Progestin-Regulated Luminal Cell and MyoepithelialCell-Specific Responses in Mammary Organoid Culture
Sandra Z. Haslam, Alexis Drolet, Kyle Smith, May Tan, and Mark Aupperlee
Department of Physiology (S.Z.H., A.D., M.T.) and the Cell and Molecular Biology Program (S.Z.H., K.S., M.A.), MichiganState University, East Lansing, Michigan 48824
Normal mammary gland development requires the coordi-nated proliferation and morphogenesis of both mammary lu-minal epithelial cells (LECs) and myoepithelial cells (MECs).Cell proliferation in cultured mammary organoids containingboth LECs and MECs is not increased by progestin (R5020) or17�-estradiol (E2) alone or R5020�E2 but is increased by E2-regulated, mammary stroma-derived Hepatocyte growth fac-tor (HGF) and further increased by HGF�R5020. We investi-gated the effects of HGF and/or R5020 on morphology andLEC- and MEC-specific in vitro proliferation in organoids.HGF-induced tubulogenesis was initiated and carried out byLECs starting with cellular extensions, followed by the for-mation of chains and cords, and culminating in tubule for-mation. MECs did not appear to have an active role in thisprocess. Whereas HGF by itself caused maximal proliferationof LECs, HGF�R5020 produced a synergistic and specific in-
crease in MEC proliferation. Because only LECs expressedprogesterone receptors (PRs), we investigated the role of re-ceptor activator of nuclear factor-�B ligand (RANKL), a pro-gestin-induced paracrine factor, in mediating increased MECproliferation. Quantitative RT-PCR showed that RANKLmRNA was induced by R5020 or HGF�R5020 and RANKL pro-tein colocalized with PRs in LECs. The increased proliferationof MECs in response to HGF�R5020 could be blocked by neu-tralizing antibody to RANKL and reproduced by treatmentwith HGF plus exogenous RANKL in place of R5020. NeitherR5020, nor exogenously administered RANKL increased pro-liferation of LECs. These results led us to conclude thatRANKL, induced by progestin in PR-positive cells, is secretedand interacts with HGF to specifically increase proliferationof PR-negative MECs. (Endocrinology 149: 2098–2107, 2008)
THE EPITHELIAL UNIT of the murine mammary glandis composed of luminal epithelial cells (LECs) and basal
myoepithelial cells (MECs). LECs form the lining of ductsand alveoli and are the cells responsible for milk synthesisand secretion during lactation. LECs are encircled basally bya layer of MECs, contractile cells that facilitate export of milkfrom the gland during lactation (1). The LEC-MEC unit issurrounded by a basement membrane that separates it fromthe adjacent mammary stroma (2). An integrated response tospecific growth factors and peptide and steroid hormones isrequired for the development of the ductal tree from pubertythrough sexual maturation of the gland and for lobuloal-veolar development and function during pregnancy and lac-tation. Normal ductal development and alveologenesis invivo requires the coordinated proliferation and morphogen-esis of both LECs and MECs.
The rodent mammary gland provides a useful model tostudy LEC and MEC regulation and function. The focus ofmost studies has been on the regulation of LEC proliferation,morphogenesis, and differentiation. Fewer studies have fo-cused on the regulation of MECs (1, 3). Most often the celltype-specific behavior and responses of LECs or MECs have
been studied in vitro, separately (4, 5). This approach may notaccurately reflect the coordinated responses of these two celltypes in vivo. In vitro reconstitution experiments in whichLECs have been cultured separately or recombined withMECs have demonstrated an important role of MECs in theestablishment of LEC polarity (6–8). In the present report, weinvestigated the regulation and cell type-specific responsesof LECs and MECs in vitro when both are present togetherin mammary organoids.
Hepatocyte growth factor (HGF) is a mesenchyme-derivedgrowth factor that is synthesized in the stroma in vivo andstimulates the proliferation, motility, and morphogenesis ofnearby epithelium (9, 10). HGF is important for normal mam-mary ductal development in vivo and has also been shown tobe important for side branching leading to alveologenesis(11, 12). We have shown that HGF is produced by mammarystromal cells in vitro, in response to 17�-estradiol (E2) treat-ment (13) Thus, whereas E2 is not directly mitogenic inmammary cells in vitro, its mitogenic effects are likely me-diated in part, indirectly through HGF. Whereas HGF isproduced by stromal cells, it acts on mammary epithelialcells that express c-Met, the cognate receptor for HGF (11, 12,14). Numerous in vitro studies have shown that HGF caninduce proliferation and produce a tubulogenic response ina wide variety of epithelial cells and cell lines, includingmammary epithelial cells, when the cells were culturedwithin a collagen gel matrix (11, 15, 16). We reported thatorganoids obtained from adult mice and containing bothLECs and MECs proliferate and produce tubules in responseto treatment with HGF. Neither treatment with the progestin,promogestone (R5020), nor R5020�E2 induce proliferation.
