[CANCER RESEARCH 38, 2486-2491, August 1978]0008-5472/78/0038-0000$02.00

Multicellular Tumor Spheroid Formation by Breast Cancer CellsIsolated from Different Sites1

John M. Yuhas, Anne E. Tarleton, and Karen B. Molzen

Cancer Research and Treatment Center and Department of Radiology, University of New Mexico. Albuquerque, New Mexico 87131

ABSTRACT

Fourteen breast cancer lines (8 human, 5 rat, and 1mouse) have been studied in terms of their ability to formmulticellular tumor spheroids (MTS) with the agar-basemethod. Only 8 of the lines formed MTS in contrast to a100% efficiency in a series of 11 varied tumors reported inthe initial studies with this method. We have comparedthe lines that do and do not form MTS in terms of a varietyof characteristics (e.g., estrogen receptors, time in serialpassage, growth in nude mice, etc.), and only one characteristic, the source of the original tumor cells, waspredictive of MTS-forming ability. All 8 of the breastcancer lines (and the original 11 lines) that formed MTShad been obtained from solid growths (primaries or métastases), while the 6 breast cancer lines that did not formMTS were all derived from pleural effusions. Similarly,artificial selection for an ascites variant of the MTS-forming rat 13762 adenocarcinoma line produced the 13762-Aline, which could no longer form MTS. These results suggest that breast cancer cells derived from pleural effusions are genetically different from the bulk of the tumorcells in solid breast cancer samples, that they are unableto grow in true solid form, and that these differencespersist in spite of prolonged propagation in tissue culture.

INTRODUCTION

We recently described a method for producing and growing MTS3 based on the plating of tumor cells in liquidmedium over an agar-medium base (19). Within 3 days to 3weeks after plating, spherical aggregates of tumor cellsappear and continue to grow to sizes of 2 to 3 mm at whichtime further study is impractical due to their fragility. TheseMTS possess a growing outer shell, an intermediate viablebut nondividing shell, and at large enough sizes a centralnecrotic core (19). Analysis of a series of murine tumors,which possessed similar monolayer-doubling times, revealed a 5-fold range of growth rates in the MTS form (18).This wide range of growth rates was largely the product ofMTS line-dependent differences in the depth of the dividingshell and therefore in the growth fraction (18). In preliminary studies it appeared that MTS growth rates correlatedwith the growth rates of the respective in situ transplantedtumors (19).

1 Research supported by National Cancer Institute Contract N01-CB-74203and by Grant CA20174-01 from the National Cancer Institute.

1To whom requests for reprints should be addressed, at Cancer Researchand Treatment Center, University of New Mexico, 900 Camino de Salud N.E.,Albuquerque, N. M. 87131.

3The abbreviation used is: MTS, multicellular tumor spheroids; i.d.,intradermally.

Received February 20, 1978; accepted May 5, 1978.

Defining an MTS as a 3-dimensional cellular aggregatethat is able to grow under these conditions, it was observedthat all 11 solid tumor lines tested did so. A similar methoddeveloped by Putman ef al. (9) and Steuer ef al. (14-16) forthe purpose of distinguishing between normal and transformed cells in in vitro carcinogenesis experiments wasused to compare 13 normal and 25 transformed lines. Inthese studies it was demonstrated that 24 of the 25 transformed lines fit our definition of MTS and that the cells fromthe 25th line, although failing to grow in the aggregateform, remained viable during the 5-day observation period(9, 14-16). None of the 13 normal cell lines tested formedaggregates that grew or remained viable for 5 days (9, 14-16). We know of only 1 case (13) where it was claimed thata normal cell line could form MTS.

