[CANCER RESEARCH 50, 717-721. Februar) I, 1990)

Tumorigenicity and Metastasis of Human Breast Carcinoma Cell Lines in NudeMice1

Janet E. Price,2 Aristidis Polyzos,3 Ruo Dan Zhang, and Lisa M. Daniels

Department of Cell Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT

There are few reports describing experimental models of the growthand metastasis of human breast carcinomas. This article discusses thetumorigenic and metastatic properties of two estrogen receptor-negativebreast carcinomas injected into nude mice.

Tumor growth in the mammary fatpad (m.f.p.) and the subcutis wascompared in female nude mice. The injection of 10s viable cells of twohuman breast carcinoma cell lines (MDA-MB-231 amd MDA-MB-435)gave a 100% tumor take rate in the m.f.p., whereas only 40% of the s.c.injections produced tumors and these occurred several weeks after theappearance of the m.f.p. tumors. Thus, the m.f.p. of nude mice is afavorable site for the growth of human breast carcinomas. MDA-MB-435 tumors produced distant métastasesin 80% to 100% of recipients.The most common sites for metastasis were the lymph nodes and lungs,with a lower incidence of métastasesin muscle (chest wall and thigh),heart, and brain. New variant cell lines were isolated from métastasesinthe lungs, brain, and heart. All the cell lines were tumorigenic in them.f.p., and the lung- and heart-derived metastasis lines produced slightlymore lung métastasesthan the original cell line. However, the brainmetastasis variant produced significantly fewer lung métastases.Intravenous inoculation of the spontaneous metastasis-derived cell lines produced few lung colonies. Only cell variants isolated from experimentallung métastasesshowed enhanced lung colonization potential when rein-jected i.v.

Our results suggest that the estrogen receptor-negative MDA-MB-435 cell line injected in the m.f.p. of nude mice could be a valuable toolfor analysis of the cellular and molecular basis of the metastasis ofadvanced breast cancer.

INTRODUCTION

Since the initial observation made by Rygaard and Povlsen(1) that a human colon adenocarcinoma grew progressively innude mice, this athymic mutant strain of mouse has beenfrequently used in human cancer research. Numerous studieshave reported that, although not all human tumors can besuccessfully xenografted, the histology and biochemical properties of the tumors that do grow in nude mice closely resemblethose of the original tumor specimens (2). A major use of nudemice is to prescreen chemotherapeutic agents that act againsthuman tumors proliferating in vivo, rather than using in vitroassays or murine tumor models (3, 4).

The nude mouse also provides a means to study the biologyof human tumor metastasis (5). Whereas some early reportsnoted that the incidence of metastasis of xenografted humantumors in the nude mouse was low (2, 6), more recent studiesreveal that the metastatic phenotype can be expressed in thismodel system. Variables affecting whether metastasis occursinclude the health and housing conditions of the mice (7, 8)and the routes of tumor cell inoculation employed (5, 9) in

Received 7/7/89; revised 10/5/89; accepted 10/24/89.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.

1Supported in part by a grant from Triton Biosciences. Inc.2To whom requests for reprints should be addressed, at The University of

Texas M. D. Anderson Cancer Center. Department of Cell Biology. 1515 Hoi-combe Blvd.. Box 173. Houston. TX 77030.

'Current address: Athens University School of Medicine, 1st Department of

Propedeutic Medicine, Laiko General Hospital. 115 27 Athens. Greece.

addition to the intrinsic properties of the tumors under investigation (10, 11). Many human tumors can proliferate wheninjected s.c. into nude mice, but metastasis from this site is rare(2, 6).

However, the implantation of human tumor cells into anatomically appropriate (orthotopic) sites, rather than into ectopiesites, has been shown to allow the expression of the metastaticphenotype, e.g., colon carcinoma cells into the cecum wall (11,12), renal cell carcinoma cells into the renal subcapsule (13),and bladder carcinoma cells into the bladder (14). In addition,the delivery of cells directly to a target organ by injection intothe blood stream, into the carotid artery, or into the spleen toproduce métastasesin lung (15), brain (16), and liver (10),respectively, is a method used to study the metastatic colonization potential of human tumors.

