0360-3016/91 $3.00 + .M Copyright 0 1991 Pergamon Press plc

Inl. J. Radiarmn 0ncolog.v Biol Phys.. Vol. 20. pp. 1273-1280 Pnnted ,n the U.S.A. All rights reserved.

??Original Contribution

EFFECTS OF HYPERTHERMIA AND/OR HYPERGLYCEMIA ON pH AND pOz IN WELL OXYGENATED XENOTRANSPLANTED HUMAN SARCOMA S. ROSZINSKI,

M.D.,* G. WIEDEMANN, M.D.,+ S. Z. JIANG, M.D.,* G. BARETTON, TH. WAGNER, M.D.+ AND C. WEISS, M.D., PH.D. (LoND.)*

M.D.,$

Medical University of Liibeck, D-2400 Liibeck, Federal Republic of Germany

The heat-induced interdependent changes of tumor blood flow, pOz, and pH decisively influence the therapeutic effect of hyperthermia (HT). This fact has induced us to determine simultaneously the frequency distribution of local p02 values and the intratumoral pH in a xenotransplanted human sarcoma cell line at a normal blood glucose level and under hyperglycemic conditions before, during, and after HT. Two groups, one of 10 and one of 9 congenitally athymic nude rats with a subcutaneously implanted S117 human sarcoma into the right hind paw (mean tumor volume 5.3 cm3) were treated with local waterbath HT (tumor temperature 43”C, 1 hr) alone or in combination with i.p. glucose injections (6 g/kg, 2 hr before the onset of HT). Tumor oxygenation remained improved throughout HT. Tumor pH did not decrease during HT. Hyperglycemia alone elicited a decrease of intratumoral pH and p02, probably mainly due to hemoconcentration. The additional warming of the tumors (43°C) during hyperglycaemia did not further decrease p02 and pH. Silver stained sections of the tumors showed only a few very small necrotic areas, even in tumors of volumes up to 8 cm3. Our results indicate a well oxygenated tumor. In contrast to most tumors studied so far, hyperthermia in this tumor induces not only an initial increase of oxygenation but a lasting elevation of mean tumor pot for the duration of HT (up to 60 min). Tumor, Human sarcoma xenografts, Oxygen partial pressure, p02, pH, Hyperthermia, graphic needle electrode.

INTRODUCI’ION

Hyperglycaemia,

Polaro-

imals carrying implanted tumors selectively reduces tumor blood flow and extracellular tumor pH (11). Hyperglycemia enhanced the response of rodent tumors to hyperthermia ( 19, 27). In the study reported here, an attempt was made to increase further the hyperthermia-induced intratumoral drop of pH by the application of glucose eliciting hyperglycemia. Before, during, and after hyperthermia and/or hyperglycemia the frequency distribution of intratumoral pOz values and the intratumoral pH were determined in human tumor xenografts on nude rats.

Results of studies carried out on cultured tumor cells indicate that hyperthermia can suppress tumor growth by inhibiting the synthesis of DNA and proteins (14,29,32). It was also shown that in vitro the sensitivity of tumor cells against hyperthermia increases with falling pH and p02 (4,7- 10, 15). In vivo, the functionally interdependent parameters of blood perfusion, tissue pH, tissue pOz , and tissue temperature are major determinants of the “milieu interne,” which, in turn, decisively influences tumor growth (17). Detailed information on these parameters and their changes during hyperthermia is a prerequisite for the effective therapeutic use of hyperthermia. Most data obtained from rodent tumor systems indicate that hyperthermia is followed by a shut-down of the tumor perfusion rate and a drop of intratumoral pH (22). It has been demonstrated that administration of glucose to an-

METHODS

AND MATERIALS

Animals and tumors Six- to 8-week-old genetically athymic Rowett nude rats* of 220-280 g b.w. were used. The animals were kept on normal chow with free access to drinking water. Point

Presented in part at the 10th Conference of the European Society for Hyperthermic Oncology, Amsterdam, The Netherlands, September 2 l-23, 1989. * Department of Physiology. + Department of Medicine. * Department of Pathology. Reprint requests to: Dr. S. Roszinski, Institut ftir Physiologie, Med. Universittit Liibeck, Ratzeburger Allee 160, D-2400 Liibeck, F.R.G.

Supported by grants from the “Gesellschaft der Freunde und Fijrderer der Medizinischen UniversitPt zu Liibeck e.V.“, Eppendorf-Netheler-Hinz GmbH., Hamburg, F.R.G. and by the “Werner und Klara Kreitz-Stiftung Kiel”, Miinkeberg, F.R.G. Accepted for publication 14 December 1990. * Mollegaard, Denmark.

