evidencethatintravenouslyderivedmurinepulmonarymelanomamÃ...

[CANCER RESEARCH 46, 5167-5171, October 1986)

Evidence That Intravenously Derived Murine Pulmonary Melanoma MÃ©tastasesCan Originate from the Expansion of a Single Tumor CellIsaiah J. Fidler1 and James E. Talmadge

Department of Cell Biology, The University of Texas, M. D. Anderson Hospital and Tumor institute, Houston, Texas 77030 Â¡I.J. F.], and Preclinical Screen Laboratory,Program Resources, inc., NCI-Frederick Cancer Research Facility, Frederick, Maryland 21701 [J. E. TJ

ABSTRACT

The purpose of these studies was to determine whether hematogenousdonai pulmonary melanoma mÃ©tastasesoriginate from the expansion ofa single cell and if so, by extrapolation, metastasis can be considered acloning process. Three different cell lines of murine K-1735 melanomawith different metastatic properties and unique karyotypes were injectedi.v. into syngeneic C3H/HeN mice as multiceli aggregates of individualcell lines or combinations of cell lines. Resultant solitary lung mÃ©tastaseswere isolated in culture as individual lines and then karyotyped. Evenwhen heterogeneous clumps of tumor cells were injected, the individualmÃ©tastasesexhibited a karyotype unique to one metastatic cell type.Furthermore, when cellular aggregates were composed of metastatic cellsadmixed with cells that were tumorigenic but nonmetastatic, the resultantmÃ©tastasesexhibited only the karyotype of the metastatic cells. Thisfinding suggests that the presence of metastatic cells did not change theinability of nonmetastatic cells to proliferate in a distant organ. Collectively, the results indicate that the resultant mÃ©tastaseswere of clonalorigin owing to the expansion of a single metastatic tumor cell in the lungparenchyma.

INTRODUCTION

Multiple mÃ©tastases,even those within the same organ, oftenexhibit a diversity in their various biological properties, such asantigenicity, immunogenicity, hormone receptors, metastaticpotential, or response to various chemotherapeutic agents (1-

6). This diversity is due to both the nature of the metastaticprocess (7-9) and to the evolution and progression of the tumorcells (10-14). Malignant neoplasms are heterogeneous and

therefore contain cells with different biological and metastaticproperties (1-9). To produce a metastasis, a tumor cell must

complete a series of steps which are potentially lethal to thecells. The outcome of this process is therefore dependent onboth the responses of the host and the intrinsic properties ofthe tumor cells (7-9). Metastasis begins with the detachment

of tumor cells from the primary lesion. After they invade theblood vessels and/or the lymphatics, single tumor cells ormulticeli emboli circulate and eventually reach the capillarybeds of distant organs, where they arrest. After tumor cellsextravasate into the organ parenchyma, they must proliferatein order to give rise to secondary lesions. MÃ©tastasesare notproduced by the random survival of circulating tumor embolibut rather by the selective growth of specialized subpopulationsof metastatic cells that preexist in the parent tumor (15-17).

We recently demonstrated that individual lung mÃ©tastasesproduced from a mouse melanoma can be clonal in origin (18).We based this conclusion on karyotype analyses of cells fromindividual lung mÃ©tastasesproduced in syngeneic C3H/HeNmice by f.p.2 growth of the K-1735 melanoma. We irradiated

Received 4/7/86; revised 6/12/86; accepted 6/13/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Department of Cell

Biology, University of Texas System Cancer Center, M. D. Anderson Hospitaland Tumor Institute, 6723 Bertner Avenue (173), Houston, TX 77030.

2The abbreviations used are: f.p., footpad; M-2, K-1735-M-2 line; X-21, K-1735-X-met-21 line; C-19, K-1735 clone 19.

the parent'tumor prior to f.p. implantation with 650 R to

induce random chromosome breaks and rearrangements (19).Although the results of this experiment demonstrated that somemÃ©tastaseshave a clonal origin and that different mÃ©tastasescan originate from different progenitor cells, it did not resolvethe question as to whether clonal growth at the metastatic siteresults from a single cell or from the implantation of a homogeneous clump of cells. Primary neoplasms are known to bezonal (20, 21), and tumor emboli entering the circulation canbe either homogeneous (composed of one subpopulation) orheterogeneous (composed of multiple subpopulations). Theclonality of melanoma mÃ©tastasescould therefore be merely thereflection of the zonal origin of the tumor embolus. Since it isunlikely that only homogeneous tumor cells circulate, wewished to determine whether the process of metastasis per secould be a cloning process; that is, if the clonal origin of ametastasis could be due to the multiplication of a single cellwithin a heterogeneous tumor embolus arrested in the capillarybed of a distant organ. Experiments presented in this reportaddress these possibilities and demonstrate that, in the murineK-1735 melanoma system, mÃ©tastasescan result from a prolif

eration of a single cell.

