bit.J. Cancer: 52, 270-285 ( 1 992) C 1992 Wiley-Liss, Inc.
9-b
Publication of the International Union Against Cancer Publication de I Union lnternationale Contre le Cancer
INFLUENCE OF GLUCOSE ON METABOLISM AND GROWTH OF RAT GLIOMA CELLS (C6) IN MULTICELLULAR SPHEROID CULTURE H. ACKER'J,G. HOLTERMANN', B. BOLLING'and J. CARLSSON~ 'Mi.u-Plritick-lnstititf ,Fir Svstetnpli,vsiologie, Rheinlunddamm 201, 0 - 4 6 0 0 Dortmund I , Germany; and 2Departtm~titof Radiation ScienceJ, Uppsala Uriiversity, Box 535,S-751 21 Uppsala. Sweden. The metabolism and growth of rat glioma C6 cells in multicellular spheroid culture depended strongly on the glucose supply. A low glucose level (0.1 g/l) in the culture medium reduced lactate production, increased oxygen consumption and diminished hydrogen ion production under normoxia as well as hypoxia. A high glucose level (10 g/l glucose) increased lactate production, had no significant influence on oxygen consumption and increased the hydrogen ion production under hypoxia. Hydrogen ion release from cells under normoxic and hypoxic conditions could be significantly diminished by amiloride (I mM), indicatingthe involvement of the Na+/H+exchanger. The growth of the C6 spheroids was enhanced under low glucose conditions, possibly due to the more physiological extracellular pH in the deeper regions of the spheroids. The growth was inhibited under high glucose conditions, which seemed to be toxic due to a massive hydrogen production giving acidosis. The glucose supply strongly influencedthe local hydrogenion production inside the C6 spheroids and this might in turn lead to changes in the response to different therapeutic modalities. 10
1992 Wilt?>-Liss,I n ( .
Multiccllular sphcroids are nearly spherical aggregates of cultured tumor cells. They are used as models of poorly vascularized tissue and have radial PO*, extracellular pH (pH,) and proliferation gradients (Miiller-Klieser and Sutherland, 1982; Acker et al., 11987~;Carlsson and Acker, 1988; Sutherland, 1988). The steepness of, for example, the PO*gradients is dependent mainly on the cell type and not so much on the method of cultivation (Acker et al., 1987b). Since spheroids rcceive their nutrition from the incubation medium by diffusion only, the p 0 2 gradients are caused mainly by oxygen consumption and the p H gradients mainly by the glycolytic breakdown of glucose to lactate and the release of lactate and H + ions from the cclls. Large differences in oxygen consumption and lactate production have been found between different types of human tumor spheroids (Carlsson and Acker, 1988). It has also been shown that E M T 6 spheroids increase cellular oxygen consumption and decrease growth rate in response to high lactate concentration in the medium, indicating a complex relationship between metabolism and growth in tumor cells (Bourrat-Floeck et al., 1991). Thc original idea of Warburg (1930) was that aerobic glycolysis, i.e. lactate production in spite of an adequate oxygen supply. takcs place in the metabolism of tumor cells and might be onc step in, or a result of, the transformation to malignancy. Normal tissues from retina, kidney medulla and fetal tissue have, however, this particularity without showing any signs of pathologic growth control (Ramaiah, 1974). Furthermore, cultured normal cells under varying conditions of oxygenation vary their lactate production according to the glucose supply (Busa and Nuccitelli, 1984). Thus, lactate production under oxidizcd conditions is not a characteristic of malignancy. Lactatc production is related to extracellular p H (pH,) and intracellular pH (pH,) and it has been shown that increases in pH, are related to the stimulus of growth factors and to the onset of DNA synthesis as well as proliferation (for review, see Grinstein et al., 1989). Acid production by aerobic glycolysis, together with membrane-bound H+ ion extrusion systems in 3-dimensional arrangements of tumor cells. probably influences the intracelluiar pH and division of the individual cells.
The aim of this work was to analyse the effects on growth of variations in the glucose concentration and the interconnection between acid production and growth in the C6 spheroid model. The use of spheroids is favorable since the local proliferation gradient in spheroids, with most peripheral cells in the cell cycle and the inner cells in the Go- phase (Sutherland, 1988), makes it possible to relate growth changes to local variations in metabolism. For this purpose, measurements of the local p 0 2 and pH, in spheroids with microelectrodes (Carlsson and Acker, 1988) were compared with biochemical investigations of oxygen consumption, lactate dehydrogenase enzyme activity and lactate production. The dependence of these parameters on variations in the glucose and oxygen supply was analysed, along with induced changes in growth rate. MATERIAL AND METHODS
Cell culture The rat glioma cells C6 (ATCC No: CCL 107) were cultured as spheroids with the liquid overlay technique (Carlsson and Acker, 1988), meaning that the spheroids were cultured at 37°C in plastic trays with agarose-coated wells. One spheroid was cultured in each well which contained 0.3-0.4 ml medium. The culture medium was Ham's FIO with 10% fetal bovine serum supplemented with L-glutamine ( 2 mM), penicillin (100 Uiml), and streptomycin (100 pgiml) (Flow, Bonn, Germany). The diameters of the spheroids varied in the range of 400 to 900 pm. Each spheroid was allowed to attach to a round cover-slip (13 mm diameter; Lux, Munich, Germany) for 10 to 12 hr before microelectrode and photometric measurements.
Microelectrodes rind experimental set-up The microelectrodes used in these experiments, as well as the experimental protocol and the perfusion chamber, have been described in detail elsewhere (Acker et al., 19876; Carlsson and Acker, 1988) and only a brief description is given below. Oxygen measurements were carried out in spheroids with double-barrelled electrodes having a tip diameter of 3-5 p n . One channel was filled electrolytically with gold, forming a recess of 1-3 pm. This channel measured the oxygen pressure according to the polarographic principle. whereas the second channel which was filled with magnesium acetate (1 mM) served for potential measurements. For extracellular pH measurements in spheroids, double-barrelled pH-sensitive microelectrodes with a tip diameter of 3-5 p n were used. The pH channel was manufactured by introducing a thin-pulled p H glass (Ingold. Steinbad, Germany) into one channel of the microelectrode. The p H glass was then fixed to the inner wall of the channel by applying heat from the outside of the electrode and blowing up the melted p H glass by connecting it to compressed air, so that the p H glass formed a close contact with the heated glass of the microelectrode. For pO2 or pH measurements, Ch spheroids were transferred from tissue culture to a perfusion chamber as previously .'To whom correspondence and reprint requests should he sent. Received: February 13, 1992 and in revised form May 13. lYY2.
280
ACKER E T A L .
described (Acker et af., 19876; Carlsson and Acker, 1988). During the experiments the perfusion chamber gave stable, controlled and reproducible conditions of temperature, p 0 2 and pH in the superfusion medium for the spheroids. The medium used was Locke's solution, consisting of NaCI, 128 mM; KCI, 5.6 mM; CaCI2, 2.1 mM; D-glucose, 5.5 mM; NaHC03, 10 mM and 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 7 mM. The medium flowed through the chamber at a rate of 10 mlimin. For calibration, pol-microelectrodes were introduced into the medium, by means of a hydraulic micromanipulator (David Kopf, Albrecht, Munich, Germany). Then, the pO1 in the medium was changed by equilibrating the medium with different gas mixtures containing O%,, 10% or 20% 0 2 in 5% CO:. the rest being N2. For a pOz of 140 Torr these electrodes gave a current of 1.1-0.2 nA and with a pOz of 0 Torr a current of 0.001-0.005 nA. The p H microelectrode was calibrated for each trial in the following way. First it was calibrated with 3 different phosphate buffers (pH: 6,8,7.2 and 7.6) at room temperature. The p H microelectrode was then led into flowing medium of the perfusion chamber and again calibrated at a temperature of 37°C by changing the pCOz in the medium. The calibration of the PO:- and pH-microelectrode was done before and after each measurement. In some cascs a drift in the calibration levels could be observed. In these cases a linear drift was assumed throughout the measurements. After calibration, the electrode was positioned in an accurate relation to the spheroid. This positioning was made by using 2 independent optical systems working along different axes. The electrode was adjusted to hit the spheroid nearly from above. The electrode had a deviation of only 30" from the vertical axis. Aftcr final adjustment of the position, the electrode was moved by the hydraulic microdrive in steps of 25-50 Krn along an axis through the center of the spheroid. When the electrode tip hit the spheroid surface, a signal was recorded in the potential channel of the double-barrelled electrode. The position of the hit was determined by this signal. The movement was stopped when the electrode tip was located in the central part of the spheroid. This position was determined by measurements of the spheroid diameter, careful positioning of the electrode tip, and continuous reading of the electrode position on a multichannel recorder during the clectrode insertion.