Human breast carcinomas have a low tumor take rate in nudemice (2), and there are few reports of metastasis of thesexenografted tumors. ER"-positive MCF-7 cells, growing in the

m.f.p. of female nude mice supplemented with estrogen, canmetastasize to the lungs and lymph nodes (17). Ozzello andSordat (18) reported two transplant able human breast carcinomas that grew rapidly in nude mice and also formed métastasesin the lungs and lymph nodes. In addition, one ER-negativecell line, MDA-MB-231, that is commonly used for in vitrostudies has been reported to form lung métastasesafter i.v.injection (19). In this article we describe the tumorigenic andmetastatic behavior of another ER-negative cell line, MDA-MB-435, which has not been previously reported as tumorigenicin nude mice. Our results show that implantation and growthof this cell line in an orthotopic site, the m.f.p., producemétastasesin several different organs in the mice and thusprovide a model for the metastasis of a highly aggressive, humanbreast carcinoma.

MATERIALS AND METHODS

Animals. Female athymic nude mice (NCr-/iu/nu), 6 to 8 weeks old,were obtained from Simonsen Laboratories (Gilroy, CA). The animalswere housed under specific pathogen-free conditions.

Cell Lines. MDA-MB-231 and MDA-MB-435 were generous giftsfrom Dr. R. Cailleau, Department of Medicine, M. D. Anderson CancerCenter (Houston, TX). Both are ER-negative cell lines isolated fromthe pleural effusions of patients with breast carcinoma (20, 21). HT-29is a human colon carcinoma cell line (22). A375 is a human melanomacell line (23), and SN12C was established from a renal cell carcinoma(13). All the cell lines were confirmed as human by karyotypic orisoenzyme analysis.

All cell lines were examined for and found to be free of reovirus type3, pneumonia virus of mice, mouse adenovirus, murine hepatitis virus,lymphocytic choriomeningitis virus, ectromelia virus, k-virus, Theiler's

virus. Sendai virus, and láclatedehydrogenase virus (assayed by Microbiological Associates, Walkersville, MD) and Mycoplasma (tested usinga Gen-Probe Mycoplasma detection kit; Gen-Probe, Inc., San Diego,CA).

Cell Culture. The cells were maintained in monolayer culture inEagle's minimum essential medium supplemented with 5r< fetal bovine

'The abbreviations used are: ER. estrogen receptor; HBSS, Hanks' balanced

salt solution; m.f.p.. mammary fatpad.

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BREAST CARCINOMA METASTASIS IN NUDE MICE

serum, sodium pyruvate, L-glutamine (2 HIM),nonessential amino acids,and 2x vitamin solution (GIBCO, Grand island, NY). The cultureswere incubated at 37°Cin a humidified atmosphere of 5% CO2 and

95% air. Tumor cells were harvested for passage or inoculation bywashing the monolayer with HBSS, followed by a brief incubation in0.25% trypsin and 0.02% EDTA. The tissue culture flask was tappedto dislodge the cells, which were then resuspended in minimum essentialmedium supplemented with 5% fetal bovine serum. Tumor cells intended for inoculation into mice were washed by centrifugation andresuspended in HBSS. Cell number and viability were determined bystaining a small volume of cell suspension with 0.2% trypan blue salinesolution and examining the cells in a hemocytometer.

