Influence of Glucose and Buffer Capacity in the - Cancer Research

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[CANCER RESEARCH 47, 3504-3508, July 1, 1987]

Influence of Glucose and Buffer Capacity in the Culture Medium on Growth and pH in Spheroids of Human Thyroid Carcinoma and Human Glioma Origin1 H. Acker,2 J. Carlsson, G. Holtermann, T. Nederman, and T. Nylén Max Planck InstituÃ-fitr Systemphysiologie, Rheinlanddamm 201, D-4600 Donmund 1, Federal Republic of Germany [H. A., G. H.] and National Defense Research Institute, S-90I82, Umea, and Department of Physical Biology, Gustaf Werner Institute, S-75121 Uppsala, Sweden [J. C., Th. N., T. N.]

ABSTRACT pH gradients were measured with microelectrodes in cellular spheroids of human glioma (U-118 MG) and thyroid carcinoma (I Hh7) origin. pH decreased outside the spheroids and then continuously decreased when the electrode was moved through the spheroid towards the center. The lowest pH values inside the spheroids were in the range 6.7-6.8 when grown under standard conditions with FIO medium. When medium with stronger buffer capacity (Dulbecco's minimum essential medium or Locke's) was used, the gradients in both types of spheroids were less steep. Less steep pH gradients were also obtained in both types of spheroids when the concentration of glucose was lowered to 0.1 g/liter in the medium. In the case of 111h7 spheroids the low pH inside the spheroids under standard culture conditions seemed toxic because the growth rate increased when the spheroids were cultured under conditions giving higher central pH values (high buffer capacity or low glucose concentration). No such growth-stimulating effects could be seen for the U-118 MG spheroids. The growth rate of both types of spheroids was retarded when they were grown in medium with very high glucose concentration (10 g/liter). The thickness of the viable cell layer increased for H I h7 spheroids when the concentration of glucose was lowered to 0.1 g/liter. A decrease in the thickness of the viable layer of U-118 MG spheroids was observed when they were grown at a high glucose concen tration (10 g/liter).

INTRODUCTION Cell spheroids have become an often used model of poorly vascularized tumor tissue in different studies in cancer research (1). It has been clearly shown that spheroids have radial prolif eration and pO2 gradients (2-6). It is reasonable to assume that spheroids also have radial pH gradients. This has been dem onstrated for two types of spheroids in a previous preliminary report from our laboratory (7). It has recently been shown that increases in intracellular pH are closely related to the stimulus of growth factors (8) and that these changes also are related to the onset of DNA synthesis and proliferation (9,10). Intracellular pH seems to be regulated in relation to extracellular pH with Na+-H+ and C1~-HCO3~ exchange systems ( 11,12). It is therefore of interest to measure extracellular pH inside cell spheroids and to analyze to what extent the pH values correlate with proliferation disturbances and induction of degenerative changes. The possibilities for the tumor cells to withstand, for example, low pH might vary between different types of cells and also between cells in differ ent areas of the spheroids. The purpose of the present study was to investigate the influence of glucose and buffer capacity in the culture medium on growth and pH gradients in two types of spheroids. A detailed knowledge about growth and metabolic gradients in Received 7/15/86; revised 12/17/86, 4/1/87; accepted 4/2/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was financially supported by the Max-Planck-Gesellschaft, München;the Herrmann und Lilly Schilling Stiftung, Essen; BMFT Grant 0318908A, Bonn; the Swedish Cancer Society; and the Swedish National Defense Research Institute. 2 To whom requests for reprints should be addressed, at the Max Planck Institut Tür Systemphysiologie, Rheinlanddamm 201, D-4600 Donmund I, Fed eral Republic of Germany.

the spheroid model might give valuable knowledge for the understanding of tumor growth. We chose human glioma U118 MG and human thyroid carcinoma HTh7 spheroids since these types have been previously studied regarding morphology, growth, and p().. gradients (4). Previous studies on transplanted tumors have shown that tumor tissue is only slightly more acidic than normal tissue (13-15), but that the tumor tissue can selectively be made more acidic by hyperglycemia (13, 16, 17). Some changes in the concentration of glucose in the culture medium (both hyperand hypoglycemia) were made in this study to evaluate the effects in the spheroid system. MATERIALS

