summary hepatoc - journal of cell science · pregnancy, ductal elongation and branching resume, and...

INTRO

Embryonic and postnatal development of the rodent mammarygland involves a precise sequence of morphogenetic events thatresult in the formation of an arborized system of epithelialducts embedded in a connective tissue stroma. Duringpregnancy, ductal elongation and branching resume, andclusters of alveoli subsequently bud off from the growing ducts(reviewed by Daniel and Silberstein, 1987). These processesare thought to be driven by a complex interplay of bothsystemic and local regulatory factors, including circulatinghormones and epithelial-mesenchymal (stromal) interactions(Daniel and Silberstein, 1987; Borellini and Oka, 1989;Donjacour and Cunha, 1990; Sakakura, 1991; Haslam, 1991).

While the multihormonal requirements for mammary glanddevelopment have been defined in considerable detail (Topperand Freeman, 1980; Imagawa et al., 1990), the role of thestroma is less well understood. The inducing effect of mes-enchyme (during embryonic development) or stroma (during

1969; Donjacour and Cunha, 1990; Sakakura, 1991) is likelyto be mediated in part by extracellular matrix components, assuggested by the ability of reconstituted matrices to promotealveolar morphogenesis and differentiation in vitro (BarcellosHoff et al., 1989; Aggeler et al., 1991). However, the findingthat culture of mammary gland epithelial cells in collagen gelsallows only limited growth and branching of duct-like struc-tures (Yang et al., 1980; Bennett, 1980; Ormerod and Rudland,1982; Danielson et al., 1984; Reichmann et al., 1989) raisesthe possibility that additional signals from living stromal cellsare required to support formation of an extensive ductal tree invitro. The observation that conditioned medium frommammary fibroblasts enhances the proliferation of mammaryepithelial cells in monolayer culture (Enami et al., 1983) alsosupports a role for paracrine stromal-epithelial interactions inmammary gland development. To date, however, solublestromal factors that stimulate duct formation and branchinghave not been identified.

Althougare thopostnatlying mthis issufusible ties of anormalembedd2 mamcollagengates orconditiodramatresultinarborizpriate seffect oabrogat(also kpolypep

SUMM

Hepa xt

duct ry

J. V. S an1Depar ol,2Divisio Un

*Author f

Journal ofPrinted in

postnatal life) on mammary gland development (Kratochwil,

413

logenesis by Madin-Darby canine kidney epithelial cells.Addition of exogenous recombinant human hepatocytegrowth factor to collagen gel cultures of TAC-2 cellsmimicked the tubulogenic activity of fibroblast conditionedmedium by stimulating formation of branching duct-likestructures in a dose-dependent manner, with a maximal 77-fold increase in cord length at 20 ng/ml. The effect of eitherfibroblast conditioned medium or hepatocyte growth factorwas markedly potentiated by the simultaneous addition ofhydrocortisone (1 µg/ml), which also enhanced lumenformation. These results demonstrate that hepatocytegrowth factor promotes the formation of branching duct-like structures by mammary gland epithelial cells in vitro,and suggest that it may act as a mediator of the inducingeffect of mesenchyme (or stroma) on mammary glanddevelopment.

Key words: scatter factor, morphogenesis, extracellular matrix,corticosteroid hormone, growth factor, epithelial-mesenchymalinteraction, c-met protooncogene

ensive development of branching

gland epithelial cells

d R. Montesano1,*

1 rue Michel-Servet, CH-1211 Geneva 4, Switzerlandiversity Medical School, Suita, Osaka 565, Japan

ARY

tocyte growth factor stimulates e

-like structures by cloned mamma

oriano1, M. S. Pepper1, T. Nakamura2, L. Orci1

tment of Morphology, University of Geneva Medical Schon of Biochemistry, Biomedical Research Center, Osaka

or correspondence

Cell Science 108, 413-430 (1995) Great Britain © The Company of Biologists Limited 1995

DUCTION

h epithelial-mesenchymal (stromal) interactionsught to play an important role in embryonic andal development of the mammary gland, the under-echanisms are still poorly understood. To addresse, we assessed the effect of fibroblast-derived dif-

factors on the growth and morphogenetic proper- clonally derived subpopulation (clone TAC-2) of

murine mammary gland (NMuMG) epithelial cellsed in collagen gels. Under control conditions, TAC-mary gland epithelial cells suspended within gels formed either irregularly shaped cell aggre- short branching cord-like structures. Addition ofned medium from Swiss 3T3 or MRC-5 fibroblasts

ically stimulated cord formation by TAC-2 cells,g in the development of an extensive, highlyed system of duct-like structures, which in appro-ections were seen to contain a central lumen. Thef fibroblast conditioned medium was completelyed by antibodies against hepatocyte growth factornown as scatter factor), a fibroblast-derivedtide that we have previously shown induces tubu-

414

Hepata pleiotrserum oculturedal., 1984spectrum1991; Kidenticaacterizedand miget al., 19et al., 19is thougto a memthe c-me1991b; Hal., 1993et al., 19et al., 1reportedcells forthe pres(CM) (Mfactor (Montesproposephogenedevelopa hypothments (S

The pfusible fof duct-embeddeffect cohave uti(NMuMprevioussandwicto unde1981). Wformatioa clonaland thatthat HGdevelop

MATER

ReagenRecombi(rrHGF) culture ma human(EGF) wMA). Huporcine pMannhei(TGF-β1growth fHuman

J

ocyte growth factor (HGF) (Nakamura et al., 1989) isopic cytokine, which was originally identified in thef partially hepatectomized rats as a potent mitogen for hepatocytes (Nakamura et al., 1984; Michalopoulos et), and later shown to promote the growth of a broad of epithelial cells and other cell types (Rubin et al.,

an et al., 1991; Matsumoto and Nakamura, 1993). It isl to scatter factor, an independently isolated and char- fibroblast-derived protein which induces dispersion

ration of epithelial cells (Stoker et al., 1987; Gherardi89; Weidner et al., 1991; Naldini et al., 1991a; Furlong91; Konishi et al., 1991; Bhargava et al., 1992). HGFht to elicit its various biological activities by binding

brane-spanning tyrosine kinase receptor encoded byt protooncogene (Bottaro et al., 1991; Naldini et al.,iguchi et al., 1992; Giordano et al., 1993; Weidner et

) expressed by epithelial cells (Chan et al., 1988; Iyer

insulin-like growth factor-II (IGF-II), nerve growth factor (NGF), andhuman recombinant keratinocyte growth factor (KGF) were kindlyprovided by Dr P. Sarmientos (Farmitalia Carlo Erba, Milan, Italy),Dr N. Yanaihara (Laboratory of Bioorganic Chemistry, Shizuoka-shi,Japan), Dr L. Aloe (Consiglio Nazionale Ricerche, Rome, Italy) andDrs J. Rubin and S. Aaronson (Laboratory of Cellular and MolecularBiology, NIH, Bethesda, MD), respectively. Rabbit polyclonalantiserum against rhHGF and anti-rhHGF IgGs, produced as previ-ously described (Montesano et al., 1991b), were generously providedby Dr K. Matsumoto (Biomedical Research Center, Osaka, Japan).Rabbit polyclonal antiserum directed against rat proalbumin decapep-tide was kindly provided by Drs K. Davidson and T. Peters (Cooper-stown, NY). Rabbit polyclonal antiserum against secretin was a giftfrom Dr R. S. Yalow (V.A. Medical Center, Bronx, NY). Rabbit IgGsagainst mouse IgG fraction were from Cappel Laboratories (Philadel-phia). Type I collagenase (from Clostridium histolyticum) waspurchased from Worthington Biochemical Corporation (Freehold,NJ). Laminin and type IV collagen were from Bethesda ResearchLaboratories (Gaithersburg, MD), and human plasma fibronectin from

. V. Soriano and others

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Collaborative Research. All hormones used in this study, as well as
92; Sonnenberg et al., 1993). We have recently
that Madin-Darby canine kidney (MDCK) epithelial branching tubules when grown in collagen gels in

nce of fibroblasts or fibroblast conditioned mediumontesano et al., 1991a), and that the fibroblast-derivedsponsible for epithelial tubulogenesis is HGFno et al., 1991b). On the basis of these findings, we that HGF may act as a paracrine mediator of mor-ic epithelial-mesenchymal interactions during theent of parenchymal organs (Montesano et al., 1991b),sis supported by recent in situ hybridization experi-nnenberg et al., 1993).

esent study was undertaken to determine whether dif-ctors released by fibroblasts could promote formationike structures by mammary gland epithelial cells in collagen gels and, if so, to ascertain whether thisld be mediated by HGF. To address this question, wezed the established normal murine mammary gland) epithelial cell line (Owens et al., 1974), which has

y been reported to form cavitary structures whened between two collagen layers (Hall et al., 1982) ando gland-like morphogenesis in vivo (David et al.,e demonstrate here that fibroblast CM induces the of a very extensive system of branching tubules by

cis-hydroxy-proline and L-proline were purchased from SigmaChemical Co. (St Louis, MO).