Tumor Cell Inoculation. In preparation for inoculation into the m.f.p.,the mice were anesthetized with Metofane (Pitman Moore Inc., Washington NJ) and a 5-mm incision was made in the skin over the lateralthorax. The m.f.p. was exposed, anda volume of 0.1 ml of cell inoculumwas injected into the tissue through a 27-gauge needle. By exposing thefat pad, we were able to ensure that the cells were injected into thetissue and not into the s.c. space. Tumor cells were also injected s.c.(lateral flank) or i.v. (lateral tail vein) in separate experimental groups.The growth of m.f.p. and s.c. tumors was monitored by weekly examination, and growth rates were determined from caliper measurementsof two diameters. In some experiments the tumors were excised at amean diameter of 1.5 cm; in other experiments, they were excised atvariable times after cell inoculation. The mice were anesthetized, thetumors were removed, and the skin incisions were closed with woundclips. Experiments were terminated 4 weeks after tumor excision orwhen the mice were moribund. For the i.v. injection experiments, themice were killed 12 weeks after injection or when moribund.

Isolation of Metastasis-derived Variants. At autopsy, métastasesweredissected from different organs (lungs, brain, heart, and lymph nodes),minced finely, and plated in tissue culture flasks in culture medium.The resulting cell lines were tested for contamination with mouse cellsby isoenzyme analysis (Authentikit: Innovative Chemistry'. Marshfield,MA). All of the metastasis-derived lines were of human origin, with nomouse cell contamination.

Growth of Human Breast Carcinoma Cells on Cytodex Beads. Cyto-dex-3 beads (Pharmacia) were rehydrated, following the manufacturer'sinstructions, and sterilized. Beads (2 x 104/rnl) and cells of the twobreast carcinoma cell lines (2 x 105/rnl) were plated in 10 ml of culturemedium in 100-mm Petri dishes (i.e., not tissue culture-treated dishes;the tumor cells adhered to the beads and not to the dishes). After 3 to5 days, when the beads were covered with cells, a sample was taken andtrypsinized to estimate the numbers of cells per bead. For MDA-MB-231, this number was 75 cells/bead and for MDA-MB-435, 28 cells/bead. Beads coated with cells were washed with HBSS and injected i.v.into groups of nude mice, approximately 5 x IO3 beads in 0.2 ml of

HBSS/mouse. Eight to 10 weeks after injection, the mice were killedand the lungs were examined for tumor colonies.

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Fig. 1. Growth curves of four human tumor cell lines injected into two sitesin nude mice. Tumor cells (10' cells/site) were injected s.c. (•)or into the m.f.p.

(A) and tumor growth was measured for up to 10 weeks. The cell lines wereMDA-MB-435. breast carcinoma: A37S, melanoma: HT-29, colon carcinoma:and SN12C, renal cell carcinoma.

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Fig. 2. MDA-MB-435 tumor in the mammary footpad of a nude mouse, x100.

RESULTS

Site of Tumor Cell Injection: s.c. versus m.f.p.. To test whetherthe site of injection influenced the growth of human breastcarcinoma cell lines in nude mice, we injected cells s.c. and intothe m.f.p. of mice and compared the tumor take and subsequentgrowth rates. For two human breast carcinoma cell lines, MDA-MB-231 and MDA-MB-435, three inoculum doses were used.At the two higher doses (IO6 and 5 x IO6 cells), tumors devel

oped in all mice at both sites of injection. However, at thelowest cell dose (10s cells), tumors grew in 2 of 5 mice injected

s.c., whereas 5 of 5 mice injected into the m.f.p. developedtumors; this result was the same for both cell lines in tworepeated experiments. Fig. 1 shows that the latent interval forappearance of MDA-MB-435 m.f.p. tumors was less than thatfor s.c. tumors, following injection of IO5 cells; the growthcurves for MDA-MB-231 tumors were similar to those ofMDA-MB-435 shown here. At the two higher cell doses, tumors

appeared in both sites after similar intervals.To determine whether the trophic effect of the m.f.p. was

specific for breast carcinoma cells, we tested the growth of threedifferent human tumor cell lines injected s.c. or into the m.f.p.For a melanoma (A375), a colon carcinoma (HT-29), and arenal cell carcinoma (SN12C), there was no difference in tumortake or growth rate following injection into the m.f.p., ascompared with growth of the same number of cells (IO5 cells)

injected s.c. (Fig. 1). A375 and SN12C cells produced tumorsin all mice given injections in either site (5 of 5), and HT-29cells produced tumors in 4 of 5 mice given injections s.c. orinto the m.f.p. The experiment was performed twice, withidentical results. Based on the results from these experimentswith MDA-MB-231 and MDA-MB-435, we adopted the m.f.p.as the site of injection in developing the model of human breastcarcinoma metastasis in nude mice.