In combination, our results (18, 19) and those of Putmanef a/. (9) and Steuer ef a/. (14-16) have demonstrated that35 solid tumor lines formed MTS. 1 solid tumor line formedviable but nongrowing aggregates, and none of the 21normal cell lines formed either growing or viable aggregates. While this near-100% MTS-forming efficiency bysolid tumors is impressive, we pointed out (19) that weconsidered it likely that solid tumors would eventually befound that failed to form MTS. The basis of our suspicionwas the surface morphology of the initial 11 MTS lines wehad studied. At one extreme (e.g., MCa-11, a murine mammary carcinoma), the cells on the surface were plate-likeand tightly packed, providing an overall smooth MTS surface. At the opposite extreme (e.g., the murine line 1alveolar cell carcinoma), the cells on the surface werespherical and loosely packed yielding a MTS that resembleda spherical bunch of grapes. The remaining lines formed aspectrum of appearances between these 2 extremes, and asimilar wide range of morphologies has been noted byPutman ef al. (9) and Steuer ef al. (14-16). Reasoning thatwe might eventually encounter lines the cell-cell cohesiveforces of which were even lower than in the lines with aloosely packed surface, we considered it possible that theselines might not be able to maintain the 3-dimensionalorganization of MTS.

In the course of mass-producing cells from our original11 tumor lines in suspension culture, we obtained our firstsuggestion as to what type of solid tumor cells would notform MTS. Lines with loosely packed surfaces adaptedreadily to suspension culture, while those that adapted tosuspension culture poorly or not at all possessed tightlypacked surface cells in the MTS form. This observationsuggested the possibility that strong natural or artificialselection for growth in suspension might produce lines thatwere unable to form MTS. Rather than initiate artificialselection experiments in vitro, we elected to study cell linesthat had been selected in vivo for the ability to grow as a

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Breast Cancer Source and MTS

suspension, i.e., malignant pleural effusions, and to compare them to similar cell lines derived from solid growths.The data presented below demonstrate that monolayercultures of 8 different breast cancer lines derived from solidgrowths were able to form MTS in our system but that 6similar cultures of breast cancer cells derived from pleuraleffusions were unable to do so. Further, artificial selectionfor an ascites-adapted variant of a MTS-forming line resulted in the loss of its MTS-forming ability.

MATERIALS AND METHODS

Cell Lines. A total of 16 cell lines (15 transformed" and 1normal) were used in this study. Table 1 summarizes thecharacteristics of these lines in terms of species of origin,site from which the line was isolated, pathological diagnosis, and the number of passages through which the lineshad been carried at receipt in our laboratory.

Lines Hs578T, Hs578Bst (normal), MCF-7, and SKBR + 3were received as frozen samples.5 All of the lines with theprefix "MDA-" were received5 as monolayer cultures and

have been maintained as such in our laboratories. The 6 ratbreast cancer lines were obtained5 as s.c. (13762, R3230AC,3M2N, DMBA 1, and DMBA 14) or i.p. (13762-A) transplantsin female Fischer 344 rats. MCa-11 (18, 19) is a mousemammary tumor and was developed in our own laboratory.

Monolayer Culture. The standard medium used throughout these experiments was Eagle's basal medium supple

mented with 10% fetal calf serum; penicillin, 50 units/ml;streptomycin, 50 ¿¿g/ml(Grand Island Biological Co., GrandIsland, N. Y., or Flow Laboratories, Rockville, Md.); and sodium insulin, 10 /¿g/ml(Elanco Products Co., Indianapolis,Ind.). This addition of insulin to our standard medium (18,19) was found to be necessary since at least 1 line, MDA-361,failed to grow in monolayer in its absence, as will bedescribed elsewhere.6 Cultures were harvested by mild

trypsinization (0.25% w/v; 3 to 5 min). All cultures weremaintained at 37°in a humidified incubator (5% CO-, plus

95% air).Frozen samples were thawed rapidly to 37°and inocu

lated in standard 75 sq cm tissue culture flasks along with10 ml of complete medium. Tumors s.c. were removedaseptically, minced, prepared as single-cell suspensions ina Teflon-glass tissue grinder, and then handled as previously. The ascites tumor, 13762-A, was aspirated from theabdominal cavity and then inoculated directly into tissueculture flasks. All of the cell lines adapted to monolayerculture had doubling times ranging from 16 to almost 100hr.

