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[CANCERRESEARCH 37, 3639-3643, October1977] SUMMARY A new technique, based on the growth of tumor cells in liquid media over an agambase, has been developed for the formation and growth of multicellular tumor spheroids. All of the 11 transformed cell lines tested formed multicellular tumor spheroids, while none of the 8 normal cell types tested did so. The advantages of the present technique over olden methods include its simplicity, generality, and expeni mental flexibility. INTRODUCTION MTS2 offer many of the characteristics of in vivo tumors, which are unavailable in monolayer or suspension culture (8). These include intimate cell-cell contacts (2), chronically hypoxic cell populations (7), and cycle times that range from comparable to exponential monolayer rates through essentially nondividing (3). In brief, they combine the rele vance of organized tissues with the accuracy of in vitro methodology. Of the method proposed for their production and growth, the most adequate is the spinner flask method (9). In this method, tumor cells are maintained in spinner flasks, and the constant movement prevents their attachment to the walls of the vessel and allows them to attach to each other and grow. While this spinner flask method overcame many of the limitations [e.g., the diffusion limitations of colonies grown in semi-solid agar (5)], it has not been used by a large number of investigators, nor has it been used in areas of cancer research other than tumor radiobiobogy (9). Presum ably, the reason for the lack of general interest in MTS is primarily a technical problem. The technique is difficult, and those who have mastered it are primarily interested in tumor radiobiobogy. Furthermore, the empirical methods (9) required to adapt any given tumor to this method have limited the array of tumors that are available for study. Finally, the need for large volumes of reagents, the inability to study individual MTS for prolonged periods, and other requirements of the system make it ill adapted for certain types of investigations. Since MTS could prove useful in many areas of cancer I Supported by the Division of Cancer Centers and Resources, National Cancer Institute, through Grant 1-P30-C-21074.01. 2 The abbreviations used are: MTS, multicellular tumor sphcroids; EBME, Eagle's basal medium; HBSS, Hanks' balanced salt solution. Received April 11, 1977; accepted June 30, 1977. research, we initiated attempts to develop a simple method forproducingand growingMTS, and our resultsareme ported below. MATERIALS AND METHODS Cells. A total of 19 different cell types were used in the present investigations, 8 normal and 11 transformed (Table 1). Cells were classified as normal unless they produced tumors in appropriate hosts, formed colonies in soft agam, or lacked contact inhibition in monolayer. All normal cell samples were obtained from apparently normal tissues in vivo, whereas transformed cells were obtained from ob vious tumors. Monolayer cultures were maintained (100% relative hu midity; 95% aim+ 5% CO2; 37°)in either EBME or F-12, supplemented with 10% fetal calf serum, 50 units of penicil bin per ml, and 50 @g per ml of streptomycin (Grand Island Biological Co., Grand Island, N.Y.). For the data presented below, all cultures were harvested by mild trypsinization (0.25% w/v, 3 to 5 mm at 37°),but similar results can be obtained by scraping the cells off the surface. The single primary normal tissue studied (adult lung from a C3H mouse) and both transplanted tumors (FSA and Line 1) were harvested by mincing in 0.9% NaCI solution, fol bowed by cell dissociation with a Teflon and glass tissue grinder. The single-cell suspension was pelleted and resus pended either in 0.9% NaCI solution for transplant or com plete EBME for attempted production of MTS. MTS Production.Arguingthatthe lackof an appropriate surface for cell attachment might promote MTS formation, just as constant agitation does in the spinner flask system (9), we compared 3 methods for producing MTS, all of which involved stationary plates maintained in a standard tissue culture incubator (100% relative humidity; 95% air + 5% CO2; 37°). Approximately 10@cells in 10ml of EBME (as above) were added to 100-mm plastic Petri dishes (Falcon Plastics, Oxnard, Calif.)that (a) had not been treated for cell attachment, henceforth referred to as bacteriological plates; (b) had been base-coated (2 to 3 mm) with 0.5% Noble agar (Difco Laboratories, Inc., Detroit, Mich.) in HBSS, henceforth referred to as agar-HBSS plates; or (C) had been base-coated (2 to 3 mm) with 0.5% Noble agar in complete EBME, henceforth referred to as agar-EBME plates. The plates were then returned to the incubator and observed for up to 30 days. No agitation or mocking was used in any of the experiments. OCTOBER1977 3639 A Simplified Method for Production and Growth of Multicellular Tumor Spheroids John M. Yuhas, Albert P. LI, Andrew 0. MartInez, and Aaron J. Ladman1 CancerResearchandTreatmentCenter(J.M. Y.,A. P.L.,andA. 0. M.JandDepartmentsof Radiology(J.M. Y.,A. P.L.J,andAnatomy(A.J. L.J,Universityof New Mexico, Albuquerque, New Mexico 87131 on May 21, 2021. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
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Page 1: A Simplified Method for Production and Growth of ... · Similar results were obtained with tumor cells in the bac teriologicalplates,butintheagam-HBSSplatesQT-A31,K- A31, MCa-11,