The histology of the MDA-MB-435 cell line is that of apoorly differentiated adenocarcinoma (Figs. 2 and 3). When

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BREAST CARCINOMA METASTASIS IN NUDE MICE

Fig. 3. MDA-MB-435 s.c. tumor in the lateral flank of a nude mouse, x 100.

Table 1 Metastasis of MDA-MB-435: excision of the mammary fat pad tumorsat different times

Thirty female nude mice were given injections of 5 x 10' MDA-MB-435 cellsinto the m.f.p. At 4, 8, and 12 weeks after injection, the tumors were excisedfrom a group of 10 mice. At 16 weeks after the initial injection, all the mice werekilled and examined for metastasis.

MetastasisTime

ofexcision(wk)4

812Weight

oftumors(g)0.13

±0.04°

0.8 ±0.242.7 ±0.7Lungs2/10*0(0-10)'

6/106(0-47)10/10 16(3-34)Lymph

nodes3/10*

7/1010/10Other

organsBrain/

ascites

Chest wall, brain'

°Mean ±SD.* Mice with lung metastases/mice with m.f.p. tumors.' Median number (and range) of lung métastases.''Mice with lymph node metastases/mice with m.f.p. tumors. Lymph nodes

involved included axillary, inguinal, iliac, cervical, and mediastinal.' Found in histological sections.

tumors growing in the m.f.p. and s.c. sites were compared, themost striking differences were the poorer vascularity and moreextensive necrosis in the s.c. tumors and a reduced fibrouscapsule around the m.f.p. tumors.

Metastasis from Tumors Growing in the Mammary Fat Pad.Both MDA-MB-231 and MDA-MB-435 cell lines formed progressively growing tumors following injection of cells (10* toIO6cells) into the m.f.p. Of 7 mice with MDA-MB-231 tumors

that were killed 20 weeks after tumor cell injection, only 1 wasfound to have macroscopic lung métastases.In contrast, inrepeat experiments, 80 to 100% of mice with MDA-MB-435tumors developed métastases,primarily in the lymph nodes andlungs. Métastasesalso were found at a lower frequency in thebrain, heart, adrenal glands, and muscle (chest wall and thigh)(Tables 1 and 2). The histological appearance of a brain metastasis is shown in Fig. 4. The lesions are of a poorly differentiatedcarcinoma, and bizarre mitotic figures are not uncommon.Subcutaneous MDA-MB-435 tumors produced fewer lung métastases, in 20 to 40% of tumor-bearing mice.

To determine the time course of metastasis, MDA-MB-435m.f.p. tumors were excised at 4, 8, and 12 weeks after injection.At 16 weeks, all mice were killed and the numbers and locationsof métastaseswere recorded. Table 1 shows that by 4 weekstumor cells had disseminated in 3 of 10 mice and by 8 weeks,at which time the mean weight of the m.f.p. tumors was 0.8 g,distant métastaseswere already established in 70% of recipients.

Table 2 Metastasis of MDA-MB-435 and selected variants from mammary fatpad tumors to the lungs of nude mice

Cells from each line were injected into the m.f.p. of female nude mice (5 x 10*

in O.I ml HBSS). The tumors were excised up to 12 weeks after injection, andthe mice were killed at the times indicated.

CelllineMDA-MB-435435-Lunglr435-Lung2'<435-Brl'435-Hl/IncidenceExpt.14/5°Expt.