CellsNMuMG cells (CRL 1636) (Owens et al., 1974) were purchased fromthe American Type Culture Collection (ATCC, Rockville, MD) androutinely grown in tissue culture flasks (Falcon, Becton-Dickinsonand Co., San José, CA) in high glucose Dulbecco’s modified Eagle’smedium (DMEM, GIBCO, Basel, Switzerland) supplemented with10% fetal calf serum (FCS) (Flow Laboratories, Baar, Switzerland).The NMuMG TAC-2 clone was established as described below andcultured in DMEM supplemented with 10% FCS in collagen-coatedflasks. Swiss 3T3 (ATCC, CCL 92) mouse embryo fibroblasts andMRC-5 (ATCC, CCL 171) human embryonic lung fibroblasts werecultured according to the instructions provided in the ATCCCatalogue of Cell Lines and Hybridomas (7th edition, Rockville, MD,1992). All culture media were supplemented with penicillin (500i.u./ml) and streptomycin (100 µg/ml).

Collagen gel culturesParental NMuMG or clonal TAC-2 cells (see below) were harvestedusing trypsin-EDTA, centrifuged, and embedded in three-dimensionalcollagen gels as described (Montesano et al., 1983, 1991a). In brief,8 volumes of rat tail tendon collagen stock solution (approximately1.5 mg/ml) were mixed with 1 volume of 10× concentrated minimalessential medium (Gibco) and 1 volume of sodium bicarbonate (11.76

derived subpopulation of NMuMG epithelial cells,his effect is mediated by HGF. These results suggest is an important stromal mediator of mammary glandent.

ALS AND METHODS

ant human HGF (rhHGF) and recombinant rat HGFere purified as described (Nakamura et al., 1989) fromdium of CHO cells transfected with a plasmid containingr rat HGF cDNA. Natural mouse epidermal growth factors purchased from Collaborative Research Inc. (Bedford,

an recombinant epidermal growth factor (rhEGF) andatelet-derived growth factor (PDGF) were from Boehringer (Rotkreuz, Switzerland), transforming growth factor-β1from R&D Systems Europe (Oxon, UK), and transformingctor-α (TGF-α) from Bachem (Bubendorf, Switzerland).combinant basic fibroblast growth factor (bFGF), rat

mg/ml) in a sterile flask kept on ice to prevent premature collagengelation. Cells were resuspended in the cold mixture, and either 400or 1500 ml aliquots of cell suspension were dispensed into 16-mmwells or 35-mm dishes (Nunc, Kampstrup, Roskilde, Denmark),respectively. After the collagen solution had gelled, 1.5 ml ofcomplete medium (DMEM + 10% FCS) was added to each dish orwell. Media were changed every 2-3 days, and the cultures incubatedat 37°C for the times indicated.

Cocultures with fibroblasts were prepared as described (Montesanoet al., 1991a) by suspending NMuMG or TAC-2 cells within acollagen gel cast on top of a preformed gel layer containing Swiss3T3 cells. To prevent contact between the upper and lower cell pop-ulations, a cell-free gel layer was interposed between the two cell-containing collagen gels.

Establishment of the TAC-2 cloneClone TAC-2 was established from a single colony of epithelial cellsformed in a collagen gel coculture of NMuMG cells and Swiss 3T3fibroblasts, as follows. Swiss 3T3 cells were seeded into 35-mmplastic dishes at 2×105 cells/dish. After overnight attachment andspreading, the fibroblasts were treated with 10 µg/ml mitomycin C

(Sigmaa cell-frtaining on top of collatubulo-under sdigestedminutessingle cties of repeatecast on cocultuhaving taining nase, thresultinmm wecells wecoated dcocultuwas sub7 and alveolasolutionbidistill100 µl/solution

QuantTAC-2 ml) casculture control describ7 or 9 d30 randat leastmitted focusinof all ep(Tektrogrammemeters.cyst-likwas lescountinprints. Vcolonieproportmeasurcomparlength icomparsample

ProlifeTo measeeded at 37°Cwith fremedia awere hater.

To mTAC-2

415HGF and mammary gland morphogenesis

) for 4 hours, washed three times with PBS, and overlaid withee collagen gel (1 ml). A second collagen layer (700 µl) con-a suspension of NMuMG cells (100 cells/ml) was then layeredof the cell-free gel. After 10 days of coculture, a small piecegen gel containing an individual NMuMG colony with a

alveolar organization (see Results) was manually removedterile conditions with the aid of fine needles, and subsequently by incubation with collagenase (4 mg/ml) at 37°C for 10. The released epithelial colony was then dissociated intoells with trypsin-EDTA. To assess the morphogenetic proper-the cells thus recovered, the procedure described above wasd by suspending the isolated epithelial cells in a collagen geltop of mitomycin C-treated Swiss 3T3 cells. After 10 days ofre, this resulted in the development of numerous coloniesa similar tubulo-alveolar organization. The collagen gel con-the tubulo-alveolar colonies was then digested with collage-e released colonies dissociated with trypsin-EDTA and theg cell suspension plated in a collagen-coated (see below) 16-ll. After 5 days, when the culture had attained confluence, there trypsinized, expanded by successive passages in collagen-

solution (400 µl) cast into 16-mm wells. After the collagen had gelled,500 µl of complete culture medium (DMEM + 10% FCS) was addedto each well, with or without the indicated growth factors. Mediumand growth factors were changed every 2-3 days. After 9 days, thecollagen gels were removed from the wells and digested by incuba-tion with collagenase (4 mg/ml) at 37°C for 15 minutes. The releasedcell clumps were recovered by centrifugation and dissociated by incu-bation with trypsin-EDTA. The isolated cells thus obtained werecounted with a hemocytometer.

To measure proliferation on three-dimensional collagen gels, TAC-2 cells were seeded at 100 cells/cm2 on the surface of collagen gelsin 16-mm wells and allowed to attach at 37°C for 2 hours, at whichtime medium was aspirated and replaced with fresh medium with orwithout the indicated treatment. Culture media and treatments wererenewed every 2-3 days. After 9 days, cells were harvested withtrypsin-EDTA and counted with a hemocytometer.

All data are expressed as mean ± s.e.m. and are compared byStudent’s unpaired t-test.

Collagen binding assay

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ishes, and subjected to a second cycle of cloning by the samee procedure described above. One of the clones thus obtainedcultured in collagen-coated flasks and used between passages3. This clone will be referred to as TAC-2 (for ‘tubulo-’ colony-2). For collagen coating, the rat tail tendon collagen described above was diluted to 300 µg/ml in cold steriled water and poured into plastic culture flasks (approximatelym2). After 5-10 minutes incubation at 37°C, the collagen

was aspirated and the flasks washed with PBS.

fication of cord length and branchingcells were suspended at 5×103 cells/ml in collagen gels (1.5 into 35-mm dishes or 22-mm wells of 12-well plates (tissueCluster, Costar, Cambridge, MA) and incubated with eithermedium, fibroblast conditioned medium (CM) (prepared asd by Montesano et al., 1991a), or the indicated agents. Afterays, the cultures were fixed as described below, and at leastmly selected colonies per experimental condition in each ofthree separate experiments were photographed under trans-ight in a Nikon Diaphot TMD inverted photomicroscope, by at the level of the major axis of each colony. The total lengthithelial cords in each colony was measured on a graphic tabletix, 4953 MOD AA) connected to a XT IBM computer pro-d for the semiautomatic evaluation of individual mean para-Cord length was considered as 0 in: (a) colonies showing a shape; and (b) structures in which the length to diameter ratios than 2. Quantification of branching was performed by

Collagen-coated dishes were prepared by incubating 60-mm bacteri-ological plastic dishes (Falcon, cat. no. 1016) with 3 ml of a solutionof rat tail tendon collagen (prepared as described above and dilutedto approximately 50 µg/ml in bidistilled water) for 20 minutes at37°C, at which time the collagen solution was aspirated and the disheswashed three times with PBS. The dishes were subsequentlyincubated with 0.5% BSA in PBS for 2 hours at 37°C to saturate non-specific cell binding sites, and washed again three times with PBS. Incontrol dishes, the collagen coating step was omitted. TAC-2 cellswere harvested with trypsin-EDTA from confluent stock culturesfollowing a 48 hour pre-incubation in the presence or the absence of10 ng/ml rhHGF, centrifuged and resuspended in serum-free DMEMsupplemented with 0.5% BSA (DMEM-BSA). The cells were thenseeded into triplicate collagen-coated bacteriological dishes preparedas described above at a concentration of 2×104 cells/ml (4 ml of cellsuspension per dish). After a 30 minute incubation at 37°C, themedium was aspirated, the dishes gently washed two times withDMEM-BSA, and the adherent cells fixed in 2.5% glutaraldehyde inPBS. Ten randomly selected fields were photographed in each dishusing a Nikon Diaphot TMD inverted photomicroscope and a 4×objective, and the number of attached cells per field was counted.Values are expressed as mean number of cells/cm2 ± s.e.m. and arecompared by Student’s unpaired t-test.

