(CANCER RESEARCH 43, 5314-5320, November 1983]

Metastatic Heterogeneity of Cells from Lewis Lung Carcinoma1

Anne-Louise van Lamsweerde,2 Nicole Henry, and Gilbert Vaes3

Laboratoire de Chimie Physiologique, Universitéde Louvain, and International Institute ot Cellular and Molecular Pathology, Avenue Hippocrate, 75, 8-1200 Brussels,Belgium

ABSTRACT

To allow investigations of the role of tumor cell proteases ininvasion and metastasis, an attempt was made to obtain well-

defined homogeneous populations of Lewis lung carcinoma cellsdiffering widely in their metastatic potential. From a single Lewislung carcinoma, a parental line of cells was established andsubsequently cloned so as to provide 18 clonal tumor cell lines.These clones differed in their ability to produce spontaneous,macroscopically visible métastases in the lung after i.m. inoculation into syngeneic C57BL/6 mice. Several of them were less

metastatic than the parental line. The parental line expressed ametastatic behavior close to that of the high-metastatic cell

subpopulations that it contained. There was, within certain limits,a good correlation between the potential for spontaneous lungmétastasesarising from a primary tumor and that for "artificial"

lung colonies obtained after i.v. injection of the Lewis lungcarcinoma cells.

Although positively correlated with the growth rate of thetumor cells, the metastatic ability of the clones could not beconsidered as a mere reflection of the proliferation rates of thecells constituting the primary tumors. Differences in metastaticbehavior observed among clones persisted in several cases afterthe cells had been maintained in culture for prolonged periods.However, this stability of the clones in vitro was not absolute.Indeed, some subclones isolated from the low-metastatic clone

H122 displayed metastatic abilities which were lower than thatof the parent clone. Furthermore, a significant increase in metastatic potential was once observed after a prolonged cultureperiod of that same clone, H122. Thus, new metastatic pheno-

types can emerged under in vitro culture conditions. However,the relative rarity of this event suggests that some metastaticheterogeneity already preexisted in vivo among the tumor cells.

INTRODUCTION

The ability of tumor cells to metastasize to distant sites mightbe dependent on their capacity to secrete proteases that affectcomponents of the surrounding extracellular matrices (26). Tostudy these possible relationships, we undertook experimentswith a spontaneously metastasizing tumor, the LLC4 (3). How

ever, as it became more and more evident that cancerous tumorsmay contain subpopulations of cells with widely differing metastatic potential (6, 20), it appeared that our investigations attempting to correlate the metastatic potential to specific properties of LLC cells would require the use of well-defined homo

geneous populations of cells, since the properties peculiar to a

1Supported by grants from the Cancer Research Funds of the Caisse Généraled'Epargne et de Retraite, Brussels, and from the Belgian Fonds de la Recherche

Scientifique Médicale.2 Boursière de lï.R.S.I.A.3To whom requests for reprints should be addressed.4The abbreviation used is: LLC, Lewis lung carcinoma.

Received November 10,1982; accepted July 13,1983.

small subpopulation of highly metastatic cells could be maskedby poorly metastatic cells.

We therefore undertook, as reported here, to derive a seriesof cloned subpopulations from a parent culture of LLC cells.These clones differed in their ability to produce spontaneouslymetastatic colonies macroscopically visible in the lung after i.m.inoculation into syngeneic mice. Homogeneous LLC subpopula-tions of cells with either low or high metastatic potential couldthus be selected to be used in our further studies on proteasesecretion. One of these studies, establishing that macrophagesmay cooperate with LLC cells for collagen degradation and thatdifferent subpopulations of LLC cells differ in their response tothat stimulation, is reported in the accompanying paper (11). Apreliminary account of the present work has already been presented in abstract form (30).

MATERIALS AND METHODS

Conditions of Culture. LLC cells, obtained from a parent tumor asdescribed below, were grown at 37° in Falcon Plastics (Cockeysville,

Md.) Retri dishes under a water-saturated atmosphere of air:CO2 (9:1) ina "basal" medium made of Dulbecco's modified Eagle's medium (from

Gibco-Biocult, Glasgow, Scotland), supplemented with penicillin (105 III/

liter) and streptomycin (100 mg/liter) to which 10% (v/v) heat-inactivated(30 min at 56°)fetal calf serum (Gibco-Biocult) was added. They were

passaged 3 times a week at a split ratio of 1:12 to 1:64 after dispersionwith EDTA (2 mw) in phosphate-buffered saline (8 rtiM Na2HPO.<:1 rtiM

KH2PCv140 mw NaCI, pH 7.4).During this investigation, cells were frozen at different times and stored

at -80° or in liquid nitrogen for later use. Reference to "culture length"

indicates the total, cumulative length of the different culture periods ofthe cells, determined, for the parental line, from the day on which thetumor was put in culture and, for the clones, from the day of cloning.

Establishment of the Parental Cell Population. A LLC (27), maintained in vivo by passage in syngeneic C57BL/6 mice, was receivedfrom Dr. G. Atassi (Institut Bordet, Brussels, Belgium). Fifteen days aftertransplantation, the tumor was aseptically removed, minced, and mechanically dispersed by repeated pipeting. The recovered cells wereadapted to grow in culture so as to obtain, after 10 passages, a cell linewhich was well able to grow on plastic and which was frozen at -80°

or in liquid nitrogen for later use. After thawing, the cells were furthercultured for 20 passages to provide our parental cell population. Samplesof the parental population were either used to produce clones or furthercultured for 5 passages and then frozen.

