(CANCER RESEARCH 46, 3105-3110, June 19861

ABSTRACF

Spheroids derived from the human bladder cancer cell line MGH-U1were initiated in spinner culture and then transferred to multiwell plateswhich contained medium with varying concentrations of glucose andpyruvate. Growth of individual spheroids was monitored, and at differenttimes after transfer spheroids were sectionedand the diameter of thenecrotic center and the thickness of the viable nm were determined. Innormal mediumcontainingglucose(1 g/liter, 5.5 mM), spheroiddiameterincreasedlinearly with time, growing from —400@mto —@12®gsmin 8days, and most spheroids did not develop central necrosis. Increase inglucoseconcentration up to 5 g/Iiter had no effect on spheroid growth.Lower glucoseconcentrationdecreasedthe rate of spheroid growth, butlarge effects wereobservedonly at glucoseconcentrationlower than 100mg/liter. Spheroidsdevelopedcentral necrosisat 2—4daysafter transferto glucose-deficient medium, and the diameter of the necrotic centerincreased more rapidly than the diameter of the spheroid. There was anapproximately linear relationship betweenthickness of the viable rim in5—6-dayspheroids and glucose concentration in the range of zero (rimthickness, -@450gim) to 500 mg/liter (rim thickness, @-400aim). Thepresenceor absenceof pyruvate (110 mgfliter) in the medium had noeffect on spheroidgrowth or formation of necrosis.Theseresults suggestthat limited penetration of glucose may be one of the factors thatcontribute to cell death in solid tumors.

INTRODUCI1ON

Cell death and necrosis occur commonly in solid tumors, butthe mechanisms which lead to cell death remain largely unknown (1). In several types of human and rodent tumors theedge of a region of necrosis has been observed to be parallel toa neighboring blood vessel, leading to “cords―of tumor cellswith a central blood vessel and surrounding necrosis; alternatively, tumor nodules may occur with surrounding stroma andcentral necrosis (2—4).These structures imply that limited diffusion of essential nutrients from tumor blood vessels or theaccumulation of toxic catabolites may be involved in the causation of cell death. The distance between a blood vessel and aneighboring edge of necrotic tissue has been reported to be inthe range of 100—200tim. These distances are consistent withthe expected diffusion distances for oxygen (2—5)and suggestthat hypoxia may be involved in the causation ofnecrosis. Also,tumors were observed to grow more slowly and there was adecrease in the radius of tumor cords when animals were placedin a hypoxic environment (6). However, cells in tissue culturecan survive hypoxia for long periods if other nutrients arepresent at physiological concentration. Thus limited supply ofseveral other nutrients probably contributes to the process ofnecrosis.

The complex anatomy of tumor blood vessels and the physiological regulation ofcomponents of the blood make it difficultto study the relationship between the concentration ofany given

Received6/I 1/85; revised I 1/6/85, 2/1 3/86; accepted2/17/86.The costs of publication of this article were defrayed in part by the payment

of pagecharges.This articlemustthereforebeherebymarkedadvertisementinaccordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

â€S̃upported by research grants CA 29526 and CA 36913 from the National

Cancer Institute, NIH, and by a grant from the National Cancer Institute ofCanada.

2To whom requestsfor reprints should be addressed.

nutrient in a blood vessel and the process of cell death inneighboring tumor cells. Spheroids are multicellular aggregatesoftumor cells (7, 8), which may be propagated in tissue culture.Spheroids resemble tumor nodules since they may developcentral necrosis, have a decreasing gradient of cell proliferationfrom the periphery, and form tight junctions between cells.They provide a useful model for studying the penetration ofnutrients into solid tissue and their influence on central necrosis(9—12),although they cannot model effects that are influencedby blood flow in vivo. In the present study we have varied theconcentration of glucose and pyruvate in medium surroundingspheroids derived from a human bladder cancer cell line todetermine the influence ofthese metabolites on spheroid growthand formation of necrosis.

MATERIALS AND METhODS

Cell Culture. The MGH-U1 cell line was derived originally from apatient with bladder cancer and is of the same origin as cell linesdesignated U and T24 (13) which have been shown to express anactivated Ha-ras oncogene(14). The cell line was kindly provided to usby Dr. G. Prout and colleagues, Massachusetts General Hospital,Boston, MA. MGH-Ul cells will grow as a monolayer on plastic andwill also form colonies in agar and xenografts in immunodeprived mice(15). We have confirmed the identity of the cells by the presence ofmarker chromosomes in their karyotype and by isoenzyme analysis.