AND

METHODS

Cell Culture Two human tumor cell lines were used in this study; the human glioma cell line U-118 MG and the human thyroid cancer cell line HTh7. Both these cell lines are usually grown as monolayer cultures, but have previously been studied when growing as spheroids (4). The diameter of the studied spheroids varied in the range of 300 to 1000 urn. Each spheroid was allowed to attach to a round cover slip (13-mm diameter; Lux Scientific Corporation, Munich, FRG) for 12-20 h before the microelectrode measurements. Only cells at the lower end of the spheroids attached to the cover slips. The cell organization inside the spheroids was not changed as was seen in histological sections (3, 4). The culture medium was Ham's FIO with 10% fetal bovine serum supplemented with i.-giùtamine (2 HIM), penicillin (100 U/ml), and streptomycin (100 jig/ml) (Flow Laboratories, Bonn, FRG). For chronic cultivation of spheroids under different glucose conditions, the glucose concentration of the FIO medium was changed between 0.1 g/ liter, 1 g/liter, and 10 g/liter (0.55, 5.5, and 55 HIM). The size of the spheroids was measured before, during, and after incubation in the different glucose concentrations. The procedure to measure the volume of individual spheroids has previously been described (3, 4). Most of the microelectrode measurements were carried out in FIO medium without serum, since the serum caused too much foam during gas equilibration. Furthermore, DMEM3 (Flow Laboratories) and Locke's solution (NaCl, 128 HIM;KC1, 5.6 HIM;CaCl2, 2.1 HIM;D-glucose, 5.5 mM; NaHCO3, 10 HIM;4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, 7 mM) having other pH buffer properties were also used during the microelectrode measurements. Microelectrodes Double-barreled pH-sensitive microelectrodes with a tip diameter of about 3 /im, manufactured for extracellular measurements, were used. The electrodes have previously been described in detail (18,19). Briefly, the pH channel was made by introducing a thin-pulled pH glass (Ingold). The pH glass was then glued to the inner wall of the channel with epoxy glue, building a recess of about 40 urn from the end of the channel. The second channel was filled with magnesium acetate (1 mM) for registration of potential changes. This channel was used to correct the signal from the pH channel, so that only changes due to changed H ' ion activities were recorded. It is important to measure bioelectrical potentials directly together with pH in the tissue; since each bioelec trical potential also is picked up by the pH electrode, some mV potential changes can influence the pH reading. This is electronically compen3 The abbreviation used is: DMEM, Dulbecco's minimum essential medium.

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sated for in our case. The potentials measured in the magnesium acetate channel also helped to judge the position at which the electrode hit the spheroid surface (4, 18). The mean response time of the pH electrode was about 15 s. The steepness per 10-fold change of the II* ion concentration varied between individual electrodes in the 47-58-mV range. Experimental Setup. The cover slip with the attached spheroids was transferred to a perfusion chamber, as described in detail previously (3, 4, 18). Culture medium without serum (10 ml/min) was circulating through the perfusion chamber. The flow rate of the medium was highest in the upper pan of the chamber while the medium flow was close to zero near the bottom. The spheroids were positioned at the bottom of the chamber (see Ref. 18 for details). The oxygen tension in the medium (about 140 mm Hg), the pH (7.3-7.4), and the temperature (37'C), were in the upper part of the chamber (in the flowing medium) continuously controlled. The electrode measurements were made in the chamber under microscopic control. The movement of the microelectrode was electronically measured through a potentiometer attached to the manipulator system (David Kopf Instruments, Munich, FRG) (3). The pH microelectrode signal and the position of the electrode together with the oxygen tension and the pH in the medium were continuously recorded on a multipen recorder (Rikadenki, Freiburg, FRG). Microelectrode Measurements. The pH microelectrode, connected to a differential preamplifier (input resistance, 10I50), was calibrated for each trial in the following way: first it was calibrated with three different phosphate buffers (pH: 6.8, 7.2, and 7.6) at room temperature. The pH microelectrode was then led into the superfusion medium and again calibrated at a bath temperature of 37'C by changing the p( 'O: in the medium. This was done before and after each measurement. In some cases a drift of the calibration levels could be observed. In these cases a linear drift was assumed throughout the measurements. The drift of the electrodes was less than 2%/h. After calibration, the electrode was positioned in an accurate relation to the spheroid. This positioning was made by using two independent optical systems working along different axes. The electrode was adjusted to hit the spheroid nearly from above. The electrode had only 30°deviation from the vertical axis (4, 18). After final adjustment of the position, the electrode was moved by the hydraulic microdrive in steps of 25-50 ^m 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-barreled 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 measurement of the spheroid diameter, careful positioning of the electrode tip, and continuous reading of the electrode position on the multichannel re corder during the electrode insertion.