Establishment of Clonal Subpopulations. Cells from the parentalpopulation, taken after 69 days of total culture length, were cloned by atechnique based on the dilution plating principle (19). Single-cell suspensions were seeded into the 96 wells of a Microtest II (Falcon Plastics)tissue culture plate at 1 cell/well. Six hr later, wells containing one singlecell were identified under the light microscope (x 100 magnification) andmarked. Colonies were transferred after sufficient growth to vessels ofincreasing size to expand the cell population. The cloned subpopulationsobtained after 6 to 11 passages (14 to 26 days of culture after cloning)were divided into multiple samples; some were used for the experimentreported in Table 1 and Chart 1, and the others were frozen until theywere needed for further testing. Some samples were also thawed at

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Metastatic Heterogeneity of LLC Cells

various time inu vals thereafter, grown in culture for 5 to 43 days (2 to18 passages) to allow their expansion, divided again into multiple ali-

quots, and frozen.Except for the experiment reported in Table 1 and Chart 1, the culture

length of the clones was not continuous, being interrupted by one or 2periods of storage at -80° or in liquid nitrogen.

In Vivo Assays for Spontaneous and Artificial Metastasis. Afterthawing, the parental cell population and the clones were cultured for 14to 84 days (6 to 36 passages) before being used for the in vivo tests.The cells were harvested from subconfluent cultures after a 1-mintreatment with phosphate-buffered saline containing EDTA (0.5 mw) and

trypsin (2.5 mg/ml); this medium was then removed by suction, and thecells were resuspended by gentle pipeting in basal medium supplementedwith 10% fetal calf serum. They were then washed by sedimentation andresuspension in serum-free basal medium and counted before their

injection into the mice. At that time, 80 to 93% of the collected cellsappeared viable, as established by their exclusion of trypan blue. FemaleC57BL/6 mice (obtained from Proefdierencentrum, KUL, Heverlee, Belgium), 10 to 16 weeks old and weighing 18 to 22 g, were used.

For the experiments on spontaneous métastases, 5 x 105 viable cells

(0.2 ml) were injected i.m. into the hind leg of syngeneic mice. The localgrowth of the primary tumor was monitored one to 2 times a week bycaliper measurements of 2 perpendicular diameters of the tumor massand by evaluating the volume of the tumor from these figures (14, 23).In most experiments, the mice were followed until their spontaneousdeath; in a few experiments, they were killed 26 days after the tumorinoculation. The primary tumors were then dissected out and weighed.The lungs were removed and rinsed in water, and the métastaseswerecounted at the lung surface (4) and measured with calipers under adissecting microscope (x 10); their volumes and weights were calculatedby assuming that each metastatic nodule was spherical and that itsdensity was equal to 1. For each experimental group, the number ofmice having lung métastases was recorded as well as, whenever appropriate, the survival time of each animal. In some experiments, 105 viable

cells (in 0.05 ml of medium) were also injected into the hind footpad ofsyngeneic mice. The time of appearance and the local growth of theprimary tumors were then monitored. When the primary tumors reacheda diameter of 6 to 8 mm, they were removed by amputation of the legabove the knee joint under sodium pentobarbital anesthesia (60 mg/kg¡.p.).The mice were then kept until their spontaneous death or, for thosethat survived, for 7 months after tumor implantation; they were thenexamined for the presence of lung métastases.

"Artificial" métastaseswere obtained by injecting 105 or 2 x 105 viable

tumor cells (in 0.5 ml of medium) in the lateral vein of the tail of syngeneicmice. The mice were either killed at various times after this i.v. injectionor kept until their spontaneous death. Tumor cell colonies were thencounted and measured in the lungs as described above.

Other Techniques and Statistical Analysis. Screening for possiblemurine virus and Mycoplasma pulmonis infection of the various cell lineswas done by Dr. J. T. M. Van der Logt at the Reference Centre forRodent Viruses from the International Committee on Laboratory Animals,Institute of Medical Microbiology, University of Nijmegen, Nijmegen, TheNetherlands, following the mouse antibody production test (2, 21). Testfor general mycoplasmal contaminations of the cell lines were done byDr. G. Collinet at the Smith-Kline-Rit Laboratories, Genval, Belgium,

following the microbiological culture method as proposed by the UnitedStates Code of Federal Regulations, Food and Drug Administration,Paragraph 610.30.

The protein content of the cells was determined following the method

of Lowryefa/. (13).All data were analyzed by nonparametric statistical methods (22). The

Kruskal-Wallis test was used to evaluate the possibility that the various

experimental groups were different in terms of their survival times,weights of primary tumors, or numbers and weights of lung métastases.Multiple comparisons were done with the Kruskal-Wallis test by usingDunn's approximation, to compare groups 2 by 2 or to compare each

group with the parental line; the level of significance selected was fixedat p «0.05. The Mann-Whitney U test was used when there were only

2 groups to be compared. Pairwise correlations between parameterswere made with Kendall's rank correlation test to produce coefficient T.