NADH Puorescence measurements A microspcctrophotometer as described by Boldt et ul. (1980) was used for recording NADH fluorescence. The spheroid surface was illuminated by an Ultropac System (Lutz, Wetzlar, Germany, 11011/0.25) for NADH fluorescence excitation with light of 366 nm, which originated from a mercury lamp XBO 100 W (Osram. Berlin, Germany) filtered by a 366-nm interference filter (Schott, Mainz, Germany). 366-nm reflected light as well as NADH fluorescence of the tissue peaking at 465 nm passed the Ultropac System and were beam-split. One beam was recorded after passing a Veril-SFilter (Lcitz) adjusted to 366 nm by a photomultiplier (EM, Hayes, UK) and the second beam was recorded simultaneously after passing another Veril-S-filter adjusted to 465 nm by a second photomultiplier. Both photomultiplier signals were recorded separately on a multi-pen ink recorder (Rikadenki, Freiburg, Germany). Ovgen consumption measurements For determination of the oxygen consumption, the method of Carlsson and Acker (1988) was used. Briefly, rabbit hemoglobin diluted with Locke's solution resulting in an oxygen solubility coefficient of cr = 2.4 ml 02/m1/760 Torr was used as an oxygen indicator. Single spheroids were incubated under air-tight conditions, together with this hemoglobin solution, in a glass chamber with a volume of 0.74 pl. The hemoglobin light-absorption spectrum was continuously measured between the wavelengths 500 nm and 620 nm using a photomicro-
scope (Zeiss, Cologne. Germany) with a motor-driven monochromator (M20, Zeiss). The oxygenation of the hemoglobin solution was automatically calculated from the recorded spectrum according to the method described by Hoffmann et al. (1984) with the aid of an online process computer (Honeywell 716, Cambridge, UK). Rabbit hemoglobin showed the first deviation from 100% oxygenation at a pOz of 42 Torr. 100% oxygenation was achieved at room air with a pOz of 146 Torr. We measured the time which a spheroid needed to consume the available oxygen with a decrease of the ambient p 0 2 in the glass chamber from about 146 Torr to 42 Torr. The oxygen consumption of a single spheroid can then be calculated by multiplying the measured time with the pOz decrease of 104 Torr by the oxygen solubility coefficient corrected for the glass chamber volume. Since the measurements were carried out at room temperature to avoid water loss by evaporation from the glass chamber, the calculated values have to be multiplied by a factor of 2-3 to obtain comparable values with measurements carried out at 37°C.
Determination of lactate production and lactate dehydrogenase activiry (LDH) Spheroids of known size were incubated for 1 hr in microwells containing 50 ~1 Locke's solution. The spheroids were washed directly before incubation 3 times in the solution to remove excess lactate. After incubation under different conditions, the spheroids, together with the solution in the microwells, were directly frozen and freeze-dried. Thereafter, each sample was homogenized in 50 ~1 perchloracid and centrifuged at 5,000 U/10 min, then the supernatant was used to determine the lactate content according to the method of Hohorst (1970). For determination of LDH, spheroids were directly homogenized in 100 pl of a 50-mM phosphate buffer (pH 7.5) after incubation under different 0 2 conditions. By the method of Bergmeyer and Bernt (1974), LDH was determined at 37°C with an NADH concentration of 11.3 mM and a pyruvate concentration ranging between 0.05 and 0.63 mM. To determine K, and the maximal turnover, the data were drawn in the Lineweaver-Burk plot. The Elko 111 photometer (Zeiss) was used to measure NADH fluorescence. Growth curves Spheroids were individually placed in agarose-coated multidishes (Nunclon, Labassco, Stockholm) in 1.5-ml culture medium with different concentrations of glucose. Between 10 and 12 spheroids were used in each experiment. Measurement of diameters, followed by medium change, was performed 3 times a week. The volume of each spheroid was calculated from the equation V = 4131~(a x b)3'2,where a and b were the observed minimum and maximum radii measured at right angles (Carlsson and Acker, 1988). Histology Spheroids were fixed for 1 hr in 10% neutral buffered formalin solution (Sigma, St Louis, MO), embedded in glycolmethacrylate (Historesin LKE3 2218-500, LKB, Bromma, Sweden) and sectioned at 2 p m thickness. The sections were stained with hematoxylin. Autoradiography The incorporation of "-TdR (methyl-3H-thymidine, Amersham, Aylesbury, UK) was analysed in the spheroids after measurement of growth. The spheroids were incubated with 0.27 MBq/ml 3H-TdR for 20 min. Fixation, histological processing and autoradiography were carried out as described (Acker et al., 1987a). Statistics The significance of the differences was assessed using the U-test according to Mann and Whitney (1947). Differences
281
GLUCOSE AND GROWTH RELATIONSHIP IN C6 SPHEROIDS I,ul 02 g-’ h-’I
6.4
B diameter [,urn]
10 glucorc/l
log glucorc/l
pno 7.3
PO2 ITorrl
721{
PHO
surface
I
I
7.1
1501
Dr
t
i
i
L
B
6.9
16.7 amllorld@(1mM) 6.6
C +
+
600
----c
800 distance from t h e center I f l m l
FIGUREI - ( a ) Oxygen consumption (B) and lactate production (0)of C6 multicellular spheroids as a function of the spheroid diameter. (h) pOz gradient ( 0 )and pH gradient (0)as a function of the depth of puncture (X-axis) as measured with microelectrodes in a single C6 spheroid. *a = 0.001, +a = 0.025.
with a level of significance a 2 0.025, which equals a confidence coefficient 2 97.596, were considered to be significant. RESULTS
p 0 2 and p H gradients Figure 1 shows the simultaneous occurrence of oxygen consumption and lactate production in C6 spheroids as measured by different techniques. Spheroids of different diameters, incubated for 1 hr in Locke’s solution, showed a clear dependencc of the oxygen consumption on size. Small spheroids had a statistically higher oxygen consumption (a = 0.001, shaded bars) than larger ones. Lactate production (open bars) under the same experimental conditions did not reveal this clear size dependence but spheroids with a diameter between 700 and 900 k m had statistically the lowest lactate production with a = 0.025 (U-lest). Degenerative changes in the center of the large spheroid might explain the size-dependent variations. Figurc 16 demonstrates extracellular pOz and p H profiles measured with microelectrodes in a single C6 spheroid, of about 800 pm diameter, superfused with Locke’s solution. To characterize these profiles in different spheroids, the difference in the p 0 2 and p H values between the culture medium and the cells 200 pm inside the spheroid was calculated. In 12 experiments a pOz difference of 132 Torr 2 1.5 and in 33 experiments a p H difference of 0.46 0.027 could be measured. The diameter of these spheroids varied in the range 600 and 900 km. No clear dependence of the measured p 0 2 and pH profiles on the diameter variations could be seen. Figure 2 shows examples of the changes in the extracellular pH (measured in 3 different spheroids) due to lowering the p 0 2 for 10 min in the superfusion medium to values of about 10 Torr (hypoxia). Under normal glucose conditions, the hypoxiainduced pH decrease seemed to be dependent on the depth of
*
10 glucorr/l
10 glucorc/l 0
10 t [mln]
20
0
10 t [mln]
20
FIGURE 2 - (a) Changes in the extracellular pH in a C6 spheroid due to acute hypoxia under normal and low glucose conditions. (b) Changes in the extracellular pH in a C6 spheroid due to acute hypoxia under normal and high glucose conditions. (c) Changes in the extracellular pH i n a C6 spheroid due to acute hypoxia under control and amiloride conditions with a constant background of 1 g glucoseil. Depth of puncture as well as time are indicated throughout. Arrows indicate time of hypoxic stimulation.
puncture, with a more distinct acidification at 250 and 300 IJ-m than at 350 pm. Under 0.1 g/l glucose conditions, the normoxic control value was, after 1 hr, significantly alkaline and the 10-min hypoxic exposure resulted in a slightly stronger alkalinization. Superfusing the spheroid with 10 g glucose/l for 1 hr caused a minor acidification under normoxic conditions but gave a strong acidification under 10-min hypoxia. Applying amiloride (1 mM) to the superfusion medium for 1 hr to inhibit acid extrusion from the cells via the N a + / H + exchanger (Grinstein et a/., 1989) resulted in a distinct alkalinization under normoxic conditions and a smaller decrease in p H during hypoxia. Figure 3 summarizes the experiments with spheroids exposed to different glucose concentrations. On the X-axis the tissue pH in spheroids under normoxic conditions is shown. This value decreases with the depth of puncture (towards the center of the spheroids). The Y-axis shows the change in the amount of hydrogen ions released from the cells to the extracellular space during the 10-min hypoxia as calculated from p H recordings of the type shown in Figure 2. The degree of the hypoxic acidification increased significantly (a = 0.001, U-test) with decreasing tissue pH peaking at 6.95 as shown in Figure 30. The hypoxic response leveled off towards more acid tissue pH values. i.e. towards the center of the spheroids. Figure 36 shows the hypoxic acidification under normal glucose conditions (filled circles) in 10 additional experiments of the same type as in Figure 3a. The result was not significantly different from that seen in ( a ) .Also, a peak at about 6.95 could be seen here. Carrying out the same experiments under high glucose conditions in the medium (10 g/l) resulted in a linear increase in the hypoxia-induced acidification (open circles) towards the center of the spheroids (n = 7). Under glucose 0.1 g/l conditions (n = 3) the normoxic control values shifted, regardless of the depth of puncture, to 7.2 and the hypoxic pH
282
ACKER E T A L .