First Published Online January 24, 2008Abbreviations: BrdU, 5-Bromo-2�-deoxyuridine; CT, cycle threshold;
3-D, three-dimensional; E2, 17�-estradiol; ER, estrogen receptor; HGF,hepatocyte growth factor; K18, cytokeratin 18; LEC, luminal epithelialcell; MEC, myoepithelial cell; PBSA, PBS containing BSA; PR, proges-terone receptor; R5020, progestin; RANK, receptor activator of nuclearfactor-�B; RANKL, RANK ligand; SMA, smooth muscle actin.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.
When treated with R5020 alone, the organoids form cyst-likestructures. Treatment with the combination of HGF�R5020results in proliferation that was increased above treatmentwith HGF alone, and the organoids exhibited blunting of thetubulogenic response (17). However, in these studies it wasnot determined which cell types, LECs and/or MECs, pro-liferated in response to HGF or HGF�R5020. The purpose ofthe present study was to investigate the cell type-specificmorphologic and proliferative responses of LECs and MECsto treatment with either HGF or R5020 by themselves orwhen combined (HGF�R5020).
In vivo in the adult virgin mammary gland, progesteronereceptor (PR)-A is expressed in a subset of LECs but not inMECs; PRB expression is undetectable before pregnancy (14).PRA-containing cells rarely proliferate and the mitogeniceffect of progesterone in the virgin gland is believed to bemediated by paracrine factors such as receptor activator ofnuclear factor-�B ligand (RANKL) (14, 18, 19). Thus, the roleof RANKL in mediating increased proliferation in organoidstreated with HGF�R5020 was also investigated. To identifythe responses occurring in LECs vs. MECs, an immunocy-tochemical approach was used.
Materials and MethodsAnimals
BALB/c female mice from our own colony were the source of mam-mary glands at the following ages and developmental stages for c-Metdevelopmental analysis: puberty (5 wk), sexual maturity (12–16 wk),pregnancy (14 d), and lactating (7 d postpartum). Adult virgin mam-mary glands (18–20 wk old) were used to prepare primary culturemammary epithelial organoids for all other experiments. Animal ex-perimentation was conducted in accord with accepted standards ofhumane animal care, and approved by the All University Committee onAnimal Use and Care at Michigan State University.
Preparation of primary mammary gland organoids
Mammary epithelial organoids were isolated using mechanical andenzymatic dissociation methods as previously described (17). Briefly,mouse mammary glands were excised, minced, and digested sequen-tially in collagenase (Worthington, Lakewood, NJ) and pronase (Cal-biochem, San Diego, CA) solutions, followed by differential centrifu-gation and Percoll (Amersham Biosciences, Piscataway, NJ) gradientsedimentation to remove mammary stromal cells and obtain mammaryepithelial organoids consisting of both LECs and MECs. At the time ofplating, MECs represent 26 � 4% of the total cells in organoids based ondouble-immunofluorescent labeling with anti-smooth muscle actin(SMA) and cytokeratin 18 (K18) antibodies to detect MECs and LECs,respectively. Cell viability was approximately 95% as determined bytrypan blue exclusion. Total cell number was determined by countingnuclei in a hemocytometer after cell disruption with 2.1% citric acidcontaining 0.4% crystal violet (20).
To retain the three-dimensional (3-D) architecture of the organoids invitro, they were cultured within a collagen I gel matrix and in serum-freemedia. Then 96-well culture dishes were coated with an underlay of 40�l/well of neutralized rat tail collagen I (2 mg/ml; BD Biosciences,Bedford, MA) before plating organoids. Freshly isolated organoids weresuspended in neutralized collagen I (2 mg/ml, 75 �l/well) at a densityof 1 � 105 cells/well and allowed to set for 30 min at 37 C before theaddition of media. All cultures were carried out in serum-free mediumwith or without growth factors or hormones [basal medium (BM): serumand phenol red free DMEM/F12, supplemented with 0.1 mm nones-sential amino acids, 2 mm l-glutamine, 100 ng/ml human recombinantinsulin, 1 mg/ml fatty acid-free BSA (fraction V), 100 �g/ml penicillin,and 50 �g/ml streptomycin]. Treatments with growth factors and hor-mones were added at the time of plating and included 50 ng/ml HGF(Calbiochem), 10 nm 17�-estradiol (Sigma, St. Louis, MO), 20 nm of the
synthetic progestin, R5020 (PerkinElmer, Boston, MA) (13, 17), and 200ng/ml RANKL (R&D systems; Minneapolis, MN) (21). Organoid cul-tures were maintained in 5% CO2 at 37 C for up to 3 d and culture mediawas replaced every 48 h. For RANKL inhibition experiments, rat anti-mouse RANKL-neutralizing monoclonal antibody (IgG2a clone IK22–5;BioLegend, San Diego, CA; 10 �g/ml final concentration) or control ratIgG2a (22) was added to the various treatment media at the time ofplating and maintained in the media throughout the treatment period.For analysis of proliferation, 5-bromo-2�-deoxyuridine (BrdU; 10 mm;Sigma) was added for 18–20 h before termination of the cultures.
