Tumor oxygenation in a transplanted rat rhabdomyosarcoma during fractionated irradiation

Int. J. Radiation

Pergamon

Oncology

Biol.

Phys.,

Vol. 32. No. 5, pp. 1391- 1400. 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0360.3016/95 $9.50 + .OO

0360-3016(94)00653-9

l

Biology TUMOR

Original

Contribution

OXYGENATION IN A TRANSPLANTED RAT RHABDOMYOSARCOMA DURING FRACTIONATED IRRADIATION

FRIEDRICH

ZYWIETZ,

PH.D.,*

WOLFRAM

REEKER,

M.D.t

AND EBERHARD

KOCHS,

M.D.’

*Institute of Biophysics and Radiobiology, University Hospital Eppendorf, D20246 Hamburg, Germany, ‘Institute of Anaesthesiology, Technische Universitslt Munchen, D-81 664 Mtinchen, Germany Purpose: To quantify the changes in tumor oxygenation in the course of a fractionated radiation treatment extending over 4 weeks. Methods and Materials: Rhabdomyosarcomas RlH of the rat were irradiated with 60Co-y-rays with a total dose of 60 Gy, given in 20 fractions over 4 weeks. Oxygen partial pressure (PO,) in tumors was measured at weekly intervals using polarographic needle probes in combination with a microprocessor-controlled device (PO*-HistographKIMOC). The pOz measurements were carried out in anesthetized animals under mechanical ventilation and in respiratory and hemodynamic steady state. Tumor pOt values were correlated to the arterial oxygen pressure p.Oz, arterial pC02, and pH determined with a blood gas analyzer. Results: Tumor oxygenation did not change significantly during the 3 weeks of irradiation (up to 45 Gy), from a median p02 of 23 + 2 mmHg in untreated controls to 19 + 4 mmHg after the third week. The decrease of the number of pOr values between 0 and 5 mmHg indicated that an improved oxygenation in the tumors occurred. However, with increasing radiation dose (fourth week, 60 Gy) a significant decrease in tumor oxygenation to a median pOz of 8 + 2 mmHg and a rapid increase in the frequency of p02 values (35 + 4%) between 0 and 5 mmHg was found. Conclusion: Improved oxygenation in rhabdomyosarcomas RlH was only present in the early phase of the fractionated irradiation. Radiation doses above 45 Gy led to a considerable decrease of tumor oxygenation in the later phase of irradiation. Rat rhabdomyosarcoma,

Fractionated

irradiation,

Tumor

INTRODUCTION

oxygenation,

pOz measurements.

fractionated treatment (2, 20, 21). The process of reoxygenation has until now not been well understood, but it was assumedto depend on radiation-induced changes in the tumor vasculature and in tumor blood flow, which is closely related to tumor tissue oxygenation (18, 41). Radiation-induced tumor shrinkage due to death of parenchymal tumor cells may reduce the intratumoral pressure (5) and allow previously nonfunctional (or compressed) capillaries to be reopened, resulting in an increasedtumor blood flow. Tumor shrinkage may also reduce the distance between capillaries and viable tumor cells and, thus, increase tumor oxygenation. However, relatively large dosesof radiation may also damagetumor capillaries (48) reduce tumor blood flow (27), and, consequently, tumor oxygenation. Most of the experimental studies on reoxygenation of tumors were carried out with single or fractionated doses of x-rays not exceeding 30 Gy (2, 9, 20, 21, 33). Clinical

It is generally accepted that tumor hypoxia is of great importance in radiation therapy and may be responsible for the failure of conventional x-ray treatment (1, 8, 10, 22, 36). Studies on various rodent tumors (29, 30, 43) human tumor xenografts (32, 34, 39), and human tumors (25,41,45) have shown that a certain proportion of malignant cells is in a hypoxic state and, therefore, radioresistant. Tumor hypoxia originates from structural (6, 26) and functional abnormalities (15, 41, 44) in the tumor microcirculation resulting in local areasof the tissue with inadequate oxygen supply, nutrient delivery, and cellular waste clearance. It has been proposed for some tumors that the radioresistant hypoxic cells are the source of local recurrencies following radiotherapy (8, 10,22). It is often suggested that tumors, which are controlled locally by radiotherapy, reoxygenate efficiently during the course of

Presented in part at the Joint Meeting of the European Societies for Radiation Biology ESRB and Hyperthermic Oncology ESHO, Amsterdam, The Netherlands, June l-4, 1994. Reprint requests to: Friedrich Zywietz, Institute of Biophysics and Radiobiology, Martinistr. 52, D20246 Hamburg, Germany.

