[CANCER RESEARCH 45, 6005-6009, December 1985]

Experimental Metastatic Ability of H-ras-transformed NIH3T3 Cells

Gregory P. Bondy,1 Sylvia Wilson, and Ann F. Chambers2

Ontario Cancer Foundation, London Clinic and Department of Radiation Oncology, University of Western Ontario, London, Ontario, Canada N6A 4G5

ABSTRACT

We have used a quantitative "experimental" metastasis assay

in the embryonic chick, an immunodeficient host, to examine invivo growth properties of ras oncogene-transformed NIH3T3

cells. We found that two independently derived populations ofNIH3T3 cells that had been morphologically transformed withthe T24 human H-ras oncogene were able to grow in vivo

following i.v. injection. Nontransformed control NIH3T3 cells withnormal morphology did not grow in this assay. Spontaneouslyarising morphological transformants from control NIH3T3 cellpopulations were also tested and did not grow in this assay. Weconclude that the H-ras gene can confer experimental metastatic

ability on nonmetastatic NIH3T3 cells, that the ras gene altersthe cells in some way beyond in vitro morphological transformation, and thus that the in vitro transformation assay detectsonly part of the malignant phenotype of these cells.

INTRODUCTION

Members of the ras oncogene family have been shown totransform NIH3T3 cells, as detected by foci of morphologicallyaltered cells in tissue culture, following transfection with ras DNAfrom cloned or genomic sources (1-6). Morphological transfor

mation in vitro appears to be one step on the path to formationof malignant cells. It is clear, however, that generation of highlymalignant cells is usually a multistep process (7-11 ). It is thus

important to determine the relationship between in vitro measures of transformation and malignant properties as detected witha variety of in vivo assays. Cells transformed with ras or otheroncogenes have been shown in some cases to be positive forlocal tumor growth when injected into nude mice (12-14), but

quantitative measures of tumorigenic growth ability and aspectsof metastatic behavior of oncogene-transformed cells are just

beginning to be examined (15).We have recently developed an experimental metastasis assay

that is well suited to the testing of cells transformed with foreigngenes (16). This assay uses the embryonic chick, a naturallyimmunodeficient host that has been shown to permit the testingof heterologous cells (16-18), DMA transfectants (19), and virally

transformed cells (20). In this assay cells are injected i.v. intochick embryo chorioallantoic membrane veins. Cellular ability tosurvive in the circulation and to be trapped and grow in the liveris measured, using an assay dependent on the differential sensitivities of rodent and chick cells to the cytotoxic drug ouabain,as described (16). This assay parallels the "experimental" metas

tasis assay in mice (21, 22) but offers advantages of (a) animmunodeficient host and (b) a sensitive, quantitative, and rapidassay in which small changes in growth ability can be readily

Received 5/10/85; revised 8/15/85; accepted 8/20/85.1Supported by a Terry Fox Training Centre Establishment Grant.2 Supported by the National Cancer Institute of Canada and the Ontario Cancer

Treatment and Research Foundation. To whom requests for reprints should beaddressed

detected (16, 19, 20). Because growth is monitored immediatelyfollowing injection, genetic instability observed following DNAtransfection (23-25) is potentially less of a problem in this assay

than it may be in longer assays involving macroscopic tumorformation. The assay requires cells to survive i.v. injection andtransit in the circulation, to be trapped in a target organ (theliver), and to grow and thus models the latter portion of hema-

togenous metastatic spread. We have used this assay here totest the experimental metastatic ability of NIH3T3 cells transformed with the activated H-ras oncogene (4) originally cloned

from human T24 bladder carcinoma cells.

MATERIALS AND METHODS

Cells and Cell Culture. NIH3T3 cells (originally obtained from D. Blair)were a kind gift from D. Fujita, University of Western Ontario, London,Ontario, Canada. Cells were grown routinely in tissue culture in medium(Dulbecco's modified Eagle's medium, Grand Island Biological Co., Grand

Island, NY) with 10% calf serum (Grand Island Biological Co.), werepassaged twice per week by trypsinization, and were replenished fromfrozen stocks after a maximum of 6 weeks in culture.

Experimental Metastasis Assay in the Chick Embryo. Experimentalmetastatic ability was assayed as described previously (16). Briefly cells(5 x 105/embryo, in a volume of 0.1 ml of medium plus calf serum) were

injected into chick embryo chorioallantoic membrane veins of 11-day-old

chick embryos (White Leghorn, obtained from a local hatchery). Followinginjection, embryos were returned to a humidified, 37°C chick embryo

incubator for the times stated. Embryonic livers were then dissected,enzymatically dissociated into a single-cell suspension, and plated inmedium plus calf serum (as above) supplemented with 2 x 10~5 M

ouabain (Sigma Chemical Co., St. Louis, MO). This concentration ofouabain permits the growth of any rodent cells present, while killing allchick cells (16). The number of rodent cells present in the chick livers isthus obtained.