A Lactate production pnol g-I h-I
Glucose 1 g/l Glucose 10 gil
B 124 10
\qp
Glucose 0.1 g/l
Normoria
Hvnoxia
204 f 12.7 n = 21 209 t 9.2 n = 24 2 r 0.1' n = 12
241 f 19.52 n = 18 293 t 20.12 n = 24 Not measured
\
Mean values & standard error of the mean are shown. n = number of analysed spheroids.-'a = O.OOl.-?a = 0.025.
-t A IH+I
'
1(
-
pH (control) towards the center
no C
2
'Iv 300
f
=O 99 / (n=L) r
, , , , , , c , ,-
4 ,
6.5
-
6.7
6.9
7.1
7.3
r=O 99
towards the center
F~GURE 3 - (a) Amount of H+ ions (Y-axis) released under 10 min acute hypoxia to the extracellular space of C6 spheroids as a function of the extracellular pH at normoxic conditions (X-axis). High pH values were measured in the periphery of the spheroids, whereas low pH values were measured in deeper regions. n = number of spheroids. (6) Amount of H+ ions (Y-axis) released under hypoxia to the extracellular space of C6 spheroids under 0.1 g/l (A),1 g/l ( 0 )and 10 g/l (0)glucose conditions. The X-axis shows the extracellular pH at normoxic conditions. The solid line is a superimposition of the curve in (a). ( c ) Amount of H+ ions (Y-axis) released under acute hypoxia to the extracellular space of C6 spheroids using normal medium ( 0 )or medium supplemented with 1 mM amiloride (0). The X-axis shows the extracellular pH at normoxic conditions. The solid line is a superimposition of the curve in (a).
FIGURE4 - Lineweaver-Burk plots showing the LDH activity (Y-axis) drawn as the reciprocal vcrws the reciprocal of pyruvate concentration (X-axis). K,,, values can be read from the intercept of the regression lines with the X-axis. Spheroids were measured under control conditions ( O ) , after 10 min of hypoxia (0)and after 30 min of hypoxia (A). r = correlation coefficient, n = number of spheroids.
response gave a slight decrease (triangles) corresponding to a slight alkalinization. Figure 3c summarizes the effect of amiloridc (1 mM) on the hypoxia-induced extracellular acidification. The values without amiloride are shown by filled circles and also peak at an extracellular pH value of about 6.9. In each of the 7 experiments in which the cells were exposed to amiloride (open circles), the hypoxic pH response decreased and the extracellular pH values also shifted to higher values during normoxia.
Lactate production Table I summarizes the lactate values of single spheroids incubated for 1 hr under normoxic or hypoxic conditions with glucose. The lactate level of spheroids incubated under normoxic conditions (PO? = 140 Torr) was equal with 1 g/l and 10 g/l glucose, whereas 0.1 g i l glucose resulted in a significant decrease ( a = 0.001) in the lactate production. Hypoxia (pOz = 10 Torr) for 1 hr induced a significant increase in the lactate production ( a = 0.025) with the highest level at 10 g/l glucose. To explain the lactate production characteristics under normoxic as well as hypoxic conditions, the lactate-dehydrogenase activity (LDH) was investigated under different stimulatory conditions. Figure 4 shows the LDH dependence on pyruvate in a Lineweaver-Burk plot. The filled circles repre-
FIGURE5 - Changes in NADH fluorescence, measured at 465 nm, due to acute hypoxia in the superfusion medium of a single C6 spheroid (diameter 600 pm). Note that the reflected light of 366 nm does not show any reaction. The time course of the p 0 2 in the superfusion medium (P,Oz) is indicated by bars. The time of 5 min is given. The maximal deviation of NADH fluorescence is taken as 100% as a reference to the data in Table 11.
283
GLUCOSE AND GROWTH RELATIONSHIP IN C6 SPHEROIDS TABLE I1 - DEPENDENCE OF THE NADH FLUORESCENCE I N C6 SPHEROIDS ON DIFFERENT GLUCOSE AND ANTIMYCIN LEVELS I N THE SUPERFUSION MEDIUM Glucose
Antimycin
0.1 gil
*
111.2 -229.6 n=4
NADH change %
i n gii
1 PM
11) +M
-39.1 2 16.8 n=3
+78.3 2 10.9 n=2
+225 r 120 n=2
The changes are expressed as a percentage of the hypoxic values obtained at the normal concentration of lgil glucose (see Fig. 6). n = number of spheroids. Mean values r standard error of the mean are shown. TABLE 111 S CHANGES IN OXYGEN CONSUMPTION OF C6 SPHEROIDS INCUBATED FOR 1 HR UNDER DIFFERENT GLUCOSE OR
AMILORJDE CONDITIONS
1 """""~"""""""'1
Chdngt: in 0: consumption as percentage ot control
50.0
: vl
-
3 W
glucose 0.1 gil +122.8 t 28.3' n = 10
glucose 10 gil +70 2 28.7 n=4
amiloride (1 mM) + glucose 1 gil -20 19 n=9
*
Values are given as differences in percentage between control and treated spheroids. The control value was 272 60 pl g-' h-' (n = 23). Mean values standard error of the mean are given. n = number of spheroids.-la = 0.001.
*
sent measurements of 13 spheroids under normoxic conditions (PO? = 140 Torr) and these data give a K,,, value of 0.22 mM pyi-uvate. After 10 min exposure to hypoxia (pOz = 10 Torr) thc maximal turnover rate of pyruvate decreased and the K, value becamc about 0.12 mM (open circles n = 7). The open triangles ( n =: 4) indicate the values for 30 min hypoxia which gave a K,,, value of 0.14 mM pyruvate. Thus, the affinity of pyruvate for LDH was higher under hypoxia than under normoxia, but the maximal turnover capacity decreased.
NA DHJuorescerrce Figure 5 shows .as a typical example the reaction of the NADH fluorcscence to a decrease in the p 0 2 in the superfusion medium. The NADH fluorescence increases whereas the constantly reflected 366-nm light signal indicates the constant light cnergy supply for NADH fluorescence excitation. Variations in the rclative NADH fluorescence due to changes in the glucose or antimycin level in the superfusion medium were analysed. The hypoxia-induced NADH change was set to 100% in each experiment. Table I1 shows that lowering the glucose content to 0.1 g/l markedly oxidized NADH, whereas 10 g/l glucose induced a small change only. Blocking the elcctron transfer in the respiratory chain by antimycin resulted in a significant increase in the NADH fluorescence. O.vgen coilsumptior[ Tablc 111 shows the changes in oxygen consumption of C6 spheroids incubattd for 1 hr under different glucose or amiloridc conditions. Values are given as the difference between control and treated spheroids. Exposure of the spheroids to low glucose for 1 hr led to a substantial increase in oxygen consumption ( a = 0.001). High glucose exposure for 1 hr resulted in a smaller increase in oxygen consumption, which was not significant. Amiloride decreased the oxygen consumption but this change also was not significant.
Growth ciine,s Growth curves were analysed for the C6 spheroids growing in liquid overlay (Fig. 6). Upon reaching a diameter of 500 pm they were randomly divided into 3 groups and thereafter continuously cultured in different media. The controls continued to grow in normal culture medium with 1.0 g/l glucose, reaching approx. 700 pm diameter after about 2 weeks'
2
2
-Y 3
10.0 : 5.0
:
s 0
5
10 15 TIME (DAYS)
20
25
FIGURE6 -Growth curve of C6 spheroids under 1 gil glucose ( O ) ,10 g/l glucose (V)and 0.1 g/l glucose (0).Twelve spheroids
were analysed in each group. Mean values t standard error of the mean are shown. treatment. Those that grew continuously in 10 g/l were not only growth-arrested but started to decrease in size. After 2 weeks they had a diameter of about 400 pm. Those that were given 0.1 gil glucose grew better than the controls since they reached an average diameter of nearly 800 p m after 2 weeks.
Autoradiography After 2 weeks' treatment as above (see Fig. 6) the spheroids were incubated with W T d R for 20 min. As shown in Figure 7 (a,b),no labelled cells were found in the control spheroids. There were, however, several labelled cells and even mitotic cells, especially in the deeper regions of the spheroids that were grown in 0.1 g/l glucose (Fig. 7c,d). Thus, there were cycling cells in these spheroids, in conformity with the improved growth under low glucose conditions. Figure 7 ( e f ) shows that cells under 10 g/l glucose are not labelled but several pyknotic cells can be detected, explaining the inhibition of growth under these conditions. DISCUSSION
Oxygen consumption and lactate production, measured per volume unit and per hour, decreased as a function of spheroid size. This was, in the larger spheroids, probably due to degeneration causing central necrosis. Oxidative metabolism does not lead to a net production of hydrogen ions, while glycolysis, giving lactate, produces, by ATP hydrolysis, an average of 2 hydrogen ions per glucose molecule consumed. Lactate production and intracellular hydrogen ion concentration are therefore directly related (Busa and Nuccitelli, 1984). Several experiments were made in which the glycolysis or the oxidative metabolism were disturbed. It was then found that temporary hypoxia influenced the extracellular pH in the spheroids. However, hardly any
284
ACKER ET AL.