Acknowledgements-The authors would like to express their thanks to S. Scheibner, I. Stark, I. Dwenger, and D. Droese for their expert technical assistance. Accepted for publication 16 December 1994. 1391

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l

Tumor

Physics

RlH

F, 02 : 0.33 N .12 n I2288 median

median

Tumor

~02

a 23 mmlig

pO2 s 1OmmHg

~02 (mmHg)

Fig. 1. Pooled ~0, histograms of RlH tumors (1.8 ? 0.3 cm’) in rats (258 5 12 g) under artificial ventilation at an inspiratory oxygen fraction (F,O,) of 0.21 or 0.33, respectively. N: number of tumors, n: number of pOz readings, broken line: median ~0,.

studies provide conflicting data on whether there are significant changes in tumor oxygenation during or in the early phase of radiotherapy (3, 4, 7). The aim of this study was to measure the oxygenation status in a tumor model (rhabdomyosarcoma RlH of the rat) in the course of a fractionated radiation of 4 weeks with a clinically relevant total dose of 60 Gy. Tumor oxygenation was measured with pOZ microelectrodes in anesthetized animals under well-defined respiratory and hemodynamic parameters.

METHODS

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32. Number

5, 1995

Irradiation Tumors were irradiated with “‘Co-y-rays at a therapy unit (Gammatron 2, Siemens, Germany) with a total dose of 60 Gy, given in 20 fractions of 3 Gy each, five times a week for 4 weeks. The dose rate was 1.00 Gy/min and the field size 3 x 3 cm2. Irradiation was carried out in air-breathing animals under anesthesia, using a combination of 50 mg/kg (body weight) ketamine (Ketavep, Parke-Davis, Germany) and 6 mg/kg (body weight) xylazine (Rompun@, Bayer, Germany), which were injected (intramuscular) in the left thigh. For irradiation, the animals were placed on a small temperature-controlled treatment table kept at 37.O”C to prevent a decrease of the core temperature during anesthesia. Measurement of tumor tissue oxygenation Tumor tissue oxygenation (PO,) was measuredpolarographically using needle electrodes with stainless steel shafts (350 pm outer diameter, 12 pm diameter of the cathode) in combination with a microprocessor-controlled PO,-device (p02-Histograph/KIMOC, Eppendorf-Netheler-Hinz, Hamburg, Germany). This method has been used for ~0, measurementsin various experimental and human tumors (14, 19, 24, 37, 43, 46, 47). The probes were calibrated before and after the p02 measurements using a buffered 0.9% saline solution equilibrated with air or with 100% nitrogen at room temperature (22 2 2°C). In vivo, an Ag/AgCl electrode (Red dot@@, 3M, USA, 7.0 mm in dia.) served as anode. Tumor ~0, values were calculated from the measured current, the air pressure, and the temperature of the tumor tissue (33.0 -+ 0.5”C).

AND MATERIALS

Experimental tumor line The tumor used for these studies was the superficially growing rhabdomyosarcoma RlH of the rat. This is an undifferentiated tumor with necrotic foci, which is enclosed by a fibrous capsule (48). Tumors were serially transplanted into the flank of male WAG/Rij/H albino rats between 12- 13 weeks of age (200 2 10 g). Animals were fed a standard diet (altromin@‘1324), and acidified vitamin C-fortified water was given ad lib. Details of the tumor/host system have been described previously (17). Forty-one tumors with a volume of 1.8 ? 0.3 cm3 and a diameter of 1.5 cm at days 21 to 26 after implantation (mean body weight 258 + 12 g) were selected for the study. Tumor volume (V,) was determined by caliper measurementsof three orthogonal diameters a, b, and c, and was calculated assuming an ellipsoid morphology V,= (rr/6(axbxc). The studies were approved by the Ethics Committee of the Hamburg Ministry of Health, and were conducted according to the guidelines for good laboratory practice including supervision according to the German Law for Animal Protection from 1987.