Processing for light and electron microscopyCollagen gel cultures were fixed in situ overnight with 2.5% glu-taraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). After

all identifiable branch points in each colony on positivealues of cord length and branching obtained from the largest are underestimates, since in these colonies a considerableon of cords were out of focus and therefore could not bed. The mean values for each experimental condition wered with controls using Student’s unpaired t-test. Values of cord

n cultures treated with MRC-5 or Swiss 3T3 CM were alsod with controls by applying the Kolmogorov-Smirnov two-

test to the cumulative frequency distribution of each sample.

ration assaysure proliferation in collagen-coated dishes, TAC-2 cells weret 50 or 100 cells/cm2 in 16-mm wells and allowed to attachfor 2 hours, at which time medium was aspirated and replacedsh medium with or without the indicated treatment. Culturend treatments were renewed every 2-3 days. After 9 days, cellsrvested with trypsin-EDTA and counted with a hemocytome-

easure proliferation within three-dimensional collagen gels,cells were suspended at 5×103 cells/ml in a gelling collagen

extensive rinsing in the same buffer, the collagen gels were cut into3 ×3 mm fragments. These were postfixed in 1% osmium tetroxide inVeronal-acetate buffer for 45 minutes and processed as described(Montesano et al., 1991a). Semi-thin (1 µm) sections were cut withan LKB ultramicrotome, stained with 1% methylene blue, and pho-tographed under transmitted light using an Axiophot photomicroscope(Zeiss, Germany). Thin sections were stained with uranyl acetate andlead citrate, and examined with a Philips EM 300 electron micro-scope.

Immunofluorescence microscopyCollagen gel cultures were removed from the culture dishes and fixedovernight with 4% paraformaldehyde in PBS. After extensive rinsingin the same buffer, the collagen gels were incubated in 15% sucrosein PBS for at least 15 hours and frozen in liquid nitrogen-cooledmethylbutane. 4-6 µm-thick sections were cut with a Jung Cryocut3000 cryostat (Leica Cambridge Ltd., Cambridge, England) andstained by indirect immunofluorescence. Briefly, sections wereincubated for 2 hours at room temperature with either sheep antiserumto mouse type IV collagen (1:200 dilution), rabbit antiserum to mouse

416

laminin (Martin, Bdilution) (sections weither a 1IgGs (By1:400 dil(Byosis) with 0.03%were moutaining 0.600 laser-Richmondpercentagwas deterusing a grControls ione of theserum; (2antiserumduring thresulted i

RNA extTotal celTAC-2 cechloroforczynski atrophoresovernightby ThomUV lightand 28 Shours at labelled mcRNA prwere was0.015 M 1982), ansodium pamount owith a 32

nase (GAfilms at rAutoradio(Genofit,relative trelative tocondition significan

RibonucRNase prAmbion Iprepared flabelled p15 hours protected performedwere exposcreens.

PlasmidpSP65mmpair mous1988) inpSP64cGA

J

1:50 dilution) (both gifts from Drs H. Kleinman and G.ethesda) or rabbit antiserum to mouse entactin (1:50Carlin et al., 1981; gift from Dr A. Chung, Pittsburgh). Theere then washed in PBS, further incubated for 1 hour with:200 dilution of FITC-conjugated rabbit IgGs anti-sheep

osis, Compiègne, France) (for anti-type IV collagen) or aution of FITC-conjugated goat IgGs anti-rabbit IgGs(for anti-entactin and anti-laminin) before counterstaining

Evans Blue. After extensive washing in PBS, the sectionsnted with a glass coverslip in PBS-glycerol (1:2, v/v) con-02% paraphenylenediamine and photographed in a MRC-scanning confocal imaging system (Bio-Rad Laboratories,, CA) connected to a Zeiss Axiophot photomicroscope. The

e of colony perimeter occupied by immunoreactive materialmined on at least 15 colonies per experimental conditionaphic tablet (Tektronix) connected to a XT IBM computer.ncluded exposure of sections during the first incubation to following reagents: (1) non-immune goat, sheep or lamb) preimmune rabbit serum; (3) an unrelated (anti-insulin)

chicken muscle GAPDH cDNA (Dugaiczyk et al., 1983), into the PstIsite of pSP64 (Melton et al., 1984). pRcMET#7 was constructed bysubcloning a 462 base pair rat c-met cDNA into pBluescript KS−. Therat c-met cDNA was amplified by PCR from IEC-6 (ATCC, CRL1592) rat intestinal epithelial cell total cellular mRNA with degener-ate primers designed from conserved regions in human and mouse c-met cDNA sequences (M.S. Pepper et al., unpublished data).pSP64rHGF5′ was constructed by subcloning a 336 base pair HincII-EcoRI fragment of clone pRBC1, a 1.4 kilobase pair rat HGF cDNAderived from rat liver exposed to CCl4 (Tashiro et al., 1990), into theHincII-EcoRI sites of pSP64 (Melton et al., 1984). pmLB1 was con-structed by subcloning a 920 base pair BglII-EcoRI fragment isolatedfrom pPE49, a plasmid containing a 1.1 kb mouse laminin B1 chaincDNA (Barlow et al., 1984) between the EcoRI and BamHI sites ofpSP65 (Melton et al., 1984). pmLB2 was constructed by subcloninga 675 base pair EcoRI-PstI fragment isolated from pPE49, a plasmidcontaining a 675 base pair mouse laminin B2 chain cDNA (Barlowet al., 1984) into pSP64 (Melton et al., 1984). p1238 was constructedby subcloning a 1.15 kb EcoRI-SSTI fragment of mouse laminin A


; and (4) the FITC-conjugated antibodies normally usede second incubation step. None of these control incubationsn specific staining of peritubular collagen matrix.

raction and northern blot hybridizationlular RNA was extracted from subclonfluent monolayers oflls according to a modification of the acid guanidine-phenol-m method of Chirgwin et al. (1979) as described by Chom-nd Sacchi (1987). RNA was denatured with glyoxal, elec-ed in a 1% agarose gel (20 µg RNA per lane), and transferred onto nylon membranes (Hybond, Amersham) as describedas (1980). RNAs were crosslinked by exposure of filters to (302 nm), and stained with methylene blue to assess 18 S ribosomal RNA integrity. Filters were prehybridized for 265°C and hybridized at 65°C with 2×106cpm/ml of 32P-ouse c-met, α1(IV) collagen, laminin A, B1 or B2 chain

obes for 18 hours as described (Busso et al., 1986). The filtershed twice at 65°C with 3× SSC (1× SSC = 0.15 M NaCl,Na citrate, pH 7.0), 2× Denhardt’s solution (Maniatis et al.,d three times at 70°C with 0.2× SSC, 0.1% SDS and 0.1%yrophosphate. As an internal control for determination of thef RNA loaded, the filters were hybridized simultaneouslyP-labelled chicken glyceraldehyde-3-phosphate dehydroge-PDH) cRNA probe. Filters were exposed to Kodak XAR-5oom temperature or at −80ºC between intensifying screens.graphs were scanned with a GenoScan laser scanner

Geneva, Switzerland); mRNA levels were normalized

chain cDNA (Sasaki et al., 1988) into pGEM2. pmα1(IV) was con-structed by subcloning a 800 base pair BamHI-HindIII fragmentisolated from pFAC, a plasmid containing a 2.1 kb mouse α1(IV)collagen cDNA (Oberbäumer et al., 1985) into pSP64 (Melton et al.,1984).

RESULTS

Morphogenetic properties of NMuMG epithelial cellsembedded in collagen gels The objective of our initial experiments was twofold: firstly,to investigate the morphogenetic properties of NMuMGepithelial cells suspended within three-dimensional collagengels; and secondly, to assess whether these properties might beinfluenced by coculture with fibroblasts. Culture of NMuMGepithelial cells in collagen gels resulted, after 7-10 days, in theformation of different types of colonies. These included essen-tially: (a) thin branching cords apparently devoid of lumen; (b)thick, stubby epithelial cords, also devoid of a visible lumen;(c) small, irregularly shaped cystic structures, and (d) bundlesof elongated cells with a fibroblastoid morphology (notshown). These observations, together with the heterogeneousmorphology of confluent monolayer cultures (Fig. 1A; see alsoHall et al., 1982), strongly suggested that the NMuMG strain

o GAPDH mRNA in the same samples, and expressed control untreated cultures. The median values for eachwere compared with controls using the median test, and at value was taken as P≤0.01.

lease protection assayotection assays were performed using an RPAII kit fromnc. (Austin, TX). A 20 µg sample of total cellular RNArom rat tissues as described above was hybridized with 32P-SP64rHGF5′ and pRcMET#7 cRNA probes (see below) forat 45°C. Hybridization, RNase digestion and detection offragments by polyacrylamide gel electrophoresis were according to the manufacturers’ instructions. Dried gelssed to Kodak XAR-5 films at −80°C between intensifying

construction and in vitro transcriptionet was constructed by subcloning fragment B, a 2.1 kilobasee c-met cDNA derived from NIH3T3 cells (Chan et al.,

to the EcoRI site of pSP65 (Melton et al., 1984).PDH was constructed by subcloning a 1.1 kilobase pair

is composed of different cell populations. To examine the effect of coculture with fibroblasts,

NMuMG cells were suspended within a collagen gel cast ontop of a gel layer containing Swiss 3T3 cells, as described inMaterials and Methods. Under these conditions, the varioustypes of colonies described above developed more rapidly and,over the same time period, reached a greater size than coloniesformed in control cultures (i.e. in the absence of fibroblasts).The most striking finding in cocultures, however, was thedevelopment of an additional, peculiar type of colony showinga high degree of structural organization. These colonies, whichwere not identified in control cultures, consisted of a centralalveolar-like cavity extending into radially disposed tubularstructures, and will therefore be referred to as tubulo-alveolarcolonies. Both the alveolar and tubular portions of thesecavitary structures were delimited by a uniformly thick epithe-lial wall (Fig. 1B). The occurrence of this type of colony incocultures suggested that the NMuMG strain contained a sub-population of cells endowed with the ability to form highly


organized duct-like and/or alveolar-like structures in responseto fibroblast-derived soluble factors. Since initial attempts toisolate this subpopulation of epithelial cells by classicalcloning procedures (i.e. limiting dilution or ring cloning) wereunsuccessful, we followed the strategy of manually removingsingle tubulo-alveolar colonies from collagen gel cocultures ofNMuMG and Swiss 3T3 cells. Cells obtained by enzymaticdissociation of the isolated colonies attached poorly and failedto spread when seeded into conventional plastic tissue culturedishes or plastic dishes which had been previously coated witheither gelatin, fibronectin, laminin or type IV collagen;however, they attached and spread on dishes coated with typeI collagen. A clonally derived cell population (clone TAC-2)established from a single tubulo-alveolar colony as describedin Materials and Methods was subcultured in type I collagencoated flasks and used in subsequent experiments. TAC-2 cellsexhibited an apparently homogeneous polygonal morphologyin confluent monolayer cultures (Fig. 1C).