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two to 0.3 ml of a suspension of washed cells from the human sarcoma S 117+were subcutaneously injected into the rats’ necks. Grown tumors were excised and cut into small pieces of a volume of approximately l-2 mm3. One piece was then implanted subcutaneously on the dorsal surface of the right hind paw of each rat. Measurements of intratumoral pH and pOZ were performed on 45- to 60-day-old tumors of a mean volume of 5.3 cm3 (k2.6). During the intratumoral measurements the animals were anesthetized by intraperitoneal injection of a pentobarbital solution (4 mg pentobarbital/lOO g b.w.). Tumor volumes were calculated on the basis of an ellipsoid approximation using the three orthogonal diameters (V = II/6 X d, X dZ X d3). Hyperthermia The anesthetized animals were fixed on a flat perspex holder, and the tumor-carrying hind paw was immersed to a depth of approximately 1 cm in a mechanically agitated, thermostatically controlled water bath* with a temperature accuracy of +O. 1“C. Blood glucose determination In a separate set of experiments, glucose (6 g/kg b.w.) was injected i.p. into 10 tumor-free rats. Blood samples (50 ~1 microsampling pipets) were drawn from the tail vein before and 80 and 150 min after the glucose injection. Blood glucose concentrations were determined spectrophotometrically using a glucose oxidase test kit.§ Measurement of temperature For the measurement of tumor temperature a chromelalumel microthermocouple** with a diameter of 250 pm was inserted into the tumor center. The signal from the thermocouple was fed into an electronically ice point compensated digital thermometer circuit.++ Placing the temperature-sensitive tip of the thermoprobe at different depths in the tumor tissue from the periphery to the center, no temperature differences exceeding 0.3”C were observed. During the periods of hyperthermia, temperature at the center ofthe tumors was held at 43°C (kO.3). Rectal temperature was continuously measured with another thermocouple and, by adjusting the distance of an infrared lamp, kept at 37°C (kO.5). Measurement of pH Intratumoral pH was measured with a needle type single barrel glass pH electrode** of 900 pm tip diameter. The electrode signal was fed into a combination digital pH and temperature meter.@ Prior to and after each single intratumoral pH measurement, the probe was two point +Krebsforschungszentrum Heidelberg, F.R.G. * Julabo V; Fa. Julabo, Seelbach, F.R.G. 5Glut-DH-Method; Fa. Merck, Darmstadt, F.R.G. ** Type K l/2, Philips, F.R.G. ++Type 871 A, Keithley, USA.

June 199 1, Volume 20, Number 6

calibrated at pH 5.5 and 7.0. For each intratumoral measurement the probe was freshly inserted into uninjured tissue. Stable readings could be obtained after 60 sec. The TgO(the time required for the pH signal to reach 90% of its final value) of the probes determined in buffer solution was at 50 sec. The probes showed no measurable drift between pre- and post tumor measurements. pH readings were corrected for changes of temperature. The same pH measuring setup was used for control pH measurements during hyperglycemia in skeletal muscle of the right hind limb of the tumor carrying animals. Measurement of pQ In this study tumor pOZ was determined with an improved method according to Weiss and Fleckenstein (30). For the polarographic determination of the pOz steel shafted needle probes with a ground lancet type tip (diameter 350 pm) were used (6, 30). The tip of the probes contained a membranized, recessed gold microcathode of 12 pm diameter. This method was used for tumor ~02 measurements also by other authors ( 12, 13,26). An Ag/ AgCl electrode glued to the neck of the animals served as anode in the polarographic circuit. The probes were inserted stepwise into the tissue by a motor driven micromanipulator. The steps consisted of a rapid forward movement of 700 pm immediately followed by an equally rapid backward movement of 200 pm (“pilgrim step”), resulting in an effective forward step of 500 pm. This type of movement was used to decrease mechanical pressure on the tip of the probes during pOZ measurements. Every 500 pm, at the end of each “pilgrim step,” a single local pOZ measurement was made during standstill of the probe. For the establishment of a pOZ histogram (frequency distribution of classified pOZ values) 100 local tissue pOZ values were taken. Because of the relatively small size of the tumors only about 50 local pOZ values could be measured in one puncturing canal. Therefore, after the first pass from the periphery through the center part to the antipode periphery of a tumor, the probes were swiftly withdrawn and inserted stepwise again. To prevent moving of the probe into previously punctured tissue, the angle of insertion was slightly changed. One second after the end of each “pilgrim step” during standstill of the probe a “local” pOZ signal was recorded. The signals were processed by a p02 histograph* and online displayed in the form of p02 versus time plots. On demand, sampled data could be displayed as pOZ histograms. The recording of 100 individual local pOZ values required 5-7 min. The pOz-probes were two-point-calibrated in buffered 0.9% NaCl solution (pH 7.4) equilibrated under atmospheric pressure with air or pure Nz, *f Type SA4, WPI, New Haven, USA. g Model Jenco 67 1P, WPI, New Haven, USA. * SIGMA pOz-Histograph/KIMOC, Eppendorf-NethelerHinz GmbH, Hamburg, F.R.G.