60

0.21 lnspiratory

0.33 oxygen fraction

Fig. ‘2. Oxygen partial pressure (pOJ in RlH tumors and in arterial blood of WAG/Rij rats (2.58 2 12 g) at an F,O, of 0.21 and 0.33, respectively (mean 5 SD).

Tumor pO1 under irradiation 0 Tumor

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distal side. Depending on tumor size, about 100 to 200 PO:, measurements were recorded in each tumor using 812 radial tracks. The recorded data were transformed into p02 histograms (PO, frequency distributions) with class widths of

RlH

2.5 mtnHg using an online computing system. Histograms

(61 \ c

> z

E 2

\

\

0

0 \ o\

os-

(71

0

\

showing negative p02 values were excluded from data analysis. Measurements of tumor pOZ were evaluated only when the arterial oxygen partial pressure p,O:! was in the range of 120 + 16 mmHg, and the acid base data was in the range of healthy rats (42). Histograms of pooled data were characterized by the mean pOZ, the median pOZ, the p0, range between the 10th and 90th percentile ApO,( lO/ 90), and by the relative frequency of p0, values falling in the two lowest classes (O-5 mmHg). Statistical analysis was performed using the Mann-Whitney test (U-test).

0 0 0

\

\

\

0

‘e 0,

\

\

0

RT’lllll 0.1 .

I 0 Weeks

lllll I 1 after

lllll start

IIIII 00

I

I

1

2

3

4

of t reatmtnt

Fig. 3. Changesin individual tumor volumesduringthe fractionatedirradiation with a total doseof 60 Gy, given in 20 fractions for 4 weeks.The numberof tumorsusedfor p02 measurements is indicated in parentheses.The horizontal line representsthe averagetumor volume. RT: eachbar denotesone fraction of 3 Gy each. The p0, measurementswere carried out in anesthetized animals under controlled mechanical ventilation and monitoring of hemodynamic parameters to minimize the

effects of anesthesia. Anesthesia was performed by an intravenous infusion of 0.05-O-l mg/kg b.wt./h fentanyl (Fentanylm, Janssen, Germany) and 0.9- 1.8 mg/kg b.wt./ h midazolam (Dormicum@@, Hoffmann-LaRoche, Germany) into the left femoral vein (perfusor secura FIYO, Braun, Melsungen, Germany). By regulting respiration rate and tidal volume at the animal ventilator (Braun, Melsungen, Germany), the end-tidal carbon dioxide of the rat was maintained at a constant level of 35 + 2 mmHg. Mean arterial blood pressure (MABP) was kept in the range of 115 -+ 20 mmHg and was monitored by a catheter in the left femoral artery and a transducer (UM3, Draeger, Germany). The same catheter was used to sample arterial blood (0.25 ml) for blwd gas analysis (ABL 2, Radiometer, Copenhagen, DK). The core temperature was kept constant at 37.0 2 0.3”C by using a temperaturecontrolled infrared irradiator. The p0, probe was automatically moved through the RlH tumor by a micromanipulator in steps of 0.7 mm (a forward movement of 1.0 mm was immediately followed by a backward movement of 0.3 mm to minimize compression effects of the probe). The PO:! measurements were performed under four defined directions: from above, from two sides under ?45 degrees, and from the