210/10Expt.3 5/75/58/93/105/8Number

ofmétastases57(0-70)*16(3-34)26

(0-47)50(9-90)64

(0-200)0(0-5)37

(0-108)Time

atautopsy(wk)18-20161615151515

" Mice with lung metastases/mice with m.f.p. tumor.* Median number (and range) of macroscopic lung métastases.' Isolated from a lung metastasis of MDA-MB-435.d Isolated from a lung metastasis of 435-Lungl.' Isolated from a brain metastasis of MDA-MB-435.'isolated from a heart metastasis of MDA-MB-435.

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Fig. 4. MDA-MB-435 metastasis in the brain of a nude mouse, x 100.

All of the mice with tumors present for 12 weeks developeddistant métastases.A similar analysis of the metastasis froms.c. tumors of weights comparable to those shown in Table 1was not performed, because most s.c. tumors did not reach thelargest weights; as noted above, the s.c. tumors became necroticat smaller sizes than the m.f.p. tumors.

The results from this experiment show that metastatic cellsdisseminate from the m.f.p. tumors at a relatively early stage(in some animals before 4 weeks; average tumor weight, 130mg) but that the frequency of metastasis is associated withtumor burden. In a related study, a smaller inoculum of MDA-MB-435 cells was injected into the m.f.p., and tumors excised8 to 10 weeks after injection weighed 0.4 to 0.55 g (i.e., lowerweights than the group reported in Table 2). Métastaseswerefound in a smaller percentage (40%) of these animals killed at16 weeks after the initial injection.

Metastatic Ability of Metastasis-derived Variants of MDA-MB-435. Variants of the MDA-MB-435 cell line were established from métastasesin nude mice that had m.f.p. tumors ofthe original cell line. 435-Lungl and 435-Lung2 were established from lung métastases,with the latter originating from ametastasis of the 435-Lungl cell line. 435-Brl was establishedfrom a brain metastasis.

When injected into the m.f.p. of nude mice, all of the metastasis-derived cell lines grew at similar rates and were not differ-

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ent from the parental cell line. The two lung metastasis-derivedlines, from one or two cycles of growth in the m.f.p. andmetastasis to the lungs, both produced high numbers of lungmétastasesand at a slightly earlier autopsy time, as comparedwith the MDA-MB-435 parental cell line (15 versus 16-20weeks) (Table 2). However, the only significantly different lungmetastasis formation resulted from injecting the brain metastasis-derived cell line into the m.f.p. Only 3 of 10 mice developed macroscopic lung métastases;in two repeat experiments,a similarly low incidence of lung metastasis from 435-Brl m.f.p.tumors was found (data not shown). Lymph node métastaseswere found in 4 of the 10 recipient mice. None, however, showedobvious symptoms of brain metastasis. Each brain was sectioned on several levels to screen for métastases,and small fociof carcinoma cells were detected in the brains of 2 mice fromthe 435-Brl group.

The ability of the MDA-MB-435 cells and the metastasis-derived variants to form lung colonies was assessed by injectingIO6 cells i.v. via the tail vein. None of the cell lines showed

marked ability to colonize the lungs, producing few lung colonies in only 2 or 3 of 10 mice injected in each group.

Experimental Lung Metastasis by Human Breast CarcinomaCells Injected as Single Cells or Attached to Cytodex Beads. Therationale for injecting breast carcinoma cells cultured on theCytodex beads was to establish whether the cells could proliferate in the lungs once they were arrested by physical trappingof the large beads (lOO-^m diameter). The results in Table 3show that MDA-MB-231 cells delivered to the lungs of nudemice on the beads proliferated and formed macroscopic lesionsin 7 of 7 mice injected. The estimated dose of MDA-MB-231cells injected on the beads was 4x10' cells; double this cell

dose injected as a monocellular suspension failed to form anyexperimental métastaseswithin 12 weeks of the injection. Thecell line established from the lung tumor in mice given injectionsof MDA-MB-231 cells grown on Cytodex beads (231-Beadl)also did not form lung métastasesafter injection as single cells.