MGH-U1 cells are maintained as a monolayer culture in 75-cm2plastic flasks containing a-medium + 10% FCS.3a-medium containsglucose (I g/liter) and pyruvate (110 mg/liter) but no other sugars; themedium alsocontains amino acidswhich can be metabolized to providesubstratesfor glycolysis. Cells are subcultured at weekly intervals following detachment using 0.05% trypsin/0.02% EDTA. Cultures arereestablishedfrom frozen stock at 3-month intervals.

In some experiments westudied the growth ofmonolayers in glucosedeficient medium. Medium waspreparedwithout glucoseand pyruvate,and appropriate amounts of glucose were added to this medium. Fetalcalf serum was dialyzed against 40:1 NaCl:KCI with phosphate bufferto remove glucose and other small molecules. The same number ofMGH-Ul cells (-@10@)were seeded into multiple flasks containingglucose-deficientmedium + 10% DFCS with varying concentration ofglucose.Flaskswereselectedat random at 2-day intervals, and the cellswere detached and counted using a hemocytometer. In most experiments the medium was changed at 2-day intervals in the remainingflasks. Cells were also plated in Petri dishes in a-medium + FCS, andplating efficiency was assessedby counting stained colonies 10 dayslater.

Samplesof medium wereanalyzedfor glucoseconcentration using acommercial kit (Sigma Chemical Co., St. Louis, Mo). This method isbased on the conversion of glucose to glucose 6-phosphate in thepresenceof ATP and hexokinase, followed by reduction of NADP toNADPH when glucose 6-phosphate is oxidized to 6-phosphogluconicacid in the presence of glucose-6-phosphate dehydrogenase. NADPHis then measured by absorption at 340 nm using a spectrophotometer.Recently some of our samples have been analyzed with a Beckmanglucose analyzer (Beckman Instruments, Brea, CA) which measuresoxygen consumption in the presenceof glucoseand glucoseoxidase.

Spheroids.The derivation of MGH-U1 spheroidshasbeendescribedelsewhere(16). All experiments described in the present paper were

3 The abbreviations used are: FCS, fetal calf serum; DFCS, dialyzed fetal calf

serum.

3105

Influence of Glucose Concentration on Growth and Formation of Necrosis inSpheroids Derived from a Human Bladder Cancer Cell Line'

Ian F. Tannock2 and Irma Kopelyan

Departmentsof Medicine and Medical Biophysics,Ontario Cancer Institute and University of Toronto, Toronto, Ontario, Canada M4X 1K9

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GLUCOSE AND CELL DEATH IN SPHEROIDS

performed on a sublimeof MGH-Ul cells that was established afterpassageof the parental line through spheroids.This sublime,designatedMGH-Ul/OCI-l, will routinely form spheroids when placed in spinnerculture, whereasthis is more rarely observedfor the parental cells.

Spheroids usedin the presentexperiments weregrown at 31T in airin 250-mI spinner flasks containing a-medium + 10% FCS + 442-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (20 mM/liter),using a rotor speed of 130 rpm. After 4—6days, spheroids of about 400

@mdiameter were selected from the spinners and pipetted one per wellinto 24-well multiwell plates. The wells contained a 0.5-mI underlayerof I % agar diluted in a-medium without glucose and pyruvate, with 1.5ml liquid medium above. Liquid medium consisted of a-medium withor without pyruvate and with varying concentration of glucose, plus10% DFCS. In most experiments the liquid medium wasaspirated andreplaced at 2-day intervals. The aspirated medium was pooled forestimation of glucose concentration as described above. The multiwellplates were maintained at 37C in 95% air/5% CO2. This experimentaldesign allowed measurements to be made on individual and uniformspheroids, without the extensive shedding of cells that we and othershave observed from the surface of spheroids in spinner culture (17).

The growth of spheroids was studied in multiple experiments. At 2—3-day intervals each multiwell plate was removed briefly from theincubator and individual spheroids were observed through a transparentcover using an inverted microscope. The maximum and minimumdiameters of each spheroid were recorded using an eyepiecereticule,and the geometric meandiameter wascalculated. (Most spheroidswereclose to spherical; examination after removal from the agar underlayerdid not reveal any systematic deviation from spherical symmetry.) Themean diameter of multiple spheroids was then plotted against time togeneratea growth curve.