RESULTS The influence of the inspiratory oxygen fraction (FiO,) on the oxygenation status of the RlH tumor was studied first. Figure 1 shows pooled histograms of tumors of rats ventilated at a FiO, of 21% or 33% of oxygen, respectively. The histogram obtained at FiO, = 0.21 shows a rather narrow profile of pOZ values (Ap(10/90) = 28 mmHg) and a marked shift to the left. The median pOZ value was 10 + 4 mmHg (-+SEM) (mean 14 + 4 mmHg (-tSEM)). A relatively high frequency of recorded values (23%) fell into the range from 0 to 5 mmHg. For F,O, = 0.33, however, the distribution of the p0, values was much broader (Ap(10/90) = 52 mmHg) in comparison to FiOz = 0.21; maximum values reached 88 mmHg. The median p02 was 23 + 2 mmHg (mean PO,! 28 + 2 mmHg (*SEM)). The frequency of values between 0 and 5 mmHg was only 4%. Figure 2 demonstrates that the p0, values of the RlH tumors depend on the inspiratory oxygen content and correlate strongly with the arterial pOZ of the animals. For FiOs = 0.21, the arterial ~02 (67 + 7 mmHg, mean & SEM) and the median tumor pOZ ( 10 ? 4 mmHg, median f SEM) were significantly lower (p < 0.05) as compared to the arterial paOz (105 + 4 mmHg) and the tumor p02 (23 2 2 mmHg) measured for FiOz = 0.33. Because the arterial p0, of air-breathing Sprague-Dawley rats during normoxia is in the range of 98 2 29 mmHg (mean +

SD) (42), the following studies were performed at an FiOz of 0.33 to reduce the effects of anesthesia as much as possible. Figure 3 shows the volumes of the individual tumors under the radiation treatment used for the pO* measurements. After the first week of treatment, tumor growth was attenuated and tumor volume remained approximately unchanged as compared to untreated controls. In the course of fractionated irradiation, tumor volume shrank continuously and the variation of tumor volume increased. Figure 4 shows the pOZ histograms recorded at weekly

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Tumor

RlH

6OGylwcckC H., n = 990 malO” po2 i *mm”g

‘. 0

20

Tumor

LO

60

80

100

~02 (mmtig)

Fig. 4. Pooledtumor pOz histogramsrecordedbefore and at weekly intervals during the fractionated irradiation with a total doseof 60 Gy for 4 weeks.N: numberof tumors,n: numberof pOz readings,broken line: median PO?

intervals during the fractionated irradiation for 4 weeks. The pooled histogram of 12 untreated controls showed a broad distribution of ~0, values with a median pOz of 23 ZL 2 mmHg (E’ig. 1). The pOz distributions after the first and second week of treatment (30 Gy/lO f) did not change significantly. After the third (45 Gy/15 f) and especially after the fourth week of treatment (60 Gy/20 f), the following observations were made: there was a marked reduction in the range of pOz values, a progressive shift of the histograms to lower p0, values, and a steady increase in the frequency of p02 values between 0 and 5 mmHg. The median PO,?values decreased only slightly during the first 3 weeks of radiation treatment, from 23 t 2 mmHg in untreated controls to 19 t 4 mmHg. The lower value of 16 ? 2 mmHg obtained after the second week was determined from a high number of measurementsin the vicinity of 10 mmHg. After the fourth week of treatment, and following a total dose of 60 Gy, a significant decreasein tumor oxygenation was observed. Most of the recorded p0, values were in the range below 20 mmHg, the median p0, was 8 _C2 mmHg, and 35% of the values recorded were between O-5 mmHg. These results demonstrate that the oxygen status of the tumor had greatly

deteriorated. Further studies on RlH tumors treated with higher number of fractions and doses(up to 75 Gy) had been planned, but could not be performed, because an adequatenumber of pOz readings in tumors with volumes less than 0.1 cm3 could not be obtained. In Fig. 5, the median p02 values of the RlH tumors and the corresponding arterial paOz of WAG/Rij rats as a function of time after the start of radiation treatment are presented. Because all arterial oxygen values (p,O,) are in the range of untreated controls (Table l), the decrease in tumor p02, especially the significant decrease after a total dose of 4.5 Gy, is not caused by a lower arterial oxygen tension, but is the result of a radiationinduced damage to the tumor vasculature. This is further emphasized by the changesin the frequency of pOz values below 5 mmHg during the 4 weeks of treatment (Fig. 6). Up to the second week of irradiation, a decreaseof this fraction was found followed by a steep increase in the last 2 weeks of irradiation. This demonstrates that after radiation dosesof 45 Gy and more, the oxygenation status of the tumors had deteriorated. The range of the pOz distribution expressed by Ap(10/90) started to decrease after the first week until the fourth week of irradiation.