Fig. 1. Emonolaymorpholcolony focollagencollagencentral astructureof the co(C) Morptubulo-amicroscohomogen

Fibroblast conditioned medium stimulates formationof branching duct-like structures by TAC-2 cellsWhen suspended in collagen gels under control conditions (i.e.in the absence of fibroblasts), TAC-2 cells gave rise, within 7-10 days, to small slowly growing colonies with a morphologyranging from irregularly shaped cell aggregates to poorlybranched structures (see Fig. 2A). In contrast, in cocultureswith Swiss 3T3 fibroblasts, TAC-2 cells formed thickbranching cords which underwent progressive multifocal cav-itation. After 9-10 days, coalescence of the focal luminaresulted in the development of tubulo-alveolar structuresdelimited by a thick epithelial wall (data not shown).

To assess whether the effect of coculture was mediated byfibroblast-derived diffusible factors, TAC-2 cells weresuspended in collagen gels and incubated with conditionedmedium (CM) from either Swiss 3T3 or MRC-5 cells. Thisexperimental condition partially mimicked the effect ofcoculture by markedly stimulating the growth of epithelialcolonies in collagen gels. Contrary to cocultures, however,fibroblast CM did not induce formation of thick-walled tubulo-alveolar structures with patent lumina, but promoted insteadthe development of an extensive network of branching cords(Fig. 2B). A quantitative analysis revealed that Swiss 3T3 andMRC-5 CM (50%, v/v) induced a 276-fold or 210-fold
increase, respectively, in mean total additive cord length percolony (Table 1). A significant (P<0.0001) increase in cordlength was observed with a concentration of Swiss 3T3 CM aslow as 2.5% (v/v), and a half-maximal increase with a con-centration of approximately 16% (v/v) (data not shown). Afrequency distribution analysis of total cord length per colonyrevealed that 80% of colonies in control cultures did not formcords at all (i.e. they consisted of irregularly shaped cell aggre-gates), while only 20% formed short linear or branching cords.In marked contrast, 100% of colonies in cultures treated withSwiss 3T3 CM formed extensively branched cords, whose totallength sometimes exceeded 30 mm (Fig. 3A). Similar resultswere obtained with MRC-5 CM (not shown). In addition, aquantitative analysis of branching demonstrated that Swiss 3T3CM induced a 56-fold increase in mean branch points percolony (Fig. 3B). Taken together, these data indicate thatfibroblast CM stimulates not only the elongation, but also thebranching of the epithelial tubules.
stablishment of the TAC-2 clone. (A) A confluenter culture of parental NMuMG cells shows a heterogeneousogy (phase-contrast microscopy). (B) Tubulo-alveolarrmed by parental NMuMG cells grown for 7 days in a

gel cast on top of a Swiss 3T3 fibroblast-containing gel layer (bright field illumination). The colony consists of alveolar-like cavity extending into radially disposed tubulars (arrows). Notice that both the alveolar and tubular portionslony are delimited by a uniformly thick epithelial wall.hology of the TAC-2 clone established from a single

lveolar colony similar to that shown in B (phase-contrastpy). The monolayer culture is composed of apparentlyeous polygonal cells. Bars, 100 µm.

418

Semithin sections showed that TAC-2 colonies in controlcollagen gel cultures consisted of either irregularly shaped cellaggregates or short cords sometimes containing a small lumen.In contrast, sections of cultures incubated with Swiss 3T3 CMrevealed smooth-contoured branching cords, often containinga clearly identifiable lumen (Fig. 2C,D). Noteworthy, cordprofiles that were apparently devoid of lumen in a given sectionfrequently showed a lumen in adjacent sections (analysis ofserial semithin sections revealed that 58% of cords in culturesincubated with Swiss 3T3 CM contained a lumen; see Table

4). In appropriate thin sections including a lumen, TAC-2 cellsappeared well polarized and showed lateral junctionalcomplexes and numerous apical microvilli (not shown).

The tubulogenic activity of fibroblast CM ismediated by HGFWe have previously demonstrated that the tubulogenic effectof fibroblast CM on MDCK cells is due to HGF (Montesanoet al., 1991a,b). To determine whether HGF might be the factorin fibroblast CM that stimulates tubulogenesis by TAC-2 cells,

J. V. Soriano and others

Fig. 2. Fibroblast CM stimulates formation of branching tubules by TAC-2 cells grown in collagen gels. (A) TAC-2 cells grown in collagengels for 7 days under control conditions have formed small colonies with a variety of morphologies ranging from irregularly shaped cellaggregates (arrowheads) to poorly branched structures (arrows). (B) TAC-2 cells grown in collagen gels for 7 days in the presence of MRC-5CM (50%, v/v) have formed an extensive network of highly arborized branching cords (bright field illumination). (C,D) Semithin sections ofcollagen gel cultures of TAC-2 cells incubated with Swiss 3T3 CM reveal the formation of duct-like tubular structures. Bar, 50 µm in (A,B)and 100 µm in (C,D).

MRC-5 CM was preincubated with anti-rhHGF antiserum(Montesano et al., 1991b) prior to addition to TAC-2 cells incollagen gels. This resulted in a dose-dependent inhibition ofthe tubulogenic activity of MRC-5 CM (Fig. 4A). Likewise,the effect of MRC-5 CM was suppressed by pre-incubationwith Protein A-purified anti-rhHGF IgGs (Montesano et al.,1991b) but not with irrelevant IgGs (data not shown). Theseresults demonstrate that the factor in MRC-5 CM that stimu-lates tubulogenesis by TAC-2 cells is a molecule immunolog-ically related to HGF.

Addition of rhHGF to collagen gel cultures of TAC-2 cellsstimulated cord formation in a dose-dependent manner, an 8-fold increase in mean total additive cord length being observedwith 1 ng/ml rhHGF and a maximal 77-fold increase with 20ng/ml (Fig. 4B). Recombinant rat HGF (rrHGF) had a similareffect on cord formation (data not shown).

We next examined whether the ability to stimulate formationof branching duct-like structures by TAC-2 cells is a specificproperty of HGF or is shared by other polypeptide growthfactors, including EGF, TGF-α, IGF-II, bFGF and PDGF. At20 ng/ml, IGF-II, bFGF and PDGF did not significantlyenhance cord formation, whereas TGF-α and recombinanthuman EGF (rhEGF) induced a 4-fold and 8-fold increase incord length, respectively, over control values (Fig. 4C). Theeffect of rhEGF was further investigated using a wide range ofconcentrations (1, 2, 5, 10, 20, 50, and 100 ng/ml) and foundto be maximal at 20 ng/ml (data not shown). Thus, the abilityto stimulate cord formation by TAC-2 cells was not restrictedto HGF. However, the increase in cord length induced by 20ng/ml rhHGF was approximately 22- and 11-times greater thanthat induced by 20 ng/ml TGF-α or 20 ng/ml rhEGF, respec-tively (Fig. 4C). In addition, when compared at equimolar (100pM) concentrations, rhHGF was approximately 24-times morepotent than rhEGF (Table 1). In separate experiments, ker-atinocyte growth factor (KGF; Finch et al., 1989) (20 ng/ml)and nerve growth factor (NGF, 50 ng/ml) had no obvious effecton cord formation, while natural mouse EGF (20 ng/ml)induced an increase comparable to that observed in responseto rhEGF. Finally, TGF-β1 (1 ng/ml) inhibited colonyformation (data not shown). Examination of serial semithinsections showed that, in contrast to HGF, EGF does not

Table 1. Stimulation of epithelial cord formation byfibroblast CM, HGF and EGF

Cord length (mm)

A. Fibroblast CMControl 0.05±0.01Swiss 3T3 CM 13.80±1.96MRC-5 CM 10.54±0.45

B. Equimolar concentrations of HGF and EGFControl 0.31±0.07rhHGF (100 pM) 18.80±2.19*,**rhEGF (100 pM) 0.79±0.17*

TAC-2 cells were suspended in collagen gels and incubated with fibroblastCM (50%, v/v) for 7 days (A), or with either rhHGF (100 pM) or rhEGF (100pM) for 9 days (B). In each experiment, at least 30 randomly selectedcolonies per condition were photographed and total additive cord length percolony was determined as described in Materials and Methods. Values aremean cord length per colony ± s.e.m. *P<0.001 (rhEGF or rhHGF vscontrol); **P<< 0.001 (rhHGF vs rhEGF); n = at least 3 experiments for eachcondition.


stimulate lumen formation (in cultures incubated with rhEGF,22% of structures contained a lumen, versus 24% in controlcultures and 80% in cultures treated with rhHGF). Takentogether, the results described above demonstrate that HGFstimulates tubule formation by TAC-2 cells to a considerablygreater extent than EGF.