In order to study the relationship betweenglucoseconcentration andformation of necrosis, spheroids were pipetted from the multiwelldishes at varying times after exposure to normal or glucose-deficientmedium. The spheroids were exposed to mercurochrome to facilitatetheir recognition. In most experiments spheroids were sectioned usinga cryostat after rapid freezing. Serial 5-sm sections were cut, followedby fixation of the slides in 95% ethanol and staining with hematoxylinand eosin. The largest cross-sections of spheroids (i.e., those throughtheir center) were examined under the microscope and the maximumand minimum diameters of both the spheroid and its necrotic centerwere recorded. There was no net shrinkage or expansion of spheroidsections using this method. More recently, spheroids have been fixedovernight in Bouin's solution, then embeddedsequentially in 1.5%agarand paraffin, followed by serial sectioning and staining. The latterprocedure is more time consuming and leads to approximately 20%shrinkage in linear dimension, but gives higher quality sections.

RESULTS

Growth of Cells in Monolayer. The growth of MGH-Ul cellsas a monolayer in varying concentration of glucose was studiedin several experiments. Under control conditions (i.e., in a-medium with glucose, 1 g/liter), cells grew exponentially witha doubling time of 16—24h after an initial lag period of 2—3days (Fig. 1). Increase of the glucose concentration in the rangeof 2—lO-g/liter had minimal effects on cell growth. Cells wereunable to grow in the absence of glucose, regardless of thepresence ofpyruvate. Growth ofcells was slower than in controlflasks at glucose concentrations in the range of 20—100 mg/liter, although the magnitude ofthis effect varied among experiments. Growth of cells was slower than in control flasks at aninitial glucose concentration of 100 mg/liter regardless ofwhether the medium was left unchanged or was replenished at2-day intervals. The mean plating efficiency of trypan blueexcluding cells was close to I 00% and was independent ofglucose concentration about 20 mg/liter. However, plating efficiency fell rapidly for cells placed in medium containing noglucose.

Doys

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Fig. I. Growthof MGH-UI cellsasa monolayerin a-mediumcontainingdifferent concentrations of glucose plus 10% DFCS. Each point was obtainedfrom hemocytometer counts after detachment of cells in a single flask. Mediumwas replenished at 2-day intervals. Qualitatively similar results were obtained inseveral replicate experiments, except that glucose, 20 mg/liter, was sometimesunable to support growth of the cells.

The rate of depletion of glucose from medium by cells inmonolayer is shown in Fig. 2A. Glucose consumption decreasedfrom about 0.7 mg/105 cells/day to about 0.1 mg/b5 cells/dayduring exponential growth of cells under control conditions;glucose consumption was lower (@‘.-0.04mg/b5 cells/day) forcells growing at a slower rate in medium containing only 100-mg/liter amounts of glucose.

Growth of Spheroids. The growth of spheroids in varyingconcentrations of glucose was studied in multiple experiments.In medium with a glucose concentration of 1 g/liter, the spheroid diameter increased linearly with time at a rate of about100 @sm/day(Fig. 3A). Growth ofspheroids in medium containing increased concentrations of glucose in the range of 2—10g/liter was similar to that under control conditions (data notshown). Spheroids grew quite rapidly in media containing 100—1000 mg/liter glucose, but there was progressive slowing ofgrowth at glucose concentration below 100 mg/liter (Fig. 3A).However, unlike cells in monolayer, spheroids grew slowly inthe total absence of glucose. The mean plating efficiency ofcells dissociated from spheroids was about 60% and did notdepend on the glucose concentration in the medium duringspheroid growth.

The growth of spheroids in medium containing glucose, 10,20, or 100 mg/liter, with or without pyruvate (1 10 mg/liter) isshown in Fig. 3B. There was no difference in spheroid growthin the presence or absence of pyruvate.