MATERIALS AND METHODS

Animals. Adult C3H/HeN mammary tumor virus-negative mice, 6to 8 weeks old, were obtained from the Animal Production Area of theNCI-Frederick Cancer Research Facility.

Cell Lines. The original K-1735 melanoma (22) was a gift from Dr.Margaret L. Kripke, University of Texas, M. D. Anderson Hospitaland Tumor Institute, Houston, TX. The K-1735-M-2 line was derivedfrom a spontaneous pulmonary metastasis that had been produced bythe K-1735 parent tumor growing at a f.p. site (17). The K-1735-M-2cells were exposed to 650 R of X-radiation to induce random chromosome breakage and rearrangements (18). These X-irradiated cells werethen implanted into the footpads of syngeneic mice. When the resultingf.p. tumors reached an average diameter of 1 cm, the tumor-bearingleg, including the popliteal lymph node, was resected at midfemur. Sixweeks later, the mice were killed, and well-isolated, solitary spontaneouspulmonary mÃ©tastaseswere aseptically removed, grown in culture asindividual lines, and karyotyped (18). Within certain mÃ©tastases,thesame chromosomal abnormalities were detected in all of the spreadsexamined, suggesting that these mÃ©tastaseswere of clonal origin. Oneof these karyotyped lines, X-21, was used in the present study. Theoriginal K-1735 melanoma was cloned in vitro by a double cloningmethod. Clone 19, thus derived, was characterized previously to benonmetastatic (22) and was used in this study.

All tumor cell lines (M-2, X-21, and C-19) were maintained in culturein Eagle's minimal essential medium supplemented with 10% fetal

bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine,and 2-fold vitamin solution (Flow Laboratories, Rockville, MD). Cellcultures were maintained on plastic and were incubated in 5% CO2 at37Â°C.Cultures were free of Mycoplasma and the following murineviruses: reovirus type 3; pneumonia; K; Theiler's encephalitis; Sendai;

minute; mouse adenovirus; mouse hepatitis; lymphocytic choriomen-ingitis; ectromelia; and lactate dehydrogenase (assayed by M. A. Bio-products, Walkersville, MD).

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METASTASIS AS A CLONING PROCESS

Kxperimcntal Metastasis Assay. To produce experimental mÃ©tastases, tumor cells in exponential growth phase were harvested by a briefexposure to 0.25% trypsin:0.02% EDTA solution (w/v). The flask wastapped sharply to dislodge the cells, medium was added, and the cellsuspension was pipeted again to produce a single-cell suspension. Thecells were washed and resuspended in Ca2+- and Mg2*-free Hanks'

balanced salt solution to the desired cell concentration. Cell viabilitywas determined by trypan blue exclusion, and only suspensions of singlecells with greater than 90% viability were used. Aliquots (0.2 ml) oftumor cell suspensions were then injected into the lateral tail vein ofC3H/HeN mice. Four weeks after injection the mice were killed; thelungs were removed, washed, and fixed in Bouin's solution to differ

entiate the neoplastic lesions from the organ parenchyma. The lungnodules were counted by visualization with a dissecting microscope.

Experimental Design. Individual cultures of K-1735-M-2, K-1735-X-met-21, and K-1735 clone 19 in exponential growth phase were harvested after short exposure to 0.25% trypsin:0.02% EDTA solution.Medium was added, and the cells were incubated in separate test tubesor admixed at a 1:1 ratio for 30 min on a rotating platform at 37Â°Cto

produce homogeneous or heterogeneous aggregates (M-2; X-21; C-19;X-21 and M-2; X-21 and C-19). After this incubation period, the testtubes were placed in a rack, and large tumor cell aggregates (10 to 20cells) were recovered after a 30-min sedimentation at 37Â°C.Parallel

experiments using autoradiography demonstrated that the X-21 andM-2 aggregates and the X-21 and C-19 mixed aggregates were indeedheterogeneous. To demonstrate this heterogeneity, X-21 cells werecultured for 24 h in medium containing 0.2 Â¿(Ci[3H]leucine/ml (NewEngland Nuclear, Boston. MA). The aggregates of X-21 cells alone, X-21 plus M-2 cells, and X-21 plus C-19 cells were plated onto glassslides. After 1 h, the slides were fixed in 10% buffered formalin andprepared for autoradiography (23).