[CANCERRESEARCH37, 3639-3643,October1977]

SUMMARY

A new technique, based on the growth of tumor cells inliquid media over an agambase, has been developed for theformation and growth of multicellular tumor spheroids. Allof the 11 transformed cell lines tested formed multicellulartumor spheroids, while none of the 8 normal cell typestested did so. The advantages of the present technique overolden methods include its simplicity, generality, and expenimental flexibility.

INTRODUCTION

MTS2 offer many of the characteristics of in vivo tumors,which are unavailable in monolayer or suspension culture(8). These include intimate cell-cell contacts (2), chronicallyhypoxic cell populations (7), and cycle times that rangefrom comparable to exponential monolayer rates throughessentially nondividing (3). In brief, they combine the relevance of organized tissues with the accuracy of in vitromethodology.

Of the method proposed for their production and growth,the most adequate is the spinner flask method (9). In thismethod, tumor cells are maintained in spinner flasks, andthe constant movement prevents their attachment to thewalls of the vessel and allows them to attach to each otherand grow. While this spinner flask method overcame manyof the limitations [e.g., the diffusion limitations of coloniesgrown in semi-solid agar (5)], it has not been used by a largenumber of investigators, nor has it been used in areas ofcancer research other than tumor radiobiobogy (9). Presumably, the reason for the lack of general interest in MTS isprimarily a technical problem. The technique is difficult,and those who have mastered it are primarily interested intumor radiobiobogy. Furthermore, the empirical methods (9)required to adapt any given tumor to this method havelimited the array of tumors that are available for study.Finally, the need for large volumes of reagents, the inabilityto study individual MTS for prolonged periods, and otherrequirements of the system make it ill adapted for certaintypes of investigations.

Since MTS could prove useful in many areas of cancer

I Supported by the Division of Cancer Centers and Resources, National

Cancer Institute, through Grant 1-P30-C-21074.01.2 The abbreviations used are: MTS, multicellular tumor sphcroids; EBME,

Eagle's basal medium; HBSS, Hanks' balanced salt solution.Received April 11, 1977; accepted June 30, 1977.

research, we initiated attempts to develop a simple methodforproducingand growing MTS, and our resultsare meported below.

MATERIALSAND METHODS

Cells. A total of 19 different cell types were used in thepresent investigations, 8 normal and 11 transformed (Table1). Cells were classified as normal unless they producedtumors in appropriate hosts, formed colonies in soft agam,or lacked contact inhibition in monolayer. All normal cellsamples were obtained from apparently normal tissues invivo, whereas transformed cells were obtained from obvious tumors.

Monolayer cultures were maintained (100% relative humidity; 95% aim + 5% CO2; 37°)in either EBME or F-12,supplemented with 10% fetal calf serum, 50 units of penicilbin per ml, and 50 @gper ml of streptomycin (Grand IslandBiological Co., Grand Island, N.Y.). For the data presentedbelow, all cultures were harvested by mild trypsinization(0.25% w/v, 3 to 5 mm at 37°),but similar results can beobtained by scraping the cells off the surface.

The single primary normal tissue studied (adult lung froma C3H mouse) and both transplanted tumors (FSA and Line1) were harvested by mincing in 0.9% NaCI solution, folbowed by cell dissociation with a Teflon and glass tissuegrinder. The single-cell suspension was pelleted and resuspended either in 0.9% NaCI solution for transplant or complete EBME for attempted production of MTS.