Tumor

~0,

under

WAG/Rij

G ; 140 5 6100 2

t

0

0

1

2

irradiation

rat 0

0 0

3

LO

4 RlH

tumor

r

0 F.

ZYWIETZ

1395

et al.

The values of the arterial pa02, the arterial pCOp, and the arterial oxygen saturation (~0~) demonstrate that during the pOZ measurements,the animals were kept in a relatively constant metabolic and respiratoric state, which is expressed by the constant arterial blood pH of 7.40 + 0.04 (mean -C SD) during the 5 weeks of measurements. The hemoglobin values (17.5 2 0.5 g/dl) remained constant during that time indicating that the Co-y-irradiation did not induce anemia. It must be mentioned here that the hemoglobin values are calculated values for human blood and are not corrected for rat blood (42).

DISCUSSION

OL

’

0 Weeks

I

1

1

I

1

2

3

L

after

start

of treatment

Fig. 5. Changes of median p02 values (t SEM) in RlH tumors

during fractionatedirradiation for 4 weeks(lower section)and correspondingarterial paOzvalues(upper section).Numbersin parenthesesindicate the number of tumors studied. Mean of: SEM.

The reduction of the values at the 90th percentile is also an indication for a decrease in tumor oxygenation (data not shown). Table 2 summarizes the number of tumors, the average tumor volume, the number of pOZ readings, the range of the p0, distribution (Ap(10/90), the mean and median p0, values, and the frequency of values in the range O5 mmHg. The decrease of the mean and median p0, values with radiation dose as well as the increase in the frequency of pOZ values below 5 mmHg clearly demonstrate that during a fractionated radiation treatment, radiobiological hypoxia increases substantially in R 1H tumors with radiation doses of more than 45 Gy. Table 1 presents the corresponding arterial blood gas values obtained immediately after the pOZ measurements.

The main purpose of this study was to determine the oxygenation status in a tumor during a fractionated irradiation for 4 weeks with a relative high total dose. Because it is generally known that anesthesiacan influence tumor blood flow (16, 28), special care was taken to minimize the effects of anesthesia and to correlate tumor p0, to the arterial paOZof the anesthetized animals. This could be achieved by an artificial respiration of the animal at a constant Fi02, monitoring of end-tidal carbon dioxide and mean arterial blood pressure, and by a controlled infusion anesthesiawith fentanyl and midazolam. It is known that these drugs have only minor influence on circulatory parameters as compared to pentobarbital(16, 28). With this experimental setup, an arterial oxygen pressure (p,O,) of 105 ? 14 (*SD) mmHg was achieved, which is close to the normal range of 98 -+ 29 mmHg in healthy conscious rats (42). For the experiments, tumors with very similar initial volumes (1.8 ? 0.3 cm’) were selected because studies by Vaupel (38) have shown that tumor p0, depends strongly on tumor volume. This is also true for the RlH tumor where, at volumes of 1.4, 1.8, and 4.3 cm’, the median p0, values decreasedfrom 38 to 23 and to 6 mmHg, respectively (31). According to the method of the p02 measurement, the probe with a diameter of 350 pm was moved forward from one measuring site to the next in steps of 700 pm in length. This is necessary to leave the previously compressed tissue area with each step (35). Local pO* in tumors was measured 1.4 s after the backward motion of

Table 1. Blood gasvalues in the arterial blood of WAG/Rij rats determinedimmediatelyafter the ~0, measurements (Table 2) of irradiatedrhabdomyosarcomas RlH Radiation dose/time Control 15 Gy/l week 30 Gyl2 weeks 45 Gyl3 weeks 60 Gy/4 weeks

l-b* [g/d11 18.3 2 0.3? 17.0 17.7 17.0 17.6

2 k -t -c

1.2 0.3 0.5 0.4

* Values not correctedfor rat blood. ’ Mean + SEM.