MEDIUM

spheroid the electrode penetrated extracellular matrix, cyto plasm, and cell nuclei. The electrode might have passed through the center of some cells, while others were probably only touched upon. Some cells probably ruptured immediately, while others were intact for longer periods. The recordings must be considered as mean values for a local region of about one or two cell diameters in size. It is supposed that the pH in such a region is measured in a mixture of cytosol and extracellular matrix. Therefore, we call the measured pH values "extracel lular values" to clearly state that true intracellular values are not measured. Fig. 1 shows a typical extracellular pH microelectrode regis tration in an U-l 18 MG spheroid. A pH gradient of about 0.3 pH units could be measured. It can be seen how the position of hit was determined. When the electrode first touched the sphe roid surface a very small prepotential was registered (Fig. 1; arrow). This prepotential indicated the position for the electrode when it hit the outer surface of the spheroid. Upon penetration of the spheroid, usually negative potentials were seen. This occurred, when the electrode penetrated cells which had a membrane potential. The position at which the negative signals were obtained varied from case to case. The pH readings were stable and seemed neither to be significantly influenced by the electrode penetration (repeated penetrations along the same track gave similar profiles) nor by any medium leakage along the electrode track (the pH readings in each position were stable for several minutes). pH gradients were constructed from the registered pH values and the electrode position readings (Fig. 2). The position of hit was taken as the penetration depth of O ¿¿m. The automatically monitored electrode position allowed a further determination of the penetration depth. Fig. 2 gives examples of several pH gradients measured in one U-l 18 MG spheroid. Note that the pH started to decrease already in the medium outside the spheroids. The pH differences between medium pH and the pH value 200 urn inside the spheroids was called ApH. All ApH values measured in U-l 18 MG and HTh7 spheroids which were longterm cultured in liquid overlay culture in FIO medium [normal pH

Autoradiography

Most of the spheroids were incubated in medium containing ('111 thymidine (l /iCi/ml; Radiochemien I Center, Amersharn, England) for l h after the measurements. These spheroids were fixed in glutaric aldehyde (2% glutaric aldehyde in 0.1 M sodium cacodylate and 0.1% sucrose, pH adjusted to 7.2 with HC1) for 1 h. The spheroids were then stored in 70% ethanol. Preparation for light microscopy, autoradiography, and evaluation of labeling index were carried out as described previously (2, 5). Four or five sections from each of four spheroids were analyzed in each group. The thickness of the viable rim was measured in an ocular grid. Growth Curves

The diameters of individual spheroids were repeatedly measured as previously described (5). Volumes were calculated and growth curves were drawn for spheroids cultured in medium with different buffer capacity or cultured in medium containing different concentrations of glucose. Twelve spheroids were analyzed in each group.

Dcpttt of puncture yum

0-|

RESULTS The pH electrode had a tip diameter of about 3 urn. With this tip diameter it is not possible to discriminate between intraand extracellular pH values. During the passage through the

Fig. 1. Original registration of a pH gradient in a U-l 18 MG spheroid (diameter, 590 urn ). From lop to bottom, course of the pH signal, the bioelectrical potential, and the electrical signal of the hydraulic microdrive giving the depth of puncture. Arrow, electrode hits spheroid surface.