MTS Production.Arguingthat the lackof an appropriatesurface for cell attachment might promote MTS formation,just as constant agitation does in the spinner flask system(9), we compared 3 methods for producing MTS, all ofwhich involved stationary plates maintained in a standardtissue culture incubator (100% relative humidity; 95% air +5% CO2;37°).Approximately 10@cells in 10 ml of EBME (asabove) were added to 100-mm plastic Petri dishes (FalconPlastics, Oxnard, Calif.)that (a) had not been treated for cellattachment, henceforth referred to as bacteriologicalplates; (b) had been base-coated (2 to 3 mm) with 0.5%Noble agar (Difco Laboratories, Inc., Detroit, Mich.) inHBSS, henceforth referred to as agar-HBSS plates; or (C)had been base-coated (2 to 3 mm) with 0.5% Noble agar incomplete EBME, henceforth referred to as agar-EBMEplates. The plates were then returned to the incubator andobserved for up to 30 days. No agitation or mocking wasused in any of the experiments.

OCTOBER1977 3639

A Simplified Method for Production and Growth ofMulticellular Tumor Spheroids

John M. Yuhas, Albert P. LI, Andrew 0. MartInez, and Aaron J. Ladman1

CancerResearchand TreatmentCenter(J.M. Y.,A. P.L., andA. 0. M.JandDepartmentsof Radiology(J.M. Y.,A. P.L.J,andAnatomy(A.J. L.J,UniversityofNew Mexico, Albuquerque, New Mexico 87131

on May 21, 2021. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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J. M. Yuhas et a!.

MTSGrowth.MTS(n = 24)wereharvestedfrom9- to 14-day-old agar-EBME plates and were transferred individuallyinto 16-mm agar-EBME wells (Costar, Cambridge, Mass.)containing 1 ml of EBME. All MTS, within a group, were thesame size at the time of harvest: MCa-11, 140 @m;FSA, 224

@m;line 1, 168 @m.Subsequent experiments (data notshown) have demonstrated that estimated growth matesdonot vary as a function of size at harvest over the mangeof 100to 600 @m.For this growth study, media were changeddaily, at which time the MTS were sized on a dissectingmicroscope (x40).

The s.c.TumorGrowth.Approximately10@tumorcells(in0.2 ml of 0.9% NaCl solution) from 3 tumor lines wereinjected s.c. into the night leg of 16-week-old, female, syngeneic hosts (BALB/c mice for line 1 and MCa-11 and C3Hfor FSA). Tumors arose within 7 to 8 days and were sizedwith vernier calipers through the 32nd day posttnansplant.Tumor size is expressed as the average of the 2 perpendiculamdiameters.

Autoradlography. Individual MTS were placed in 16-mmagar-EBME wells containing 1 ml of EBME + 2 @Ciof[3H]thymid me (Amersham-Searle, Arlington Heights, III.;specific activity, 6.7 Ci/mmole). Twenty-four hr later theMTS were fixed and processed according to standard techniques.

RESULTS

MTS Production and Morphology. Normal cells (Table 1),when added to bacteriological, agar-HBSS, or agar-EBMEplates, formed cellular aggregates (slOO sm), which failedto grow and broke apart within 72 hr. These normal cellclumps contained viable cells through 48 hm,as evidencedby the ability of these aggregates to reestablish monolayercultures when placed in Petmidishes that had been treatedfor cell attachment. By 72 hm,the number of surviving nonmal cells in the few clumps remaining was insufficient toreestablish monolayer growth under appropriate conditions.

Similar results were obtained with tumor cells in the bacteriologicalplates,butintheagam-HBSS platesQT-A31,K-A31, MCa-11, FSA, and line 1 formed cellular aggregates(Fig . 1A) that continued to grow, while SV-A31 and BP-A31

formed aggregates that grew slowly then broke apart. Withthe agar-EBME plates, all 11 tumor lines formed cellularaggregates that continued to grow, while repeated testingof the 8 normal cell types yielded consistently negativeresults (Table 1). We refer to these growing cellular aggregates,therefore,as MTS.

The MTS that developed do not attach to the agar-EBMEbase but are freely movable. For the tumor lines listed inTable 1, MTS (100 @m)appear within3 to 14 days andcontinue to appear through the time (3 to 7 days) at whichsubcultuming into new agam-EBMEplates is required due tomedia exhaustion. In general, large cell inocula (10@to 106)result in more rapid MTS development, while lower numbers yield a greater number of MTS per cell inoculated, butthey take longer to appear. For 1 tumor, at least, the line 1lung carcinoma, MTS can be produced with as few as 100cells/100-mm agan-EBME dish. In none of the tumor lines

tested were fewer than 100 MTS produced within the 1st 3weeks with an inoculum of 106 cells.