In most of the above experiments the liquid medium inmultiwells was replenished at 2-day intervals, although therewas no effect on spheroid growth rate in one experiment wherethe medium was left unchanged (Fig. 3A). Representative values

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GLUCOSE AND CELL DEATH IN SPHEROIDS

the Beckman glucose analyzer indicate that this method isaccurate, to ±2%(SD), and this accuracy leads to a confidencerange for glucose consumption of about 0—0.02mg glucose/spheroid/day. Glucose consumption by MGH-U1 spheroids isabout 10-fold lower than values reported by Li (10) for 9L ratbrain tumor spheroids grown in suspension culture. Since theamount of glucose removed from medium (Fig. 2B) was of thesame order as the uncertainty in the most accurate detectionprocedure available to us, we were unable to@investigate therelationship between glucose consumption by spheroids, theirvolume, and the concentration of glucose in medium. Sincespheroids could grow slowly for a limited time in mediumwithout glucose, the lower limit of consumption of exogenousglucose is zero. Spheroids may, however, consume glucose thatwas trapped between cells on transfer from initial spinnerculture and/or glucose that is released from cells which diewithin spheroids.

Other investigators have studied the influence of glucoseconcentration in the medium when spheroids were maintainedin spinner culture (10—12).We therefore compared spheroidgrowth and glucose consumption by single spheroids in multiwells with those placed in new spinners after their initial establishment. This comparison was made in medium containing arange of glucose concentration. The number of spheroids wasadjusted to provide approximately one spheroid per 2 ml medium (i.e., 100 spheroids per spinner), the same ratio of spheroids to medium as in multiwells. Under these conditionsspheroids grew slightly more rapidly in spinner culture, although the reverse is observed at a higher concentration ofspheroids in the spinners. Examination of the medium fromspinners showed the presence of numerous single cells as wellas aggregates and small spheroids which were formed by cellsshed from spheroids; this was not found in multiwells. Thenumber of cells shed from larger spheroids (@1 mm diameter)was estimated by allowing spheroids to settle under gravity,followed by counting of cells in medium that was aspirated andtrypsinized. The number of such cells in three spinner flaskscontaining @-100spheroids ranged from 2.8—7.3x 106 cellsafter 6 days in culture. The total extraction of glucose in

0

Fig. 2. A, measured glucose concentration in flasks containing MGH-U 1 cellmonolayers. Cells (1—2x 10') were seededinitially in 20 ml medium containingeitherglucose,I g/liter (V,0, @,0), or glucose,100mg/liter(, •).Themediumwas not replenished. Different symbols represent different experiments. B, measuredglucoseconcentrationin multiwells(0, •)or spinners(0, U) containingMGH-Ul spheroids (approximately 1 spheroid/2 ml medium). The medium wasreplenishedat 2-dayintervals(0, 0) or left unchanged(N,•).

of glucose concentration in the medium are shown in Fig. 2B.Despite the strong influence of glucose concentration in themedium on spheroid growth, the data of Fig. 2B indicate a verylow rate of consumption of exogenous glucose. From the measured change in glucose concentration in media taken beforeand after spheroid incubation in multiwells, the best estimateof glucose consumption by spheroids growing in media containing glucose, 200—1000 mg/liter, is about 0.01 mg glucose!spheroid/day (equivalent to @0.02mg/105 cells/day). Replicatemeasurements of glucose concentration on a single sample with

Days

Fig.3. Increase in mean diameter ofMGH-U1 spheroids in multiwells containingmedium with different concentrations of glucose and pyruvate. In A, wells contained 110mg/literpyruvateandtheindicatedconcentration of glucose.Mediawerereplenishedat 2-day intervals(0, L@,0, V) or left intact (Uglucose,20 mg/liter). In B, wellscontainedpyruvate(110 mg/liter) or no pyruvatewithvarying glucoseconcentrations (10, 20, or 100mg/liter)asindicatedin thekey.Meandiameter ±SE (bars) is indicated for at least 10spheroids.

Days

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GLUCOSEAND CELL DEATH IN SPHEROIDS

spinners was greater than in multiwells at an initial glucoseconcentration of ‘@@500mg/liter or 1 g/liter, but not at 100 mg/liter (Fig. 2B). This increase in glucose extraction could beaccounted for by consumption by single cells and clumps at arate of about 0.2 mg/105 cells/day, well within the range ofglucose consumption found for MGH-U1 cells in monolayer.Thus we did not find evidence for major differences betweenspheroid growth and glucose extraction by MGH-U1 spheroidsunder stirred conditions in spinner culture and unstirred conditions in multiwells.

