a stereomicro- scope during heat treatment. Changes in the microcir- culatory bed can be observed and recorded with a mi- crocamera. The time to produce stoppage of the microcirculatory flow (ST) is used as a parameter. This stoppage is mostly limited to the center of the tumor, leaving a small outer ring of circulating vessels. All tumors were exposed for 3 hours after the exposure temperature of 42”, 42.5”, 43” or 43.5”C was reached. For treatment, the animals were anesthetized with “Hypnorm” (Philips- Duphar) at I ml kg- ’ i.p. and only the skin flap with the “sandwich” chamber was inserted into an isolated perspex box in which heating was performed with warm air.27 The temperature of the air at the start of heating was 37°C and a temperature of 43.5”C was reached within 10 minutes. Temperature control and temperature mea-

Reprint requests to: A. E. van den Berg-Blok, Radiobiological Institute TNO, P.O. Box 58 15, 2280 HV Rijswijk, The Neth- erlands.

Accepted for publication 14 February :3x1

738 Radratlon Oncology 0 Biology 0 Physics May 1984. Volume IO. Number 5

surements were done using thermocouples placed in ther- mal contact with the coverslip next to the tumor by ap- plying adhesive gum. 27.28 The temperature of the ther- mocouples was regulated via a proportional temperature controller. The temperature of the air in the box in the immediate vicinity of the coverslip was usually about O.S”C higher than that of the indicated thermocouple needle temperature. The recording ofchanges in the tumor was made by observation (every 15 minutes) and pho- tography (every hour). This report deals with I22 exper- iments divided over the four temperature levels.

RESULTS

The results of the 3 hr treatment of “sandwich” tumors at various temperatures are shown in Table I. The 50% stoppage time (ST50) with the 95% confidence limit in- tervals was calculated according to the least-squares best fit to the Weibull distribution.4 The ST50 at 42°C rep- resents an extrapolated value. The ST50 values are tab- ulated in Table 2. Fig. 1 shows the same results graphically. The relationship between heating time and treatment temperature is represented by the expression t = a X bT (t = stoppage time, T = temperature, a = constant).

Using the results presented in Fig. I, b was calculated to be 0.455 1 (95% confidence limits 0.425 1 and 0.485 1) and a was 5.1 X 1016. This indicates that the bIological effect of heat treatment remains about the same upon reduction of the treatment temperature by I “C if the exposure time is doubled.

DISCUSSION

The endpoint used in these investigations is a somewhat unusual one in the field of tumor biology. The score is the time required for the microcirculation in the tumors to stop as determined by “continuous” (every 15 minutes) observations. On the following day. the tumors exhibiting

Table I. Stoppage of tumor mlcrocirculatlon as a result of hyperthermia

Number of tumors with Intact Time of

observation (min) 42°C

microcirculation

42.5”C 43°C -

0 50 41 I6 I5 50 41 I6 30 50 41 16 45 50 41 I5 60 50 41 I4 75 50 41 II 90 50 39 IO

105 50 35 7 120 49 33 4 135 47 29 4 150 46 20 3 I65 44 I5 2 180 43 I3 2

43.5”C

I5 I5 15 15 II 3 2 0 0 0 0 0 0

Table 2. Calculated 50% stoppage times (ST50)*

Treatment ST50 95% C.L. No. of temperature (mm) interval tumors

42 226 198-297 50 42.5 I52 148-157 41 43 I01 96- 106 I6 43.5 70 65-75 I5

* ST50 = time necessary to produce in 50% of the treated tumors microcirculation stoppage.

microcirculation stoppage almost inevitably show necrosis in the parts affected. Despite this seemingly subjective endpoint, it is not possible to influence the data. as the ST50 values are calculated retrospectively and only once. The four data points in Fig. I show a remarkably good fit. The slope is 0.4551 S 0.03 (SD). The data do not indicate the presence of the breaking point that has been reported on similar plots by various authors. Such break- ing points, as observed by Baker et al. at 41.5”C for the

ischaemic mouse tail’ and for CHO cells in vitro at 43°C by Bauer and Henle’ and Dikomey,’ are indicative of the operation of more than one temperature dependent mechanism. The absence of an indication of a breaking point in the isoeffect curve depicted in Fig. 1, therefore. suggests that, in the Rhabdomyosarcoma BA 1112 in our “sandwich” chambers, a single mechanism is responsible for the microcirculation stoppage. The same can be in- ferred from the data of Crile as analysed by Suit and Schwayder32 and recently by Leith EI al.*’ Whether a breaking point would emerge in our system with the ap- plication of lower temperatures cannot be presently de- termined, in view of the excessive prolonged exposure times which would be required. This would in itself in- troduce a substantial number of complications and/or artifacts. It should be noted, however, that the wide con- fidence interval of the (extrapolated) 42°C point does not exclude other interpretations. One could argue. for in- stance. that at this moderate temperature the heterogeneity has increased, which could then be interpreted as being due to thermotolerance. However, the array of ST50 val- ues as depicted in Fig. I and which are all derived from the same statistical model are not at all suggestive for such a deviation.

Exposure of the tumors to temperatures higher than 43°C sometimes resulted in a stoppage of the microcir- culation of the tumor bed, i.e., the subcutis in the ob- servation chamber surrounding the tumor. ST50 values for 43 and 43.5”C were 208 and 179 minutes, respectively, using the same method of calculation as with the tumor tissue.4 At temperatures below 43”C, no damage to the subcutis was observed. At temperatures above 43.5”C. the stoppage of the circulation in the tumor bed might influence the microcirculation in the tumor. so that the ST50 is determined by more than one factor. This might explain the breaking points sometimes observed in tumors ,it around 33°C On the othc>r hand. similar breaking

Stoppage of mvXoclrculation in tumors by hqxrthermla 0 A. E. VAU DE% BERG-BLOC; ANO H S. R!-Ihtit)I [I 739

n = 50 n = 41 n = 16 n = 15

- 220 R =axbT 5 .- E a z.5.1 x1016

- 180 0, b = 0.4551 E ._

a, 0) K 140

? *

?+

5: 100

300

260

60 I I 1

42.0 42.5 43.0 43.5

temperature (OC )

Fig. 1. Relationship between treatment temperature and 50% microcirculation stoppage time in Rhabdomyosarcoma BA I I I2 in “sandwich” observation chambers.

points have been observed in vitro ‘.’ Generally, at tem- many in viva normal tissues’ I”’ ” ” 2h as well as for peratures above 43°C. a temperature reduction of 1°C tumors.22.‘6 requires a doubling of the exposure time to obtain an. It can be concluded therefore that microcirculation

isoeffect. in our study, this factor of two apparently ex- stoppage time under hyperthermic treatment follows the tends to 42°C. The same factor has been observed for same pattern as that of other in \‘/\‘o and [II VI[~O systems.

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