Fig. 1A is a scanning electron micrograph of a 420-smMTS derived from the highly malignant line 1 lung carcinoma (10). Individual cells possessmultiple microvilli andare loosely packed within the MTS. A further description ofthe surface morphology of this and other types of MTS willbe provided elsewhere.

Figs. lB through iF are autoradiographs of sectionstaken through the center of line 1 MTS of increasing size(280 to 840 sm). At the smallest size shown (Fig. 18, 280pm), virtually all of the nuclei are labeled, indicating that allof the cells are in cycle and had passed through DNA synthesis during the 24 hr of exposure of [3H]thymidmne. AsMTS size increases (Fig. 1, C to F), a nondividing but viablecentral region develops followed by, with further growth, acentral necrotic come.As shown in Fig. 1C, the viable portion of the MTS can be divided into 3 general areas: (a) theoutermost shell in which almost all cells are labeled; (b)immediately beneath that a shell in which approximately50% of the cells are labeled; and (C) the innermost viableshell, immediately adjacent to the necrotic core, in whichnone of the cells are labeled. As suggested elsewhere (1),we interpret this depth dependence for percentage of cellslabeled as being the product of declining oxygen concentrations between the periphery and center of the MTS. Direct evidence supporting this conclusion will be providedelsewhere.3

MTS versusin Vivo Growth Rates. One of the majoradvantages of MTS is that they simulate in vivo tumorsmomphobogically and hopefully should simulate them functionally. To test this possibility, we compared the in vivogrowth matesof line 1, FSA, and MCa-11 with their growthmatesas MTS. Chart 1 is a plot of the mean diameter of the 3tumors as a function of time after s.c. transplantation. Aspointed out elsewhere (11) the growth matesof FSA and line1 are indistinguishable and averaged 0.56 ±0.07 mm/day(Chart 1) in spite of the fact that they differ markedly in theirimmunogenicity, with the former (12) being far more immunogenic than the latter (6). The growth mateof MCa-11 (0.23±0.04 mm/day) is far bower. Chart 2 is a plot of mean MTS

diameter as a function of time for the same 3 tumor celllines. In this experiment, media were changed daily in orderto avoid the possibility of nutrient limitation. Subsequentexperiments (data not shown) have demonstrated that media can be changed as infrequently as once per week without reducing the growth rate for most of the MTS studied.As was the case for the tumors growing in vivo (Chart 1), thegrowth matesfor line 1 and FSA (Chart 2), when grown asMTS in vitro, were indistinguishable and averaged 83 ±3pm/day, while the growth matefor MTS derived from MCa11 was far lower at 32 ±5 pm/day (Chart 2). Monolayergrowthmatesforthese3 linesdo notcorrelatewitheitherthein vivo on MTS growth mates,since all 3 lines show doublingtimes of 16 to 18 hr.

This lack of correspondence between monolayer andMTS growth matesis a reflection of the fact that not all cells

3 J. M. Yuhas, and A. P. Li. In Vitro Studies on the Radioresistance of

Oxic and Hypoxic Cells in the Presence of Both Aadioprotective and Radiosensitizing Drugs, submitted for publication to Radiation Research.

3640 CANCERRESEARCHVOL. 37

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Table1Capabilities of normal and transformed cells' from 3 species to form MTS. None of the normal cells formed MTS, while all of the

transformedcells formed them readilyA. Normal cells

SourceCell typeDesignationbPropagationRemarksBALB/c

mouseEmbryonic lungAL-iMCTested at passages0, 1, and2BALB/cmouseEmbryoAL-2MTested at passages0, 1, and2BALB/cmouseAdult lungAL-3MTested at passages0, 1, and2C3H

mouseLung fibroblastsAL-4MTested at passages0, 1, and2C3HmouseAdultlungPBALB/c

mouse3T3, cloneA31A31MHuman,newbornFibroblast75-69MHuman,

fetusFibroblast75-86B. TransformedcellsMSourceOriginal

cell typeTransforming agentDesignationPropaga tionRemarksC3H

mouseConnective tissueSpontaneousL-cellsM All formedMTSC3HmouseFibroblastsMethylcholanthreneFSAMC3HmouseFibroblastsMethylcholanthreneFSATPBALB/c

mouseType II lung alveolar cellSpontaneousLine1MBALB/cmouseType II lung alveolar cellSpontaneousLineITPBALB/cmouseMammaryepitheliumRadiationMCa-11MBALB/cmouse