FIGURE7 -Histological sections of C6 spheroids. The spheroids were incubated with 3H-TdR, fixed, embedded in plastic and sectioned. The sections were processed for autoradiography and thereafter stained with hemotoxylin. The situation is depicted (a) and (b) under 1 g/l glucose, (c) and ( d ) under 0.1 g/l glucose, ( e ) and (f, under 10 g/l glucose. 100 pm is given by bars.
hypoxia-induced increase in H+ ion concentration could be measured in the peripheral regions of the spheroids (Fig. 3). This might have been due to a low capacity for increased glycolysis in the outer cell layers, or to fast diffusion of H+ ions out into the culture medium. The hypoxia-induced increase in H + ion concentration was high at the inner cell layers, starting at a depth of approx. 300 bm. The strongest increase was obtained when high conccntrations of glucose were administered in the culture medium. This indicated that there is a lack of glucose in the central areas under hypoxia, when there is a higher demand for glucose due to increased anaerobic glycolysis in the C6 cells, as shown by Ercinska and Silver (1987). A decrease of approx. 60% in the glucose concentration in the innermost part of the spheroids could be directly measured in EMT6 spheroids (Gollner et nl., 1991) meaning that an additional increase in the glucose consumption, as under hypoxia, can only be covered by an increase in the glucose supply. The results also showed that there are no severe diffusion limitations for glucose, in conformity with previous determinations of glucose diffusion coefficients in spheroids (Freyer and Sutherland, 1986; Casciari et nl., 1988; Nederman et nl., 1988). The pH gradients were less stcep when the glucose concentration was decreased (Fig. 2a). Furthermore,
no extra H+ ions were released as a response to hypoxia under low glucose conditions. The pH measurements were in good accordance with the lactate production determinations. Hypoxia increascd the lactate production at both 1.0 and 10.0 g/l glucose while low glucose levels decreased the lactate production. Changes in glucose supply between 1 and 10 g/l under normoxic conditions did not influence the lactatc production, in accordance with the nearly unchanged p H values. Analysis of the LDH activity in Lineweaver-Burk plots showed that the LDH activity was proportional to the pyruvate concentration and that the K,,, values for L D H decreased under hypoxic conditions, i.e. the enzyme became more efficient in converting pyruvate to lactate under hypoxic conditions. LDH exists in 5 diffcrent isoenzymes, which are tetramers of 2 subunits: H and M. The synthesis of different forms of the enzyme from L D H l to LDHS can be influcnced by oxygen changes. In lymphocytes and chicken embryos, chronic hypoxia induces the formation of M types of subunits, which have a higher affinity for pyruvate (Hellung-Larsen and Andersen, 1970). It would bc of interest to investigate, in further experiments, whether the observed changes in affinity and turnover of pyruvate induced by acute hypoxia (as shown in
285
GLUCOSE AND GROWTH RELATIONSHIP IN C6 SPHEROIDS
Fig. 5 ) are due to a :shift in the subunit composition or to other causes. The oxygcn consumption was higher at 0.1 than at 1.0 g/l glucose. meaning that the metabolic pathway was shifted from lactate production to oxidative metabolism so that more energy could be obtained from the Krebs cycle and the electron transport chain. The oxygen consumption also increased somewhat at 10.0 g/l glucose. This increase, however, was statistically not significant. The lower turnover in the glycolytic pathway ‘IS also indicated by the decreased NADH fluorescence under low glucose conditions. The increased oxygen consumption under these conditions could also decrease NADH fluorescence due to a higher turnover in the respiratory chain, since blocking of the respiratory chain by antimycin leads to increased NADH fluorescence (Table 11). This means that the coenzyme NADH in the C6 cells is equally involvcd in the respiratory chain as well as in the glycolytic breakdown of glucose. Amiloride gave extracellular alkalization in the spheroids and a lower p H response at hypoxia. This might be the result of a block of the amiloride-sensitive N a + / H + antiport (Grinstein et ul., 1989), which should give a decreased capacity for release of H + ions from the cells. This might in particular be the case under hypoxia, which highlights the importance of this exchanger to avoid intracellular acidification under hypoxia. The involvement of the lactate/proton symporter in the hypoxiamediated proton release was not investigated due to the high efficacy of the amiloride treatment. Low glucose (0.1 gil) led to an improvement in growth capacity in that the saturation diameter increased. This was probably due to the fact that the extracellular acidification was less dramatic at low glucose concentration. A similar correlation between lowered glucose concentration and increased growth potential has been reported for human thyroid carci) the growth rate noma spheroids (Acker et al., 1 9 8 7 ~whereas
of human glioma U 118 MG spheroids remained unchanged (Acker et al., 1987a) and mouse mammary carcinoma EMT6/Ro as well as human bladder carcinoma MGH-U1 spheroids decreased their growth rate at low glucose levels (Freyer and Sutherland, 1986; Tannock and Kopelyan, 1986). High glucose levels (10.0 g/l) seemed toxic to the spheroids because they started to decrease in size. This may have been the result of excessive extracellular acidification. Decreased growth capacity and degeneration due to hyperglycemia have been reported for tumors in vivo (Wike-Hooley et al., 1984). This study shows that C6 rat glioma spheroids normally produce large amounts of lactate and that they can change their metabolism depending on the glucose supply. For example, they changed from LDH-mediated lactate production to more efficient oxidative metabolism in response to decreased glucose levels. These changes also imposed modifications in the H+ ion release and, consequently, in the extracellular pH. Such changes might modify the response to different therapeutic agents. For example, increased acidosis might sensitize for hyperthermia treatment but at the same time protect against radiation (Wike-Hooley et al., 1984). Metabolic changes can possibly be exploited in the development of new anti-tumor therapeutic principles. ACKNOWLEDGEMENTS
We thank the staffs of the Department of Physical Biology, Uppsala University, and of the Max-Planck-Institut fur Systemphysiologie, Dortmund, for assistance in cell culturing and microelectrode measurements. This work was financially supported by the National Board of Laboratory Animals 88-22, Swedish National Board for Technical Development 8605103P, Swedish Cancer Society 1079-B90-01XA, Max-PlanckGesellschaft, Munich, the Hermann and Lilly Schilling Stiftung, Essen, and the BMFT, Bonn.
REFERENCES
ACKER,H.. CARLSSON, J., HOLTERMANN, G., NEDERMANN, T. and GRINSTEIN. S., ROTIN, D. and MASON,J.M., Na+/H exchange and NYLEN. T., Influence of glucose and buffer capacity in the culture growth factor-inducedcytosolic pH changes. Role in cellular proliferamedium on growth and pH in spheroids of human thyroid carcinoma tion. Biochinl. biophvs. . . Acia, 988.73-97 (1989). and human glioma origin. Cancer Res., 47,3504-3508 (1987~). HELLUNG-LARSEN. P. and ANDERSEN, V., Studies on PO?-dependent changes in lactate dehydrogenase isoenzyme distribution. FEBS Lett., ACKER,H.. CARLSSOV, J., MUELLER-KLIESER, W. and SUTHERLANU, R.M.. Comparative ~ ‘ - 3 2measurements in cell spheroids cultured with 18, 363-167(1970). different techniques. Brit. J. Cancer, 56,325-327 (1987b). HOFFMANN, J., WODICK, R., HANNEBAUtK, F. and LUBBERS, D.W., BERGMLYER. H.U. and BERNT.E., Lactate dehydrogenase, UV assay Quantitative analysis of reflection spectra of the surface of the guinea with pyruvate and NADH. In: H.U. Bergmeyer (ed.), Methods of pig brain. Advanc. e.rp. Med. Biol., 169,831-839 (1984). eri,-vmatic unu/vsis 11, pp. 574-579. Academic Press, New York (1974). HOHORST. H.J., L-(+)-Lactat.112: H.U. Bergmeyer (ed.), Merhoden der BoLw, M., H A R B I G . K., WEIDEMANN, G. and LUBBERS, D.W., A etzqvniutischen Analyse II, pp. 1425-1429, Verlag Chemie, Bergstrasse sensitive dual wavelength microspectrophotometer for the measure- (1970). ment of tissue fluorescence and reflectance. PflUgers Arch., 385, MA”, H.B. and WHITNEY, D.R., On a test of whether one of two 167-173 (1980). random variables is stochastically larger than the other. Ann. Math. Statist, 18,50-60 (1947). BOURRAT-FLOECK. B.. GKOEBE. K. and MUELLER-KLIESER. . W.., Bioloeical response of multicellular EMT6 spheroids to exogenous lactat;. MULLER-KLIESER, W. and SUTHERLAND, R.M., Influence of convecItit. J. Cancer, 47, 792--799(1991). tion in the growth medium on oxygen tensions in multicellular BLISA.W.B. and NuC(’ITEI.LI. R., Metabolic regulation via intracellular spheroids. Cancer Res., 42,237-242 (1982). pH. Anier. J. Pliyssiol.. 246, R409-R438 (1984). NEDERMANT., CARLSSON, J. and KUOPPA,K., Penetration of subCARLSSON, J. and ACKER, H., Relations between pH. oxygen partial stances into tumour tissue; model studies using saccharides, thymidine pressure a n d growth in cultured cell spheroids. Int. J. Cancer, 42, and thymidine-5’-triphosphatein cellular spheroids.Cancer Chernother. +
~~~~~
715-720 (1988).