To produce lung mÃ©tastases,C3H/HeN mice were given injectionsi.v. of 1,000 to 2,000 tumor cell aggregates totaling 10,000 to 20,000tumor cells. The injections lasted 2 to 3 minutes to avoid pulmonaryembolism and death. Preliminary studies showed that this long injectiontime also reduced the possibility of reducing the size of the clumps dueto shear forces as the aggregates passed through the injection needle.Six weeks later, the mice were killed and individual well-isolated lungmÃ©tastases(I mm in diameter) were recovered aseptically, dispersedthrough a stainless steel sieve (Cellector, E. C. Apparatus, St. Petersburg, FL), and cultured. Individual cell lines were established from eachmetastatic lesion, as described previously (16, 17). Chromosome analyses of at least 60 consecutive spreads from each individual line werecarried out by two independent observers as described in detail previously (24). Briefly, confluent cultures were split 1:3 and harvested 48 hlater. Cell cultures were exposed to Colcemid (2.5 jig/ml) for 1 h. Afterbrief (3 min) trypsinization, the suspended cells were swollen in 0.38%potassium chloride, fixed with 2 x Carnoy's fixative (methanol:glacial

acetic acid) and dropped onto cold, wet slides.Whenever possible, 60 to 80 cells from each tumor population were

analyzed completely for breakage and recombinant formation. Includedamong unstable breaks are acentric fragments, chromatid and chromosome breaks, and complex rearrangements. Slides were stained withGiemsa, and metaphases were examined by phase microscopy at amagnification of x 1250. While this limited the definition of recombi

nants, it optimized recognition of breakage. Small unmatched centricchromosomes were scored as minutes and included in the chromosomecounts. Double minute fragments were scored but not counted aschromosome. Dicentric and multicentric chromosomes were countedas single chromosomes.

RESULTS

The tumorigenic and metastatic properties of the 3 K-1735lines used in these studies are summarized in Table 1. By thecriteria used, C-19 cells are tumorigenic but nonmetastatic,whereas cells of the M-2 and X-21 lines are both tumorigenicand highly metastatic. Karyotype analyses of the 3 culturedlines revealed that M-2 cells have a fairly stable mode of 44chromosomes, most of which are telocentric; while C-19 ispseudotetraploid with a mode of 54 chromosomes, most ofwhich are telocentric (18). The X-21 cells have a chromosomemode of 42, and all of the cells (in >1000 spreads) express thesame unique submetacentric chromosome marker with a 3:4arm length ratio (1, 25).

Multiceli aggregates (> 10 cells/clump) were injected i.v. intomice. The aggregates were either homogeneous (M-2, X-21,and C-19) or heterogeneous (X-21 and M-2; X-21 and C-19),as shown by autoradiography of the admixed clumps (Fig. 1).Visual examination of more than 50 clumps revealed that allwere heterogeneous and contained both radiolabeled and unla-beled cells. In mice given injections i.v. of clumps consistingonly of C-19 cells, we found no lung colonies, and therefore nofurther studies were carried out. From mice given injections ofclumps of M-2 cells, 10 cell lines were established from isolatedindividual lung mÃ©tastases.Ten individual cell lines were alsoestablished from lung mÃ©tastasesisolated from mice giveninjections of clumps of X-21 cells. Twelve individual cell lineswere established from solitary mÃ©tastasesisolated from micegiven injections i.v. of clumps consisting of both X-21 and M-2 cells, and 9 individual cell lines were established from lungmÃ©tastasesharvested from mice given injections i.v. of clumpsconsisting of cells from both the X-21 and C-19 lines. Allcultures were established in 16-mm culture wells. After 2 passages (2 weeks), the cultures had expanded sufficiently to allowkaryotypic analysis. All cell lines established from mÃ©tastasesproduced by M-2 cells contained cells with the M-2 karyotype,i.e., all had telocentric chromosomes and no marker chromosome. All cell lines established from mÃ©tastasesproduced byX-21 cells contained only cells with the unique submetacentricmarker chromosome with a 3:4 arm length ratio (Table 2).