BALB/c mouse3T3,clone A31

3T3, clone A31MethylcholanthreneSV4OQT-A31 SV-A31MMBALB/cmouse3T3, clone A31Kirsten muninesarcomavirusK-A31MBALB/cmouse3T3, cloneA31BenzopyreneBP-A31MChinesehamster

Chinese hamsterFibroblast Ovarian epitheliumMethylcholanthreneSpontaneousBi4-150 CHO-K,MMHumanCervicalepitheliumSpontaneousHeLaM

Spheroid Growth

a Cells were classified as normal, unless they produced tumors in animals, grew in soft agar, or showed a piled up morphology in

monolayer culture.b Cell lines were derived in our own laboratory (AL-i , AL-2, AL-3, AL-4, Line 1 , MCa-1 1 , and primary C3H lung) or were a gift from Dr. G.

Martin, Universityof Washington(75-69,75-86,L, Bi4-150, and HeLa);Dr. A. Tennantand Dr. A. W. Hsie,OakRidgeNational Laboratory(A31,QT-A31,SV-A31,K-A31,and BP-A3i); or Dr. H. A. Withers, M. 0. Anderson Hospital and Tumor Institute (FSA).

C M, monolayer; P, primary; TP, transplant.

in the MTS are in division just as is the case in vivo, inaddition to possible differences in the growth rate of therespective individual cells.

DISCUSSION

The data presented above have demonstrated 3 points: (a)MTS can be produced very simply from a variety of tumorcell sources; (b) 8 normal cell types do not form cellularaggregates that are capable of growth; and (C)growth ratesof MTS show a better correlation with in vivo tumor growthrates than do monolayer cultures.

The technique is far simpler than any of the other methods (5, 9) presently used to produce MTS, and it is far moreadaptable to a variety of problems in cancer research. Wehave successfully produced MTS using agam-EBME Petmidishes, ranging in size from 35 to 100 mm, and made ofglass (Pyrex) or plastic, from a number of manufacturers(Falcon; Linbro Chemical Co., New Haven, Conn.; and Costar), using fetal calf serum from various batches and suppliers (Grand Island Biological Co. ; Microbiological Associates, Inc., Bethesda, Md.; Pacific Biobogicals, Richmond,Calif. ; Kansas City Biological, Lenexa, Kans.), using cellconcentrations of 102to 10' per 100 mm agar-EBMEplate,using media from various formulations (EBME, F-12, F-10,

L-15, and Dulbecco's Modified Eagle's Medium), and usingat least 2 sources of agan, Noble agar and special Nobleagam(Difco). The reason for our success is not only the lackof a surface for attachment; but it must also include nutritional factors from the agam-EBMEcombination, since bactemiobogical plates did not support growth of the tumor cellaggregates that formed. We point out the range of expemimental conditions that have allowed MTS production inorder to emphasize the fact that successful use of thismethod does not require unique conditions.

The experimental flexibility of the agar-EBME (or agarmedia) system is readily apparent, as is its adaptability tomany areas of cancer research. With relative ease 11 different tumors are now available as MTS, but this does notnecessarily mean that all solid tumors will form MTS in oursystem. In fact, we are presently attempting to select both invitro and in vivo for variants of MTS-forming tumors that canno longer do so, in much the same way that Fidler (4) hasselected for greater metastatic potential. In brief, thismethod allows the long-term study (30 to 60 days) of individual MTS from a variety of solid tumors in medium volumesas small as 1 ml. Hopefully, the combination of simplicityand experimental flexibility will allow the use of this methodfor a variety of experimental procedures.

The fact that the 8 normal cell types studied could notform aggregates that were capable of growth suggests that

OCTOBER1977 3641

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J_ M. Yuhas et a!.

a basic difference exixts between normal and transformedcells and that our MTS system relies on this difference for itsfunction. Whether or not other methods can produce MTSfrom normal cells is not the problem, however, since theagar-EBME system appears able to discriminate betweenthe 2 types of cells.