Pliarniucol., 22,21-25 (1988).
CASCIARI, J.J., SOTIKCHOS, S.V. and SLITHERLAND, R.M., Glucose diffusivity in multict:llular spheroids. Cancer Res., 48, 3905-3909
Regul., 8,297-345 (1974).
( 1988).
EKCINSKA, M. and SILVER, I.A., Energy relationships between ATP synthesis and K + griidients in cultured glial-derived cell line. Acia hrochim. Pol.. 34, 195--203(1987). FREYER, J.P. and SUTHERLAND, R.M., Regulation of growth saturation and the development of necrosis in multicell spheroids by the oxygen and glucose supply. Cancer Rex, 46,3504-3512 (1986). GOLLNER. A., GERLACH, S., MULLER-KLIESER, W. and TEUTSCH. H.F., Glucose levels in microregions of multicellular EMT6IRo tumor spheroids. Progr. Historhem. Cytocheni., 23,73-76 (1991).
RAMAIAH. A,, Pasteur effect and phosphofructokinase. Cirrr. Top. Cell SUTHERIAND, R.M., Cell and environment interactions in tumor microregions. The multicell spheroid model. Scietice, 240, 177-184 (1988). TANNOCK, I.F. and KOPELYAN, I., Influence of glucose concentration on growth and formation of necrosis in spheroids derived from a human bladder cancer cell line. CancerRes., 46,3105-3110 (1986). WARBURG, O., The rnctaholrsm oftiunors. Arnold Constable, London (1930).
WIKE-HOOLEY, J.L., HAVEMANN, J . and REINHOLD, S.H.. The rele-
vance of tumour pH to the treatment of malignant disease. Radiother.
Oncol., 27,343-366 (1984).
9-b
Publication of the International Union Against Cancer Publication de I Union lnternationale Contre le Cancer
INFLUENCE OF GLUCOSE ON METABOLISM AND GROWTH OF RAT GLIOMA CELLS (C6) IN MULTICELLULAR SPHEROID CULTURE H. ACKER'J,G. HOLTERMANN', B. BOLLING'and J. CARLSSON~ 'Mi.u-Plritick-lnstititf ,Fir Svstetnpli,vsiologie, Rheinlunddamm 201, 0 - 4 6 0 0 Dortmund I , Germany; and 2Departtm~titof Radiation ScienceJ, Uppsala Uriiversity, Box 535,S-751 21 Uppsala. Sweden. The metabolism and growth of rat glioma C6 cells in multicellular spheroid culture depended strongly on the glucose supply. A low glucose level (0.1 g/l) in the culture medium reduced lactate production, increased oxygen consumption and diminished hydrogen ion production under normoxia as well as hypoxia. A high glucose level (10 g/l glucose) increased lactate production, had no significant influence on oxygen consumption and increased the hydrogen ion production under hypoxia. Hydrogen ion release from cells under normoxic and hypoxic conditions could be significantly diminished by amiloride (I mM), indicatingthe involvement of the Na+/H+exchanger. The growth of the C6 spheroids was enhanced under low glucose conditions, possibly due to the more physiological extracellular pH in the deeper regions of the spheroids. The growth was inhibited under high glucose conditions, which seemed to be toxic due to a massive hydrogen production giving acidosis. The glucose supply strongly influencedthe local hydrogenion production inside the C6 spheroids and this might in turn lead to changes in the response to different therapeutic modalities. 10
1992 Wilt?>-Liss,I n ( .
Multiccllular sphcroids are nearly spherical aggregates of cultured tumor cells. They are used as models of poorly vascularized tissue and have radial PO*, extracellular pH (pH,) and proliferation gradients (Miiller-Klieser and Sutherland, 1982; Acker et al., 11987~;Carlsson and Acker, 1988; Sutherland, 1988). The steepness of, for example, the PO*gradients is dependent mainly on the cell type and not so much on the method of cultivation (Acker et al., 1987b). Since spheroids rcceive their nutrition from the incubation medium by diffusion only, the p 0 2 gradients are caused mainly by oxygen consumption and the p H gradients mainly by the glycolytic breakdown of glucose to lactate and the release of lactate and H + ions from the cclls. Large differences in oxygen consumption and lactate production have been found between different types of human tumor spheroids (Carlsson and Acker, 1988). It has also been shown that E M T 6 spheroids increase cellular oxygen consumption and decrease growth rate in response to high lactate concentration in the medium, indicating a complex relationship between metabolism and growth in tumor cells (Bourrat-Floeck et al., 1991). Thc original idea of Warburg (1930) was that aerobic glycolysis, i.e. lactate production in spite of an adequate oxygen supply. takcs place in the metabolism of tumor cells and might be onc step in, or a result of, the transformation to malignancy. Normal tissues from retina, kidney medulla and fetal tissue have, however, this particularity without showing any signs of pathologic growth control (Ramaiah, 1974). Furthermore, cultured normal cells under varying conditions of oxygenation vary their lactate production according to the glucose supply (Busa and Nuccitelli, 1984). Thus, lactate production under oxidizcd conditions is not a characteristic of malignancy. Lactatc production is related to extracellular p H (pH,) and intracellular pH (pH,) and it has been shown that increases in pH, are related to the stimulus of growth factors and to the onset of DNA synthesis as well as proliferation (for review, see Grinstein et al., 1989). Acid production by aerobic glycolysis, together with membrane-bound H+ ion extrusion systems in 3-dimensional arrangements of tumor cells. probably influences the intracelluiar pH and division of the individual cells.
The aim of this work was to analyse the effects on growth of variations in the glucose concentration and the interconnection between acid production and growth in the C6 spheroid model. The use of spheroids is favorable since the local proliferation gradient in spheroids, with most peripheral cells in the cell cycle and the inner cells in the Go- phase (Sutherland, 1988), makes it possible to relate growth changes to local variations in metabolism. For this purpose, measurements of the local p 0 2 and pH, in spheroids with microelectrodes (Carlsson and Acker, 1988) were compared with biochemical investigations of oxygen consumption, lactate dehydrogenase enzyme activity and lactate production. The dependence of these parameters on variations in the glucose and oxygen supply was analysed, along with induced changes in growth rate. MATERIAL AND METHODS
Cell culture The rat glioma cells C6 (ATCC No: CCL 107) were cultured as spheroids with the liquid overlay technique (Carlsson and Acker, 1988), meaning that the spheroids were cultured at 37°C in plastic trays with agarose-coated wells. One spheroid was cultured in each well which contained 0.3-0.4 ml medium. The culture medium was Ham's FIO with 10% fetal bovine serum supplemented with L-glutamine ( 2 mM), penicillin (100 Uiml), and streptomycin (100 pgiml) (Flow, Bonn, Germany). The diameters of the spheroids varied in the range of 400 to 900 pm. Each spheroid was allowed to attach to a round cover-slip (13 mm diameter; Lux, Munich, Germany) for 10 to 12 hr before microelectrode and photometric measurements.
Microelectrodes rind experimental set-up The microelectrodes used in these experiments, as well as the experimental protocol and the perfusion chamber, have been described in detail elsewhere (Acker et al., 19876; Carlsson and Acker, 1988) and only a brief description is given below. Oxygen measurements were carried out in spheroids with double-barrelled electrodes having a tip diameter of 3-5 p n . One channel was filled electrolytically with gold, forming a recess of 1-3 pm. This channel measured the oxygen pressure according to the polarographic principle. whereas the second channel which was filled with magnesium acetate (1 mM) served for potential measurements. For extracellular pH measurements in spheroids, double-barrelled pH-sensitive microelectrodes with a tip diameter of 3-5 p n were used. The pH channel was manufactured by introducing a thin-pulled p H glass (Ingold. Steinbad, Germany) into one channel of the microelectrode. The p H glass was then fixed to the inner wall of the channel by applying heat from the outside of the electrode and blowing up the melted p H glass by connecting it to compressed air, so that the p H glass formed a close contact with the heated glass of the microelectrode. For pO2 or pH measurements, Ch spheroids were transferred from tissue culture to a perfusion chamber as previously .'To whom correspondence and reprint requests should he sent. Received: February 13, 1992 and in revised form May 13. lYY2.