Formation of Necrosis. Spheroids which grew in mediumcontaining a concentration ofglucose in the range of0.5—10g/liter grew to a diameter of 1.0—1.2mm without central necrosis,or with minimal necrosis occurring in the largest spheroids(Fig. 4A). Spheroids which grew in lower concentration ofglucose developed central necrosis (Fig. 4B) and the relationship between the thickness of the viable rim and glucose concentration in the medium is shown in Fig. 5. These data wereobtained for spheroids that were sectioned at 5—6days aftertransfer to glucose-deficient medium and demonstrate an approximately linear relationship between rim thickness and glucose concentration up to 500 mg/liter. This relationship wasnot influenced by the presence or absence of pyruvate at aconcentration of 110 mg/liter.

The rate offormation ofnecrosis was estimated by sectioningofspheroids at varying intervals after placing them into glucosedeficient medium (Fig. 6). Necrosis was first observed at 3—4days, and the diameter of the necrotic center then increased ata rate that was faster than the rate of increase in spheroiddiameter, so that the thickness of the viable rim decreased.These data were then used to generate the relationship betweenthe volume of morphologically viable tissue and time in varioustypes of media (Fig. 7). The growth of viable tissue in controlspheroids deviated only slightly from exponential. Even in thetotal absence of glucose there was initial expansion of the viablecompartment, and growth of spheroids was not due simply tothe accumulation of dead cells. However, in glucose-deficientmedia the volume of morphologically viable tissue increasedtoward a maximal value, so that later growth in larger spheroids

was due to a balance between peripheral cell production andaccumulation of necrotic tissue centrally.

DISCUSSION

We have demonstrated that the glucose concentration in themedium influences the growth rate and formation of necrosisin spheroids derived from the MGH-U1 human bladder cancercell line. Our results are in qualitative agreement with the workof Freyer, Mueller-Klieser and their colleagues (12, 18) whofound that glucose concentration was a major determinant ofnecrosis formation in EMT6/Ro spheroids of murine origin.

An unexpected finding was the failure ofpyruvate to influencespheroid growth or formation of necrosis. Pyruvate is knownto cross cell membranes (19); pyruvate is also formed normallyfrom glucose by glycolysis and is a substrate for production ofATP by the Krebs cycle. Failure of pyruvate to influence spheroid growth or morphology may suggest limited involvementof the Krebs cycle in the energy metabolism of spheroid cells.It was also surprising to find that cells in spheroids, but not inmonolayer, could proliferate in the total absence of exogenousglucose. Viable cells may have utilized some carbohydrates thatwere released from damaged cells; this might occur more readilywhen cells are in close proximity in spheroids as compared towhen they are dispersed in a monolayer. Small amounts ofglucose may also have been transferred within spheroids whenthey were placed initially into multiwells.

Li (10, 11) studied the relationship between glucose consumption and concentration for 9L rat brain tumor cells inculture and then used this relationship to solve the diffusionequation to obtain the expected distribution of glucose concentration in spheroids derived from the same cell line. He foundthat the relationship between the radius of the necrotic zoneand the radius of 9L spheroids could be fitted by assuming thatnecrosis occurred when the glucose concentration fell to 6 xiO-@mg/mi. His calculations required the assumption, however,that glucose consumption was independent of the concentrationof oxygen and other key metabolites, and this seems ratherunlikely.

;.;@:@@.::7. ‘: : @‘. .

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Fig. 4. Cross-sectionsof MGH-Ul spheroids.Left, spheroidgrownfor 4 daysin multiwellsundercontrol conditions(glucose,1 g/liter) containsno centralnecrosis.Right, spheroid of similar size grown for 7 days in medium containing glucose, 100 mg/liter, contains a large region of central necrosis.

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0 I 2 3 4 5

GLUCOSEAND CELL DEATH IN SPHEROIDS

500

E

@@400

@ 300a

@200

00

0 200 400 600 800Glucoseconcentrationtmg/iiter)

I, Spheroids

Relationship between thicknessof viable rim and glucoseconcentration in 5-6 dayMGH- UI spheroids

000

Fig.5. Relationshipbetweenthicknessof theviablerim andconcentrationofglucosefor MGH-Ul spheroidsafter 5—6daysin multiwellscontainingthespecifiedconcentrationof glucose.Differentsymbolsrepresentdifferentexperiments.

0 I 2 3 4Days

Fig.6. Increasein meandiameterofMGH-Ul spheroidsandoftheir necroticcenters during growth in multiwells containing (a) glucose, 20 mg/liter, and nopyruvate. or (b) glucose, 100 mg/liter, and pyruvate, 110 mg/liter. Each pointrepresentsa separatespheroid, except mean values ±SE (bars) are indicated for>6 spheroidsat sometimeintervals.