4 All of the transformed lines studied were derived from frank tumors invivo, and the derived cultures possess at least 1 of the following characteristics: ability to form colonies in soft agar; piled up morphology in monolayerculture; low serum requirement; high saturation density; and ability to formtumors in immunosuppressed mice. The characterization of the singlenormal line. Hs578Bst. is given in Ref. 6.

'The Hs578T. Hs578Bst, MCF-7. and SKBR + 3 lines were provided byContract E-73-2001-N01 within the Special Virus-Cancer Program NIH,USPHS. through the courtesy of Dr. Walter A. Nelson-Rees. The MDA-157,MDA-231, MDA-330, MDA-361. and MDA-436 lines were provided by Dr.ReídaCailleau of the M. D. Anderson Hospital and Tumor Institute. The 6 ratmammary cancers were provided by Dr. Arthur Bogden of the MasonResearch Institute.

•¿�J. M. Yuhas and A. E. Tarleton. Dormancy and Spontaneous Regrowthof Human Breast Cancer In Vitro, submitted for publication to CancerResearch.

MTS Formation. For the comparison of MTS-formingability, approximately 106 cells harvested from monolayersor from transplanted tumors (19) were seeded, along with10 ml of complete medium into 100-mm Retri dishes thathad been base coated (2 to 3 mm) with 0.75% Noble agar(Difco Laboratories, Inc., Detroit, Mich.) in complete medium. The increase in the agar concentration from 0.5%(18, 19) to 0.75% was made due to occasional detachmentof the agar from the plate at the lower concentration. Theplates were returned to the incubator and were observedfor up to 60 days for MTS formation.

The methods for measuring monolayer and MTS growthrates have been detailed elsewhere (19) as has the[125l]iododeoxyuridine incorporation method for estimatingthe depth of the dividing shell in a MTS (18).

Samples for scanning electron microscopy were handledaccording to standardized techniques (17).

RESULTS

MTS Formation. Table 2 summarizes the results of ourattempts to produce MTS from monolayer cultures of 14breast cancer lines (13762-A, the ascites tumor discussedbelow) and 1 normal myoepithelial breast line. In accordwith our prior experience (19) and that of others (9, 14-16),the normal myoepithelial cell line derived from the breast.Hs578Bst, did not form MTS. Eight of the 14 breast cancerlines formed MTS, and all of these had been isolated fromeither solid tumors or métastases.This required anywherefrom 1 to 3 weeks (Table 2). The remaining 6 breast cancerlines, which were originally isolated from malignant pleuraleffusions, failed to form MTS, and with one exception,MDA-231, failed to form aggregates larger than 10 to 50cells. MDA-231 cells formed aggregates of up to 100 /¿m,but these aggregates failed to grow and rapidly degenerated. However, these pleural effusion-derived cell linescontinued to grow in the agar-base cultures as single cellsand clusters of up to 10 cells, providing a simple means ofsimulating suspension culture conditions.

In an attempt to provide conditions that would allow thepleural effusion-derived lines to form MTS, a variety ofculture conditions were tested. These included observationfor up to 60 days, use of 2 additional types of medium(Dulbecco's modified Eagle's medium and Liebovitz L-15),

use of 3 different batches of fetal calf serum, reduction ofthe number of cells added to each culture to as few as 102,

and the removal of insulin and/or the addition of otherhormones (dibutyryl cyclic adenosine 3',5'-monophos-phate, 17/3-estradiol, prolactin, etc.) to the medium. Whilewe were not able to test all permutations of these variablesor test each variable in all 6 lines, none of these alterationsallowed any of the 6 pleural effusion-derived lines to formMTS.