Antibody labeling of organoids in collagen gels
The method for antibody labeling of mammary organoids in collagengels was adapted from O’Brien et al. (23). After 24, 48, or 72 h in culture,collagen gels containing mammary epithelial organoids were removedwith forceps, placed in glass vials, and rinsed for 10 min in PBS (pH 7.2)containing 1 mm CaCl2 and 0.5 mm MgCl2 (PBS�). The gels were thenfixed in 4% paraformaldehyde in PBS� and permeabilized for 30 minin 0.025% saponin in PBS� (P buffer). Gels were again rinsed in PBS�for 10 min, followed by quenching for 10 min in PBS� containing 75 mmNH4Cl and 20 mm glycine, and blocking for 10 min in a 0.025% saponin,0.3% gelatin solution in PBS� (B buffer). All rinsing and blocking stepswere carried out at room temperature on a gently rocking platform. Gelswere then incubated by gently rocking for 3 d at 4 C simultaneously withmouse monoclonal primary antibody against K18 (catalog no. ab668-100;Abcam, Cambridge, MA) diluted 1:200 and with rabbit polyclonal an-tibody against �SMA (catalog no. ab5694; Abcam) diluted 1:400 in Bbuffer. Tissue sections from adult virgin mammary gland served aspositive controls. For negative controls, samples were treated only withsecondary antibody. Samples then were washed 4 � 15 min in P bufferand washed 1 � 15 min in B buffer. Samples were incubated overnightat 4 C with goat antimouse Alexa 488 and goat antirabbit Alexa 546(Molecular Probes, Eugene, OR), each diluted 1:100 in B buffer. Samplesthen were washed 4 � 15 min in P buffer. Samples were postfixed in 4%paraformaldehyde in 0.1 m sodium cacodylate buffer (pH 7.2) andmounted on glass slides using fluorescence-mounting media. Imageswere captured using a Pascal laser scanning confocal microscope (CarlZeiss Inc., Thornwood, NY). The images were captured in a 3-D Z planeseries with a step size of 4 �m between planes. Individual planes wereanalyzed from the Z-stack.
Antibody labeling of organoid sections
To obtain mammary gland organoid sections for immunohistochem-ical analyses, organoid cultures were removed from 96-well plates andfixed for 1 h in 10% phosphate-buffered formalin [0.4% sodium phos-phate monobasic and 0.65% sodium phosphate dibasic (anhydrous) in10% formalin]. Fixed gels were then processed overnight through aseries of dehydrating alcohols, followed by xylenes and a xylene/par-affin (1:1) mixture on a Tissuematon (Fisher Scientific, Vernon Hills, IL)before embedding in paraffin blocks for sectioning. Five-micrometersections were mounted onto coverslips treated with 3-aminopropyl tri-ethoxysilane. Whole mammary glands were fixed overnight in 10%phosphate-buffered formalin and processed in the same way to obtainsections for c-Met immunohistochemical analysis. Paraffin sections werefirst deparaffinized and rehydrated sequentially through xylenes andgradient alcohols (100–50% EtOH). Sections were then immersed in 10mm sodium citrate solution (pH 6.0) and autoclaved at 15 lb/square in.2
for 20 min for antigen retrieval.For double labeling of BrdU and SMA, sections were first incubated
with goat antimouse IgG Fab fragments [Jackson Laboratories, WestGrove, PA) (diluted 1:100 in PBS containing 1% BSA; Sigma; 1% PBSA)60 min], rinsed with PBS, and then blocked with normal goat serum(Vector Laboratories, Burlingame, CA; diluted 1:1 in PBS, 30 min). BrdUimmunoreactivity was detected using a mouse monoclonal anti-BrdU/nuclease solution (catalog no. RPN202; Amersham Biosciences, Buck-inghamshire, UK; undiluted from kit, 1 h at room temperature), followedby incubation with goat antimouse Alexa 546 (Molecular Probes; diluted1:400 in PBS, 30 min). Sections were then blocked again in a 2% PBSAsolution and incubated with rabbit polyclonal antibody against �SMA,(catalog no. ab5694; Abcam; diluted 1:400 in PBS, 60 min) followed bygoat antirabbit Alexa 488 (Molecular Probes; diluted 1:200 in PBS, 30
Haslam et al. • Progestin and Proliferation of Mammary Cells Endocrinology, May 2008, 149(5):2098–2107 2099
min). Cell nuclei were counterstained using either 4�,6�-diamino-2-phe-nylindole or ToPro 3-iodide (Molecular Probes), and coverslips weremounted using fluorescence-mounting media. Tissue sections fromadult virgin mammary gland were used as a positive control. BrdU-positive LECs or MECs are expressed as a percentage of total LECs orMECs counted, respectively.
For cell type-specific detection of PR isoforms, the protocol wasfollowed as above; however, either mouse monoclonal hPRa7 (PRAspecific) or hPRa6 (PRB specific) (24) (Neomarkers, Fremont, CA; diluted1:50 in PBS/0.5% Triton X-100, overnight at 4 C) was used in the placeof the BrdU antibody. Tissue sections from adult virgin mice and mid-pregnant mice were used as positive controls for PRA- and PRB-specificstaining, respectively (14).