Data from the admixing experiment are shown in Table 3.Twelve lung mÃ©tastasesproduced by the X-21 and M-2 aggregates were analyzed. In 4 of the lines (established from individual mÃ©tastases),all the spreads uniformly exhibited the M-2

Table I Tumorigenic and metastatic properties of K-1735 melanoma M-2, X-21, and C-19 cells

MetastaticpotentialCell

lineC-19M-2

X-21Tumorigenicity"6/1010/1010/10Spontaneous*

(no. positive/total)0/10

10/1010/10Experimental

(Median(range)]0(0)

160(80-200)280(100->300)Marker

chromosomeNone

None3:4 SM*Chromosome

mode54

4442

" Footpad tumors measuring 1 cm in diameter were produced by the injection of 2 x III' cells. Number of positive mice/number of mice given injections.* Mice were given injections of 2 x 10* cells into the hind footpad and killed 8-12 weeks later when visceral mÃ©tastaseswere determined. Number of mice with

metastasis/number of mice given injections.c Mice were given injections of C-19 cells: 5 x 10*cells/mouse. M-2 or X-21 cells: 5x10* cells/mouse. Mice were killed 4 weeks after the i.v. injection of tumor

cells, and lung tumor colonies were counted by visualization with a dissecting microscope (more than 300 colonies/lung cannot be counted with accuracy). Median(range) of lung tumor colonies.

* SM. submetacentric chromosome. 3:4 arm length ratio.

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Fig. 1. Autoradiography of multiceli aggregates. Unlabeled M-2 cells (A),radioactive labeled X-21 cells (B), and a mixture of M-2 cells and X-21 cells (Q.Cells of the X-21 line were incubated for 24 h with medium containing 0.2 Â»iCiof [3H]leucine/ml. Aggregates were produced as described in "Materials andMethods." Cell aggregates were deposited on glass slides and fixed. The slideswere covered with NBT-2 emulsion (Eastman Kodak. Rochester, NY) and developed 10 days later.

karyotype. In the remaining 8 of the 12 isolated lines, all thespreads uniformly exhibited the X-21 karyotype.

In the final set of experiments, mice were given injections i.v.of tumor cell aggregates that consisted of both C-19 (nonme-tastatic) and X-21 (metastatic) cells. Karyotype analyses of celllines established from 9 solitary lung mÃ©tastasesrevealed thepresence of only X-21 cells, i.e., cells containing a submetacen-

tric marker chromosome with a 3:4 arm length ratio (Table 4).

Table 2 Karyotype analysis of cells from experimental lung mÃ©tastasesproducedby the i.v. injection of multiceli aggregates consisting of only M-2 or only X-21

cellsKaryotype0

1 UllllHHill,M-2

parentMet*1Met

2Met3Met4MetSMet

6Met7MetSMet

9Met10X-21

parentMetIMet2Met3Met4MetSMetoMet?MetSMet

9Met10Telocentric6063636664666465646600000010000Submetacentric

marker00064

tetraploid00000006765646565626067645363

" M-2 cells have a chromosome mode of 44, and all the chromosomes aretelocentric. X-21 cells have a chromosome mode of 42, and all the cells expressthe same Submetacentric chromosome (3:4 arm length ratio). The numbersrepresent the number of spreads analyzed for each line.

* Met. metastasis.

Table 3 Karyotype analysis of cells from experimental lung mÃ©tastasesproducedby the i.v. injection of multiceli aggregates consisting of both X-21 and M-2 cells

Karyotype0

TumorlineMet*lMet

2Met3Met

4MetSMet

6Met?MetSMet

9Met10Met11Met

12M-26060000000006060X-2100636065606143616100Â°Number of spreads per line. M-2 karyotype: all telocentric chromosomes. X-

21 karyotype: all spreads with Submetacentric marker chromosome (3:4 armlength ratio).

Met, metastasis.

Table 4 Karyotype analysis of cells from experimental lung mÃ©tastasesproducedby the i.v. injection of multiceli aggregates consisting of X-21 and C-19 cells

Karyotype0

Tumor line C-19 X-21Met*l

Met 2Met 3Met 4MetSMetoMet?MetSMet 9

606267646463616564

'Number of spreads per line. C-19 karyotype: all telocentric chromosomes.X-21 karyotype: all spreads with Submetacentric marker chromosome (3:4 armlength ratio).

* Met, metastasis.