The results with the MDA-MB-435 cell line were essentiallysimilar (Table 3), with an increase in the incidence and numberof experimental lung métastaseswhen the cells were injectedon Cytodex beads (estimated dose of 1.4 x IO6 cells) ratherthan injected as a monocellular suspension. However, the 435-Beadl variant cell line (from lung tumors of mice given injections of MDA-MB-435 cells grown on Cytodex beads) did showan increased lung colonization potential when injected as singlecells. A cell line established from the few lung colonies ofMDA-MB-435 injected i.v., designated MDA-435-PM1, also

Table 3 Production of experimental métastasesby human breast carcinoma cellsinjected as single cells or attached to Cytodex heads

MDA-MB-231 and MDA-MB-435 cells were cultured on Cytodex-3 beads.Mice were given i.v. injections of 5 x 10'beads in 0.2 ml HBSS or of monocellularsuspensions of 10' cells of the different cell lines.

CelllineMDA-MB-231.single cells (10')

MDA-MB-231 on beads (4 x 10')"MDA-231-Beadl*, single cells(10')MDA-MB-435.

single cells (10')MDA-MB-435 on beads (1.4 x 10')°MDA-435-Beadl*. single cells (10')MDA-435-PM1', single cells (10')Experimental

lungmetastasisNo.

of coloniesIncidence(range)0/10

7/7 90(5-150)0/51/10

0(0-8)5/6 30 (0-70)5/5 17(10-36)5/6 11 (0-23)

" Estimated cell dose on the Cytodex beads.* Cell lines established from the lung tumors in mice given injections of cells

growing on the beads.'Cell line established from lung colonies in mice given injections of MDA-

MB-435 cells.

had a higher lung colonization potential than the original cellline. Thus, for the MDA-MB-435 cell line, growth in the lungof nude mice appeared to select for cells with enhanced lung-colonizing properties; these cells were possibly only a smallsubset of the parent cell line, indicated by the result that 1 ofthe 10 mice given injections of single cells developed lungcolonies (Table 3).

Essentially, the experiments with the Cytodex beads showedthat the human breast carcinoma cells were capable of growthin the nude mouse lungs and that the poor lung colonizationafter i.v. injection of IO6 single cells was possibly due to few

cells arresting in the lungs, rather than an inability to grow inthis organ.

DISCUSSION

Use of athymic nude mice has allowed many and diversestudies of human tumor biology (1-3), including in relativelyrecent years the analysis of metastatic properties (6, 9-15). Inthis report, we describe a new experimental model of themetastasis of an ER-negative human breast carcinoma cell linefollowing implantation and growth in the m.f.p. of nude mice.

The initial question was whether the route of injection ofbreast carcinoma cells in the nude mice would influence tumor-igenicity and metastasis, as seen for different types of humantumors (11-14). The m.f.p. is a more favorable site for thegrowth of mouse mammary tumors, compared with the subcutis(24), and also for the development of métastases,with a higherfrequency of metastasis from the m.f.p. tumors (25). It has beenshown previously that both cleared and intact m.f.p. in nudemice support the growth of human neoplastic breast tissue (17,26). In this study of five human tumor cell lines injected s.c.and into the m.f.p., only the two breast carcinoma cell linesshowed enhanced growth in the m.f.p. at the low inoculumdose. Preferential growth in the m.f.p. site for all tumors wouldsuggest a general effect, resulting, for example, from a goodblood supply in the glandular tissue as compared with thesubcutis. The trophic effect on breast carcinomas and not theother tumors may suggest specific tumor-host cell interactionsthat create a favorable environment for neoplastic breast cellsand allow growth of tumors at a lower inoculum dose than isrequired in the subcutis. Conversely, the s.c. site is comparatively restrictive to the growth of breast carcinoma cells and,thus, the threshold tumorigenic dose is higher, as comparedwith that of the m.f.p. site. At higher inoculum doses, possiblyabove the threshold for s.c. growth, differences in tumor takeand latent interval in the two sites were not found. The resultsof our study recommend the m.f.p. site for the inoculation offreshly isolated human breast carcinomas to hopefully improvethe tumor take in nude mice, which is low for these cancers ascompared with colon carcinomas and melanomas (2).