Differential effect of HGF and EGF on TAC-2 cellproliferation in two-dimensional and three-dimensional culturesThe finding that HGF and EGF enhance formation ofbranching cords by TAC-2 cells prompted us to assess theeffect of these growth factors on TAC-2 cell proliferation inboth monolayer culture and three-dimensional collagen gels.In cultures of TAC-2 cells grown on a collagen coating, rhHGF

Fig. 3. Quantitative analysis of cord length and branching. TAC-2cells were embedded in collagen gels and incubated with eithercontrol medium or Swiss 3T3 CM at 50% (v/v) for 9 days. In eachexperiment, at least 30 randomly selected colonies per conditionwere photographed, and either the total additive cord length percolony (A) or the number of branch points (B) was determined asdescribed in Materials and Methods. (A) Frequency distribution oftotal cord length per colony in control and Swiss 3T3 CM-treatedcultures. Values are relative frequencies of cord length per colony;P<0.001, compared by Kolmogorov-Smirnov two-sample test to thecumulative frequency distribution of each experimental condition.n=3 experiments for each condition. (B) Number of branch pointsper colony. Branch points were quantified as described in Materialsand Methods. Values are mean number of branch points per colony ±s.e.m. P<0.001. n=3 experiments for each condition.

420

(10 ng/ml) induced an approximately 2-fold increase in cellnumber with respect to cultures without HGF, whereas incultures on three-dimensional collagen gels it did not signifi-cantly increase cell number. In marked contrast, in cultures ofTAC-2 cells grown within three-dimensional collagen gels,rhHGF induced a 13-fold increase in cell number (Table 2).rhEGF (10 ng/ml) did not significantly modify cell number oneither collagen coatings or collagen gels, but induced an almost3-fold increase in the number of TAC-2 cells grown withinthree-dimensional collagen gels (Table 2). These resultsindicate: firstly, that these growth factors have little or no effecton the proliferation of TAC-2 cells in two-dimensional culturesbut have a clear mitogenic effect on the same cells grown in athree-dimensional collagen matrix; and secondly, that HGFstimulates proliferation of TAC-2 cells in collagen gels to aconsiderably greater extent than EGF.

HGF has been reported to induce scattering of many, but notall, epithelial cells grown in monolayer culture (reviewed byMatsumoto and Nakamura, 1993). In cultures of TAC-2 cells

grown either in collagen-coated dishes or on top of dimensional collagen gels, rhscattering.

HGF decreases the adhecollagenSince HGF-induced tubulogchanges in cell-matrix interaHGF could affect the bindingshown in Table 3, pre-treatmdecreased their attachmentapproximately 46%. This fiHGF, by loosening the adhe


Fig. 4. Demonstration that the tuis due to HGF and comparison ofactors. (A) Antiserum to rhHGFMRC-5 CM. MRC-5 CM was prthe indicated dilutions of rabbit aanti-proalbumin antiserum (open(open circle) or non-immune serto cultures of TAC-2 cells embedconcentration of 50% (v/v). Treadays, and the cultures fixed afterper colony ± s.e.m. Values for Mirrelevant anti-proalbumin antisefrom MRC-5 CM preincubated with anti-rhHGF antiserum, whvalues for MRC-5 CM preincubated with either irrelevant anti-secretin antiserum or non-immune serum were not. Inverted optriangle: complete culture medium (without MRC-5 CM) pre-incubated with anti-rhHGF antiserum; filled square: complete cmedium alone. n = at least 3 experiments. (B) Dose-dependentinduction of epithelial cord formation by rhHGF. TAC-2 cells wsuspended in collagen gels and incubated with the indicatedconcentrations of rhHGF for 9 days. Values are mean cord lengcolony ± s.e.m. Values for all concentrations of rhHGF aresignificantly different from control (P<0.001); n = at least 3experiments for all conditions. (C) Comparison of the effect ofdifferent growth factors on cord formation by TAC-2 cells. TAcells suspended in collagen gels were incubated with the indicagrowth factors at a concentration of 20 ng/ml for 9 days. Valuemean cord length per colony ± s.e.m. Values for PDGF, IGF-IIbFGF were not significantly different from control; *P<0.001,**P<<0.001; n = at least 3 experiments for all conditions. In A,and C, at least 30 randomly selected colonies per condition werphotographed in each experiment, and total additive cord lengthdetermined as described in Materials and Methods.

Table 2. Effect of HGF and EGF on TAC-2 cellproliferation in two-dimensional and three-dimensio

culturesControl HGF EGF

On coating 100 225.06±57.95 130.53±2On gel 100 117.64±34.11 62.67±2Within gel 100 1334.74±500.49 283.52±8

TAC-2 cells were seeded in triplicate wells on two-dimensional collacoatings (On coating), on the surface of three-dimensional collagen gelgel) or within collagen gels (Within gel), and subsequently incubated wcontrol medium, rhHGF (10 ng/ml) or rhEGF (10 ng/ml). After 9 days which time cells grown in two dimensions had not yet attained confluencells were harvested and counted as described in Materials and MethodValues are expressed as mean percentage of the number of cells in cont± s.e.m. n = at least 4 experiments per condition.

three-

nal

0.996.997.73

gens (Onith(atce),s.rols

HGF did not induce a detectable

sion of TAC-2 cells to

enesis may conceivably involvections, we investigated whether of TAC-2 cells to collagen. Asent of TAC-2 cells with rhHGF

to collagen-coated dishes bynding raises the possibility thatsion of TAC-2 cells to collagen

bulogenic activity of fibroblast CMf the effect of different growth inhibits the tubulogenic activity ofeincubated at 4°C for 4 hours withnti-rhHGF antiserum (filled circles), square), anti-secretin antiserumum (open triangle) prior to additionded in collagen gels at a finaltments were renewed every 2-3 9 days. Values are mean cord lengthRC-5 CM preincubated withrum were significantly different

ereas

en

ulture

ere

th per

C-2teds are and

B,e was

HGF and mamm

fibrils, allows the occurrence of morphogenetic cell rearrange-ments which are essential for tubulogenesis. Further studieswill be required to determine whether HGF decreases TAC-2cell adhesion to collagen by modulating the expression orfunction of specific integrins, or by producing increasedamounts of matrix-degrading proteases.

Hydrocortisone enhances lumen formation andpotentiates the tubulogenic effect of HGFSince the postnatal development of the mammary gland isregulated by a number of hormones, including estrogens, prog-esterone, insulin, hydrocortisone (HC) and prolactin (Topperand Freeman, 1980; Imagawa et al., 1990), we next investi-gated whether these mammotrophic hormones might modulatethe effect of either fibroblast CM or HGF on epithelial cord

formation by TAC-2 cells2 cells were suspended ieither Swiss 3T3 CM (5motrophic hormones alonmammotrophic hormonesesterone (1 µg/ml), insulinhad no detectable effect whether added alone or i(data not shown). In contreffect on TAC-2 cells groadded alone or together wFor the sake of clarity, wealone and subsequently theither Swiss 3T3 CM or H

Effect of HC aloneAs already mentioned abcontrol conditions consistcords, which sometimes 5A,B,D,E). In contrast, thethe presence of HC (1 µgshaped cysts containing delimited by a palisade o(Fig. 5F). A quantitativmarkedly increased both thlike organization (Fig. 6A(data not shown), with a ethasone (a synthetic glucticosteroid with predominsimilar effect on cyst for

Table 3. Effect of HGF on adhesion of TAC-2 cells tocollagen-coated dishes

Cells/cm2

Control 5429±929rhHGF 2941±233

TAC-2 cells were harvested from confluent cultures following a 48 hourpre-incubation in the presence or the absence of 10 ng/ml rhHGF and seededinto collagen-coated bacteriological dishes. After a 30 minute incubation at37°C, the number of attached cells was determined as described in Materialsand Methods. Values are mean number of cells/cm2 ± s.e.m.; P<0.001; n = 3experiments per condition. Non-specific adhesion to dishes that had not beencoated with collagen was negligible (<10 cells/cm2 ).

Fig. 5. Hydrocortisone stimulates lumenformation in colonies of TAC-2 cells.TAC-2 cells suspended in collagen gelswere grown for 6 days in the absence(A,B,D,E) or the presence (C,F) of HC(1 µg/ml). (A-C) Phase-contrastmicroscopy. (D-F) Semi-thin sections.(A,B,D,E) Small irregularly shaped cellaggregates and cords formed by TAC-2cells grown under control conditionscontain a very small lumen (arrows inD,E). (C,F) Large alveolar-like cysticstructures formed by TAC-2 cellsincubated with HC. The cysts contain awidely patent lumen and are delimitedby a palisade of cubic or columnarepithelial cells (F). Bars, 50 µm.