Estrogen receptor (ER)-� immunoreactivity was detected usingmouse monoclonal anti-ER�, NCL-L-ER-6F11 (Novocastra, Newcastle,UK; diluted 1:10 in PBS, overnight at 4 C) using identical blocking stepsand secondary antibody conditions as previously described for BrdU,hPRa6, and hPRa7. Sections were then blocked with 2% PBSA andincubated with rabbit antihuman PR antibody (catalog no. A0098; Dako,Carpinteria, CA; diluted 1:200 in 2% PBSA, overnight at 4 C), followedby incubation with secondary antibody and nuclear counterstain, aspreviously described for rabbit polyclonal antibodies. Tissue sectionsfrom adult virgin mammary gland were used as a positive control.
For RANKL, receptor activator of nuclear factor-�B (RANK), andPRA staining, nonspecific staining was blocked using normal rabbitserum (Vector Laboratories; diluted 1:1 in PBS, 30 min) and then incu-bated with goat antimouse RANKL antibody (R&D Systems) or rabbitantihuman RANK (Santa Cruz Biotechnology, Santa Cruz, CA; diluted1:100 in PBS, overnight at 4 C), followed by incubation with rabbitantigoat Alexa 488 (Molecular Probes; diluted 1:100, 30 min) or second-ary antibody and nuclear counterstain as previously described for rabbitpolyclonal antibodies. Positive controls for RANKL and RANK weretissue sections from midpregnant mammary gland (25). Negative con-trol for RANK was lack of stromal cell staining in tissue sections fromvirgin mammary gland. In the case of double staining with anti-RANKLand anti-PRA antibodies, sections were then blocked following the pro-tocols above for mouse monoclonal antibodies and incubated with pri-mary antibodies against PRA (hPRA7) and the appropriate secondaryantibodies as described above.
c-Met and SMA double labeling of cultured organoids and mammarygland tissue sections was performed using the rabbit antimouse c-Metprimary antibody SP260 (Santa Cruz Biotechnology) (diluted 1:50, 1 h atroom temperature). Blocking steps, secondary antibodies, SMA staining,and nuclear counterstaining steps were identical with rabbit/mousedouble labeling protocols as described above. For all antibody staining,negative controls received no primary antibody followed by the relevantfluorochrome-conjugated secondary antibody.
To accommodate multiple protocols, in some experiments the fluo-rochromes Alexa 488 and Alexa 546 were switched; however, identicalstaining patterns with all antibodies were observed, regardless of whichfluorochrome was used.
Image analysis and stain intensity quantification
Immunofluorescent images were captured using either a Pascal laser-scanning confocal microscope (Carl Zeiss) or a Nikon inverted epiflu-orescence microscope (Mager Scientific, Dexter, MI) with MetaMorphsoftware (Molecular Devices Corp., Downington, PA). Confocal imageswere viewed and analyzed using the Zeiss LSM image browser programand National Institutes of Health Image J (Bethesda, MD). Epifluorescentimages were viewed and analyzed using MetaMorph software. In bothcases, to analyze fluorescence intensity, the average pixel intensity of allpositively stained nuclei was determined. Images were thresholded toexclude background fluorescence and gated to include intensity mea-surements only from positively staining epithelial cells. In the case of celltype-specific c-Met intensity quantification, cells that were shown to beexpressing SMA were analyzed separately from cells not expressingSMA.
RT-PCR analysis
Cultured whole mammary organoids, containing both MECs andLECs, were harvested at 24 h after treatment with BM, HGF, R5020, or
HGF�R5020 as described above. Total RNA was extracted from or-ganoids using TRIzol (Invitrogen, Carlsbad, CA) following the manu-facturer’s suggested protocol. All extracted RNA samples were storedat �80 C until analyzed. RNA concentration was determined based onthe ratio of UV absorbance read at 260 and 280 nm. cDNA was producedby reverse transcription with random hexamer primers using the Su-perscript III first-strand synthesis system for RT-PCR (Invitrogen) fol-lowing the manufacturer’s instructions. Then 4 �l of cDNA from eachtreatment group were added to primers (Applied Biosystems, FosterCity, CA) for either murine RANKL (assay no. Mm00441980_m1) or 18SrRNA (assay no. Hs99999901_s1). Real-time PCR for each sample wasperformed in triplicate using a Prism 7500 sequence detection system(Applied Biosystems). Cycling conditions were as follows: 10 min at 95C for initial denaturation and enzyme inactivation and then 40 cycles of15 sec at 95 C and 1 min at 60 C.