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i"" •

7.0 --

H

1-

B-

-2«

-100

0

100

200

300

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experiments U-l 18 MG and HTh? spheroids with varying sizes were cultured in Ham's FIO medium for 4 days with different

A-

7J

IN CULTURE

400

DISTANCE FROM THE SURFACE Uml

Fig. 2. pH gradients in a 650 ,mi U-l 18 MG glioma spheroid under different buffer and pH conditions. The measurements sianoti with a buffer capacity of 11.3 niM/pll and a medium pH of about 7.4 (•).Then the buffer capacity was changed to 4 HIM pi I with a medium pH of first 7.3 (x) and then 7.6 (®).pH changes in the culture medium were introduced by varying the percentage of CO2 in the gas phase. The buffer capacity was then shifted back 11.3 mM/pH with a medium pH of about 7.4 (A). The medium pH, under these buffer conditions (11.3 mM/pH), finally changed to 7.7 (•).The depth of puncture is given in micrometers on the x axis. The measured curves are shown while the same data is replotted (.I) and as the change ¡npH inside the spheroids in relation to the pH of the culture medium (Hi. Table 1 bpH values in U-l 18MG ana HTh 7 spheroids as a function of the buffer capacity of the culture medium ApH is defìnedas the culture medium pH minus the pH value 200 i/m inside the spheroids. Measurements were made on U-l 18 MG spheroids and HTh 7 spheroids under different buffer capacity conditions (4.0, 5.7, and 11.3 mM/pH). Data for both small and large spheroids were pooled. The diameters were in the range of 400-800 pm.

glucose concentrations (1 g/liter, 0.1 g/liter, or 10 g/liter). After this period, the spheroids were transferred to the perfu sion chamber and investigated regarding pH gradients in the same type of culture medium in which they were precultured. All results from these measurements are summarized in Table 2. The ApH was much lower when the spheroids were cultured 4 days in glucose (0.1 g/liter) than when they were cultured the corresponding time in glucose (1 or 10 g/liter). No significant differences were seen in ApH between the 1 and 10 g/liter groups. The ApH was somewhat higher for the big spheroids than for the corresponding groups of small spheroids. Table 3 summarizes the results from measurements on four spheroids exposed to acute changes in glucose concentration. All these spheroids were long-term cultured in liquid overlay culture in normal FIO medium (glucose, 1 g/liter) before the experiments. The spheroids were then transferred to the per fusion chamber and the penetration with the microelectrode started. Leaving the electrode 200 j¿minside the spheroid tissue the glucose concentration of the superfusion medium was changed from 1.0 to 0.1 g/liter for the two first spheroids. The ApH decreased significantly for both the HTh? and the U-l 18 MG spheroid under low glucose conditions within about 20 min. When the medium was changed back to 1.0 g/liter the ApH increased to a value close to the original value. However, the same types of spheroids did not show any significant changes in ApH when changes were made between glucose, 10 g/liter, 1 g/liter, and then back to glucose, 10 g/liter. Table 2 &pH values for glioma U-l 18 Mg and thyroid HTh7 spheroids grown for 4 days in FIO medium with different concentrations of glucose Small (300-600 urn) and large (600-900 um) spheroids were analyzed.

Spheroid typeGlioma 10.29niM/pl Buffer capacityU-118MG mM/pH0.45 ±0.08" ±0.06 ±0.03 0.4 + 0.12ApH5.7 0.27 ±0.0511.3mM/pH0.27 ±0.03' HTh74.0 0.18 Mean ±SD.

diameter (Mm)300-600"g/Iiter)0.16 (0.1

(l.Og/liter)0.37 (10.0g/liter)0.41(1)

±0.04 (7)*

glucose ( 1.0 g/liter) and buffer capacity (4 m M/pH )|. transferred to the perfusion chamber, and after 30 min measured in FIO (4 mM/pH), Locke's solution (5.7 mM/pH), or DMEM (11.3 HIM/ pH) are summarized in Table 1. Both types of spheroids showed high values of ApH under low buffer capacity conditions. The values became smaller when higher buffer capacities were used. Measurements were also made in single spheroids during changes in the medium buffer capacity and some changes of the medium pH. The measurements shown in Fig. 2A on a 650-^m U-l 18 MG spheroid gave a total pH decrease of about 0.250.30 units inside the spheroid when the buffer capacity was 11.3 mM/pH. The total pH decrease amounted to about 0.45-0.50 pH units when the buffer capacity was changed to 4 mM/pH. The corresponding pH changes in relation to the culture me dium pH is shown in Fig. IB. These measurements clearly show that the extracellular pH in spheroids is a function of the buffer capacity of the culture medium. Similar measurements giving essentially the same results were also made in a HTh7 spheroid (data not shown). The influence of different glucose concentrations on the pH gradients were investigated using both chronic (during the normal liquid overlay culture the spheroids were exposed to increased or decreased glucose concentrations for several days) and acute (the exposures were made directly in the perfusion chamber for only 20-30 min) experiments. In the chronic