The 13762-A line, which was artificially derived from the13762 line, grows as an ascites (1), as opposed to the solidgrowth form seen in the parent line (2). We tested the13762-A line for its MTS-forming ability and, as predictedby the correlation given previously, it was unable to formMTS. In addition to demonstrating that it is possible toselect for MTS-nonforming cells within a solid tumor thatdoes form MTS, this observation rules out the possibility

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Table 1Species, site of isolation, diagnosis, number of passages, and etiology of the 16 cell lines used in these experiments

CelllineHs578BstHs578TMCF-7SKBR

+3MDA-157MDA-231MDA-330MDA-361MDA-4361376213762-A3M2NR323OACDMBA1DMBA

14SpeciesHumanHumanHumanHumanHumanHumanHumanHumanHumanRatRatRatRatRatRatIsolation

siteBreastBreastPleural

effusionPleuraleffusionPleuraleffusionPleuraleffusionPleuraleffusionBrainmetastasisPleuraleffusionBreastBreast

ascitesBreastBreastBreastBreastDiagnosisNormal

myoepithelialCarcinosarcomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAscites

form of13762SquamouscellcarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaForm

at areceiptFML"FM

LFMLFMLMLMLMLMLMLTPTPTPTPTPTPNo.

ofpassages8933817217573392511136821814152Etiology???7?7??DMBADMBA3-Methyl-2-naphthylamineSpontaneousDMBADMBARef.661253,43,43,43,43,4212222

MCa-11 Mouse Breast Adenocarcinoma TP + ML Radiation 18,19" FML, frozen monolayer; ML, monolayer; TP, transplant; DMBA, dimethylbenz(a)anthracene.

Table 2Comparison the ability of monolayer cultures of 14 transformed

and 1 normal breast line to form MTS

MTS formationfromLineHs578BstMCF-7SKBR

+3MDA-157MDA-231MDA-330MDA-436TypeN"TTTTTTSourceBPEPEPEPEPEPEMono

layerNoNoNoNoNoNoNoTransplant-Celldegenerationwithin

3daysClusterof 10 to50cells——

Noclusterformation—

Aggregates of100/xmthatdegenerate-

Clusters of 10to50cells-

Noclusterformation

137623M2NR323OACDMBA1DMBA

14MCa-11MDA-361Hs578TTTTTTTTTBBBBBBMBYesYesYesYesYesYesYesYesYesYesYesYesYesYes——3

wk toappear8days toappear9days toappear3wk toappear3wk toappear1wk toappear1wk toappear2wk to appear

" Remarks refer to behavior in agar-base cultures derived frommonolayers.

6 N, normal; B, breast; -, line not available in transplant; T,transformed; PE, pleural effusion; M, solid metastasis.

that inability to form MTS is found only in human lines. Weare presently attempting to select within the 13762-A line

for the ability to grow as a solid tumor; i.e., due to its recentorigin it may still be possible to proceed in the reversedirection.

The 5 rat tumor lines (13762, R3230AC, 3M2N, DMBA 1,

and DMBA 14) and the 1 mouse breast cancer (MCa-11) are

maintained in our laboratory as s.c. transplants as well asmonolayer cultures. Single-cell suspensions of these transplants produce MTS as readily as do single-cell suspensions

derived from monolayers (Table 2) and, in fact, do so morerapidly. Whether this represents more rapid or effectivetumor cell aggregation or more rapid growth remains to bedetermined.

Although the tumor line characteristic chosen for studywas the site from which the tumor was isolated, it ispossible that other characteristics would correlate withMTS-forming ability as well. If so, this would either questionour "site of isolation" argument by demonstrating a similar

correlation with an unrelated characteristic (e.g., estrogenreceptor content) or reinforce it by demonstrating a correlation with a more subtle characteristic related to the site ofisolation.