For quantification, a standard curve was first generated by makingserial 10-fold dilutions of total cDNA. At each dilution, an amplifi-cation curve was generated for RANKL and 18S RNA to obtain thecycle threshold (CT). The slope of the �CT,RANKL:18S vs. copy numberline, where �CT,RANKL:18S � CT,RANKL � CT,18S was less than 0.1,indicating that the amplification efficiency was approximately equalfor RANKL and control 18S RNA amplification. Because amplifica-tion efficiency was the same for both RANKL and 18S RNA, we usedthe comparative CT method to calculate the fold change in RANKLexpression. Briefly, the fold change in RANKL abundance in samplesstimulated with 50 ng/ml HGF, 6.525 ng/ml R5020, or both 50 ng/mlHGF and 6.525 ng/ml R5020 over the abundance of RANKL expres-sion in unstimulated samples (BM) was calculated by 2���C
Quantitation of the percentage of labeled cells or immunofluores-cence intensity was determined from a minimum of 1000 LECs or 500MECs for each treatment from three to five separate culture experiments.In each experiment, each treatment was performed in triplicate. Resultsare expressed as mean � sem, and differences are considered significantat P � 0.05 by using Student’s t test or ANOVA where appropriate. Datawere analyzed using SAS statistical analysis software (SAS Inc., Cary,NC).
ResultsMorphological responses of LECs and MECs in mammaryorganoids to treatment with HGF, R5020, or HGF�R5020
We and others have previously reported that HGF inducesproliferation and tubulogenesis of mammary cells in 3-Dcollagen gel cultures (11, 15–17). In our previous studies,treatment with the combination of HGF�R5020 resulted inincreased proliferation and the shortening and widening oftubules (17). To gain an understanding of the regulation ofproliferation of LECs and MECs and their respective roles inproducing the morphological changes in response to culturetreatments, immunofluorescence analyses were carried outon intact mammary organoids directly in collagen gels. Toaccomplish this, organoids were incubated with anti-K18antibody, which is specific for LECs (26), and anti-SMA an-tibody, which is specific for MECs (27), and the organoidswere visualized by confocal microscopy. This allowed thedetermination of the location and morphology of LECs andMECs, respectively.
A time course of labeling at 24, 48, and 72 h was carriedout to examine morphological responses of LECs and MECsto the various treatments. In control BM-treated cultures, theorganoids remained rounded in shape throughout the 72-hculture period (Fig. 1A, BM). Centrally localized LECs (Fig.1A, green) were surrounded basally by MECs (Fig. 1A, red).
2100 Endocrinology, May 2008, 149(5):2098–2107 Haslam et al. • Progestin and Proliferation of Mammary Cells
At 24 h, organoids treated with R5020 formed multicellularcyst-like structures comprised of an inner layer of LECs thatwere encircled by elongated MECs. The cysts contained awell-defined lumen (Fig. 1A, R5020). These cyst-like struc-tures were maintained throughout the 72-h culture period.
Organoids treated with HGF exhibited distinct morpho-logical changes over time (Fig. 1B). At 24 h after HGF treat-ment LECs had formed extensions leading away from theorganoid body (Fig. 1B, green arrow). Many MECs wererounded in shape and remained with the organoid body (Fig.1B, red arrow). By 48 h the extensions of LECs had formedchains consisting of multiple cells arranged in a single layerof cells (Fig. 1B, chain). The chains also contained MECs.Some of the chains progressed to form cords, comprised ofa bilayer of LECs, surrounded by elongated MECs (Fig. 1B,cord). LECs (Fig. 1B, green) formed the leading edge of ex-tensions, chains, and cords, with MECs (Fig. 1B, red) locatedposterior and external to LECs. By 72 h, numerous fullyformed tubules with lumens were present; elongated MECswere located exterior to the cuboidal LECs forming the tu-bules (Fig. 1B, tubule).
By 72 h treatment with HGF, very few MECs (Fig. 1B, red)were seen at the organoid body (Fig 1C, HGF). Organoidstreated with HGF�R5020 produced the same general mor-phologic changes and exhibited the same cellular organiza-tion as organoids treated with HGF alone, with the exceptionthat the tubules that formed were shorter, as previouslyreported (Fig. 1C, HGF�R5020) (17). In addition, there wasa greater number of MECs (Fig. 1C, red) with a roundedmorphology localized around the body of the organoid.
Analysis of proliferation in LECs and MECs
We had previously analyzed proliferation in culturedorganoids by assaying 3H-thymidine incorporation intoDNA (17). By that method we determined that BM-treatedorganoids exhibited only a low level of proliferation, andtreatment with R5020 alone did not increase proliferationabove that of BM. HGF induced significant proliferationand maximal proliferation was observed after treatmentwith HGF�R5020. Measurement of proliferation by thatmethod did not permit analysis of cell-type-specific (LECs,MECs) proliferation. To address this question and deter-mine the contribution of cell type-specific proliferation tothe different morphological responses described above,organoids were treated with BrdU for 18 h before fixationat 72 h for all treatments. Paraffin sections of organoidcultures were dual labeled with antibody to SMA andBrdU, and BrdU-labeled cells were quantitated (Fig. 2). InBM-treated cultures, a low percentage of LECs (7.2 � 0.5%)and MECs (2.4 � 0.5%) were BrdU positive (BrdU�).Treatment with HGF alone produced significant 4-fold
FIG. 1. Organoid, LEC, and MEC morphology after treatment withBM, R5020, HGF, or HGF�R5020. Organoids were treated with BMcontrol or R5020 (20 nM) (A), HGF (50 ng/ml) (B and C), orHGF�R5020 (50 ng/ml � 20 nM) (C). After 24–72 h, organoids weresubjected to in situ double-immunofluorescence antibody labelingwith anti-SMA (red) and anti-K18 antibodies (green), and imageswere captured by laser-scanning confocal microscopy as described inMaterials and Methods. Green arrows, LECs, red arrows, MECs;dotted circles, lumens. A, Comparison of morphologies after BM orR5020 treatment. B, Sequential events during HGF-induced tubuleformation. The formation of LEC cytoplasmic extensions (green
arrow) after 24 h was followed by chains of LECs after 48 h (48 hchain). Some LECs have progressed to form a bilayered cord of cellsby 48 h (48 h cord). By 72 h tubule formation by LECs nears com-pletion with the development of a lumen (72 h tubule). C, Comparisonof organoid morphology after 72 h treatment with HGF orHGF�R5020. Note longer tubules in HGF-treated organoids andelongated appearance of MECs (red). In HGF�R5020-treated or-ganoids, tubules are shorter and MECs (red) are rounded and con-centrated at the organoid body. Scale bar, 25 �m.