The last point concerns the proportionality between tumomgrowth rates in vivo (Chart 1) and the growth of thesame tumors as MTS (Chart 2). The growth rates in the 2systems are not identical but proportional, suggesting thatthe in vivo factors that affect growth rate (non-tumor cellinfiltration, stromal elements, growth factors, mateof deadcell clearance, immunological inhibition, etc.) operate similamlyon all 3 tumors. The fact that MTS grow at a rate that isproportional to their in vivo growth rate, while monolayersdo not, further suggests that the organized nature of MTSallows for greater expression of inherent growth characteristics and lessendependence on the particulars of the mediaused. In addition, growth in the MTS system is a function ofnot only cellular doubling times but also the size of thegrowth fraction. Preliminary data indicate that the size ofthe growth fraction varies widely among MTS from differenttumors. These data are presently being expanded and willbe reported elsewhere.

I IS. C. TUMORS

FSA //

I

20 -

E.! l@

LU

w

4

0

:DI-

z4w

5

@-ll

I I I I I I

10 20 30DAYS

Chart 1. The s.c. tumor diameter as a function of time after transplantation for 3 murine tumor lines. Five x 10' tumor cells were transplanted s.c. inthe right leg, and tumors were measured (2 perpendicular diameters) withvernier calipers. Line I and MCa-1I were grown in 4-month-old BALB/cfemales, while FSA cells were grown in similar C3H mice.

E

w

LU

4

z4LU

Chart 2. MTS diameter as a function of time for 3 murine tumor lines.Individual MTS were harvested from 100-mm production plates, and placed,along with 1 ml of EBME, In 16-mm agar-undcrlayed wells. Twelve MTS wereused per line, and the media were changed daily.

Fig. 1. Line 1 alveolar cell carcinoma MTS. A, scanning electron microscopic view of a 420-g.m MTS. X 200. B to F, midline autoradlographic sections ofMTS that had been exposedto (‘Hjthymidine,2 @Ci/ml,for 24 hr prior to fixation. X 250. B, 280-sm MTS. C, 420-@&mMTS. 0, 560-sm. MTS: 1, denselylabeledouter shell; 2, lightly labeled intermediate shell; 3, nonlabeled innermost shell; NC, necrotic area. E, 700-sm MTS. F, M0-@m MTS.

3642 CANCERRESEARCHVOL. 37

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2. Durand, A. E., and Sutherland, A. M. Effects of Intercellular Contact onRepair of Radiation Damage. Exptl. Cell Ace., 71: 75-80, 1972.

3. Durand, A. E., and Sutherland, A. M. Dependence of the RadiationResponse of an In Vitro Tumor Model on Cell Cycle Effects. Cancer Ris.,33: 213-219, 1973.

4. Fidler, I. J. Selection of SuccessiveTumor Line for Metastasis. NatureNew Blol., 242: 148-149, 1973.

5. Folkman, J., Hachberg, M., and Knighton, D. Self Regulation of Growthin Three Dimensions. In: B. Clarkson and R. Baserga (ads.), Control ofProliferation in Animal Cells, pp. 833-842. Cold Spring Harbor, N. V.:Long Island Biological Association, 1974.

6. MIlas, L., Hunter, N., Mason, K., and Withers, H. A. ImmunologicalResistance to Pulmonary Metastases In C3HI/Bu Mice Bearing SyngencicFibrosarcomaofDifferentSizes.CancerRes.,34:61-71, i974.

7. Sutherland, A. M., and Durand, A. E. Radlosensitization in Nifuroxime ofthe Hypoxic Cells in an In Vitro Tumour Model. Intern. J. RadiatIon Blol.22: 613-618, 1972.

8. Sutherland, A. M., and Durand, A. E. Radiation Response of MultlcellSpherolds—AnIn Vitro Tumour Model.CurrentTopics RadiationRae.,11:87-139,1976.

9. Sutherland, A. M., McCredle, J. A., and Inch, W. A. Growth of MulticellSpherolds in Tissue Culture as a Model of Nodular Carcinoma. J. NatI.Cancerlnst.,46: 113-120,1971.

10. Yuhas, J. M., and Pazmino, N. H. Inhibition of Subcutaneously GrowingLine 1 Lung Carcinomas Due to Metestatic Spread. Cancer Rca., 34:2005-2010, 1974.

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Page 6: A Simplified Method for Production and Growth of ... · Similar results were obtained with tumor cells in the bac teriologicalplates,butintheagam-HBSSplatesQT-A31,K- A31, MCa-11,

1977;37:3639-3643. Cancer Res   John M. Yuhas, Albert P. Li, Andrew O. Martinez, et al.   Tumor SpheroidsA Simplified Method for Production and Growth of Multicellular

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