7.36 7.35 7.44 7.42 7.41

+ -+ + + e

PCO, [nun&l

bi!z2g,

PH 0.02+ 0.02 0.02 0.02 0.02

105 t 119-c 136 -e 103 t 1342

4’ 1 10 14 7

38 38 32 34 31

+ + 2 5 +

2’ 3 2 2 2

so2 97 97 98 97 98

2 1+ + 1

+ 1 2 1 + 1

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Table 2. Oxygen partial pressure (PO,) measurements in rhabdomyosarcomas RlH of the rat during a fractionated irradiation with 60 Gy applied in 20 fractions for 4 weeks Radiation dose/time Control 15 Gy/l 30 Gyf2 45 Gyf3 60 Gyl4

Number of tumors

Average tumor volume, cm’

Number of readings

12 6 7 6 7

1.8 2 0.1* 1.6 + 0.1 1.0 2 0.1 0.5 i 0.1 0.3 t 0.1

2288 1080 1280 841 880

week weeks weeks weeks

APO, (10/90) (mm&9 52 55 35 32 11

+ -c 2 k -c

4* 3 3 6: 1’

~0, (mmHg) Mean 28 28 21 22 8

? k r + +

2* 4 2 4’ 2f

Median 23 21 16 19 8

+ ” ? 2 k

2* 5 2 4t 2’

pO*-values below 5 mmHg (%) 4 -+ 2* 6+4 221 12 k 3t 35 2 4’

* Mean + SEM. ’ Statistically different @ < 0.05). the probe in less than 40 ms to circumvent any significant effect on the recorded local tissue pOz, e.g., compression of vessels in the vicinity of the needle electrode and 0, consumption of the cathode. Tissue injury due to the electrode movement is rather small and has no effect on the p02 values recorded (35). The pOz values recorded with the needle electrode are strongly weighted towards a thin layer of tissue (some pm) near the surface of the cathode (1.6 x lop4 mm2). A volume of tissue measured, however, cannot be defined, because geometrical and biological alterations of the punctured canal during the backwards drive of the probe are unknown (Fleckenstein, personal communication). When comparing the pOz value obtained for the RlH tumor with pOz values of other untreated rodent tumors and of human tumor xenografts determined with the same method, a great variability between mean or median p02 values exists (Table 3). This may be based in part on the type of the tumor, its size, and anatomical site (leg, hip, foot), but also on the experimental conditions at which p0, measurements had been carried out. All measure60

H\\ #-) 9 1\

\

Weeks

after

start

of irradiation

Fig. 6. Changes in frequency of ~0, values below 5 mtnHg (closed circles) and in p0, range between the 10th and 90th percentile ApO?( 10/90), (open squares) during fractionated irradiation for 4 weeks. Mean t SEM.

ments with the exception of one (43) were performed in animals under general anesthesia with different doses of pentobarbital and unknown time intervals between anesthesia and ~0, measurement. An influence of the anesthetic drug on tumor oxygenation has been demonstrated in studies on mouse fibrosarcoma FSaII (19, 30, 43). For tumors of nearly the same size (0.13 cm’), the median pOZ value is about 17% lower when measured in tumors of anesthetized mice in comparison to unanesthetized controls. Therefore, it is necessary to correlate tumor p02 with measurements of the p0, in arterial blood and/or with tumor blood flow; otherwise, a comparison of the results of different studies is difficult to perform. The median ~0, obtained for the RlH tumor (23 + 2 rnmHg) is similar to the median p02 of a human lung tumor xenograft on a rat (23 rnmHg, 2.5 g) (18) and to the mean p0, of a DS-sarcoma of the rat (23 mmHg, 1.8 g) (38), bearing in mind the differences of the tumors, of the rat strains, and of the anesthetics used. There are only a few reports in the literature describing p0, measurements in irradiated experimental and human tumors. Vaupel et al. (40) noted an improvement in tumor tissue oxygenation in a mouse mammary carcinoma 72 to 74 h after a single dose of 60 Gy, resulting in a marked reduction of p0, values below 2.5 mmHg, indicating reoxygenation of the tumor tissue. Koutcher et al. (23) also found an increase in p02 3 to 4 days after irradiation of mouse mammary carcinomas with single doses of 32 and 65 Gy and a decrease in the frequency of p07_ values below 2.5 mmHg. Both studies were carried out with single large doses and short times after radiation. Our studies on the RlH tumor have shown that the median pO1_does not change significantly during the first 3 weeks of the fractionated irradiation (Table 2). After 2 weeks of irradiation (total dose 30 Gy), the percentage of p02 readings between 0 and 5 mmHg is reduced, indicating an improved oxygenation in the RlH tumor. Because this fraction increased rapidly after a radiation dose of 45 Gy and reached a value of 35 t 4% after 60 Gy, the improved tumor oxygenation was only transient and radiation doses of more than 45 Gy led to tumor hypoxia. It has further been shown that tumor hypoxia determined by electrode measurement of p02 can be correlated