DISCUSSION

In a previous series of experiments we showed that murineK-1735 melanoma mÃ©tastasesoften have a clonal origin andthat different mÃ©tastasescan originate from different progenitorcells (6, 8, 18). Poste et al. (26, 27) have published similar data

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for the B16 melanoma system. None of these experimentshowever, resolve the question of whether the process of metastasis is a cloning process per se. That is, do mÃ©tastasesarise asa consequence of the expansion of individual cells surviving inthe blood stream, or do they arise from the growth of homogeneous clumps of cells reaching an organ parenchyma? Thepresent studies were undertaken to clarify this question, andthe results suggest that in the K-1735 murine melanoma system,lung mÃ©tastasesresult from the expansion of a single cell.

We used three established cell lines of the K-1735 melanomawith defined metastatic properties and took advantage of theirunique karyotypic phenotypes. The X-21 cell line consistentlyexhibits a submetacentric marker chromosome, the nonmeta-static C-19 is pseudotetraploid, and the M-2 cell line consistsof telocentric chromosomes with a mode of 42. The analysis ofcell lines established from discrete lung mÃ©tastasesthat resultedfrom injection of multicellular emboli consisting of both X-21cells and M-2 cells, or X-21 cells and C-19 cells, revealed thatthe cultures were of clonal origin, i.e., all chromosome spreadsexhibited an uniform X-21 or M-2 karyotype (Table 2). Sincethe multiceli emboli injected i.v.'were heterogeneous (Fig. 1),

we conclude that the lung mÃ©tastasesresulted from the expansion of a single cell or from a few cells of clonal origin subsequent to the arrest of the embolus in the capillary bed of thelung. It is noteworthy that of the 12 lung mÃ©tastasesproducedfollowing the i.v. injection of X-21 and M-2 aggregates, 8 werethe progeny of X-21 cells and 4 were the progeny of the M-2cells. Although the cell aggregates injected i.v. were heterogeneous, the degree of this heterogeneity could well have variedfrom one aggregate to another. Variation in the ratio of oneclone to another could make the presence of one clone difficultto detect by in vitro growth techniques (28). Visual examinationof a large number of aggregates, however, did not reveal aggregates with a great excess of clone X-21. Therefore, we favor theexplanation that the disparity between the metastatic lines couldbe due to the high metastatic potential of X-21 tumor cells ascompared to M-2 tumor cells (Table 1; Refs. 6 and 17).

Studies from our laboratory (29, 30) and from others (25,31) have clearly demonstrated that circulating multiceli tumoremboli are more likely to give rise to experimental lung mÃ©tastases than single cells. In this study, the i.v. injection of heterogeneous X-21 and C-19 aggregates produced lung mÃ©tastasesthat consisted exclusively of X-21 cells (Table 4). It appearsthat cells of the C-19 line cannot proliferate in the lung parenchyma of C3H/HeN mice (32). Thus, despite the fact thatinjected tumor aggregates contained C-19 cells, they did notproduce a metastasis. These data reemphasize that the formation of a metastasis occurs subsequent to completion of amultistep process by metastatic cells. Merely reaching the organparenchyma does not ensure that a metastasis will occur (33).There are many reasons for the failure of tumor cells to producemÃ©tastases.C-19 cells are incapable of growing in the lungparenchyma and this deficiency cannot be compensated for bythe presence of metastatic X-21 cells. If the deficiency of C-19cells were associated with their inability to reach the capillarybed of the lungs, the results could have been different. Indeed,in a mouse mammary tumor system one metastatic subpopulation can induce some (34), but not all,3 nonmetastatic cells,

to produce mÃ©tastases.Primary neoplasms which are larger than 1 cm3 shed millions

of tumor cells into the circulation on a daily basis (8). Our earlystudies on the distribution and fate of radioactively labeled

metastatic tumor cells established that the majority (>99.9%)of circulating cells die before they can produce a metastasis,and this suggested that a metastasis could develop from fewsurviving cells (33). The present data demonstrate that theprocess of metastasis is a cloning process. Although manytumor cells can enter the circulation and reach the cpaillary bedof distant organs, the linai phase in the metastatic process,proliferation in distant organs, may be the result of the clonalexpansion of a single cell.

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1986;46:5167-5171. Cancer Res Isaiah J. Fidler and James E. Talmadge Single Tumor CellMelanoma Metastases Can Originate from the Expansion of a Evidence That Intravenously Derived Murine Pulmonary

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