Haslam et al. • Progestin and Proliferation of Mammary Cells Endocrinology, May 2008, 149(5):2098–2107 2101
increases in BrdU� LECs and BrdU� MECs (P � 0.05).Treatment with R5020 did not cause a significant increasein proliferation of LECs or MECs above BM treatment.Treatment with HGF�R5020 did not increase the percent-age of BrdU� LECs, compared with treatment with HGFalone. However, treatment with HGF�R5020 produced asignificant 3.5-fold increase in BrdU� MECs over thatobserved with treatment with HGF alone (P � 0.05). Theseresults demonstrate that HGF induces proliferation in bothLECs and MECs. Furthermore, whereas R5020 alone didnot cause significant proliferation of either LECs or MECs,when combined with HGF, proliferation was notably in-creased, specifically in MECs.
c-Met expression in LECs and MECs in mammary organoids
c-Met is the cognate, cell surface receptor for HGF (10). Todetermine whether increased c-Met levels could account for
the increased proliferation observed in MECs after treatmentwith HGF�R5020, we examined c-Met expression over timein culture. This was accomplished by immunofluorescentdouble labeling of organoid sections with anti-c-Met anti-body and anti-SMA antibody, a specific marker of MECs (Fig.3A). Fluorescence intensity of c-Met staining was analyzedseparately for LECs and MECs in all treatment groups at 24,48, and 72 h of culture. c-Met expression was consistentlyhigher in MECs than LECs. Neither length of time in culturenor the various treatments had a significant effect on the levelc-Met expression in either LECs or MECs. Representativelevels of c-Met expression as determined by fluorescenceintensity are shown for the 72-h time point (Fig. 3B).
FIG. 2. Regulation of proliferation of LECs and MECs. Organoidswere treated for 72 h with control BM, HGF (50 ng/ml), R5020 (20 nM),or HGF�R5020 (50 ng/ml � 20 nM), treated with BrdU (10 nM), fixed,and processed for immunohistochemistry (A) and quantitated forpercentage of total LECs and total MECs proliferating by fluorescence(B) microscopy as described in Materials and Methods. A, Prolifer-ating LECs are indicated by red staining nuclei (yellow arrows), andproliferating MECs are indicated by the presence of SMA (greencytoplasm) and red nuclei (white arrows); nonproliferating LECs andMECs are indicated by blue nuclei. B, The values represent themean � SEM from five separate experiments. *, P � 0.05; the percentof BrdU-positive LECs and MECs in HGF-treated organoids wasgreater than LECs and MECS of BM-treated organoids; **, P � 0.05;the percent of BrdU-positive MECs in HGF�R5020-treated organoidswas greater than MECs in HGF-treated organoids.
FIG. 3. c-Met expression in organoids or intact mammary glands.Organoids were treated with control BM, HGF (50 ng/ml), R5020 (20nM), or HGF�R5020 (50 ng/ml � 20 nM). Sections prepared fromcultured organoids (A and B) or intact mammary glands (C) from 5-or 10-wk-old virgin, pregnant, or lactating mice were analyzed forc-Met immunofluorescence staining intensity as described in Mate-rials and Methods. A, c-Met immunofluorescence staining is greaterin MECs that express SMA (white arrowheads, red staining cyto-plasm) than in LECs (no SMA). B, c-Met expression levels in or-ganoids at 72 h after various treatments. C, c-Met levels in tissuesections from intact mammary glands at various stages of develop-ment. The values represent the mean � SEM from five separate cultureexperiments or from three animals per developmental stage; a min-imum of 1000 LECs and 500 MECs per treatment or animal wereanalyzed. *, P � 0.05; staining intensity of c-Met MECs was signif-icantly lower in ducts during pregnancy and lactation.