280
ACKER E T A L .
described (Acker et af., 19876; Carlsson and Acker, 1988). During the experiments the perfusion chamber gave stable, controlled and reproducible conditions of temperature, p 0 2 and pH in the superfusion medium for the spheroids. The medium used was Locke's solution, consisting of NaCI, 128 mM; KCI, 5.6 mM; CaCI2, 2.1 mM; D-glucose, 5.5 mM; NaHC03, 10 mM and 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 7 mM. The medium flowed through the chamber at a rate of 10 mlimin. For calibration, pol-microelectrodes were introduced into the medium, by means of a hydraulic micromanipulator (David Kopf, Albrecht, Munich, Germany). Then, the pO1 in the medium was changed by equilibrating the medium with different gas mixtures containing O%,, 10% or 20% 0 2 in 5% CO:. the rest being N2. For a pOz of 140 Torr these electrodes gave a current of 1.1-0.2 nA and with a pOz of 0 Torr a current of 0.001-0.005 nA. The p H microelectrode was calibrated for each trial in the following way. First it was calibrated with 3 different phosphate buffers (pH: 6,8,7.2 and 7.6) at room temperature. The p H microelectrode was then led into flowing medium of the perfusion chamber and again calibrated at a temperature of 37°C by changing the pCOz in the medium. The calibration of the PO:- and pH-microelectrode was done before and after each measurement. In some cascs a drift in the calibration levels could be observed. In these cases a linear drift was assumed throughout the measurements. After calibration, the electrode was positioned in an accurate relation to the spheroid. This positioning was made by using 2 independent optical systems working along different axes. The electrode was adjusted to hit the spheroid nearly from above. The electrode had a deviation of only 30" from the vertical axis. Aftcr final adjustment of the position, the electrode was moved by the hydraulic microdrive in steps of 25-50 Krn along an axis through the center of the spheroid. When the electrode tip hit the spheroid surface, a signal was recorded in the potential channel of the double-barrelled electrode. The position of the hit was determined by this signal. The movement was stopped when the electrode tip was located in the central part of the spheroid. This position was determined by measurements of the spheroid diameter, careful positioning of the electrode tip, and continuous reading of the electrode position on a multichannel recorder during the clectrode insertion.
NADH Puorescence measurements A microspcctrophotometer as described by Boldt et ul. (1980) was used for recording NADH fluorescence. The spheroid surface was illuminated by an Ultropac System (Lutz, Wetzlar, Germany, 11011/0.25) for NADH fluorescence excitation with light of 366 nm, which originated from a mercury lamp XBO 100 W (Osram. Berlin, Germany) filtered by a 366-nm interference filter (Schott, Mainz, Germany). 366-nm reflected light as well as NADH fluorescence of the tissue peaking at 465 nm passed the Ultropac System and were beam-split. One beam was recorded after passing a Veril-SFilter (Lcitz) adjusted to 366 nm by a photomultiplier (EM, Hayes, UK) and the second beam was recorded simultaneously after passing another Veril-S-filter adjusted to 465 nm by a second photomultiplier. Both photomultiplier signals were recorded separately on a multi-pen ink recorder (Rikadenki, Freiburg, Germany). Ovgen consumption measurements For determination of the oxygen consumption, the method of Carlsson and Acker (1988) was used. Briefly, rabbit hemoglobin diluted with Locke's solution resulting in an oxygen solubility coefficient of cr = 2.4 ml 02/m1/760 Torr was used as an oxygen indicator. Single spheroids were incubated under air-tight conditions, together with this hemoglobin solution, in a glass chamber with a volume of 0.74 pl. The hemoglobin light-absorption spectrum was continuously measured between the wavelengths 500 nm and 620 nm using a photomicro-
scope (Zeiss, Cologne. Germany) with a motor-driven monochromator (M20, Zeiss). The oxygenation of the hemoglobin solution was automatically calculated from the recorded spectrum according to the method described by Hoffmann et al. (1984) with the aid of an online process computer (Honeywell 716, Cambridge, UK). Rabbit hemoglobin showed the first deviation from 100% oxygenation at a pOz of 42 Torr. 100% oxygenation was achieved at room air with a pOz of 146 Torr. We measured the time which a spheroid needed to consume the available oxygen with a decrease of the ambient p 0 2 in the glass chamber from about 146 Torr to 42 Torr. The oxygen consumption of a single spheroid can then be calculated by multiplying the measured time with the pOz decrease of 104 Torr by the oxygen solubility coefficient corrected for the glass chamber volume. Since the measurements were carried out at room temperature to avoid water loss by evaporation from the glass chamber, the calculated values have to be multiplied by a factor of 2-3 to obtain comparable values with measurements carried out at 37°C.
Determination of lactate production and lactate dehydrogenase activiry (LDH) Spheroids of known size were incubated for 1 hr in microwells containing 50 ~1 Locke's solution. The spheroids were washed directly before incubation 3 times in the solution to remove excess lactate. After incubation under different conditions, the spheroids, together with the solution in the microwells, were directly frozen and freeze-dried. Thereafter, each sample was homogenized in 50 ~1 perchloracid and centrifuged at 5,000 U/10 min, then the supernatant was used to determine the lactate content according to the method of Hohorst (1970). For determination of LDH, spheroids were directly homogenized in 100 pl of a 50-mM phosphate buffer (pH 7.5) after incubation under different 0 2 conditions. By the method of Bergmeyer and Bernt (1974), LDH was determined at 37°C with an NADH concentration of 11.3 mM and a pyruvate concentration ranging between 0.05 and 0.63 mM. To determine K, and the maximal turnover, the data were drawn in the Lineweaver-Burk plot. The Elko 111 photometer (Zeiss) was used to measure NADH fluorescence. Growth curves Spheroids were individually placed in agarose-coated multidishes (Nunclon, Labassco, Stockholm) in 1.5-ml culture medium with different concentrations of glucose. Between 10 and 12 spheroids were used in each experiment. Measurement of diameters, followed by medium change, was performed 3 times a week. The volume of each spheroid was calculated from the equation V = 4131~(a x b)3'2,where a and b were the observed minimum and maximum radii measured at right angles (Carlsson and Acker, 1988). Histology Spheroids were fixed for 1 hr in 10% neutral buffered formalin solution (Sigma, St Louis, MO), embedded in glycolmethacrylate (Historesin LKE3 2218-500, LKB, Bromma, Sweden) and sectioned at 2 p m thickness. The sections were stained with hematoxylin. Autoradiography The incorporation of "-TdR (methyl-3H-thymidine, Amersham, Aylesbury, UK) was analysed in the spheroids after measurement of growth. The spheroids were incubated with 0.27 MBq/ml 3H-TdR for 20 min. Fixation, histological processing and autoradiography were carried out as described (Acker et al., 1987a). Statistics The significance of the differences was assessed using the U-test according to Mann and Whitney (1947). Differences
281
GLUCOSE AND GROWTH RELATIONSHIP IN C6 SPHEROIDS I,ul 02 g-’ h-’I
6.4
B diameter [,urn]
10 glucorc/l
log glucorc/l
pno 7.3
PO2 ITorrl
721{
PHO
surface
I
I
7.1
1501
Dr
t
i
i
L
B
6.9
16.7 amllorld@(1mM) 6.6
C +
+
600
----c
800 distance from t h e center I f l m l
FIGUREI - ( a ) Oxygen consumption (B) and lactate production (0)of C6 multicellular spheroids as a function of the spheroid diameter. (h) pOz gradient ( 0 )and pH gradient (0)as a function of the depth of puncture (X-axis) as measured with microelectrodes in a single C6 spheroid. *a = 0.001, +a = 0.025.
with a level of significance a 2 0.025, which equals a confidence coefficient 2 97.596, were considered to be significant. RESULTS
p 0 2 and p H gradients Figure 1 shows the simultaneous occurrence of oxygen consumption and lactate production in C6 spheroids as measured by different techniques. Spheroids of different diameters, incubated for 1 hr in Locke’s solution, showed a clear dependencc of the oxygen consumption on size. Small spheroids had a statistically higher oxygen consumption (a = 0.001, shaded bars) than larger ones. Lactate production (open bars) under the same experimental conditions did not reveal this clear size dependence but spheroids with a diameter between 700 and 900 k m had statistically the lowest lactate production with a = 0.025 (U-lest). Degenerative changes in the center of the large spheroid might explain the size-dependent variations. Figurc 16 demonstrates extracellular pOz and p H profiles measured with microelectrodes in a single C6 spheroid, of about 800 pm diameter, superfused with Locke’s solution. To characterize these profiles in different spheroids, the difference in the p 0 2 and p H values between the culture medium and the cells 200 pm inside the spheroid was calculated. In 12 experiments a pOz difference of 132 Torr 2 1.5 and in 33 experiments a p H difference of 0.46 0.027 could be measured. The diameter of these spheroids varied in the range 600 and 900 km. No clear dependence of the measured p 0 2 and pH profiles on the diameter variations could be seen. Figure 2 shows examples of the changes in the extracellular pH (measured in 3 different spheroids) due to lowering the p 0 2 for 10 min in the superfusion medium to values of about 10 Torr (hypoxia). Under normal glucose conditions, the hypoxiainduced pH decrease seemed to be dependent on the depth of
*
10 glucorr/l
10 glucorc/l 0
10 t [mln]
20
0
10 t [mln]
20
FIGURE 2 - (a) Changes in the extracellular pH in a C6 spheroid due to acute hypoxia under normal and low glucose conditions. (b) Changes in the extracellular pH in a C6 spheroid due to acute hypoxia under normal and high glucose conditions. (c) Changes in the extracellular pH i n a C6 spheroid due to acute hypoxia under control and amiloride conditions with a constant background of 1 g glucoseil. Depth of puncture as well as time are indicated throughout. Arrows indicate time of hypoxic stimulation.