±0.10 U-118MG (5) 600-900 Glioma 0.24 ±0.05 0.46 ±0.14 0.42 ±0.06 U-l 18 MG (4) (6) (4)NAC0.49 300-600 Thyroid 0.20 ±0.07 0.40 ±0.11 HTh7 (4) (6) 600-900Glucose Thyroid 0.24 ±0.08 0.45 ±0.08 ±0.08 HTh7Spheroid (2)ApHGlucose (5)Glucose (5) Mean ±SD are given in all cases except for small U-l 18 MG spheroids grown in glucose, 10.0 g/liter, where only one spheroid was measured and for big HTh7 spheroids grown in glucose, 0.1 g/liter, where only two spheroids were measured. In the latter case the maximal error was instead given. Numbers in parentheses, measured spheroids in each point. c NA, not analyzed. Table 3 The effect of acute glucose concentration changes on the pH gradient in U-l 18 MG and HTh? spheroids For each case the glucose concentration, in the superfusion medium, the pH in the superfusion medium, the pH 200 pm inside the spheroid, the pH difference between medium pH and 200 urn inside (ApH), as well as the time in relation to the first measurement, is given.

anddiameterU-118Mg, Spheroid type ^mHTh7,

520

»imU-l500 urnHTh7, 18, 900

900 *mGlucose(g/liter)10.1110.111011010110pHmedium7.457.407.467.457.417.467.377

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It was investigated, in liquid overlay culture, whether varia tions of the glucose concentration had any influence on the growth rate and proliferation gradient of both types of sphe roids. Figs. 3 and 4 show the obtained growth curves and proliferation gradients. It is to be seen, that cultivation of the spheroids under glucose, 0.1 g/liter, 1 g/liter, and 10 g/liter, condition had nearly no influence on the growth rate during the first 4 days. However, longer cultivation (up to 2 weeks) showed for both types of spheroids a distinct slowing of the growth rate under glucose, 10.0 g/liter, conditions. The U-l 18 MG spheroids cultivated in a concentration of glucose, 0.1 g/ liter, had about the same growth curve as the spheroids culti vated in 1.0 g/liter (Fig. 3, A and B). However, in the case of HTh7 spheroids the situation was different (Fig. 3, C and D). These spheroids had faster growth when grown in glucose, 0.1 g/liter, than when grown in 1.0 g/liter. In addition, the labeling

3.0

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10 Time, days

10 Time, days

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Fig. 3. Growth curves for U-l 18 MG (A and B) and HTh7 (Cand D) spheroids under different glucose conditions [0.1 g/liter (O). 1 g/liter (•).and 10 g/liter (A)]. The relative volume is drawn on the y axis and the time in days on the x axis. Points, mean ±SD from measurements on 12 spheroids. The results are separated for spheroids with different diameters at the beginning of the measure ments. A, 400-500 /im; B, 300-400 /im; C, 350-450 ^m; D, 250-350 ^m.

30.0

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index (Fig. 4B) and the thickness of the viable layer (Table 4) increased, when the HTh7 spheroids were grown for 2 weeks in glucose, 0.1 g/liter. No such effect of glucose, 0.1 g/liter, was seen in the U-l 18 MG spheroids (Fig. 4/1 and Table 4). In comparison to the low glucose conditions the high glucose concentration (10 g/liter) gave a reduced labeling index in the HTh7 spheroids and a reduced thickness of the viable layer in both spheroid types. The growth rate was measured for 12 HTh7 spheroids (di ameter, 400-500 jim) grown in DMEM medium with high buffer capacity (11.3 mM/pH) and for 12 other HTh7 spheroids of the same size but growing in FIO medium with low buffer capacity (4 mM/pH). The doubling times were in the DMEM medium 6 ±1 days and in the FIO medium 14 ±1 days. DISCUSSION pH measurements in tumor tissue in vivo have been carried out by several investigators such as Vaupel et al. (20, 21), and J alide et al. (13, 14). Such measurements have recently been reviewed by Wike-Hooley et al. (22) and from this review it is clear that the range of measured pH values in the spheroids are in the same range as those which have been reported for tissue pH values in tumors. Variations in the buffer capacity of the superfusion medium had a significant influence on the pH gradient in the spheroids. This indicated that the buffer capacity inside the spheroids (in the extracellular matrix) was to a large extent determined by the culture medium. Carbonate and other buffering ions prob ably diffuse into the spheroids and exert their buffer capacity in the extracellular spaces. It is likely that this situation is similar to the situation in vivo, where buffering substances from the vascular system can penetrate into the extracellular spaces. However, no in vivo measurements have, to the authors' knowl