DNA Transfection. The cloned human H-ras-T24 oncogene in pBR322(plasmid pT24-C3, from the American Type Culture Collection) (4) was agift from D. Fujita, University of Western Ontario. H-ras-T24 DNA (1 ^g)

was mixed with 30 n§of carrier NIH3T3 DNA and used to transfect 5 x105 NIH3T3 cells per 10-cm tissue culture dish by calcium phosphate

precipitation (4, 26). Control plates were treated identically except theH-ras-T24 plasmid DNA was omitted. The plates were split 1:3 by

trypsinization 48 h after transfection and were maintained in mediumsupplemented with 5% calf serum. Focus formation was scored 21 daysafter transfection. The transfection frequency was ~300 foci/Mg H-ras-T24 plasmid DNA. Two representative plates, each containing -100 foci,

were selected from separate transfection experiments and were designated series I and II. Foci were harvested by trypsinization, were grownin vitro for 1 week in medium with 5% calf serum (as above), and wereanalyzed for experimental metastatic ability in the chick embryo.

Southern and Dot Blot Analysis. Genomic DNA (10 /.g/lano) wasdigested with SamHI (which separates the 6.6-kilobase H-ras-T24 se

quence from pBR322 sequences), subjected to electrophoresis in a0.7% agarose gel, and transferred to a nitrocellulose filter, according tothe procedures of Southern (27). The flter was hybridized for 48 h tonick-translated 32P-labeled H-ras-T24 DNA (6.6-kilobase SamHI fragmentof plasmid pT24-C3) under moderately stringent conditions (42°C,50%

formamide, 5 x standard saline citrate) and then used to expose X-ray

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METASTATIC ABILITY OF ras-TRANSFORMED CELLS

film.For quantitative dot blot analysis, 20 ng of DNA were denatured,

neutralized, serially diluted 1:2, and dot blotted onto nitrocellulose paper(28). The filters were hybridized to 32P-labeled H-ras-T24 probe as

described above. The individual dots were cut out and radioactivity wasmeasured by liquid scintillation counting. Using cloned H-ras-T24 DNA

as a standard, an estimate of gene copy number was calculated, according to procedures described by Kozbor and Croce (29).

Isolation of Spontaneously Transformed NIH3T3 Cells. Spontaneously arising, morphologically transformed NIH3T3 cells were obtainedby plating NIH3T3 cells at a density of 1 x 10s cells/10-cm plate in

medium with 5% calf serum. The medium was changed twice weekly.Foci (~100/plate) began to appear -5 weeks after plating and demon

strated features of morphological transformation, as described by Perucho et al. (26). Foci were not seen at 3 weeks, the incubation periodused to select for oncogene-transforme.d cells. Estimates of frequencyof spontaneous foci in the original populations are difficult to obtain,since the population size increased from the original 1 x 105 cells plated,

and it cannot be determined how many of the -100 resulting foci per

plate were independently derived spontaneous transformants. Spontaneous foci were not obtained from control cells during routine tissueculture. Ten clones of spontaneous foci were picked from ten independent plates and were grown in vitro for testing in the chick assay.

RESULTS

Experimental Metastatic Ability of H-ras-transformedNIH3T3 Cells. Two independently obtained pools of -100 H-ras-transformed foci were tested for experimental metastaticability in chick embryos. The /-as-transformed cells [Chart 1, b

and c; passage 0] showed enhanced experimental metastaticability relative to control cells [NIH3T3 cells transfected withNIH3T3 DNA (Chart 1a; passage 0)] and NIH3T3 cells (data notshown)]. To determine if further enhancement of growth abilitycould be achieved by passage through the chick, ~100 clones

of cells recovered from chick livers were pooled, grown briefly(~1 week) in vitro, and reinjected into chick embryos. Repeatedpassage through the chick embryo failed to enrich further forexperimental metastatic ability in either transformed series (Chart1, b and c; passages 1 and 2) and also resulted in no enhancement of metastatic growth of the control cells (Chart 1a; passages 1 and 2).