Recent studies of different human tumors have shown thatthe site of implantation can affect whether distant métastasesare formed (9-14), although it has yet to be determined howthe tissue environment affects the tumor cell phenotype. Forthe MDA-MB-435 breast carcinoma, the m.f.p. supported localgrowth, invasion, and métastases,while a lower incidence ofmetastasis was seen from s.c. tumors. In contrast, MDA-MB-231 tumors also grew in the m.f.p. but produced fewer macroscopic métastases,as compared with MDA-MB-435 tumors.Thus, whereas the m.f.p. can promote the growth of breastcarcinomas, it does not necessarily induce expression of themetastatic phenotype (or tumorigenicity) in all tumors.

Métastaseswere found in the lungs and lymph nodes of most720

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mice with MDA-MB-435 m.f.p. tumors, and the numbers oflung métastaseswere related to the tumor size. However, i.v.injection of a bolus of MDA-MB-435 cells produced few lungcolonies. Possible explanations for this are that disseminationvia the lymphatics is the principal route for MDA-MB-435cells or that the formation of lung métastasesrequired millionsof cells to be shed from the m.f.p. tumors, over a number ofweeks. A previous study using mouse mammary tumors provides similar examples of tumors that were metastatic in theautochthonous host (i.e., growing in the mammary tissue) butthat had poor lung-colonizing potential following single i.v.injections of IO6cells into syngeneic mice (27).

Selection of tumor cell populations with increased metastaticproperties can be achieved by isolating the métastasesandestablishing new variants of a tumor (11, 28, 29). In this studythe lung metastasis-derived cell lines showed a modest increasein numbers of lung métastasesformed, compared with theoriginal MDA-MB-435 cell line, which was highly metastaticfrom the m.f.p.. None of the cell lines derived from métastasesfrom m.f.p. tumors showed enhanced lung colonization potential after i.v. injection. Only cell lines from lung colonies,resulting from i.v. injection of single cells or from tumor cellsgrown on Cytodex beads, showed enhanced lung-colonizingproperties (Table 3). We can conclude from this that the tumorcell properties selected for by the process of metastasis fromm.f.p. tumors may not be exactly the same as those requiredfor successful formation of experimental lung métastasesby thehuman breast carcinoma cells (28).

Lymph nodes and lungs are the most common sites formetastasis of human tumors in nude mice. Finding brain métastases in some mice with MDA-MB-435 tumors adds to thevalue of this cell line for a model of metastatic breast carcinoma.Occasional brain métastaseswere large enough to be grosslyvisible, and the 435-Brl cell line was established from one ofthese. On reinjection into the m.f.p. of mice, this cell lineproduced few lung métastases,compared with the original orlung metastasis-selected cell lines. Current studies are comparing the 435-Lung and 435-Brl cell lines to investigate thecellular basis of the apparent organ preference of metastasis, interms of differential adhesion to extracellular matrices or capillary endothelial cells (30, 31) and responses to organ-derivedgrowth factors (30, 32, 33).

Metastatic breast carcinoma is a major cause of death inNorth America and Western Europe, yet to date few modelsystems are available for experimental studies of this importantdisease. In this initial report, we describe the growth of humanbreast carcinomas in the m.f.p. of nude mice and the dissemination of métastasesto several different organs. This modelsystem could be an important element in further studies on thecellular and molecular basis of the metastatic phenotype ofhuman breast carcinoma.

ACKNOWLEDGMENTS

We thank Patherine Greenwood for preparing the manuscript.

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