5.0

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edge, been made regarding this. The pH gradients became less steep when the glucose content of the medium was decreased from 1.0 to 0.1 g/liter. This favors the idea that the pH gradients in spheroids are mostly due to a lactate production. However, it is possible that also other factors than glucose and lactate (e.g., accumulation of other catabolic products or lack of other nutrients) might influence the pH gradient. The results shown in Figs. 3 and 4 and Table 4 indicate a relationship between pH and growth. Inhibition of acidosis by decreasing the concentration of glucose from 1.0 to 0.1 g/liter or introduction of medium with high buffer capacity gave an increased growth rate and an increasing number of proliferative cells in the HTh7 spheroids. Considering the glucose changes from 1.0 to 0.1 g/liter our results are in contrast to results recently reported for two other spheroid systems. Freyer et al. (23, 24) reported for EMT6/Ro spheroids that the growth rate, Table 4 Thickness of the viable rimfor human thyroid carcinoma (HTh7) and human glioma (U-l IS MG) cell spheroids as a function of the concentration of glucose in the culture medium Evaluations were made on the spheroids which were measured in Fig. 3. Spheroids were cultured for about 2 weeks in their different media and after the growth curves were measured they were fixed and stained. The thickness of the viable rim was in each point analyzed in 12 sections (four central sections from each of three spheroids). Mean values and standard deviations are given. In the case of U-l 18 MG spheroids there was no sharp border between the viable zone and the necrotic zone. In this case the viable rim was defined as the zone free of pycnotic nuclei.

400 O Distance from the surface, i

Fig. 4. Thymidine labeling index (y axis) versus the distance from the spheroid surface in micrometers (x axis) in spheroids grown for 14 days under different glucose conditions [0.1 g/liter (O), l g/liter (•),and 10 g/liter (A)]. The evalua tions were made on the spheroids which were measured in Fig. 3. They were incubated with thymidine and fixed at day 14. Four or five sections from each of four spheroids were analyzed in each group. A, results for U-l 18 MG spheroids; B, results for HTh7 spheroids.