An advantage of this assay is that it permits a detailed in vivodescription of the fate of injected cells in embryonic organs overtime following injection. A kinetic description of H-ras-trans

formed cell behavior in liver following injection was obtained(Chart 2). Transformed cells began to grow slowly in embryonicliver over the 7-day period after injection, in marked contrast to

the behavior of morphologically normal control NIH3T3 cellstreated with NIH3T3 DNA, the numbers of which in liver declinedsteadily after their initial trapping in liver. Transformed cells fromthe second series of transformants followed a growth patternsimilar to that of the transformed cells shown in Chart 2, andNIH3T3 cells (not treated with DNA) also declined in numbers(data not shown).

Detection of H-ras Sequences in DNA of H-ras-transformedCells. The presence of the activated H-ras-T24 oncogene was

confirmed in the DNA of the ras transformants by Southern anddot blotting and probing with the ras gene (Fig. 1). Southern blotanalysis revealed that morphological transformants arising afterras transfection and after subsequent passages through thechick embryo all contained detectable 6.6-kilobase H-ras sequences, while the controls did not (Fig. 1a). (Additional H-ras

specific sequences were also detected which were more prominent in cells recovered after several passages through the chick.These sequences may represent H-ras rearrangements or enrichments for various cellular clones present in the original ~100

transformed foci.) Estimates of gene copy number using DNAdot blots (Fig. 10) showed that H-ras-T24-transformed NIH3T3cells contained on average ~2 copies of the H-ras-T24 gene per

cell before passage through the chick, while three passages ofthese cells through the chick embryo assay reduced this numberslightly, to -1 copy per cell, for both series I and series II. It

should be stressed that these numbers are estimates due to thenature of the technique; however, the change from ~2 to ~1

copy per cell was seen consistently in the two independentseries. Successive passage through the chick did not select foreither enhanced experimental metastatic ability in chicks (Chart1) or increased numbers of copies of the ras gene per cell (Fig.1).

Experimental Metastatic Ability of Spontaneously Transformed NIH3T3 Cells. In order to determine whether the experimental metastasis assay was detecting a phenotype identical tothat of in vitro morphological transformation, we tested cells froma series of ten clones of spontaneously arising, morphologicallytransformed foci for experimental metastatic ability in the chickembryo. Such spontaneous transformants arise at very lowfrequency in the NIH3T3 cell line we are using, as described in

Chart 1. Experimental metastatic growthability in embryonic chick of NIH3T3 cells transformed with H-ras-T24 oncogene. Cells (5 x105 cells/embryo) were injected into 11-day

chick embryos, and the number of viable rodentcells present per liver was determined 7 dayslater (passage 0) as described (16). Cells (~ 100clones) recovered from livers were pooled,grown briefly, and reinjected (5 x 105 cells/

embryo) into chick embryos in order to determine if cells with further enhanced growth abilities could be obtained by such enrichmentpassage. Cells analyzed were as follows: a,control NIH3T3 cells transfected with NIH3T3DNA; b, c, two independently obtained pools(series I and II. respectively) of NIH3T3 cellstransformed with H-ras-T24 plasmid DNA. Eachpoint represents the number of viable rodentcells present in one embryonic liver, x, mediannumber of rodent cells: when not shown, themedian was less than 20 cells/liver.

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Chart 2. Kinetic description of the fate of injected cells in embryonic livers overtime following injection. Cells (5 x K^/embryo) were injected as described in Chart1. Embryos were killed and livers were processed [dissected, dissociated, andplated in ouabain (16)] immediately after injection ("0 days") or 3, 5, or 7 days later.

The number of cells recovered per liver at 0 days is ~1% of the original inoculum,

as has been reported for other cells (transformed and normal) (16, 19, 20), andrepresents the number of cells initially trapped in the liver Cells assayed wereNIH3T3 cells morphologically transformed with H-ras-T24 DMA, series I (testedafter passage twice through the chick) (•),and control NIH3T3 cells transfectedwith NIH3T3 DNA (O). Each point represents the number of rodent cells in oneembryonic liver; x, median number.

"Materials and Methods." When cells were injected i.v. into chick

embryos, the behavior of all ten spontaneous transformant lineswas indistinguishable from that of normal control NIH3T3 cells(Chart 3; d. Chart 1a).