421ary gland morphogenesis

. In a first set of experiments, TAC-n collagen gels and incubated with0%, v/v) alone, each of the mam-e, or Swiss 3T3 CM plus individual. 17-β-Estradiol (10-100 nM), prog- (10 µg/ml) and prolactin (5 µg/ml)

on cord formation by TAC-2 cells,n combination with Swiss 3T3 CMast, hydrocortisone had a prominentwn in collagen gels, whether it wasith either Swiss 3T3 CM or rhHGF. shall first describe the effect of HCe effect of HC in combination withGF.

ove, TAC-2 colonies formed undered of either cell aggregates or short
contained very small lumina (Fig. vast majority of colonies formed in/ml) consisted of large, irregularlya widely patent lumen (Fig. 5C)f cubic or columnar epithelial cellse analysis demonstrated that HCe percentage of colonies with a cyst-) and lumen size in cystic colonies
maximal effect at 1 µg/ml. Dexam-ocorticoid), and aldosterone (a cor-ant mineralocorticoid action) had amation, whereas 17-α-hydroxypro-

422

gesteronemineralocinactive m

Effect of When adpotentiateparticularcollagen Swiss 3Textensivetomosingthe 117-fcolony infourfold resulting not shownethasone terone oformationdemonstrthe growt

An addeither Swment of luincubatedwere rarethey coulcontrast, iHC (or wfied by pstep in thmultiple epithelialsively enresulting lumen (Fidemonstrcentage oand (b) lueffects ofthe area oall profile

J.

Fig. 6. Quacyst formatubulogenecollagen gconcentratselected cophotographobjective iwere arbitrdiscernibleand are comall concentcontrols (Pwere suspeincubated both HC ancord lengthexperimen

V. Soriano and others

ntitative evaluation of hydrocortisone-inducedtion (A) and potentiation of HGF-inducedsis (B). (A) TAC-2 cells were suspended in

els and incubated with the indicatedions of HC. After 6 days of culture, 100 randomlylonies per experimental condition wereed by phase-contrast microscopy using a 10×

n each of three separate experiments. Coloniesarily classified as cysts when containing a clearly lumen. All data are expressed as mean ± s.e.m.pared using Student’s unpaired t-test. Values for

rations of HC are significantly different from<0.001; n = 3 experiments). (B) TAC-2 cellsnded in collagen gels at 500 cells/ml and

with either HC (1 µg/ml), rhHGF (10 ng/ml), ord rhHGF for 9 days. In each experiment, at least 30 randomly selected colonies per condition were photographed and total additive was determined as described in Materials and Methods. Values are mean cord length per colony ± s.e.m. (*P<0.001; n = 4

ts).

(a corticosteroid devoid of both glucorticoid activity) and tetrahydroetabolite of HC) had no effect.

coaddition of HC and Swiss 3T3 Cded together with Swiss 3T3 CM, d cord formation induced by the CM. ly evident when TAC-2 cells weregels at low density, in which case the3 CM and HC resulted in the develo three-dimensional networks of branc cords. A quantitative evaluation demold increase in mean total additive cduced by Swiss 3T3 CM was furby the simultaneous addition of Hin a total 474-fold increase over contr). A similar potentiation was observeor aldosterone, but not with 17-α-hr tetrahydrocortisone. HC also po induced by rhHGF (Fig. 6B). Proliated that HC and rhHGF synergistich of TAC-2 cells in collagen gels (Taitional effect of HC, when added in coiss 3T3 CM or rhHGF, was a pronoumen formation within the epithelial co with either Swiss 3T3 CM or rhHGFly visible by phase-contrast microscd easily be detected by sectioning; cf.n all cultures incubated with both Swiith both HGF and HC), lumina werehase-contrast microscopy. The earliese process of lumen formation was thetiny circular spaces aligned along t cords (Fig. 7A). These ‘beaded lumlarged, and sometimes merged witin the formation of a wide, continug. 7B). A quantitative analysis on sem

ated that HC markedly increased bof Swiss 3T3 CM-induced cords contamen size in tubular cords (Table 5). T HC resulted in an approximately 3-fccupied by lumina with respect to ths present in a semithin section (Ta

ocorticoid andcortisone (an

MHC markedly

This effect was suspended in coaddition ofpment of veryhing and anas-onstrated that

ord length perther enhancedC (1 µg/ml),ol values (datad with dexam-ydroxyproges-tentiated cordferation assaysally stimulatedble 4). mbination withnced enhance-

sections, lumina appeared to be delimited by well polarizedepithelial cells provided with numerous apical microvilli andlateral junctional complexes (Fig. 7C).

Hydrocortisone increases mRNA levels of HGFreceptor (c-met) in TAC-2 cellsTo assess whether the potentiation of HGF-induced tubuloge-nesis by HC might be mediated by an upregulation of HGFreceptor expression, we measured levels of c-met mRNA inTAC-2 cells cultured on collagen-coated dishes by northernblot hybridization, as described in Materials and Methods. HCincreased c-met mRNA expression in a dose-dependentmanner (Fig. 8). Dexamethasone (100 nM) and aldosterone (1µg/ml) also increased c-met mRNA expression, which wassimilar in magnitude to that seen with 1 µg/ml HC. In contrast,17-α-hydroxyprogesterone (1 µg/ml) and tetrahydrocortisone(1 µg/ml) did not significantly increase c-met mRNA levels(this correlates with the lack of a potentiating effect on Swiss3T3 CM-induced cord formation, as described above). HC (1µg/ml) also induced a six-fold increase in c-met mRNAexpression in TAC-2 cells embedded in collagen gels (data notshown). Taken together, these results suggest that potentiationof HGF-induced formation of duct-like structures by HC maybe, in part, mediated by an upregulation of c-met expression in

rds. In cultures alone, luminaopy (although Fig. 2C,D). Inss 3T3 CM and readily identi-t recognizable appearance ofhe axis of the

ina’ progres-h each other,ous cylindrical

ithin sectionsth (a) the per-ining a lumen,hese combinedold increase ine total area of

ble 5). In thin

TAC-2 cells.

Hydrocortisone promotes deposition of basementmembrane proteins by TAC-2 cellsOne conceivable mechanism by which HGF and HC induce

Table 4. Synergistic effect of HGF and hydrocortisone onthe proliferation of TAC-2 cells in collagen gels

Cell number (×104)

Control 0.54±0.22Hydrocortisone 2.84±1.20rhHGF 14.96±3.07rhHGF + hydrocortisone 30.18±1.29

TAC-2 cells suspended in collagen gels at 5×103 cells/ml were incubated induplicated wells with HC (1 µg/ml), rhHGF (10 ng/ml) or both HC andrhHGF. After 9 days, cells were harvested and counted as described inMaterials and Methods. Values are mean cell number per well ± s.e.m.; n = 3experiments.


the formation of tubular or cystic structures, respectively, is thestimulation of endogenous matrix deposition by TAC-2 cells.To investigate whether HGF and/or HC modulate the produc-tion of basement membrane components by TAC-2 cells,cryostat sections of collagen gel cultures were processed forindirect immunofluorescence microscopy using antisera toeither collagen type IV, laminin or entactin. In controlcolonies, immunoreactivity for collagen type IV and lamininwas detectable as short bands of staining underlying the basalsurface of TAC-2 cells (Fig. 9A,D); staining for entactin was

either absent (not shown) or detectable as short bands alongthe periphery of the colonies (Fig. 9G). In cultures incubatedwith 10 ng/ml rhHGF, collagen type IV and lamininimmunoreactivity occupied a greater extent of colonyperimeter than in control cultures (Fig. 9B,E), whereas entactinimmunoreactivity was not detectably increased (Fig. 9H). It isnoteworthy that, in the cyst-like structures induced by HC, athick band of staining for collagen type IV (Fig. 9C), laminin(Fig. 9F) and entactin (Fig. 9I) surrounded most of colonyperimeter. In HC-treated cultures, a fluorescent signal was

Fig. 7. Combined treatment withfibroblast CM and hydrocortisoneinduces formation of tubules withwidely patent lumina. TAC-2 cells weregrown in collagen gels in the presenceof both Swiss 3T3 CM and HC (1µg/ml). (A) Formation of multiplecircular spaces aligned along the axis ofthe epithelial cords in a 6-day-oldculture (arrows). Bar, 100 µm. (B) Wide cylindrical lumen in a 12-day-old culture, resulting from thecoalescence of multiple sphericallumina. Bar, 100 µm. (C) Thin sectionof a tubule in a 7-day-old culture. Thelumen is delimited by cubic orcolumnar epithelial cells provided withnumerous apical microvilli, andcontains flocculent material and smallvesicular structures. Bar, 10 µm.