RESULTS

Parental Line and Clones. Eighteen clonal sublines wereobtained from the cloning of the parental cell population. All ofthem were tumorigenic. Cells from these sublines, injected i.m.(5 x 105 cells) into syngeneic mice, developed into primary

tumors the weights of which, at the time of the spontaneousdeath of the mice, did not differ significantly from the weights ofthe primary tumors developed from the parental cell line (Table1). The growth of the primary tumors was, however, slower withthe clones than with the parental line, and the survival time ofthe mice given injections of a clonal line was also, for most ofthe clones, significantly longer than that of the mice injected withthe parental line (Table 1).

The growth rate of the parental line and of 5 clones wasevaluated several times under our culture conditions. The generation time was 10.6 ±0.9 (S.D.) hr (n = 8) for the parental line,10.8 ±1.6 hr (n = 7) for clone H61, 11.6 ±0.9 hr (n = 5) forclone C53, 11.3 ±2.6 hr (n = 4) for clone E34, 12.6 ±1.2 hr (n= 3) for clone G92, and 13.1 ±2.3 hr (n = 10) for clone H122.Final saturation densities were 1.0 to 1.2 x 105 cells/sq cm for

the parental line and for clones E34, C53, H61, and G92 but 6to 8 x 104 cells/sq cm for clone H122. When cultured on plastic,

each of the latter 5 clonal cell lines had some distinctive morphological traits. E34 and C53 cells are small and rounded, but C53cells adhere more poorly than do E34 cells to plastic; H61 cellsare often elongated and adhere better to plastic than do any ofthe other clones; G92 cells tend to associate in multicellularagglomerates; H122 cells are rounded and significantly largerthan the cells of the other clones. The protein content of C53,H61, G92, E34, and parental cells was 500 pg/cell, but it was850 pg/cell for H122 cells.

Spontaneous Lung Métastases.All the clones were able toproduce lung métastasesafter their i.m. injection into syngeneicmice. They displayed, however, great differences in this regard,both in the number of mice with métastasesat the time of death(incidence of métastases)and in the number of métastasespermouse (Table 1). Five clones (G92, H122, E34, B103, and F61)were low metastatic. The median number of métastases permouse was zero. On the other hand, 3 clones (H61, C53, andB22) were high metastatic; they caused métastases in all themice, with a median number of métastasesper mouse equal to10.5 to 12. These 3 clones were not significantly different fromthe parental line, but they differed significantly from all the otherclones except clones A51, C11, G54, B64, and D81. As shownin Chart 1, there was a direct relationship between the survivaltime and the number of métastasesfound after death for themice given injections of the high-metastatic clones C53 and H61but not for the mice given injections of the low-metastatic clones

G92, H122, and E34. These 5 clones were selected for ourfurther studies.

Significant differences between low- and high-metastatic

clones were also found when the mice were killed at a predetermined day (Day 26) after the i.m. injection of the clones (Table2). In these experiments, clones that gave primary tumors ofsimilar weights on Day 26 were compared. The differences

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Table 1

Spontaneous metastasis and other biological properties of LLC parental cellpopulation and its donai subpopulations

C57BL/6 mice were given i.m. injections of 5 x 105 viable cells. All mice

developed a primary tumor. The weight of the primary tumors, the number of micewith pulmonary métastases,and the number of pulmonary métastasesper mousewere determined at the time of spontaneous death. Except for the incidence ofmetastasis, median values are presented followed in parentheses by the range.For each cell line, the total length of culture preceding its injection to the mice isgiven in days (for the parental line. Day 0 = day on which the tumor was put inculture; for the clones. Day 0 = day of cloning).

CellsParentClonesG92H122E34B103F61E113B82G113H42D81B64G54C11H41A51B22C53H61Culturelength(days)98303528301927302928292925293027302928Median

survival(days)21(15-30)"30

(26-34)°33(26-40)c32(26-42)c27(25-30)29(28-42f29(24-35)c31(23-39)c38(32-47)c34(29-42)c29(22-33)°32(25-33)°29(22-36)°29(26-32)34(29-40)°30(26-34)°31(23-33)°33(27-41)°35(32-41)°Median

primary tumorwt(g)12

(8-17)10

(7-13)11(4-12)12(9-16)11(7-13)11(4-15)9

(7-12)11(6-19)13(12-14)14(12-16)13(10-16)15(13-18)13(6-16)12(7-13)12(9-16)13(11-16)15(15-17)13(11-15)14

(12-16)Pulmonary

métastasesInci

dence336/411/92/103/94/94/95/106/87/96/106/107/107/108/109/108/910/1010/109/9Medianno./

mouse5

(0-22)0

(0-2)"0(0-4)"0(0-3)*0(0-4)"0(0-7)"0.5(0-7)°1.5(0-4)"1

(0-5)"1.5(0-11)2

(0-14)2(0-11)4.5(0-8)2.5(0-6)2(0-3f6

(0-21)10.5(1-19)11.5(1-25)12

(4-25)

"Number of mice with métastases/total number of mice given tumor injection."Numbers in parentheses, range.°Significantly different from the parental line (Kruskal-Wallis test, p «0.05)."Significantly different from either the parental line or clones B22, C53, and H61

(Kruskal-Wallis test, p s, 0.05)."Significantly different from clones B22, C53, and H61 (Kruskal-Wallis test, p s

0.05) but not from the parental line.

observed in the number of métastasesare thus not related todifferent growth rates of the primary tumors.