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DISCUSSION

The ras oncogene family has been the subject of intenserecent research. Much of this work has been directed towarddescribing the role of ras oncogenes on in vitro measures oftransformation. The relationship of these in vitro parameters toin vivo tumorigenic and metastatic properties remains largely tobe described. The work presented here demonstrates that transformation of NIH3T3 cells with the human bladder cancer H-ras

oncogene confers experimental metastatic ability on these cells,as detected in the embryonic chick. The presence of ~1 -2 copiesof the H-ras gene per NIH3T3 cell is sufficient to enable the cells

to undergo the latter steps of hematogenous metastasis. Thisability is not shared with control NIH3T3 cells. Furthermore whenten clones of spontaneously arising, morphologically transformedNIH3T3 cells were tested for experimental metastatic ability,their behavior was indistinguishable from that of control NIH3T3cells. Spontaneous transformation in vitro would appear to beinsufficient for experimental metastatic growth in the embryonicchick liver following i.v. injection. The in vitro morphologicaltransformation assay apparently detects only a part of the malignant properties of the ras-transformed cells, since only the

morphological transformants resulting from ras oncogene transformation (and not the spontaneously arising transformants)possess experimental metastatic ability.

Our results are consistent with similar studies reported recently by Thorgeirsson ef al. (15), using a different system. Theyfound (a) that pools of NIH3T3 cells transformed with humantumor genomic DNAs were able to form lung tumors in nudemice after i.v. injection and that those cells contained N-rassequences and (u) that H-ras-T24 transformed cells could form

lung tumors after either i.v. or s.c. injerction. They also foundthat spontaneously arising, morphologically transformed NIH3T3

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Fig. 1. H-ras-T24 sequences in DMA from NIH3T3 cells transfected with H-ras-T24 DMA. A, Southern transfer analysis. DNA was analyzed, as described in"Materials and Methods," from the following cell lines: control NIH3T3 cells trans

fected with NIH3T3 DNA (Lanes a and f): series I, NIH3T3 cells (pool of -100 foci)transformed with H-ras-T24 DNA before passage through the chick (Lane b), orafter one (Lane c), two (Lane d), or three (Lane e) passages through the chick;series II, H-ras-T24-transformed NIH3T3 cells obtained in parallel but independentlyfrom series I, analyzed either before (Lane g) or after one (Lane n) or two (Lane /')passages through the chick. B, quantitative dot blot analysis. Row "H-ras-T24"

consists of serial (1:2) dilutions of cloned H-ras-T24 DNA (600 to 4.7 pg). DNAsamples analyzed in Rows a to /' correspond to the samples described in A. Copy

number per cell was calculated according to the procedures of Kozbor and Croce(29), as discussed in the text, kb, kilobases.

cells, although tumorigenic, were nonmetastatic in nude mice.These results suggest that similar metastasis-related properties

are being measured in experimental metastasis assays in the

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Chart 3. Experimental metastatic growth ability of spontaneously arising, morphologically transformed NIH3T3 clones. Ten clones of spontaneous transformantswere obtained, as described in "Materials and Methods." Cells (5 x 105/embryo)

were injected as described in Chart 1. Each point represents the number of rodentcells in one embryonic liver 7 days after injection; median numbers were less than20 per liver unless marked with x.

two hosts, the nude mouse and the chick embryo. Recent workby Bernstein and Weinberg (30) suggests that ras-transformedcells initially may be nonmetastatic when tested in immunocom-

petent hosts. The reported subsequent acquisition of metastaticability in immunocompetent hosts by these cells may depend onthe exogenously added DMA or on selection for further pheno-typic changes in the ras-transformed cells. The significance of

these two studies is discussed below.The experimental metastasis assay in the chick embryo offers

a number of features useful in the molecular biological analysisof metastatic properties: (a) because molecular approaches ofteninvolve the generation of new cell surface antigens, hosts thatare relatively immunodeficient, such as the nude mouse or theembryonic chick, are required; (b) the chick embryo offers theunique ability to develop over a range of temperatures, permittingthe testing of temperature-sensitive mutations in a variety of

genes. We have recently exploited this property in a study of theeffect of the src oncogene on experimental metastatic ability(20); (c) the chick embryo assay permits monitoring of cell growthor death immediately after i.v. injection and permits one to followthe fate of injected cells more closely than is possible whentumor formation is the end point of the assay. Genetically manipulated cells, such as those generated during DNA transfection,are often genetically and phenotypically unstable (23-25), offer

ing advantages to assays not requiring long growth periods fortumor formation; (d) it must be considered that some of thevariability observed in experimental metastasis assays in immu-nocompetent hosts (see Refs. 31-34 for reviews) may be due

to heterogeneity of immunogenicity in populations of otherwisemetastasis-competent cells. Rapid rates of generation of experimental metastatic variants have been reported for clonal populations of several tumor types, when tested in syngeneic mice(35-37). Assays in immunocompetent hosts do not distinguish

between cells with enhanced ability to avoid immune destructionby the host and cells with increases in other metastasis-related

properties. Availability of a quantitative metastasis assay in thenaturally immunodeficient embryonic chick will permit examination of the role of modulation of immunogenicity in the generationof metastatic ability.