Tumor

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Table 3. Mean and median pO1 values determined in various rodent tumors and human tumor xenografts with the Eppendorf PO,-device and corresponding arterial p02 values and/or tumor blood flow Tumor type Mouse tumors Fibrosarcoma FSaII FibrosarcomaFSaII

Weight volume diam. 0.05 cm’ 0.15 cm? 0.56 cm’ 0.13 cm’ 0.35 cm’

Mammary adenocarcinoma FibrosarcomaFSaII

0.13 0.35 0.13

Lewis lung carcinoma CarcinomaSCCVII

1.4 cm diam. 3.1 cm diam. 0.2 cm7 2.5 cm’ 0.8 cm’ 1.4 cm?

SarcomaRIFl Rat tumors NeurinomaTV 1A Yoshida sarcoma Yoshida sarcoma DS-sarcoma

cm? 61113 cm3

Anesthesia mg/kg b.w. no no Pentogb. 50 50

POZ (mmHg) Mean Median 44 20 8 -

40 18 6 15 6 4-5 2 -14

50 60 50 50

Blood flow (ml/g/min)

-

-

Reference Vaupel et al. (1989)

Kallinowski et al. (1990)

-

Okunieff et al. -

6.8 1.4 36 2 14 8+ 2 2Ok 6

ll?

Arterial ~0, (mmHg)

(1991)

Lartigau et al. (1992)

Terris et al.

-

(1992)

5

Pentobarb. 4.5 cm’ 3.3 cm3 1 cm’ 4 cm’ 0.6 g 1.4 g 2.3 g

Rhabdomyosarcoma 1.8 % 0.3 cm’ RlH Humantumor xenograftson mice Human squamous 0.13 cm’ cell carcinoma 0.35 cm3 Humanglioma 0.13 cm3 0.35 cm’ Human melanoma 0.8 cm diam. Humancolonic 0.8 cm diam. adenocarcinoma Humanbreast 0.19 cm7 carcinoma Human sarcoma 0.18 cm’ Humantumor xenograftson rats Humanmedullary 2.1 k 0.9 g breastcarcinoma 6.0 + 1.0 g Human squamous 2.4 ? 0.5 g cell carcinoma Humanmedullary 2.5 g breastcarcinoma Humansquamous 2.5 g breastcarcinoma Humanlung cancer 2.5 g Humansarcoma 5.3 cm’

30 30 30 40

Fentanyl and midazolam

8.2 4.6 6 4 392 255 19+ 28k

Volk et al.

93 k 2 93 2 2 -

(1993)

3 2 2 2

Pentobarb. 50 50

3.7 3.7

23 f

14 7 18 19

2

-

1.08 2 0.02

-

0.73 0.52

k 0.05 k 0.08

JIhde et al. (1993) Vaupel et al. (1993)

This paper

105 2 4

-

-

Kallinowski et al. (1990)

60 60

9.4 11.4

60

6

6

-

60

7

2

-

3 2 4

80 t 2 80 k 2 83 t 2

2 0.02 0.05 0.20 t 0.03

Vaupel et al.

8 9

30-35

2

88 t

2

0.17

+ 0.02

Kallinowski et al.