Tumor cells were also implanted in the footpad of syngeneicmice. Under these conditions, the primary tumors that developedgrew faster with the parental line and significantly slower withthe low-metastatic clone H122; they were removed by amputa

tion when they reached a diameter of 6 to 8 mm (i.e., on MedianDays 24, 31, 36, and 55, respectively, for the parental line andclones H61, C53, and H122). Seven months after amputation,all the 26 mice given injections of clone H122 were still alive, andno métastaseswere found at their autopsy. Only 24 to 33% ofthe mice given injections of the parental line (7 mice of 29) orwith the high-metastatic clones C53 (5 mice of 18) and H61 (7

mice of 21) were alive at that time, all without métastases.Thisexperiment thus confirmed that, among our clones, H122 had avery low metastatic potential and that the parental line expresseda spontaneous metastatic potential that corresponded to that ofthe high-metastatic clonal cell subpopulations H61 and C53.

"Artificial" Lung Colonies. Several experiments were done

to establish whether the differences observed in the rates ofspontaneous métastasesof the clones were paralleled by differences in their ability to develop colonies ("artificial" métastases)

in the lung after their i.v. injection in the mice. As shown in Table3, the overall behavior of the clones in "artificial" metastasis was

similar to that observed in spontaneous metastasis. Again, theincidence of mice with tumor colonies in the lungs and the mediannumber of these colonies per mouse were the lowest with clone

H122. Clones E34 and G92 tended also to produce less colonies,while clones C53 and H61 were not significantly different fromthe parental line. In the mice that developed lung colonies, themean weight of the colonies was often also significantly lowerwith clones H122, E34, and G92 than with the parental line. Itwas also apparent that the number of lung colonies which couldbe counted at autopsy of the mice given injections of low-metastatic clones had a tendency to increase with the time ofsurvival of the mice after the injection. The "low"-metastatic

phenotype was most easily identified when the mice were killedat an early time after the injection. However, similar completeinvasions of the lungs, with colonies too numerous to count,were found in mice which had been given injections (105 cells

i.v.) of clones H122 or E34 or with the parental line when themice were autopsied after their spontaneous death (not shown).Nevertheless, death occurred earlier (median, 41 days; range,35 to 71 days) after the injection of the parental line than afterthat of clone H122 (median, 67 days; range, 57 to 91 days; p «s0.05); it occurred at an intermediate time with clone E34 (median,55 days; range, 42 to 60 days).

Stability of the Metastatic Phenotype of the Cloned CellSubpopulations. To evaluate the possible effect of the cloningprocedure on the stability of the metastatic phenotype (and thusalso the possibility that the heterogeneity in metastatic potentialobserved after the initial cloning of the parental cell population

uj 30w

20LU

0 10irLUm

1 O

O A

A o°

26 30 34 38 42

TIME OF DEATH (days)

Chart 1. Relationship between survival time and number of spontaneous pulmonary métastasesin mice given injections of either low- or high-metastatic clones.Data are from the experiment reported in Table 1. High-metastatic clones: C53 (O),and H61 (A). Low-metastatic clones: G92 (•),H122 (A), and E34 (•).Day 0 is the

day of tumor injection.

Table 2Spontaneous metastasis of selected clones in mice killed 26 days after tumor

inoculationThe mice had been given i.m. injections of 5 x 105 cells. All of them developed

a primary tumor. The results of 3 distinct experiments are presented in the sameway as in Table 1.

ExperimentABCCloneG92C53H122H61E34H61Culturelength(days)71693939105146Median

primary tumorwt(9)7

(5-9)"7(5-8)°5

(3-6)6(4-9)°5

(3-6)5(4-7)cPulmonary

métastasesInci

dence"13/1513/132/913/144/149/10Medianno./mouse4

(0-14)23(11-46)"0

(0-2)3(0-8)"0

(0-2)5(0-8)*

"Number of mice with metastases/total number of mice given tumor injection.

Numbers in parentheses, range.°Identical to the other experimental group within the same experiment (Mann-

Whitney U test)."Different (at p «0.002) from the other experimental group within the same

experiment (Mann-Whitney U test).

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Metastatic Heterogeneity of LLC Cells

TablesHeterogeneity in artificial métastasesamong clones from LLC

Mice were given i.v. injections of 105 or 2 x 105 viable cells either from the

parental cell population or from selected clonal subpopulations H122, E34, G92,H61, and C53. They were killed at various time intervals thereafter. The number ofmice with pulmonary colonies, the number of pulmonary colonies per mouse, andthe mean weight of the colonies per mouse with colonies were then determined.The results of several experiments are presented in the same way as in Table 1.