The MGH-Ul cells used in the present study differed in twoimportant ways from the 9L cells used by Li (10, 11): (a)glucose consumption by spheroids was about 10-fold lower (andbecause of this could not be measured accurately); and (b)glucose consumption by cells in culture varied with cellularconcentration and was greater than for the same cells in spheroids. Also, MGH-U1 spheroids grew slowly and had a viablerim of cells even in the total absence of exogenous glucose.Thus our data on spheroid growth and formation of necrosiscannot be fitted by a simple diffusion-consumption model whichconsiders glucose alone. Rather, our results demonstrate thatlack of glucose is an important factor which contributes toformation of necrosis but that the process of cell death iscomplex and depends on other factors as well. Absence ofglucose does not cause cell death in the presence of adequateconcentration of other key metabolites (e.g., oxygen, aminoacids such as glutamine, etc.) but probably does so when theseother metabolites are also depleted.

Spheroids described in the present paper appear to showsome major differences as compared to those described byothers. When individual MGH-Ul spheroids are placed under

b)@

Necrotic ACentre /

I.

4Doys

Fig. 7. Estimated volume ofviable tissue (logarithmic axis) at different times(linear axis) during growth of MGH-Ul spheroidsin multiwellscontainingdifferent concentrations of glucose. Volume was calculated from estimates ofdiameterof spheroidsandtheir necroticcenters(Figs.3, 5, and6) by assumingsphericalsymmetry.

control conditions glucose, (1 g/liter) in muitiwells, or at lowconcentration in spinner culture, their volume increases almostexponentially to a large size (1.2-mm diameter). Many of thesespheroids had no central necrosis while others developed mmimal necrosis at a diameter >1 mm. Most other types ofspheroids, including those of human origin (20), have beenreported to develop central necrosis at a mean diameter of <700zm. MGH-U1 spheroids develop central necrosis at -@500 smdiameter when grown under more crowded conditions in spinner culture, despite replenishment of the medium at 2-dayintervals. Thus formation of necrosis in spheroids may dependas much on the culture conditions (i.e., number of spheroidsper unit of medium) as on properties of the cell line. When theconcentration of spheroids is high it may be necessary to renewthe medium at least twice daily (12) to avoid depletion ofglucose and other essential nutrients. With the lower concentration of spheroids (one spheroid per 2 ml medium) used inthe present study, medium changes were probably unnecessary(multiwells)or were required at less frequent intervals in spinnerculture because of consumption of glucose and other metabolites by cells shed from the surface of spheroids.

We elected to study individual spheroids grown in multiwellson an agar underlayer since this system has the followingadvantages as compared to spinner culture: (a) it allows determination of the rate of growth of individual spheroids ratherthan that of representative spheroids obtained at random fromthe population; (b) the size of spheroids remains more uniformand does not depend critically on factors such as speed of therotor, (c) shedding of cells from the surface is minimal or does

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3. Tannock, I. F. The relation betweencell proliferation and the vascularsystemin a transplanted mouse mammary tumour. Br. J. Cancer, 22: 258—273,1968.

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bronchial and cervical squamouscell carcinomas: inferencesfor their cellularkinetics and radiobiology. Br. J. Cancer, 51: 407—413,1985.

5. Tannock, I. F. Oxygen diffusion and the distribution of cellular radiosensitivity in tumours. Br. J. Radiol., 45: 515—524,1972.

6. Tannock, I. F., and Steel,G. G. Tumor growth andcell kinetics in chronicallyhypoxic animals. J. NatI. Cancer Inst., 45: 123—133,1970.

7. Moskowitz, M., Amborski, G. F., and Wicker, C. H. Structure developmentin aggregens.Nature (Lond.), 211: 1047—1049,1966.

8. Sutherland, R. M., McCredie, J. A., and Inch, W. R. Growth of multicellspheroids in tissueculture asa model of nodular carcinomas.J. Nail. CancerInst., 46: 113—120,1971.

9. Franko, A. J., and Sutherland, R. M. Oxygen diffusion distance and developmentof necrosisin multicellspheroids.Radiat.Res.,79:439—453,1979.

10. Li, C. K. N. The glucosedistribution in 9L rat brain multicell tumor spheroidsand its effect on cell necrosis.Cancer (Phila.), 50: 2066—2073,1982.