We were unable to locate data on all 14 lines for any 1characteristic, but we were able to compare MTS-formingand -nonforming lines in terms of a variety of characteristics. None of the characteristics that we have consideredare predictive of MTS-forming ability. These characteristicsincluded growth rate in monolayer; growth in nude mice;estrogen receptor patterns; growth in soft agar; growth inthymectomized, irradiated, and bone marrow-reconstituted

mice; number of passages in serial propagation; or expression of differentiated functions.

Preliminary Characterization of Breast Cancer MTS. All8 of the breast cancer lines capable of forming MTS yieldedMTS with a smooth surface composed of interlocking cells(Fig. 1, A and C). Microvilli were sparse except in cellsundergoing mitosis (Fig. 13). As will be discussed in detailin a subsequent report, the cells on the surface of thesebreast cancer MTS are flattened and oriented perpendicularto the radius of the MTS. Chart 1 is a plot of the growth of 3types of breast cancer MTS: MCa-11, MDA-361, and

Hs578T. As pointed out elsewhere (18), the growth rates ofthe MTS do not correlate with their respective monolayer

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Breast Cancer Source and MTS

growth rates. In our studies on 7 murine tumors (18), themonolayer-doubling times ranged from 13 to 21 hr, whilethere was a 5-fold range in the MTS growth rates from thesame lines, largely due to variations in the growth fractionwithin the MTS. In this comparison a similar dissociation ofmonolayer-doubling times and MTS growth rates was observed (Table 3).

While Hs578T grows faster than does MDA-361 in mono-layer, the reverse is true for MTS growth rates. This appearsto be due to 2 factors. The MDA-361 growth fraction islarger than that for Hs578T (Table 3), and the doubling timeof MDA-361 cells is faster in the MTS than it is in monolayer,

700

MDA-361

H«578 T

300

IO 20 30

DAYS

Chart 1. Growth of 3 types of breast cancer MTS as a function of time.Individual MTS (N, 12 to 24/line) were placed in individual agar-based 16-mm wells along with 1 ml of complete medium. MTS were sized 3 timesweekly, and the medium was changed 2 times weekly.

Table 3Characteristics of the growth of 3 breast cancers lines in the

monolayer or MTS system

LineMCa-11

MDA-361Hs578TMonolayer-

doubling time(hr)11.3±2.16

91 ±4.832.3 ±4.9MTSGrowth

rate(¿im/day)31

±1.912 ±2.34.1 ±0.6Growth

fraction(%)»48

33<15r

" Calculated for a 300-/¿mMTS and expressed as a percentage

of the total volume occupied by dividing cells; depth of the dividingcells determined by [125l]iododeoxyuridine incorporation assay(18).

6 Mean ±S.E.c [125l]lododeoxyuridine incorporation assay indicates a dividing

shell depth of <8 /¿mthat is approaching the limits of detection inthis system.

as opposed to the reverse case which is observed with mostMTS (18).

DISCUSSION

The data presented previously clearly demonstrate thatthe ability to form MTS is not an immutable characteristicof all solid tumor cells. Both natural and artificial selectionhas produced stable variants that are able to grow insuspension, and in acquiring this ability they lose theirability to form MTS. Since MTS-forming and -nonforminglines are stable in spite of long-term passage in monolayerculture, it must be concluded that adaptation to MTSformation or suspension culture represents genetically determined alternate states and that monolayer culture doesnot select against either one. It is not clear, however, thatthis adaptation to 1 type of growth or the other is as clear-cut for all tumor types. The basic observation that led us toinvestigate malignant pleural effusions of breast cancerwas that our original 7 murine tumors possessed gradedadaptations to the 2 types of culture (MTS and suspension)and that the 2 adaptations were reciprocal. Breast cancercell lines represent an extreme case of the more generalinverse relationship between the 2 types of adaptation.