2102 Endocrinology, May 2008, 149(5):2098–2107 Haslam et al. • Progestin and Proliferation of Mammary Cells
To determine whether the pattern of c-Met expressionobserved in vitro reflected the c-Met expression pattern invivo, a similar analysis was carried out on tissue sections ofintact mammary gland from adult mice (Fig. 3C). In the adultmammary gland c-Met was expressed at a higher level inMECs than LECs. Fluorescence intensities of c-Met in LECsand MECs in the adult mammary gland were similar to thatobserved in vitro in cultured mammary organoids. Analysisof c-Met expression at different stages of mammary glanddevelopment revealed that the levels of c-Met expression inLECs were very similar across development and did notappear to be developmentally regulated. However, c-Metlevels in MECs were significantly reduced in ducts duringpregnancy and lactation (Fig. 3D).
PR isoform expression in mammary organoids
PR isoforms, PRA and PRB, are temporally and spatiallyseparated at different stages of mouse mammary gland de-velopment (24). PRA is the major isoform expressed in theadult, virgin mammary gland in vivo; PRB is undetectablebefore pregnancy. PRA and PRB expression is restricted toLECs (24). To investigate the role of progestin and PR in themorphological and proliferative responses observed, we ex-amined PR isoform expression in cultured mammary or-ganoids. Immunofluorescent analyses of PRA and PRB ex-pression were carried out using antibodies that detect onlyPRA or only PRB (Fig. 4). Organoids derived from adult,virgin mammary gland expressed only PRA, and only LECswere PRA positive (PRA�) (Fig. 4A). In vivo, ER� colocalizeswith PRA in LECs in intact mammary gland (18). ER� wasalso expressed in the organoids and was highly colocalizedwith PRA (Fig. 4B). Treatment of organoids with E2 did notincrease the number of PRA� cells or intensity of PRA stain-ing (data not shown). No PRB positive cells were detected
(data not shown). Time-course analysis revealed that therewere no significant differences in the percentage of PRA�cells under all treatment conditions and that the percentageof PRA� cells was similar to the percentage present in or-ganoids at the time of plating (time 0 � 30 � 3%) (Fig. 4C).We also analyzed the effect of the various treatments on PRAexpression levels (Fig. 4D). Analysis of immunofluorescenceintensity of anti-PR antibody staining revealed that treat-ment with R5020 or HGF�R5020 significantly reduced over-all PRA expression.
RANKL is a paracrine mediator of proliferation in MECstreated with HGF�R5020
Because MECs do not contain PR, this suggested that thesynergistic effect of R5020�HGF to increase proliferation inMECs was mediated in part by a progestin-induced para-crine factor. RANKL is a secreted protein induced by pro-gestin in mouse mammary gland and is thought to act in aparacrine manner to promote ductal side branching (25, 28).Therefore, we asked whether RANKL might also act as pro-gestin-induced paracrine factor in mammary organoids.mRNA levels of RANKL were analyzed by real-time RT-PCRin whole organoids at 24 h after treatment (Fig. 5A). RANKLmRNA was increased 1000-fold in R5020- and HGF�R5020-treated organoids, compared with treatment with BM orHGF alone. Protein expression was analyzed by immuno-fluorescent staining with anti-RANKL antibody (Fig. 5B).Intense RANKL staining was seen in LECs in organoidstreated with R5020 or HGF�R5020 but not in organoidstreated with BM or HGF alone. Additionally, RANKL wasexpressed specifically in PRA� cells. We also examined theexpression of RANK, the receptor for RANKL (Fig. 5C).Similar expression levels of RANK were detected in all treat-ment groups.
FIG. 4. PRA expression in organoids.Organoids were treated for 24, 48, or72 h with control BM, HGF (50 ng/ml),R5020 (20 nM), HGF�R5020 (50 ng/ml� 20 nM), or E (10 nM). Organoid sec-tions were double labeled with anti-PRA (green arrow) and anti-SMA (redarrow) antibodies (A) or anti-PRA(green arrow) and anti-ER� antibodies(red arrow) (B) and analyzed for immu-nofluorescence staining as described inMaterials and Methods. Yellow arrow,PRA�ER� coexpression in the samecells; nuclei were stained with 4�,6�-diamino-2-phenylindole (blue). Scalebar, 25 �m. C and D, The values rep-resent the mean � SEM from five sepa-rate experiments; a minimum of 1000LECs and 500 MECs per treatment oranimal were analyzed. *, P � 0.05;staining intensity of PRA was signifi-cantly lower in R5020- and HGF�R5020-treated organoids.
Haslam et al. • Progestin and Proliferation of Mammary Cells Endocrinology, May 2008, 149(5):2098–2107 2103
To determine whether RANKL contributed to the in-creased proliferation of MECs in organoids treated withHGF�R5020, we tested the effects on MEC proliferation oftreatment with exogenous RANKL�HGF or a RANKL-neu-tralizing antibody (Fig. 5D). Treatment with RANKL�HGFproduced the same degree of MEC proliferation observed inorganoids treated with HGF�R5020. MEC proliferation wassignificantly reduced by 70% (P � 0.05) in organoids treatedwith HGF�R5020�anti-RANKL-neutralizing antibody.RANKL alone or anti-RANKL antibody did not significantlyaffect proliferation of either LECs or MECs under any othertreatment.