PulmonarycoloniesNo.

ofExperi- cells in- Day of

ment jected killingCells12 x 104 25 G92

C53Parent2

5 x 104 20 H122

E34G92H61

C533

105 21 H122

E34G92H61C53

Parent4

10s 21 H122

E34Parent105

28 E34

Parent105

43H1225

2 x 105 15 G92

C53Parent2

x 106 19 G92

C53Parent6

2x105 15 H122

E34Parent2

x 105 21 H122

E34ParentInci

dence31/8

5/105/103/10

1/82/104/93/74/10

5/118/9

10/118/98/92/98/9

10/109/9

6/69/107/1110/10

10/1011/12

9/99/90/9

4/97/91/9

9/108/8Median

no./mouse0

(0-1)"

1 (0-4)0.5(0-3)0

(0-1)0 (0-1)0 (0-2)0 (0-1)0(0-9)0

(0-2)c0 (0-2)c

3 (0-11)15 (0-35)8 (0-56)3(0-15)0

(0-2)°

2 (0-6)9.5(4-28)9

(2-22)"

16.5(6-27)5.5

(0-25)"3

(0-28)c

50 (26-77)49.5(27-90)7

(0-31 )c

37 (19-62)30(5-41)0

(0-0)c0 (0-3)c

18(0-33)0

(0-4)c7 (0-1 8)d

27.5 (7-39)Median

meanwt/

mouse(mg)0.5

4.24.20.3

0.30.52.21.30.4°

0.41.713.23.20.3°

0.4C

6.24.2C

16.63.10.3C

1.62.71.3C

6.160.4

0.70.3

0.4C

9.2aNumber of mice with colonies/total number of mice given tumor injection."Numbers in parentheses, range.cSignificantly different from parental line within the same experiment (Kruskal-

Wallis test, p s 0.05)." Significantly different from the results obtained within the same experiment

and with the same clone in mice killed at Day 21 (Experiment 4) or at Day 15(Experiment 6) (Mann-Whitney test, p «0.02).

resulted from the process of in vitro cloning and did not preexistin that population), clone H122 was recloned to produce 10subclones. These subclones were tested in vivo in 2 experiments(Table 4). Within each experiment, the number of pulmonarymétastases produced by each subclone was indistinguishablefrom that of the other subclones (Kruskal-Wallis test), but sub-

clones 2 and 17 did not produce any métastasesand appearedsignificantly different in this regard from the parental cell line.Other subclones could also be distinguished by other phenotypiccharacters, such as the survival time of the mice (significantlylower with subclone 14 and higher with subclone 9) or the invivo growth rate of the primary tumors (faster with subclones 2and 14, slower with subclone 9). Interestingly, the fastest growth

Table 4

Spontaneous metastasis and other biological properties ot subclones of cloneHT22

The clonal cell subpopulation H122 was recloned to produce 10 subclones. Twoexperiments were done in which cells from the subclones and the parent clonewere harvested in parallel and injected i.m. (5 x 105 viable cells) into mice at a time

when the parent clone accumulated 64 (Experiment 1) or 71 (Experiment 2) daysof culture. All mice developed a primary tumor. The day on which the primarytumors reached an average volume of 3 cu cm (calculated by interpolation fromthe data collected by caliper measurement) is presented as an index of their growthrate. All other measurements as well as the presentation of the results were carriedout as indicated in Table 1.

PrimarytumorCellsExperiment

1ParentH122Subclone

2Subclone5Subclone7Subclone9Subclone

10Subclone14Subclone

16Subclone17Subclone

18Subclone20Experiment

2ParentH122Subclone

2Subclone9Subclone14Subclone

17Median

survival(days)35

(30-42)°32(25-39)34(29-39)38(33-53)45

(37-61f35(31-45)28

(24-32)e34(31-49)32

(25-39)33(27-43)42(35-49)39

(33-50)35(23-39)44(36-67)28(25-32)"34

(27-42)Growth

rate8

(days)26172429322620262224292916352022Medianwt(g)912129111310968111014111011(6-14)(10-16)(5-16)(8-16)(6-16)(5-17)(8-12)(4-14)(5-14)(3-13)(9-15)(7-17)(7-15)(3-20)(8-14)(6-15)Pulmonary

métastasesInci

dence"13/200/103/105/103/102/101/102/90/124/125/77/100/102/92/100/10Medianno./mouse100(0-11)(0-0)d(0-2)0.5

(0-3)000000110000(0-11)(0-4)(0-1)(0-2)(0-0)"(0-1)(0-7)(0-5)(0-Of(0-6)(0-2)(0-0)"

"Time required to reach an average volume of 3 cu cm."Number of mice with metastases/total number of mice given tumor injection.c Numbers in parentheses, range.dWithin the same experiment, significantly different (p ss0.05) from the parental

line but identical to the other subclones (Kruskal-Wallis test).* Significantly different from the parental line and from several other subclones

within the same experiment (Kruskal-Wallis test, p «0.05).

rate of the primary tumors was observed with subclone 2, whichdid not produce any métastases.

The stability of the metastatic phenotypes of several clones(H122, E34, C53, H61) was also evaluated by comparing theobservations made during the in vivo testing of cells injected intomice after culture for increasing lengths of time (Table 5). Thiscomparison included a survey of the various experiments whichhad been performed over 3 years of investigation and whichinvolved a quantitative evaluation of the capability for spontaneous metastasis of cells that had been kept in culture forvariable lengths of time prior to in vivo testing. It also includedspecial experiments (identified by A, B, or C in Table 5) whereincells were maintained in continuous culture for several monthsand tested in vivo at various time intervals for their metastaticability.