30-35

4

99 t

3

0.10

? 0.02

0.55

5 0.05

-

Lartigau et al. -

(1992)

Wiedemannet al. (1992)

Pentobarb. 30 30

0.14

(1987)

(1989)

30-35 40

12

23

81

-

t 1

Roszinskiet al. (1991)

with the radiobiological hypoxic fraction in tumors. Studies by Vaupel and his colleagues ( 19, 39, 40, 43) have strongly suggestedthat electrode measurementsof tumor oxygenation might be a good indicator of radiobiological tumor hypoxia. p0, measurementsin human tumors indicated that a low level of tumor oxygenation before treatment correlates with a poor responseto radiotherapy (8,

10, 22). Horsman et al. ( 13) studied extensively the relationship between radiobiological hypoxia and electrode measurementsin various mouse tumors. In a C3H mouse mammary carcinoma an excellent correlation between the percentage of p0, values below 5 mmHg and the radiobiological hypoxic fraction determined from the direct analysis of local tumor control data was found (12). In

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other murine tumors, however, only obvious trends between the p0, data were observed, in that as tumor hypoxia increased, the mean and median p0, decreased, while the percentage of values below 5 mmHg increased (13). This was also observed during the fractionated irradiation of the RlH tumors, but a correlation between the pOZ measurement (percentage of values below 5 mmHg) and the radiobiological hypoxic fraction has not been studied yet. The changes in tumor oxygenation are in agreement with our morphological findings in the sametumor model where under identical irradiation, a reduction of the vasculature in the tumor periphery, as well as in the tumor center was observed (48). Ultrastructural studieson tumor capillaries (49) have shown further that the capillaries preserved their endothelial lining up to a radiation dose of 45 Gy. At total doses of 60 and 75 Gy, progressive damage of tumor capillaries was found. The morphological changes might have adverse effects on tumor blood flow and, thus, oxygenation. Although the morphological status of the tumor vasculature during irradiation cannot be compared with the functional state of tumor blood vessels, the histological studies can give additional information about the injuries of tumor blood vessels during irradiation. Clinical studies on tumor oxygenation during radiation therapy are very scarce. Bergsjo and Evans (4) reported that during the early phase of external radiation (775 lU 5fr/6 days) and 1700 R/11 fr/15 days), a slight rise in the average oxygenation of tumors of the uterine cervix occurred. Quantitative p0, measurementsduring a conventional radiotherapy in various carcinomas of the oropharynx, breast, lung, and bladder, and in the tumor bed, were performed for the first time by Badib and Webster (3). At weekly intervals, a progressive increase in tumor oxygenation during irradiation was observed, which reached its maximum at the end of radiation. The analysis of the tumor pOz values measured polarographically in various tumors shows that the range of the pOZ values is

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very narrow (8-36 mmHg), and that no values below 5 mmHg have been recorded. In comparison to pOZ measurements in other human tumors (7, 11, 45), much broader pOZ distributions have been obtained. Fleckenstein et al. (7) repeately measured~0, values in a metastasisof a squamouscell carcinoma of a patient during the course of radiation therapy. Two weeks after the start of therapy (32.5 Gy), a reduction of the mean pOZ value within the entire tumor massin comparison to the value before treatment was measured. After 4 weeks of treatment (70 Gy), the mean p0, was slightly increased and locally steep p0, gradients in the center and periphery of the tumor were observed. He concluded that the reappearance of pOZgradients is a sign of recovery of the oxidative metabolism of the tissue. The results obtained for the human tumors during radiation treatment (3, 4, 7) are not in agreement with the changes of oxygenation found in the RlH tumor. This is probably due to differences in the tumorbiology, especially to differences in the vascularization of human and transplanted rodent tumors. Whereas in human tumors a normal host vasculature exists that gives rise to neovascularization in the growing tumor, transplanted animal tumors are mainly characterized by an encapsulatedgrowth, absenceof residual blood vessels,and by tumor capillaries with seriesof structural and functional abnormalities that influence tumor blood flow and, thus, tumor oxygenation (41). It, therefore, appears that the different behavior of tumor oxygenation between human and transplanted tumors is due to differences in the radiation injuries of the tumor vascularization. The p0, studies on the RlH tumor clearly have demonstrated that reoxygenation is not a reversible process during a fractionated radiation treatment, but is a dose-dependent parameter. Therefore, it is probably not valid to extrapolate the results of tumor oxygenation from relatively low doses to high doses of radiation. Further investigations with clinical relevant schedules and doses are needed to prove this finding.

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