11. Li, C. K. N. The role ofglucose in the growth of9L multicell tumor spheroids.Cancer(Phila.),50:2074—2078,1982.

12. Mueller-Klieser, W., Freyer, J. P., and Sutherland, R. M. Evidence for amajor role of glucose in controlling development of necrosis in EMT6/Romulticell tumor spheroids. In: H. I. Bicher and D. F. Bruley (eds.), OxygenTransport to Tissue, pp. 487—495.New York: Plenum Publishing Corp.,1983.

13. O'Toole, C. M., Povey,S., Hepburn, P., and Franks, L. M. Identity of somehuman bladder cancercell lines. Nature (Lond.), 301: 429—430,1983.

14. Parada,L. F., Tabin, C. T., Shih, C., and Weinberg, R. A. Human EJ bladdercarcinoma oncogeneis homologue of Harvey sarcomavirus rca gene.Nature(Lond.), 297: 474—478,1982.

15. Kovnat, A., Armitage, M., and Tannock, I. F. Xenografts of human bladdercancerin immune-deprivedmice.CancerRes.,42:3696—3703,1982.

16. Erlichman, C., and Vidgen, D. Cytotoxicity ofAdriamycin in MGH-U1 cellsgrown as monolayer cultures, spheroids,and xenografts in immune-deprivedmice.CancerRes.,44:5369—5375,1984.

17. Landry, J., Freyer, J. P., and Sutherland, R. M. Shedding of mitotic cellsfrom the surfaceof multicell spheroids during growth. J. Cell. Physiol., 106:23—32,1981.

18. Freyer, J. P., and Sutherland, R. M. The role of glucose in regulatingquiescentcell populationsin EMT6/Ro spheroids.Radiat.Res.,91: 342,1982.

19. Halestrap, A. P. Transport of pyruvate, NAD, lactate into human erythrocytes.Evidencefor the involvementof thechloridecarrier,anda chlorideindependent carrier. Biochem. J., 156: 193—207,1976.

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not occur, removing the potential artifact of consumption ofglucose and other metabolites by single cells and by smallerspheroids that form from them in suspension.

Possible disadvantages of the multiwell system are that lackof stirring might lead to depletion of nutrient metabolitesaround the surface of the spheroids and that a lower concentration of metabolites in the agar underlayer might lead to asymmetry in the vertical plane. We found no evidence for systematicdepartures from spherical symmetry either on gross examination after removing spheroids from the agar underlayer, or afterhistological sectioning. The area of contact between spheroidand agar is small, and glucose and other metabolites probablyequilibrate rapidly between agar and liquid medium. It is possible that lack of stirring led to a lower consumption of glucoseby spheroids in multiwells, but the higher glucose extractionmeasured in spinners (Fig. 2B) could be explained entirely byconsumption of glucose by cells (and their progeny) that hadbeen shed from the surface of spheroids.

In the current study we found an effect of glucose concentration on formation of necrosis at concentration in the mediumbelow 500 mg/liter. Tumors have been found to utilize about30% of the glucose supplied to them (21) and the above valueis probably close to the lower limit of glucose concentrationwhich maybe found in blood draining a tumor. Lower values ofglucose concentration might occur in tumor capillaries. Also,we have reported the effects of glucose concentration only inthe presence of normal or high concentration of oxygen andother metabolites. The PO2 in tumor blood vessels is likely tobe closer to 40 mm Hg than to 150 mm Hg (95% air), the valueto which the medium was exposed in the current experiments.Radiobiological experiments do not suggest a significant hypoxic fraction when our spheroids are irradiated in air, incontrast to results for most experimental tumors. It is probablethat formation of necrosis may occur in spheroids growing inmedium containing a higher concentration ofglucose if the PO2and/or levels of other important metabolites such as glutamineare reduced to a concentration similar to those in tumor bloodvessels (9, 12). Spheroids will provide an important model forunderstanding the relationship between the distribution of glucose, oxygen, and other metabolites and the formation of necrosis in solid tumors.

ACKNOWLEDGMENtS

We thank Dr. J. Trent, University of Arizona, for performing karyotype analysis on MGH-Ul cells.

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1986;46:3105-3110. Cancer Res   Ian F. Tannock and Irina Kopelyan  Cell LineNecrosis in Spheroids Derived from a Human Bladder Cancer Influence of Glucose Concentration on Growth and Formation of

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