Although malignant pleural effusions of breast cancerfrequently contain clusters of tumor cells (7,11,12), seldomdoes one encounter a true solid mass within the pleuralcavity. Whether these in vivo aggregates are viable or are inthe process of degeneration remains unknown. In vitrostudies with cell lines derived from these effusions, however, are instructive. MCF-7, a malignant pleural effusionderived from a human mammary adenocarcinoma (12) canbe grown on collagen-coated sponge substrates (10,11). Inthis system the cells form small aggregates but are seldommore than a few cells deep. This would suggest that theinability of clusters to continue growing to larger sizes is amembrane or cohesion defect since metabolic limitationswould allow continued growth'of the cells on the surface

and a gradually increasing necrotic center. We are currentlyinvestigating the membranes of MTS-forming and -non-forming lines, especially lines 13762 and 13762-A. Preliminary studies have indicated that the membrane glycopro-teins of these 2 lines differ significantly (M. Brysk and J. M.Yuhas, unpublished data) and that, even when the 13762-Aline is forced to grow in a confined area by i.d. transplant,it develops fewer intercellular junctions than does the 13762line (A. J. Ladman and J. M. Yuhas, unpublished data).Whether these observations are causative or the result ofdifferences in growth rate or i.d. transplant differences invascularity is presently under study.

Clearly, the tumor cells in a malignant pleural effusionare derived from the primary tumor, and it would appearthat they are variants that, although no longer able to growas a true solid tumor, have a selective advantage for growthin suspension. Since at least 1 of our lines, MDA-361, wasderived from a solid metastasis, it is clear that primaryhuman breast cancers can shed at least 2 types of "meta-static" cells, and the course of the disease is determined by

the interaction of the type of cell released and the selectiveforces at the site where the cell comes to rest. These dataprovide further support for the evolution of tumor cell

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J. M. Yuhas et al.

populations within the body, which was recently summarized and expanded by Nowell (8). In a subsequent report,6

we will demonstrate that evolution from hormone dependence to hormone independence can be observed in vitro.

It would appear, at first, that the inability of certain tumorlines to form MTS would allow for false negatives if oneused MTS formation as an index of transformation asproposed by Putman ef al. (9) and Steuer et al. (14-16). Thisis in fact not true since Steuer's index is not true MTS

formation but rather is retention of viability in the aggregateform and some of our pleural effusion lines formed viableaggregates, at least temporarily. More importantly, however, pleural effusions are a late-occurring result of transformation and appear under the influence of selectivepressure. Since the transformation assay proposed by Putman et al. (9) and Steuer ef al. (14-16) involves in vitrotransformation and no selective pressures, the lack of MTSformation by pleural effusions has no bearing on theirsystem.

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1. Bodgen, A. E., Haskell, P. M., Cobb, W. R., and Kelton, D. E. Heterogeneity in Chemotherapy Responsiveness of the Solid 13762 MammaryAdenocarcinoma and Two Derived Ascites Tumor Lines. Proc. Am.Assoc. Cancer Res., 17: 40, 1976.

2. Bodgen, A. E.. and Taylor, D. J. Predictive Mammary Tumor TestSystems for Experimental Chemotherapy. In: J. C. Heuson, W. H.Mattheiem. and M. Rozencweig (eds.), Breast Cancer: Trends in Research and Treatment, pp. 95-110. New York: Raven Press, 1976.

3. Cailleau, R M. Old and New Problems in Human Tumor Cell Cultivation,In: J. Fogh (ed.). Human Tumor Cells In Vitro, pp. 75-114. New York:

Plenum Press, 1974.4. Cailleau, R., Cruciger, Q., Hokanson, K. M., Olive, M., and Blumen

schein, G. Morphological, Biochemical and Chromosomal Characterization of Breast Tumor Lines from Pleural Effusions (Abstract). In Vitro,72:331, 1976.