424

often obThis lumthe peripimmune was appevaluatioimmunostaining cultures,in HC-trcontrol vcolony ptreated (P<0.0017.5±1.7%in HGF-(P<0.001and to a lsition byproteins.results mmembranof type I2 cells cuHC and expressiochain or addition,chain mRanother mAs no ulthin sectbasemenassemblemembran

Since

J

Table 5

A. StructurB. Lumen

with lC. Total lu

of poo

TAC-2 cSwiss 3T3HC). Afterdescribed i

A. To ascord profilsections an

B. The a50 tubular analyzer (Lthe area ofValues are

C. Semicavitary) wbetween thcalculated.sections pe


. Hydrocortisone enhances lumen formation in theepithelial cords

CM CM+HC

es with lumen (%) 58.80 98.00area (in structures 8.63±1.03 21.86±2.19umen) (%)men area per total area 4.59±0.69 14.63±1.83led structures (%)

ells were suspended in collagen gels and incubated with either CM alone (CM) or both Swiss 3T3 CM and HC (1 µg/ml) (CM + 7 days, collagen gel cultures were fixed and processed asn Materials and Methods. sess the percentage of epithelial structures containing a lumen, 50es per experimental condition were followed in serial semithind scored for the presence or absence of a lumen. rea occupied by the lumen in cavitary structures was measured onprofiles per experimental condition with a Qmet 500 imageeyca Cambridge Ltd., Cambridge, England), and the ratio between

the lumen and the total area of the entire structure was calculated.

ity as well (see Fig. 9C,I).d non-specific, since unlike detected when either non-lated antiserum (not shown)bation step. A quantitativelony perimeter occupied bythat: (a) type IV collagenolony perimeter in controld cultures, and 81.0±3.3%

1 for control vs HGF andng occupied 28.6±4.4% ofures, 38.3±3.9% in HGF- in HC-treated cultures

entactin staining occupiedontrol cultures, 13.2±3.8%

4.1% in HC-treated culturese findings indicate that HC,

cord profiles (either solid or image analyzer, and the ratio the total area of pooled structures

s.e.m.; n = at least 10 semithin

Fig. 8. Upregulation of c-met mRNA expression by corticosteroidhormones. (A,B) Steady-state levels of c-met mRNA are increasedafter 14 hours of treatment of subconfluent monolayers of TAC-2cells with either HC (0.1, 1 and 10 µg/ml), aldosterone (1 µg/ml)

served within the cyst cavinal staining was considereheral staining it was alsoserum (Fig. 9L) or an unrelied during the first incun of the percentage of co

reactive material showed occupied 27.8±3.0% of c 54.0±3.2% in HGF-treateeated cultures (P < 0.00s HC); (b) laminin stainierimeter in control cult

cultures, and 76.6±3.5% for control vs HC); (c) of colony perimeter in c

treated cultures, and 73.5± for control vs HC). Thes

mean % area ± s.e.m. thin sections containing at least 5ere examined with the Qmet 500e total area of pooled lumina and Values are mean % lumen area ±r experimental condition.

esser extent HGF as well, promote the polarized depo- TAC-2 cells of three major basement membrane To determine whether these immunocytochemical

ight correlate with an upregulation of basemente component expression, we measured mRNA levels

V collagen and laminin A, B1 and B2 chains in TAC-ltured on collagen-coated dishes. We found that bothSwiss 3T3 CM increased laminin B1 chain mRNAn. No significant modulation of either laminin B2

type IV collagen was observed (results not shown). In we found that TAC-2 cells do not express laminin ANA, which is consistent with previous findings withammary epithelial cell line (Reichmann et al., 1989).

trastructural correlate of a basal lamina was visible byion electron microscopy (not shown), we conclude thatt membrane components are secreted but notd into a morphologically identifiable basemente structure.

HC and HGF enhance the deposition of type IV collagen

by TAC-2 cells, we used cis-hydroxy-proline (CHP) to assess theeffect of inhibition of collagen formation on HC-induced cysto-genesis and HGF-induced tubulogenesis. CHP is a proline analogthat is incorporated into procollagen α-chains and prevents theformation of a normal triple helix by steric hindrance (Uitto andProckop, 1974). The conformationally defective procollagenmolecules have been shown to be degraded intracellularly (Berget al., 1980). Addition of 50 µg/ml CHP to collagen gel cultures

(ALD) or dexamethasone (100 nM) (DEX); CTR, control. (A) Eachpoint represents c-met mRNA levels from a single experimentexpressed relative to control. (B) Median values for each condition.1, P<<0.01; 2, P< 0.01; 3, P=0.01. (C) Northern blot hybridizationof total TAC-2 cellular RNA. HC (0.1, 1 and 10 µg/ml) increasessteady-state levels of c-met mRNA; 0, control. Hybridization of thesame filter with a chicken glyceraldehyde-3-phosphatedehydrogenase (GAPDH) probe reveals the presence of similarquantities of mRNA in each lane. 28 S RNAs are indicated.


totally inhibited the initial elongation and subsequent branchingof TAC-2 cell colonies induced by rhHGF. The inhibitory effectof CHP was abrogated by the simultaneous addition of a 10-foldexcess (500 µg/ml) of L-proline. In marked contrast, HC-inducedlumen formation was only slightly decreased by CHP (results notshown). This differential effect of CHP suggests that lumenformation may be dependent on factors other than deposition ofendogenous collagen, and that the two processes of lumenformation and colony elongation and branching are indepen-dently regulated. Further studies are required to determinewhether other basement membrane components, e.g. laminin, areimportant for lumen formation.

HGF and c-met mRNAs are expressed in the ratmammary glandTo ascertain whether our in vitro findings might be relevant tomammary gland development in vivo, we examined whetherHGF and c-met mRNAs are expressed in the rat mammarygland. Using an RNase protection assay, we found that bothHGF and c-met mRNAs are expressed in the mammary glandof virgin and pregnant rats (Fig. 10). These qualitative findingsindicate that the HGF/c-met pathway is likely to be operativein the mammary gland. Further studies are underway todetermine whether HGF and c-met expression are modulatedduring pregnancy and lactation.

Fig. 9. Effect of HGF and hydrocortisone on thedeposition of basement membrane proteins byTAC-2 cells. Cryosections of collagen gelcultures were stained by indirectimmunofluorescence with antisera to collagentype IV (A-C), laminin (D-F) and entactin (G-I)or non-immune lamb serum (J-L), andphotographed with a laser scanning confocalmicroscope. The red colour is due to the EvansBlue counterstaining. In control 9-day cultures(A,D,G), immunoreactivity for type IV collagen(A) and laminin (D) is detected as short bands ofstaining underlying the basal surface of TAC-2cells; (G) shows a colony with a short band ofstaining for entactin (the majority of colonies aredevoid of entactin immunoreactivity). In culturesincubated with 10 ng/ml rhHGF for 9 days(B,E,H), immunoreactivity for collagen type IV(B) and laminin (E) is detected as longer bandsof staining along the perimeter of the tubuleswhereas entactin immunoreactivity is notdetectably increased (H). In cultures treated with1 µg/ml HC for 9 days (C,F,I), staining forcollagen type IV (C), laminin (F) and entactin (I)is detected as a thick band surrounding most ofthe outer surface of cyst-like structures. Thefluorescence visible in the cavity of some cysts(C,I) is likely to be non-specific, since it is alsoobserved when non-immune serum (L) or anunrelated antiserum (not shown) is appliedduring the first incubation step. Bar, 50 µm.

426

DISCUS

Since thet al., 1mammasional st(reviewethat thisous cell Ormerod1984; R

epithelial structures described in these studies, however, wererelatively rudimentary and underwent only limited elongationand branching, which suggests that the development of anextensive ductal tree in vitro may require specific signals.

To determine whether diffusible factors produced by stromalcells might stimulate formation of branching duct-like struc-tures by mammary epithelial cells, we cocultured TAC-2 cells,a clonal derivative of NMuMG mammary gland epithelialcells, with Swiss 3T3 or MRC-5 fibroblasts in collagen gelsunder conditions precluding heterocellular contact. WhereasTAC-2 cells grown alone in collagen gels formed irregularlyshaped aggregates or short cord-like structures, when cocul-tured with fibroblasts they generated branching structures con-taining widely patent lumina. Incubation of TAC-2 cells incollagen gels with fibroblast CM partially mimicked the effectof coculture by inducing formation of a highly arborizedsystem of duct-like structures, which however enclosed con-

J

Fig. 10. Rc-met (B)follows: 1Beverly, 3, purifiehybridizayeast tRNtotal celluincubatedmammaryµg total cLanes in 2, purifiebase pairbuffer; 4,32P-labelfrom rat kµg total c32P-labelfrom 12-dare in bas


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siderably narrower lumina than those formed in cocultures.The reasons for this difference are unclear, but it is conceiv-able that labile fibroblast-derived factors whose activity is lostduring preparation or storage of CM promote the developmentof widely patent lumina while preventing extensive cordelongation and branching.

We have previously reported that CM from Swiss 3T3 orMRC-5 fibroblasts induces kidney-derived MDCK cells toform branching tubules when grown in collagen gels(Montesano et al., 1991a) and that this effect is mediated byHGF (Montesano et al., 1991b). To determine whether HGFmight be the factor in fibroblast CM that stimulates tubuloge-nesis by TAC-2 cells, CM was preincubated with anti-HGFantibodies prior to addition to TAC-2 cells in collagen gels.This completely abrogated the tubulogenic activity of fibrob-last CM. In addition, exogenous HGF mimicked the tubulo-genic activity of fibroblast CM by stimulating formation ofduct-like structures in a dose-dependent manner, with amaximal 77-fold increase in tubule length at 20 ng/ml. Takentogether, these results demonstrate that the factor in fibroblastCM that induces formation of branching duct-like structures byTAC-2 cells is HGF.