The relationship between experimental metastasis, after i.v.injection into either chick embryo or mouse, and spontaneousmetastasis is poorly understood. Work of Glaves (38) suggeststhat different processes (including survivability in circulation ver

sus shedding from a primary tumor) may be rate limiting theseassays. It is interesting to note that cells transformed with N-ras

from tumor genomic DNAs initially were metastatic from i.v. butnot from s.c. injections, while cells transformed with H-ras-T24

were metastatic in both experimental and spontaneous assays(15). Furthermore tumor formation after i.v. injection appears torequire properties different from those required for local tumorformation after i.m. or s.c. injection. The kinetics of the twoprocesses are quite different, and the ability of individual cellstrapped in an organ to grow, to invade into tissue, and to formtumors is likely to be different from the ability of a mass of cellsimplanted locally to grow to form a single tumor. Clarification ofthe interrelationships between properties that contribute to aspects of both tumorigenicity and metastatic ability are crucial toour understanding of the metastatic process.

A synthesis of the data presented here on the ras oncogene,our observations that the src oncogene (20) and genomic DNAfrom a human metastatic tumor (19) can contribute to experimental metastatic ability, and the studies mentioned above byThorgeirsson ef al. (15) and Bernstein and Weinberg (30) leadsus to propose the following model. There appear to be twoclasses of changes, beyond bulk tumor-forming ability, that are

necessary for full expression of metastatic ability in an immunocompetent host, (a) The cells must have intrinsic metastaticability. This property, which can be conferred to appropriate cellsby the activated ras oncogene, the viral src oncogene, or genomic DNA from metastatic cells, permits the invasion of andgrowth of individual tumor cells in tissue following cellular survivalin the circulation. Although obviously related to tumorigenicgrowth ability, the nonmetastatic behavior of tumorigenic, spontaneously transformed cells reported (15) suggests that thephenotypes are in fact distinct. Intrinsic metastatic ability canmost easily be assessed in immunodeficient hosts, in which thisproperty can be distinguished from the second class of changes.(b) Fully metastatic cells must escape immune rejection. Anotherwise metastatic cell may behave in an apparently nonmetastatic fashion due to rejection by various components ofthe immune system. This may explain the metastatic nature ofras-transformed NIH3T3 cells as reported here and by Thorgeirs

son ef al. (15) in immunodeficient hosts and the initially nonmetastatic nature of similar cells tested in immunocompetenthosts, as reported by Bernstein and Weinberg (30). The rangeof immune responses against métastaseshave been reviewedby Frost and Kerbel (39) and Nicolson and Poste (40). Whenassessing metastatic ability in immunocompetent hosts, it isdifficult to determine whether failure to metastasize representsan intrinsic metastatic inability of the cells or an active rejectionof otherwise metastatic cells by the host. Therefore the use of avariety of in vivo hosts with a range of immunocompetenciesand deficiencies is advocated, in order to clarify the nature ofphenotypic changes contributing to both intrinsic metastaticability and immune escape.

The mechanism by which ras oncogenes can confer intrinsicmetastatic ability in in vivo experimental metastasis assays issubject to speculation. The in vitro function of the altered H-rasgene product, p21 protein, is not known, but the GTP-bindingfunction of p21 protein has led to the suggestion that it may actlike GTP-binding coupling factors in hormone-adenyl cyclase

interactions (41). The detailed kinetic data presented here (Chart2) demonstrate the fate of injected cells following their arrival in

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METASTATIC ABILITY OF ras-TRANSFORMED CELLS

the liver via the circulation. As has been described previously fornormal cells and other "normal" cell lines (16, 19, 20), NIH3T3

cells die rapidly in the liver following i.v. injection, in markedcontrast to the behavior of the H-ras-transformed NIH3T3 cells.Obviously an understanding of how the altered H-ras oncogene

contributes to this altered in vivo behavior is important to ourunderstanding of the role of this gene in tumor progression andmalignancy.

ACKNOWLEDGMENTS

We thank D. Fujita for the kind gifts of cells and cloned ras oncogene used inthis study and D. Denhardt, D. Fujita. and J. Harris for helpful comments on themanuscript.

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CANCER RESEARCH VOL. 45 DECEMBER 1985

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1985;45:6005-6009. Cancer Res   Gregory P. Bondy, Sylvia Wilson and Ann F. Chambers  Cells

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