5. Fogh. J.. and Trempe, G. New Human Tumor Cell Lines. In: J. Fogh

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6. Hackett, A. J., Smith, H. S., Springer, E. L., Owens, R. B., Nelson-Rees,

W. A., Riggs. J. L., and Gardner, M. B. Two Syngeneic Cell Lines fromHuman Breast Tissue: The Aneuploid Mammary Epithelial (Hs578T) andthe Diploid Myoepithelial (Hs578Bst) Cell Lines. J. Nati. Cancer Inst.. 58:1795-1806, 1977.

7. Luse, S. A., and Reagan, J. W. A Histocytologic and Electron Microscopic Study of Effusions Associated with Malignant Disease. Ann. N. Y.Acad. Sci.,63: 1331-1347, 1956.

8 Nowell, P. C. The Clonal Evolution of Tumor Cell Populations. Science,194: 23-28, 1976.

9. Putman, D. L., Park, D. K., Rhim, J. S., Steuer, A. F., and Ting, R. C.Correlation of Cellular Aggregation of Transformed Cells with TheirGrowth in Soft Agar and Tumorigenic Potential. Proc. Soc. Exptl. Biol.Med., 755. 487-494, 1977.

10. Russo, J., Bradley, R. H., McGrath, C., and Russo, I. H. Scanning andTransmission Electron Microscopy Study of a Human Breast CarcinomaCell Line (MCF-7) Cultured in Collagen-coated Cellulose Sponge. CancerRes..37: 2004-2014, 1977.

11. Russo, J., Soule, H. D., McGrath, C.. and Rich, M. A. Reexpression ofOriginal Tumor Pattern by a Human Breast Carcinoma Cell Line (MCF-7)in Sponge Culture. J. Nati. Cancer Inst., 56: 279-282, 1976.

12. Soule. H. D. Vasques, J., and Long, A. S. A Human Cell Line from aPleural Effusion Derived from a Breast Carcinoma. J. Nati. Cancer Inst.,51: 1409-1416, 1973.

13. Sutherland, R., and Durand, R. Radiation Response of Multiceli Spheroids An In Vitro Tumor Model. Current Topics Radiation Res.. 11: 87-139, 1976.

14 Steuer, A. F., Hentosh, P. M., Diamond, L., and Ting, R. C. SurvivalDifferences Exhibited by Normal and Transformed Rat Liver EpithelialCell Lines in the Aggregate Form. Cancer Res., 37: 1864-1867, 1977.

15. Steuer, A. F., Rhim, J. S., Hentosk, P. M., and Ting, R. C. Survival ofHuman Cells in the Aggregate Form: Potential Index of In Vitro CellTransformation. J. Nati. Cancer Inst.. 58: 917-921, 1977.

16. Steuer, A. F., and Ting, R. C. Formation of Larger Cell Aggregates byTransformed Cells: An In Vitro Index of Cell Transformation. J. Nati.Cancer Inst., 56: 1279-1280,1976.

17. Waterman, R. E. Topographical Changes along the Neural Fold: Associated Neuration in the Hamster and Mouse. Am. U. Anat., 746. 151-172,1976.

18. Yuhas, J. M., and Li, A. P. Growth Fraction as the Major Determinant ofMulticellular Tumor Spheroid Growth Rates. Cancer Res., 38: 1528-

1532, 1978.19. Yuhas, J. M., Li, A. P., Martinez, A. 0., and Ladman, A. J. A Simplified

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Breast Cancer Source and MTS

C

Fig. 1. Scanning electron micrograph of a 340-^m MCa-11 MTS. A, 350-power view of MTS; 8, 2800-power view of cell in mitosis; C, 2800-power view ofnonmitotic cell. "Popcorn"-like material adherent to surface likely to be an artifact.

AUGUST 1978 2491

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1978;38:2486-2491. Cancer Res   John M. Yuhas, Anne E. Tarleton and Karen B. Molzen  Isolated from Different SitesMulticellular Tumor Spheroid Formation by Breast Cancer Cells

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