(¡an)Spheroid typeHTh7 U-118MGGlucose

Thickness of the viable cell layer (0.1 g/liter)286

(1.0 g/liter)150

(10.0 g/liter)142

±19 ±14 ±16 443 ±21Glucose 449 ±14Glucose 310 ±28

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the thickness of the viable cell layer, and the number of cells in S-phase decreased when the glucose concentration in the me dium decreased from 1 to 0.15 g/liter. Using the EMT6/Ro spheroids it has also been reported that oxygen consumption decreases when the glucose concentration is decreased (25). Tannock and Kopelyan (26, 27) showed that for human bladder cancer MGH-U1 spheroids growth rate and thickness of viable cell layer decreased when the glucose concentration in the medium decreased from 1.0 to 0.1 g/liter. In our case the human thyroid cancer HTh7 spheroids increased both their growth rate and the number of S-phase cells. On the other hand, the growth pattern of the human glioma U-118 MG spheroids was neither stimulated nor inhibited after such changes. The EMT6/Ro and MGH-U1 spheroids were cultured in spinner flasks while the U-118 MG and HTh7 spheroids were cultured in liquid overlay culture. It is not known whether the culture method influences the response to hypoglycemia. It is possible that acidosis. nutrient depletion, and catabolic clear ance are different in the unstirred liquid overlay culture than in spinner flask cultures. However, the different responses to hypoglycemia are probably due to differences between the cell types. EMTo/Ro cells are of mouse origin and probably have a faster metabolism (high growth rate, high oxygen consumption, etc.) and therefore probably need high levels of glucose for optimal growth. Three different responses to hypoglycemia were observed for the three human tumor cells (U-118 MG, HTh7, and MGH-U1). It is possible that the different responses reflect differences in phenotype. Increasing the glucose concentration 10-fold above the nor mal level in our experiments did not have a significant influence on the pH gradients although it retarded the growth rate for both types of spheroids. It seems likely that the high level of glucose is toxic (see Figs. 3-4). Recent studies have indicated that DNA synthesis and cell proliferation is inhibited at intracellular pH values below 7.2 (9, 10). The pH values measured in this study are to be consid ered as extracellular pH values. The electrode tip diameter was 3 firn, which is too big to allow intracellular measurements. It is difficult to extrapolate from extracellular pH values to cytosolic pH values. From some investigations it is shown that the cytosolic pH is regulated in relation to the extracellular pH by II ion extrusion (8, 11, 28). Investigations from isolated rat diaphragm, isolated perfused turtle heart, and ascites tumor cells suggest that the cytosolic pH is fairly stable in the range of extracellular pH values of 7.2 to 7.5, but either above or below this range the cytosolic pH changes significantly. In these cases an extracellular pH below 6.6-6.7 gave intracellular pH values nearly equal to the extracellular values (29). This leads to the assumption that also the cytosolic pH decreases along the pH gradient in the spheroids. Thus, it is reasonable to assume that cells deep inside the spheroids suffer from a low cytosolic pH leading to an inhibition of the cell proliferation. This proliferative inhibition could (at least for HTh7 spheroids) be overcome by low glucose or high buffer capacity in the culture medium which gave more physiological pH values inside the spheroids. Future experiments are needed to clarify cytosolic pH regulation in relation to the extracellular pH and the importance of cytosolic pH for tumor cell growth. ACKNOWLEDGMENTS The authors thank the staffs at the Max-Planck-Institut für System physiologie, Dortmund; the Department of Radiobiology, National

Defense Research Institute, Umea; and the Department of Physical Biology, Gustaf Werner Institut, Uppsala, for assistance in cell culturing and microelectrode measurements.