puncture, with a more distinct acidification at 250 and 300 IJ-m than at 350 pm. Under 0.1 g/l glucose conditions, the normoxic control value was, after 1 hr, significantly alkaline and the 10-min hypoxic exposure resulted in a slightly stronger alkalinization. Superfusing the spheroid with 10 g glucose/l for 1 hr caused a minor acidification under normoxic conditions but gave a strong acidification under 10-min hypoxia. Applying amiloride (1 mM) to the superfusion medium for 1 hr to inhibit acid extrusion from the cells via the N a + / H + exchanger (Grinstein et a/., 1989) resulted in a distinct alkalinization under normoxic conditions and a smaller decrease in p H during hypoxia. Figure 3 summarizes the experiments with spheroids exposed to different glucose concentrations. On the X-axis the tissue pH in spheroids under normoxic conditions is shown. This value decreases with the depth of puncture (towards the center of the spheroids). The Y-axis shows the change in the amount of hydrogen ions released from the cells to the extracellular space during the 10-min hypoxia as calculated from p H recordings of the type shown in Figure 2. The degree of the hypoxic acidification increased significantly (a = 0.001, U-test) with decreasing tissue pH peaking at 6.95 as shown in Figure 30. The hypoxic response leveled off towards more acid tissue pH values. i.e. towards the center of the spheroids. Figure 36 shows the hypoxic acidification under normal glucose conditions (filled circles) in 10 additional experiments of the same type as in Figure 3a. The result was not significantly different from that seen in ( a ) .Also, a peak at about 6.95 could be seen here. Carrying out the same experiments under high glucose conditions in the medium (10 g/l) resulted in a linear increase in the hypoxia-induced acidification (open circles) towards the center of the spheroids (n = 7). Under glucose 0.1 g/l conditions (n = 3) the normoxic control values shifted, regardless of the depth of puncture, to 7.2 and the hypoxic pH
282
ACKER E T A L .
A Lactate production pnol g-I h-I
Glucose 1 g/l Glucose 10 gil
B 124 10
\qp
Glucose 0.1 g/l
Normoria
Hvnoxia
204 f 12.7 n = 21 209 t 9.2 n = 24 2 r 0.1' n = 12
241 f 19.52 n = 18 293 t 20.12 n = 24 Not measured
\
Mean values & standard error of the mean are shown. n = number of analysed spheroids.-'a = O.OOl.-?a = 0.025.
-t A IH+I
'
1(
-
pH (control) towards the center
no C
2
'Iv 300
f
=O 99 / (n=L) r
, , , , , , c , ,-
4 ,
6.5
-
6.7
6.9
7.1
7.3
r=O 99
towards the center
F~GURE 3 - (a) Amount of H+ ions (Y-axis) released under 10 min acute hypoxia to the extracellular space of C6 spheroids as a function of the extracellular pH at normoxic conditions (X-axis). High pH values were measured in the periphery of the spheroids, whereas low pH values were measured in deeper regions. n = number of spheroids. (6) Amount of H+ ions (Y-axis) released under hypoxia to the extracellular space of C6 spheroids under 0.1 g/l (A),1 g/l ( 0 )and 10 g/l (0)glucose conditions. The X-axis shows the extracellular pH at normoxic conditions. The solid line is a superimposition of the curve in (a). ( c ) Amount of H+ ions (Y-axis) released under acute hypoxia to the extracellular space of C6 spheroids using normal medium ( 0 )or medium supplemented with 1 mM amiloride (0). The X-axis shows the extracellular pH at normoxic conditions. The solid line is a superimposition of the curve in (a).
FIGURE4 - Lineweaver-Burk plots showing the LDH activity (Y-axis) drawn as the reciprocal vcrws the reciprocal of pyruvate concentration (X-axis). K,,, values can be read from the intercept of the regression lines with the X-axis. Spheroids were measured under control conditions ( O ) , after 10 min of hypoxia (0)and after 30 min of hypoxia (A). r = correlation coefficient, n = number of spheroids.
response gave a slight decrease (triangles) corresponding to a slight alkalinization. Figure 3c summarizes the effect of amiloridc (1 mM) on the hypoxia-induced extracellular acidification. The values without amiloride are shown by filled circles and also peak at an extracellular pH value of about 6.9. In each of the 7 experiments in which the cells were exposed to amiloride (open circles), the hypoxic pH response decreased and the extracellular pH values also shifted to higher values during normoxia.
Lactate production Table I summarizes the lactate values of single spheroids incubated for 1 hr under normoxic or hypoxic conditions with glucose. The lactate level of spheroids incubated under normoxic conditions (PO? = 140 Torr) was equal with 1 g/l and 10 g/l glucose, whereas 0.1 g i l glucose resulted in a significant decrease ( a = 0.001) in the lactate production. Hypoxia (pOz = 10 Torr) for 1 hr induced a significant increase in the lactate production ( a = 0.025) with the highest level at 10 g/l glucose. To explain the lactate production characteristics under normoxic as well as hypoxic conditions, the lactate-dehydrogenase activity (LDH) was investigated under different stimulatory conditions. Figure 4 shows the LDH dependence on pyruvate in a Lineweaver-Burk plot. The filled circles repre-
FIGURE5 - Changes in NADH fluorescence, measured at 465 nm, due to acute hypoxia in the superfusion medium of a single C6 spheroid (diameter 600 pm). Note that the reflected light of 366 nm does not show any reaction. The time course of the p 0 2 in the superfusion medium (P,Oz) is indicated by bars. The time of 5 min is given. The maximal deviation of NADH fluorescence is taken as 100% as a reference to the data in Table 11.
283
GLUCOSE AND GROWTH RELATIONSHIP IN C6 SPHEROIDS TABLE I1 - DEPENDENCE OF THE NADH FLUORESCENCE I N C6 SPHEROIDS ON DIFFERENT GLUCOSE AND ANTIMYCIN LEVELS I N THE SUPERFUSION MEDIUM Glucose
Antimycin
0.1 gil
*
111.2 -229.6 n=4
NADH change %
i n gii
1 PM
11) +M
-39.1 2 16.8 n=3
+78.3 2 10.9 n=2
+225 r 120 n=2
The changes are expressed as a percentage of the hypoxic values obtained at the normal concentration of lgil glucose (see Fig. 6). n = number of spheroids. Mean values r standard error of the mean are shown. TABLE 111 S CHANGES IN OXYGEN CONSUMPTION OF C6 SPHEROIDS INCUBATED FOR 1 HR UNDER DIFFERENT GLUCOSE OR
AMILORJDE CONDITIONS
1 """""~"""""""'1
Chdngt: in 0: consumption as percentage ot control
50.0
: vl
-
3 W
glucose 0.1 gil +122.8 t 28.3' n = 10
glucose 10 gil +70 2 28.7 n=4
amiloride (1 mM) + glucose 1 gil -20 19 n=9
*
Values are given as differences in percentage between control and treated spheroids. The control value was 272 60 pl g-' h-' (n = 23). Mean values standard error of the mean are given. n = number of spheroids.-la = 0.001.
*
sent measurements of 13 spheroids under normoxic conditions (PO? = 140 Torr) and these data give a K,,, value of 0.22 mM pyi-uvate. After 10 min exposure to hypoxia (pOz = 10 Torr) thc maximal turnover rate of pyruvate decreased and the K, value becamc about 0.12 mM (open circles n = 7). The open triangles ( n =: 4) indicate the values for 30 min hypoxia which gave a K,,, value of 0.14 mM pyruvate. Thus, the affinity of pyruvate for LDH was higher under hypoxia than under normoxia, but the maximal turnover capacity decreased.
NA DHJuorescerrce Figure 5 shows .as a typical example the reaction of the NADH fluorcscence to a decrease in the p 0 2 in the superfusion medium. The NADH fluorescence increases whereas the constantly reflected 366-nm light signal indicates the constant light cnergy supply for NADH fluorescence excitation. Variations in the rclative NADH fluorescence due to changes in the glucose or antimycin level in the superfusion medium were analysed. The hypoxia-induced NADH change was set to 100% in each experiment. Table I1 shows that lowering the glucose content to 0.1 g/l markedly oxidized NADH, whereas 10 g/l glucose induced a small change only. Blocking the elcctron transfer in the respiratory chain by antimycin resulted in a significant increase in the NADH fluorescence. O.vgen coilsumptior[ Tablc 111 shows the changes in oxygen consumption of C6 spheroids incubattd for 1 hr under different glucose or amiloridc conditions. Values are given as the difference between control and treated spheroids. Exposure of the spheroids to low glucose for 1 hr led to a substantial increase in oxygen consumption ( a = 0.001). High glucose exposure for 1 hr resulted in a smaller increase in oxygen consumption, which was not significant. Amiloride decreased the oxygen consumption but this change also was not significant.