A given clone, sampled after similar culture lengths and assayed repeatedly for spontaneous metastasis, provided, as arule, very reproducible results characteristic of its metastaticphenotype. The number of métastasesper mouse appeared tobe a better index of these metastatic phenotypes than was theincidence of metastasis in the mice (ratio of the number of micewith métastases over the total number of mice given tumorinjection), the latter being more subject to apparently randomfluctuations with low-metastatic clones. Moreover, the numberof métastasesper mouse obtained with both the high-metastaticclones, H61 and C53, and the low-metastatic clone, E34, did not

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Table 5Phenotypic stability ot low- and high-metastatic donai cell lines

Clones with low (H122 and E34) and high (H61 and C53) metastatic ability weregrown for varying lengths of time (Day 0 = day of cloning), and their ability forspontaneous metastasis was again tested after i.m. injection of 5 x 106 viable cellsin mice. The results of several experiments over a 3-year period are presented inthe same way as in Table 1; each experiment is identified by a number, thesignificance of which is explained below. All mice developed a primary tumor; fora given clone, the weights of the primary tumors (not shown) did not varysignificantly over the time of culture. The growth rate of these tumors was evaluatedas indicated in Table 4.

Culturelength

Clone(days)H61

285064656699149217C53

29344453129197E34

28629199148217H122

35506468727699161162170226230233265330Experi

ment87907"811182058202A800481048205A8207A7907d80018202A81118205A8207A7907"8202A800681048205A8207A7907a81118205B80048205C8202A8104820888205A8208C821

OB8207A821

OC8208A8210AMedian

survival(days)35

(32-41f31(28-44)28(21-39)29(21-35)31

(22-34)35(23-48)35(26-43)31(22-43)33(27-41)39(31-42)33

(29-40)30(26-35)30(25-39)29(22-31)32

(26-42)38(30-50)34(25-41)39

(32-43)37(32-60)41(34-53)33

(26-40)39(34-45)36(30-50)33(26-42)37(32-53)38(33-49)46(40-58)42(37-53)29(26-42)37(32-47)42(33-52)33(28-48)36(32-58)33(30-44)37

(29-45)Primary

tumorav.

growthrate"

(days)ND'20211721221915252322211914272325293027ND2927262829372922262922262423Pulmonary

métastasesInci

dence09/910/109/910/1010/1115/1510/109/1010/1010/1010/109/1010/1010/103/98/109/1212/158/103/82/104/1020/300/1210/205/106/158/1118/217/107/1310/108/157/913/14Medianno./

mouse12253018122034.53511.5(4-25f(4-49)(3-48)(1-48)"(0-51)(2-57)(11-49)(0-51)(1-25)918.5

(14-33)127.5232603.53.542000100.50.5014116156.5(2-31)"(0-27)(4-40)(1-36)(0-3f(0-8)"(0-7)(0-16)(0-14)(0-7)(0-4)(0-2)(0-11)(0-0)(0-8)"(0-11)"(0-5)(0-6)(0-24)'(0-8)(0-6)(2-24)'(0-4>

„(0-10)*(0-25)'

"The first 2 digits of the experiment number refer to the year, and the last 2

digits refer to the month of the experiment. A, B, and C refer to distinct experimentsinvolving cultures extended over several months after their initiation with 3 differentbatches of frozen cells and tested in vivo at various time intervals.

"Time required to reach an average volume of 3 cu cm.c Number of mice with metastases/total number of mice given tumor injection."Experiment 7907 is that reported in Table 1 and Chart 1."Numbers in parentheses, range.'ND, not determined9Not different (Kruskal-Wallis test, p > 0.05) from the number of métastases

obtained with the same clone when assayed after any one of the longer culturelengths.

"Not different (Kruskal-Wallis test, p > 005) from the number of métastases

obtained with the same clone assayed within the same experiment (A, B, or C)after longer culture lengths.

'Significantly different (Kruskal-Wallis test, p s 0.05) from the results obtained

with the same clone in Experiments 7907. 8004, and 8104 only.'Significantly different (Kruskal-Wallis test, p s 0.05) from the results obtained

with the same clone in Experiments 7907, 8111, 8004, 8205C, and 8104 only.* Significantly different (Kruskal-Wallis test, p «0.05) from the results obtained

with the same clone in Experiment 8004 only.'Significantly different (Kruskal-Wallis test, p s 0.05) from the results obtained

with the same clone in Experiments 7907, 8111, 8004, 8205C, 8104, and 8210Conly.

vary significantly for a given clone, when the cells were testedafter cumulative periods in culture, of up to 197 or 217 days (i.e.,about 85 to 92 passages), according to the clone (Table 5). Moreambiguous results were obtained with clone H122. Within eachof the 3 special experiments, A, B, or C, the numbers of métastases obtained with this clone after short culture lengths (i.e.,respectively, 76, 64, or 72 days of culture) did not vary significantly from those obtained after longer culture lengths (i.e., 162,230, 265, and 330 days for Experiment A; 161 and 226 days forExperiment B; 170 and 233 days for Experiment C). However,when all the experiments presented in Table 5 were compared,significantly more métastases were obtained in Experiment Awith cells inoculated after 162, 230, 265, and 330 days of culture(Experiments 8205A, 8207A, 8208A, and 821OA) than in severalother experiments (as indicated in Table 5) with cells that hadbeen cultivated for shorter times. It is therefore likely that achange towards a slightly higher metastatic potential has occurred in the metastatic phenotype of clone H122 between Days76 and 162 of its culture in this experiment. This interpretationis further supported by the fact that changes appeared also atthat time in several other phenotypic characters of the clone inthis experiment only. Indeed, the growth rate of the primarytumor increased (Table 5). Furthermore, the cells lost their characteristic morphological appearance. They became similar in sizeto the other clones, losing their distinctive larger size. Simultaneously, their higher protein content (850, 880, or 900 pg/cell,respectively, in Experiment 7907, 8205C, or 8208C) regressedtowards lower values (610 pg/cell in Experiment 8208A), morecomparable to that of the other clones.