While this work was in progress, two papers appeared thatare relevant to the present study. Kanazawa and Hosick (1992)

ibonuclease protection assay for detection of HGF (A) andmRNAs in rat mammary glands. Lanes in (A) are as, SP6 control template marker (New England Biolabs,

A); 2, purified 336 base pair 32P-labelled rat HGF cRNA; 336 base pair 32P-labelled rat HGF cRNA incubated withion buffer; 4, 32P-labelled rat HGF cRNA incubated with

; 5, 32P-labelled rat HGF cRNA incubated with 20 µgar RNA from rat lung; 6, 32P-labelled rat HGF cRNAwith 20µg total cellular RNA from 3-month virgin rat gland; 7, 32P-labelled rat HGF cRNA incubated with 20llular RNA from 12-day pregnant rat mammary gland.) are as follows: 1, SP6 control template marker (pSP64);

462 base pair 32P-labelled rat c-met cRNA; 3, purified 4622P-labelled rat c-met cRNA incubated with hybridization2P-labelled rat c-met cRNA incubated with yeast tRNA; 5,

SION

pioneering work of Nandi and collaborators (Yang80), many investigators have shown that primary epithelial cells have the ability to form three-dimen-

uctures resembling branching ducts in collagen gels by Levay Young et al., 1987; Durban, 1987), and

morphogenetic property can be retained by continu-nes established from either neoplastic (Bennett, 1980;and Rudland, 1982) or normal (Danielson et al.,

ichmann et al., 1989) mammary gland tissue. The

reported increased formation of branching structures bymammary gland organoids cocultured in collagen gels withmesenchymal tissue, but the molecules responsible for thiseffect were not identified. Tsarfaty et al. (1992) showed thatHGF induces colon and breast carcinoma cell lines grown onglass to arrange in two-dimensional rings that bear someresemblance to glandular lumina. Although the latter observa-tion is in agreement with our previous demonstration that HGFcan act as an epithelial morphogen (Montesano et al., 1991b),it does not support a role for HGF in the development of themammary gland ductal tree. Our study therefore provides thefirst experimental evidence that HGF promotes the develop-ment of branching duct-like structures by normal mammarygland epithelial cells in vitro.

An additional novel finding to emerge from our study is thedemonstration of a cooperative interaction between HGF andcorticosteroid hormones in the induction of tubulogenesis byTAC-2 cells. Among a number of hormones known to promotemammary gland development and functional differentiation in

d rat c-met cRNA incubated with 20 µg total cellular RNAdney; 6, 32P-labelled rat c-met cRNA incubated with 20llular RNA from 3-month virgin rat mammary gland; 7,d rat c-met cRNA incubated with 20 µg total cellular RNA

ay pregnant rat mammary gland. Molecular size markers pairs.

vivo (Tβ-estraobvioucells, wCM. In(HC) activitya furthcultureenhancticosterwhile ethe absduct grHGF-inby the collageulationHC.

Intergenesistheir eabsencHC or resultedwidelydescribcells. Tticostervivo an1980). formatithe depcells, primary1981). secretiotherefomembrprocesslumen cocortial., 199al., 199ulationenlargeoccurs

In ou1991b)result oMDCKexclusihowevegels unshowinshapedbe arguas a moproliferform bculture


opper and Freeman, 1980; Imagawa et al., 1990), 17-diol, progesterone, insulin and prolactin did notsly modify the morphogenetic properties of TAC-2hether added alone or in combination with fibroblast contrast, the corticosteroid hormones hydrocortisone

and aldosterone greatly potentiated the tubulogenic of either fibroblast CM or HGF, as evidenced by: (a)er significant increase in tubule length with respect tos incubated with CM or HGF alone; and (b) a markedement of lumen formation. These in vitro effects of cor-oids are consistent with the in vivo observation thatxtensive growth of mammary gland ducts can occur inence of glucocorticoids, these are required for maximalowth (Topper and Freeman, 1980). The potentiation ofduced tubulogenesis by HC may be explained in partsynergistic stimulation of TAC-2 cell proliferation inn gels by HGF and HC, and possibly also by the upreg- of the HGF receptor (c-met) mRNA in TAC-2 cells by

number of TAC-2 cells than control cultures, it seems reason-able to assume that stimulation of cell proliferation by HGFcontributes to the formation of an extensive system of duct-like structures. Nonetheless, several findings indicate that HGFis not acting solely as a mitogen on TAC-2 cells. Firstly, wehave shown that fibroblast CM modifies colony morphologyby markedly increasing both the percentage of branchedcolonies (from 20% in control conditions to 100% in treatedcultures) and the number of branch points per colony (see Fig.3). Secondly, in long-term collagen cultures without HGF,76% of colonies consisted of irregularly shaped cell aggregatesdevoid of tubular extensions (unpublished observations),which indicates that an increase in cell number per se does notnecessarily result in the formation of branching cords. Finally,we have recently found that TAC-2 cells grown in collagengels in the presence of both HC and the cAMP-elevating agentcholera toxin almost exclusively form cystic structures, andthat addition of HGF to these cultures induces formation of

estingly, while HC and aldosterone enhanced tubulo- only when added together with fibroblast CM or HGF,ffect on lumen formation was also observed in thee of CM or exogenous HGF. Thus, addition of eitheraldosterone to cultures of TAC-2 cells in collagen gels in the formation of alveolar-like cysts enclosing a

patent lumen. To our knowledge, this is the first reporting an effect of HC on lumen formation by epithelialhis phenomenon may be related to the ability of cor-oid hormones to promote alveolar development, both ind in mammary gland explants (Topper and Freeman,The mechanisms by which HC promotes lumen

on are not known. We have shown that HC enhancesosition of basement membrane components by TAC-2

which is in agreement with previous studies using cultures of mammary epithelial cells (Salomon et al.,

However, we have also found that inhibitors of collagenn do not prevent lumen formation. Further studies will

re be required to establish whether other basementane components, e.g. laminin, play a role in this. Other mechanisms that may conceivably contribute toformation by TAC-2 cells include the ability of glu-coids to decrease tight junctional permeability (Zettl et2) and to promote functional polarization (Sjaastad et

branching tubules (unpublished observations). Taken together,these findings show that HGF, in addition to stimulating theproliferation of TAC-2 cells in collagen gels, modifies theirspatial arrangement. It is therefore likely that HGF promotesthe development of branching duct-like structures by TAC-2cells by acting both as a mitogen and as a morphogen. It isnoteworthy that we have found that HGF also stimulatesformation of branching cords by the human mammary epithe-lial cell line HBL-100 (unpublished observations), whichindicates that this effect is not restricted to a particular cell line.

Among a number of well characterized growth factors whichwe examined, EGF and TGF-α significantly enhanced cordformation by TAC-2 cells. This finding is consistent with thereported ability of these polypeptides to stimulate proliferationof mammary gland epithelial cells in vitro (Tonelli and Sorof,1980; Yang et al., 1980; Taketani and Oka, 1983; Imagawa etal., 1985; Smith et al., 1989; Sakthivel et al., 1993) and devel-opment of the mammary gland ductal tree in vivo (Vonderhaar,1987; Coleman et al., 1988; Snedecker et al., 1991). However,the increase in cord length induced by maximally effectiveconcentrations of EGF was at least one order of magnitudelower than that elicited by HGF, and when compared atequimolar (100 pM) concentrations, EGF was approximately24-times less potent than HGF (see Fig. 4C and Table 1).Moreover, addition of EGF to TAC-2 cells grown in the

3) in cultures of mammary gland epithelial cells (upreg- of c-met by HC is unlikely to account for lumenment, because this phenomenon, as described above,also in the absence of exogenously added HGF).r previous study with MDCK cells (Montesano et al.,

, induction of epithelial tubulogenesis by HGF was thef a morphogenetic activity of the growth factor, since cells grown in collagen gels under control conditionsvely formed spherical cysts. In the present study,r, TAC-2 mammary epithelial cells grown in collagender control conditions gave rise to discrete coloniesg a variety of morphologies, ranging from irregularly aggregates to short branching cords. It could thereforeed that in this experimental system HGF is not actingrphogen, but promotes tubulogenesis by stimulating theation of epithelial cells that have the inherent ability toranching ducts. Since our data show that collagen gels incubated with HGF contain a considerably greater

presence of both HC and cholera toxin did not elicit tubuloge-nesis, in contrast to what was observed with HGF (unpublishedobservations). Finally, EGF did not stimulate lumen formationin the epithelial cords. This suggests that, in contrast to HGF,EGF lacks morphogenetic activity in this experimental systemand may enhance cord formation by TAC-2 cells exclusivelyby virtue of its mitogenic properties.

Although it has long been recognized that the developmentof the mammary gland requires an interaction with the adjacentmesenchyme (or stroma) (Kratochwil, 1969; Donjacour andCunha, 1990; Haslam, 1991; Sakakura, 1991), paracrinefactors that mediate these interactions have not yet been iden-tified. Our findings that fibroblast CM induces the formationof a highly arborized system of duct-like structures by TAC-2cells in vitro, and that this activity is due to HGF, suggest thatthe inducing effect of mesenchyme or stroma on mammarygland development in vivo is mediated in part by HGF.Although HGF was originally thought to have a narrow target

428

cell spenow coimportaSonnenb1991; Is1993) o(NakamNakamua morphThe resuboth HGvivo, stimportagland. Fstandingonly ducarcinogpriate eresponsovergroepitheliureportedimportaet al., 1

In conthat HGhighly gland epit may amorpho

We arCancer R(fragmenLondon,(Nationachain cDMartinsrA. P. SaGeneva)and M. Aanalysis Di SanzaP. Gerbesupporte(number

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(Received 27 June 1994 - Ac


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, DeVault, M. R., Wellings,
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cepted 21 October 1994)

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