Growth ciine,s Growth curves were analysed for the C6 spheroids growing in liquid overlay (Fig. 6). Upon reaching a diameter of 500 pm they were randomly divided into 3 groups and thereafter continuously cultured in different media. The controls continued to grow in normal culture medium with 1.0 g/l glucose, reaching approx. 700 pm diameter after about 2 weeks'
2
2
-Y 3
10.0 : 5.0
:
s 0
5
10 15 TIME (DAYS)
20
25
FIGURE6 -Growth curve of C6 spheroids under 1 gil glucose ( O ) ,10 g/l glucose (V)and 0.1 g/l glucose (0).Twelve spheroids
were analysed in each group. Mean values t standard error of the mean are shown. treatment. Those that grew continuously in 10 g/l were not only growth-arrested but started to decrease in size. After 2 weeks they had a diameter of about 400 pm. Those that were given 0.1 gil glucose grew better than the controls since they reached an average diameter of nearly 800 p m after 2 weeks.
Autoradiography After 2 weeks' treatment as above (see Fig. 6) the spheroids were incubated with W T d R for 20 min. As shown in Figure 7 (a,b),no labelled cells were found in the control spheroids. There were, however, several labelled cells and even mitotic cells, especially in the deeper regions of the spheroids that were grown in 0.1 g/l glucose (Fig. 7c,d). Thus, there were cycling cells in these spheroids, in conformity with the improved growth under low glucose conditions. Figure 7 ( e f ) shows that cells under 10 g/l glucose are not labelled but several pyknotic cells can be detected, explaining the inhibition of growth under these conditions. DISCUSSION
Oxygen consumption and lactate production, measured per volume unit and per hour, decreased as a function of spheroid size. This was, in the larger spheroids, probably due to degeneration causing central necrosis. Oxidative metabolism does not lead to a net production of hydrogen ions, while glycolysis, giving lactate, produces, by ATP hydrolysis, an average of 2 hydrogen ions per glucose molecule consumed. Lactate production and intracellular hydrogen ion concentration are therefore directly related (Busa and Nuccitelli, 1984). Several experiments were made in which the glycolysis or the oxidative metabolism were disturbed. It was then found that temporary hypoxia influenced the extracellular pH in the spheroids. However, hardly any
284
ACKER ET AL.
FIGURE7 -Histological sections of C6 spheroids. The spheroids were incubated with 3H-TdR, fixed, embedded in plastic and sectioned. The sections were processed for autoradiography and thereafter stained with hemotoxylin. The situation is depicted (a) and (b) under 1 g/l glucose, (c) and ( d ) under 0.1 g/l glucose, ( e ) and (f, under 10 g/l glucose. 100 pm is given by bars.
hypoxia-induced increase in H+ ion concentration could be measured in the peripheral regions of the spheroids (Fig. 3). This might have been due to a low capacity for increased glycolysis in the outer cell layers, or to fast diffusion of H+ ions out into the culture medium. The hypoxia-induced increase in H + ion concentration was high at the inner cell layers, starting at a depth of approx. 300 bm. The strongest increase was obtained when high conccntrations of glucose were administered in the culture medium. This indicated that there is a lack of glucose in the central areas under hypoxia, when there is a higher demand for glucose due to increased anaerobic glycolysis in the C6 cells, as shown by Ercinska and Silver (1987). A decrease of approx. 60% in the glucose concentration in the innermost part of the spheroids could be directly measured in EMT6 spheroids (Gollner et nl., 1991) meaning that an additional increase in the glucose consumption, as under hypoxia, can only be covered by an increase in the glucose supply. The results also showed that there are no severe diffusion limitations for glucose, in conformity with previous determinations of glucose diffusion coefficients in spheroids (Freyer and Sutherland, 1986; Casciari et nl., 1988; Nederman et nl., 1988). The pH gradients were less stcep when the glucose concentration was decreased (Fig. 2a). Furthermore,
no extra H+ ions were released as a response to hypoxia under low glucose conditions. The pH measurements were in good accordance with the lactate production determinations. Hypoxia increascd the lactate production at both 1.0 and 10.0 g/l glucose while low glucose levels decreased the lactate production. Changes in glucose supply between 1 and 10 g/l under normoxic conditions did not influence the lactatc production, in accordance with the nearly unchanged p H values. Analysis of the LDH activity in Lineweaver-Burk plots showed that the LDH activity was proportional to the pyruvate concentration and that the K,,, values for L D H decreased under hypoxic conditions, i.e. the enzyme became more efficient in converting pyruvate to lactate under hypoxic conditions. LDH exists in 5 diffcrent isoenzymes, which are tetramers of 2 subunits: H and M. The synthesis of different forms of the enzyme from L D H l to LDHS can be influcnced by oxygen changes. In lymphocytes and chicken embryos, chronic hypoxia induces the formation of M types of subunits, which have a higher affinity for pyruvate (Hellung-Larsen and Andersen, 1970). It would bc of interest to investigate, in further experiments, whether the observed changes in affinity and turnover of pyruvate induced by acute hypoxia (as shown in
285
GLUCOSE AND GROWTH RELATIONSHIP IN C6 SPHEROIDS
Fig. 5 ) are due to a :shift in the subunit composition or to other causes. The oxygcn consumption was higher at 0.1 than at 1.0 g/l glucose. meaning that the metabolic pathway was shifted from lactate production to oxidative metabolism so that more energy could be obtained from the Krebs cycle and the electron transport chain. The oxygen consumption also increased somewhat at 10.0 g/l glucose. This increase, however, was statistically not significant. The lower turnover in the glycolytic pathway ‘IS also indicated by the decreased NADH fluorescence under low glucose conditions. The increased oxygen consumption under these conditions could also decrease NADH fluorescence due to a higher turnover in the respiratory chain, since blocking of the respiratory chain by antimycin leads to increased NADH fluorescence (Table 11). This means that the coenzyme NADH in the C6 cells is equally involvcd in the respiratory chain as well as in the glycolytic breakdown of glucose. Amiloride gave extracellular alkalization in the spheroids and a lower p H response at hypoxia. This might be the result of a block of the amiloride-sensitive N a + / H + antiport (Grinstein et ul., 1989), which should give a decreased capacity for release of H + ions from the cells. This might in particular be the case under hypoxia, which highlights the importance of this exchanger to avoid intracellular acidification under hypoxia. The involvement of the lactate/proton symporter in the hypoxiamediated proton release was not investigated due to the high efficacy of the amiloride treatment. Low glucose (0.1 gil) led to an improvement in growth capacity in that the saturation diameter increased. This was probably due to the fact that the extracellular acidification was less dramatic at low glucose concentration. A similar correlation between lowered glucose concentration and increased growth potential has been reported for human thyroid carci) the growth rate noma spheroids (Acker et al., 1 9 8 7 ~whereas
of human glioma U 118 MG spheroids remained unchanged (Acker et al., 1987a) and mouse mammary carcinoma EMT6/Ro as well as human bladder carcinoma MGH-U1 spheroids decreased their growth rate at low glucose levels (Freyer and Sutherland, 1986; Tannock and Kopelyan, 1986). High glucose levels (10.0 g/l) seemed toxic to the spheroids because they started to decrease in size. This may have been the result of excessive extracellular acidification. Decreased growth capacity and degeneration due to hyperglycemia have been reported for tumors in vivo (Wike-Hooley et al., 1984). This study shows that C6 rat glioma spheroids normally produce large amounts of lactate and that they can change their metabolism depending on the glucose supply. For example, they changed from LDH-mediated lactate production to more efficient oxidative metabolism in response to decreased glucose levels. These changes also imposed modifications in the H+ ion release and, consequently, in the extracellular pH. Such changes might modify the response to different therapeutic agents. For example, increased acidosis might sensitize for hyperthermia treatment but at the same time protect against radiation (Wike-Hooley et al., 1984). Metabolic changes can possibly be exploited in the development of new anti-tumor therapeutic principles. ACKNOWLEDGEMENTS
We thank the staffs of the Department of Physical Biology, Uppsala University, and of the Max-Planck-Institut fur Systemphysiologie, Dortmund, for assistance in cell culturing and microelectrode measurements. This work was financially supported by the National Board of Laboratory Animals 88-22, Swedish National Board for Technical Development 8605103P, Swedish Cancer Society 1079-B90-01XA, Max-PlanckGesellschaft, Munich, the Hermann and Lilly Schilling Stiftung, Essen, and the BMFT, Bonn.
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