When considered altogether, the data reported in Table 5further show that the average growth rate of the primary tumorswas correlated positively with the median number of métastasesper mouse (Kendall's rank correlation test: r = 0.70; p < 3 x10~5),and negatively with the median time of survival of the mice(T = -0.62; p < 3 x 10~5). It was also positively correlated with

the median weight of the primary tumors (not shown in the table)at the time of the spontaneous death of the mice (T = 0.28; p <0.012). A negative, inverse correlation was also noted betweenthe median number of métastases per mouse and the mediansurvival time of the mice (r = -0.42; p < 25 x 10~5).

Status of Mycoplasma and Virus Infection in the ParentalLine and in Clonal Sublines. Screening for possible Mycoplasma infection was done on the parental line, on the low-

metastatic sublines (E34, B103, and the 3 H122 cell populations,A, B, and C, which were kept under prolonged culture for theexperiment reported in Table 5), and on the high-metastatic

sublines (C53, H61, B22); all were free of mycoplasms. In addition, tests for possible murine virus contaminations were doneon the parental line and also on the low-metastatic [E34, B103,

H122, subclones 2 and 17 of H122 (see Table 4), Population Aof H122 (see Table 5)] and on the high-metastatic (C53, H61,

B22) sublines. All these cell populations were free of the followingmurine viruses: mouse adenovirus, lymphocytic choriomeningitisvirus, mouse hepatitis virus, Sendai virus, vaccinia virus, REO-3virus, K-virus, polyoma virus, and mouse pneumonia virus; they

were also free of M. pulmonis.

DISCUSSION

The present experiments were undertaken to provide us withhomogeneous lines of cells with differing metastatic potential, in

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Metastatic Heterogeneity of LLC Cells

order to facilitate the study, using suitable material, of the roleof tumor cell proteases in invasion and metastasis. LLC (27) waschosen because it constitutes a well-documented case of natu

rally metastasizing cancer in mice that is widely used for thescreening of chemotherapeutic drugs and other studies on metastasis (24). After i.m. inoculation, primary tumors rapidly develop and spontaneously disseminate numerous métastasestothe lungs.

From a single LLC, a parental line of cells, well adapted toculture, was established and subsequently cloned so as toprovide 18 clonal tumor cell lines. Five clones which differedwidely in their metastatic potential were selected for our furtherstudies. Three of them had a low metastatic potential, with acapacity to form either spontaneous lung métastasesor "artificial" lung colonies which was significantly lower than that of the

parental line. The other 2 clones had a high metastatic potentialwhich was also manifested in the formation of either spontaneousmétastasesor "artificial" lung colonies, but which was not signif

icantly different, in most experiments, from that of the parentalcell line. The parental line apparently expressed a metastaticpotential which corresponded to that of the high-metastatic cell

subpopulations that it contained. Thus, the present report demonstrates the existence of LLC subpopulations that differ in theirability to produce spontaneous, macroscopically visible métastases in the lung. It cannot, however, be extrapolated from thesedata that the clones also differ in metastatic frequency or ininvasiveness. These properties of the clones are presently beinginvestigated, as well as the possible role played by immunefactors in the different metastatic behaviors which were observed. These additional studies are indeed essential to allowthe proper use of this cell material for the evaluation of the roleof cell proteases in tumor invasion and metastasis.

Both in our study and in that of Olsson et al. (18), there wasa good correlation between the potential for spontaneous lungmétastasesarising from a primary tumor and that for "artificial"

lung colonies obtained after i.v. injection of the LLC cells. Others(12) have observed similar correlations for other types of cancercells, but the existence of distinct lung-colonizing and lung-

metastasizing cell populations has also been reported (25). Thepossibility that some of the cells injected i.m. or s.c. colonize thelung due to their accidental introduction into blood vessels (25)cannot totally be excluded, but it appears unlikely that it couldplay a significant role in our experiments. Indeed, in all likelihood,only a limited number of cells could be introduced directly intoblood vessels during the i.m. injections, and, in our experiments(see Table 3, Experiments 1 and 2), the number of lung coloniesobserved after the i.v. injection of either 2 x 10" or 5 x 10" cells

(i.e., 4 or 10% of the total number of cells injected i.m. in theexperiments on spontaneous metastasis) from the parental lineor from C53 and H61 clones, all highly metastatic, was muchlower than the number of spontaneous métastases observedafter i.m. implantation of the tumor (see Tables 1, 2, and 5). Thecorrelation that we observed between spontaneous lung metastasis and "artificial" lung colonies was not, however, absolute.