REFERENCES 1. Acker, II., Carlsson, J., Durand, R., and Sutherland, R. M. (eds.) Spheroids in Cancer Research, Berlin-Heidelberg-New York-Tokyo: Springer Verlag, 1984. 2. Carlsson, J. A proliferation gradient in three-dimensional colonies of culture human glioma cells. Int. J. Cancer, 20:129-136, 1977. 3. Carlsson, J., Stalnacke, C-G., Acker, H., Haji-Karim, M., Nilsson, S., and Larsson, B. The influence of oxygen on viability and proliferation in cellular spheroids. Int. J. Radiât.Biol. Phys., 5:2011-2020, 1979. 4. Carlsson, J., and Acker, H. Influence of the oxygen pressure in the culture medium on the oxygénationof different types of nuil licci lula r spheroids. Int. J. Radiât.Biol. Phys., //: 535-546, 1985. 5. Haji-Karim, M., and Carlsson, J. Proliferation and viability in cellular spheroids of human origin. Cancer Res., 38:1457-1464, 1978. 6. MOller-Klieser, W. F., and Sutherland, R. M. Oxygen tensions in multiceli spheroids of two cell lines. Br. J. Cancer, 45: 256-264, 1982. 7. Acker, H., Holtermann, (.;., and Carlsson, J. Microelectrode measurements of pH in cellular spheroids. Pfluegers Arch. Eur. J. Physiol. 394:199, 1982. 8. Moolenaar, W. H. Effects of growth factors on intracellular pH regulation. Ann. Rev. Physiol., 48: 363-376, 1986. 9. Pouysségur,J., Sardet, C, Franchi, A., L'Allemain, G., and Paris, S. A specific mutation abolishing Na'/l r antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc. Nati. Acad. Sci. USA, XI: 4833-4837, 1984. 10. L'Allemain, G. L., Franchi, A., Cragoe, E., and Pouysségur,J. Blockade of the Na'/H* antiport abolishes growth factor induced DNA synthesis in fibroblasts. J. Biol. Chem., 259:4314-4319, 1984. 11. Pouysségur,J. The growth factor activatable Na*/H* exchange system: a genetic approach, Trends Biochem. Sci., 2:453-455, 1985. 12. Roos, A., and Boron, W. F. Intracellular pH. Physiol. Rev., 61: 296-434, 1981. 13. Jähde, E., and Rajewski, M. F. Tumor-selective mollification of cellular microenvironment in vivo: effect of glucose infusion on the pH in normal and malignant rat tissues. Cancer Res., 42: 1505-1512, 1982. 14. Jähde,E., Rajewsky, M. F., and Baumgärtl,H. pH distributions in trans planted neural tumors and normal tissues of BDIX rats as measured with pH microelectrodes. Cancer Res., 42:1498-1504, 1982. 15. Ardenne, v. M., Reitnauer, P. G., Rohde, K., and Westmeyer, H. In vivo pHmessungen in Krebs-mikrometastasen bei optimierter Übersäuerung. Z. Na turforsch., 24:1610-1619, 1969. 16. Voegtlin, C., Fitch, R. H., Kahler, H., Johnson, J. M., and Thompson, J. W. The influence of parenteral administration of certain sugars on the pH of malignant tumors. Nati. Inst. Health Bull., 164:1-14, 1935. 17. Rauen, H. M., Friedrich, M., and Norpoth, K. Messungen zur manipulation der glucose-abhängigen tumorübersäuerungin vivo. Z. Naturforsch., 23: 1461-1475, 1968. 18. Acker, H., Holtermann, G., Carlsson, J., and Nederman, T. Methodological aspects of microelectrode measurements in cellular spheroids. Adv. Exp. Med. Biol., 759:445-451, 1983. 19. Delpiano, M. A., and Acker, J. Extracellular pH changes in the reperfused cat carotid body during hypoxia and hypercapnia. Brain Res., 342:273-280, 1985. 20. Vaupel, P. W., Frinak, S., and Bicher, H. I. Heterogeneous oxygen partial pressure and pH distribution in C3H mouse mammary adenocarcinoma. Cancer Res., 41: 2008-2013, 1981. 21. Vaupel, P., Müller-Klieser,W., Otte, J., Manz, R., and Kallinowski, F. Blood flow, tissue oxygénation,and pH distribution in malignant tumours upon localized hyperthermia. Strahlentherapie, 759: 73-81, 1983. 22. Wike-Hooley, J. I... Haveman, J., and Reinhold, H. S. The relevance of tumour pH to the treatment of malignant disease. Radiother. Oncol., 2:343366, 1984. 23. Freyer, J. P., and Sutherland, R. M. Regulation of growth saturation and development of necrosis in EMT6/Ro multicellular spheroids by the glucose and oxygen supply. Cancer Res., 46: 3504-3512, 1986. 24. Freyer, J. P., and Sutherland, R. M. Proliferative and clonogenic heteroge neity of cells from F.MTfi/Ro multicellular spheroids induced by the glucose and oxygen supply. Cancer Res., 46: 3513-3520,1986. 25. Mueller-Klieser, W., Freyer, J. P., and Sutherland, R. M. Influence of glucose and oxygen supply conditions on the oxygénationof multicellular spheroids. Br. J. Cancer, 53: 345-353, 1986. 26. 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. Cancer Res., 46: 3105-3110,1986. 27. Tannock, I. F., and Kopelyan, I. Variation of pO2 in the growth medium of spheroids: interaction with glucose to influence spheroid growth and necrosis. Br. J. Cancer, 53: 823-827, 1986. 28. Busa, W. B., and Nucitelli, R. Metabolic regulation via intracellular pH. Am. J. Physiol. 24: R409-R438, 1984. 29. Wadell, J. W., and Bates, R. G. Intracellular pH. Physiol. Rev., 49: 285329, 1969.

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Influence of Glucose and Buffer Capacity in the Culture Medium on Growth and pH in Spheroids of Human Thyroid Carcinoma and Human Glioma Origin H. Acker, J. Carlsson, G. Holtermann, et al. Cancer Res 1987;47:3504-3508.

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Influence of Glucose and Buffer Capacity in the - Cancer Research

[CANCER RESEARCH 47, 3504-3508, July 1, 1987] Influence of Glucose and Buffer Capacity in the Culture Medium on Growth and pH in Spheroids of Human T...

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