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respectively. After the recording of 100 local pOz values the probes were recalibrated. PO* readings were corrected for changes of temperature. Main technical data of the probes when immersed in 0.9% NaCl solution at 37°C and at a polarisation potential of -700 mV: O2 sensitivity: 6.0 * 3.0 PA/mm Hg ~02; Response time (TgO): ~500 msec.; pH effect on pOz signal: + 1%/pH unit (within a pH range from 6.5-9.5) Temperature effect: 2.44%/Y of temperature change; Drift: < 1O%/hr; Stirring effect at 37°C (as defined by (1)) ~4% Usable time after first polarisation: >30 hr. Tumor histology Histological examination of tumors was carried out either before or 45 min following hyperthermia. Silver impregnation of the reticulum fibers for detection of tumor blood vessels was performed according to Wilder (3 1). Experimental protocol In the first group (hyperthermia group) of 10 rats, the tumor-carrying hind paw was heated in a water bath to 43°C for 1 hr. In the second group (hyperglycemia/hyperthermia group) of 9 rats, 2 hr prior to the beginning of the period of 43°C hyperthermia the animals received 6 g glucose/kg b.w. by intraperitoneal injection of a 50% glucose solution. Additionally, in this group the pH was measured during hyperglycemia in the skeletal muscle of the right hind limb. In both groups intratumoral pH and p02 were measured simultaneously before, during, and after hyperthermia. Statistical evaluation Data are expressed as the mean + standard error. Dunnett’s test was used to compare a control mean with several treatment means. Differences were considered significant at the p < 0.05 level.

rel. frequency

(Xl n = 1900 jf= 12.6

25 -

I

15 -

0

20

40

60 80 100 tissue p0, (mmHq)

Fig. 1. Frequency distribution of 1900 local ~02 values (pooling histogram) from 19 human sarcoma S 117 xenografts of different sizes (5.3 + 2.6 cm3).

Figure 3 shows p02 histograms obtained from the pooled local pOz values measured before, during, and after hyperthermia in all 10 animals of the first group. Each histogram is made up of 1000 single pOz values. In the pooling histogram which contains the p02 values measured before hyperthermia (Fig. 3), a relatively large number of pOz values between 5 and 15 mm Hg exists. During the actual pOz measurements only small pOz differences between subsequent local measurements were observed (small gradients of tissue p02) (Fig. 4). During hyperthermia a marked broadening of the pOz profile and a shift to the right, that is, to higher pOz values, can be pti

7.2 7.1 ;::

RESULTS

Intratumoral pOz before, during, and after hyperthermia and/or hyperglycemia The mean of 1900 local p02 values measured in 19 tumors before treatment at normal body temperature and at a mean tumor temperature of 33.2”C lay at 12 mm Hg (Fig. 1). No pOz values below 2.5 mm Hg occurred. In Figure 2 the time course of the changes of the mean tumor pOz and of the mean tumor pH in 10 tumors during a I-hr period of 43°C hyperthermia is depicted. It can be seen that initially, with the onset of hyperthermia, the mean ~0, rose steeply. Subsequently, during hyperthermia the mean ~02 declined only slightly. Forty-five minutes after the end of hyperthermia when, at unchanged ambient and rectal temperatures, and with a dry tumor surface, tumor temperature had spontaneously dropped to 26°C mean intratumoral pOz decreased to 9 mm Hg.

0

15

start HT 43'C

30

60

15

45 tlminl

stop HT

Fig. 2. Time course of the changes of mean tumor pH (upper section) and of mean tumor p02 (lower section) before, during, and after 1 hr of 43°C hyperthermia (HT) in normoglycaemic animals. Mean f SE, n = 10. (*) values significant

or hyperglycemia on pH and pO2 in well oxygenated xenotransplanted human sarcoma.

The heat-induced interdependent changes of tumor blood flow, pO2, and pH decisively influence the therapeutic effect of hyperthermia (HT). This fact h...
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