Indeed, in our experiments, the number of artificial lung coloniesobtained with the low-metastatic clones had a tendency to

increase with increasing time after the i.v. injection, so that noclear differences between low-metastatic clones and parental

line could still be found if the mice were kept until their spontaneous death; however, death occurred earlier with the parental

line than with low-metastatic clones. The explanation of this

progressive increase with time in the number of visible lungcolonies obtained after i.v. administration of low-metastatic

clones is not directly evident. It is possible that cancer cellsremained trapped somewhere, perhaps in perivascular sheaths,after their i.v. injection and succeeded in crossing that barrieronly at later times, the hosts being still alive, to colonize thelungs secondarily. Alternatively, cells of low-metastatic clones,

although present somewhere in the lungs from the early timesfollowing their i.v. injection, may have developed more slowlyinto visible colonies ("artificial" métastases)than did the cells of

the parental line or those of the high-metastatic clones.

As further discussed below, the differences in metastaticbehavior observed among our clones persisted in several casesafter prolonged periods of cell culture, indicating that the variousmetastatic phenotypes expressed in these homogeneous linesare sufficiently stable to allow their use in further work onmetastasis. The metastatic ability of the clones was also positively correlated with the growth rate of the tumor cells in vivo(Table 5), at least when all the data concerning all the cloneswere considered together. This correlation was also apparent invitro, inasmuch as the generation time of the clones under cellculture conditions was, grossly, inversely proportional to theirmetastatic ability. However, the number of métastasesthat couldbe counted in the lungs of the mice at the time of their spontaneous death cannot be considered as a mere reflection of theproliferation rate of their constitutive cells. Indeed, differences inmetastatic behavior between clones persisted when comparisons were made at a time when primary tumors of similar weightswere present, as for instance in the experiments reported inTable 2, or, even more significantly, in the experiment wheretumor cells were implanted in the footpad of the mice and thetumor was excised by amputation when it reached a predetermined volume. Also, in the subcloning experiment reported inTable 4, the fastest growth rate of the primary tumor wasobserved with subclone 2 which did not produce any metastasis.

Our data may be added to the growing amount of evidencewhich supports the view (5, 8) that neoplasms contain a varietyof subpopulations of cells with differing metastatic potential andthat the process of metastasis results from the survival andgrowth of specialized subpopulations of cells. Fidler and Kripke(9) first demonstrated that cloned sublines isolated from a B16melanoma syngeneic to the C57BL/6 mouse differed markedlyin their metastatic potential, and Kripke ef al. (12) made similarexperiments and observations with a newly UV-induced fibrosar

coma of a C3H mouse. Similar heterogeneities in metastaticpotential of cloned cancer cell populations have also been reported for a methylcholanthrene-induced mouse fibrosarcoma(28), a murine sarcoma virus-transformed 3T3 fibrosarcoma (16),a dimethylnitrosamine-transformed rat liver epithelioid cell line(29), a newly UV-induced murine melanoma (10), and a dietary-

induced rat mammary adenocarcinoma (15). This metastaticheterogeneity has been considered as the result of the clonalevolution of tumor cell populations in which the acquired geneticlability of the tumor cells combined with selection pressuresexerted by the host permit stepwise selection of variant sublines(1,7,8,17).

Metastatic heterogeneity among LLC cells was recently shownby Olsson et al. (18), who isolated from a lung metastatic focusa cloned cell line that had a higher metastatic potential than 2

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A-L van Lamsweerde et al.

other cloned lines isolated from a primary tumor. Although ourpresent observations suggest that this heterogeneity preexistsamong cells isolated from the primary tumor and adapted togrow in culture, they do not, however, allow us to completelyexclude the possibility that the differences observed in the met-

astatic potential of our selected clones result from mutationsarising during the selection process, due to the cell cultureconditions.

Indeed, although the metastatic phenotypes of several of ourcloned cell populations, either high (H61, C53)- or low (E34)-

metastatic, remained remarkably stable when these cells weregrown in culture for about 7 months (i.e., 90 passages or 420generations), a significant change (increase) in metastatic potential was once observed after a prolonged culture period of thelow-metastatic clone H122. It appeared in Experiment A only

(Table 5) between 76 and 162 days of culture (i.e., between 32and 69 passages or between about 140 and 300 generations),together with changes in several other phenotypic characters ofthese cells, suggesting that, in all likelihood, variant sublines withhigher metastatic potential had appeared in that initial clonal cellpopulation and had been selected during this prolonged cultureperiod. Furthermore, some subclones isolated from clone H122displayed metastatic abilities which were significantly lower thanthat of the parent clone, indicating that the process of in vitrocloning could be at the source of at least part of the metastaticheterogeneity observed after the initial cloning of the parentalcell population. However, much less metastatic variability wasfound after that subcloning (Table 4) than after the initial cloningof the parental cell population (Table 1), suggesting that someheterogeneity already preexisted in that parental population. If,as indicated above, a stepwise selection of variant sublines (17)is the cause of a metastatic heterogeneity among tumor cells invivo, it is likely that this selection process can exert itself also invitro, although possibly at a different rate, under the pressure ofthe cell culture or cloning conditions. It is therefore not surprisingto observe, as we did, the emergence of new phenotypes underin vitro conditions.

ACKNOWLEDGMENTS

We are grateful to L. Lamury-Maisin for her excellent technical assistance, toDrs. E. Le Boulenge and J. Bouharmont (Facultédes Sciences. UniversitédeLouvain, Louvain-la-Neuve,Belgium) for useful advice in the statistical treatmentof the data, and to Dr. G. Colline! (Smith-Kline-Rit Laboratories) for performingassays for mycoplasmal infections in our cell lines.

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