Discussion
In this report we present novel observations about therespective behaviors of LECs and MECs during in vitro tu-bulogenesis. Additionally, we provide evidence for syner-gism between the progestin-induced paracrine factor,RANKL, and HGF to specifically increase MEC proliferation.
Mechanism of tubule formation by HGF
We found that under all treatment conditions, LECs werecentrally located and MECs were located external to LECs.This in vitro organization of the two cell types is similar tothat observed in the intact mammary gland in vivo. Time-course analysis of HGF-induced tubule formation revealedthat the initial event was the formation of LEC cellular ex-tensions followed by the formation of chains of LECs, whichthen developed into cords composed of two layers of LECs.
The final stage was the formation of a lumen within the cordof LECs. MECs were observed to migrate behind the leadingedge of LEC during all stages of tubule formation.
The observation that LECs produced a morphologic re-sponse leading to tubule formation is in contrast to a previouslypublished report of the behavior of primary cultures of purifiedprimary human mammary LECs (4). In that study only purifiedMECs, and not purified LECs, exhibited a morphologic/mo-togenic response to HGF. However, in other studies of tubu-logenesis using mouse mammary epithelial lines devoid ofMECs, such as NMuMg and EpH4, tubulogenic responses toHGF have been reported (15, 29). The reasons for the differencesreported for human and mouse LEC response to HGF could bedue to differences between the responses of primary cells andcell lines or due to species differences in cell-type-specific re-sponses in the human vs. the mouse.
The process of tubule formation observed in HGF�R5020-treated organoids followed the same basic process observedin HGF-treated organoids; however, the tubules were shorterin length and frequently larger in diameter as previouslyreported (17), suggesting that combined treatment withHGF�R5020 had an inhibitory effect on tubule elongation.The mechanism(s) mediating the R5020-induced inhibitionof tubule elongation is currently under investigation. Addi-tionally, there was a higher concentration of MECs aroundthe organoid body, and the MECs were rounded rather thanelongated in appearance. The increased number of MECsunder this treatment condition is likely due to the increasedproliferation of MECs.
FIG. 5. RANKL and RANK expressionand the effect of RANKL inhibition onMECproliferation.Organoidsweretreatedfor 24 or 72 h with control BM, HGF (50ng/ml), R5020 (20 nM), HGF�R5020 (50ng/ml�20nM).A,Real-timeRT-PCRanal-ysis of RANKL mRNA expression in mam-mary organoids after 24 h. B, After 72 h ofculture, organoid sections were double la-beled with anti-PRA (red arrow) and anti-RANKL (green arrow) antibodies or anti-RANK antibody (C) and analyzed forimmunofluorescence staining as describedin Materials and Methods. White arrowsshow punctuate RANK antibody stainingconsistent with the pattern of RANK mem-brane expression. Mammary stromal cellsin tissue section from intact mammarygland serve as a negative control for RANK(C, inset). Scale bar, 25 �m. D, Organoidswere treated with control BM, HGF (50 ng/ml),R5020(20nM),HGF�R5020(50ng/ml� 20 nM) with (red text) or without RANKLneutralizing antibody (10 �g/ml medium),with RANKL (200 ng/ml), or with HGF �RANKL(50ng/ml�200ng/ml)(greentext).After 72 h organoid sections were quanti-tated forpercentageof totalLECsandtotalMECsproliferatingbyfluorescencemicros-copy as described in Materials and Meth-ods. *, P � 0.05; the percentage of MECproliferation in HGF�R5020�antibody(Ab)-treated organoids was significantlylower than the HGF�R5020-treated or-ganoids.
2104 Endocrinology, May 2008, 149(5):2098–2107 Haslam et al. • Progestin and Proliferation of Mammary Cells
Studies of in vitro tubule formation have identified fivegeneral mechanisms to date: 1) wrapping (neural tube), 2)cavitation (salivary gland), 3) cell hollowing, 4) budding(ureteric bud) and 5) cord hollowing (30). The sequence ofevents observed herein during HGF-induced tubule forma-tion in mammary organoids most closely resembles the pro-cess described by cord hollowing. Cord hollowing is seenwhen a linear array of cells, a chain of cells, polarizes todelineate an apical membrane and a central lumen. Althoughtubule formation in mouse mammary organoids composedof LECs and MECs in 3-D collagen gels have previously beeninvestigated (31), the behavior of MECs during tubule for-mation has not been previously described. To the best of ourknowledge, this is the first report of an analysis of LECs andMECs proliferative and morphological responses in culturedmouse mammary organoids that contain both LEC and MEC.
Proliferation of LECs and MECs
Treatment with HGF or HGF�R5020 produced the sameamount of proliferation of LECs and was due to the actionof HGF because treatment with R5020 alone did not induceproliferation in LECs. In contrast, treatment with HGF vs.HGF�R5020 significantly increased MEC proliferation.These results reveal a novel role for progestin in promotingMEC proliferation. Additionally, MECs had a more roundedmorphology and were concentrated around the cell body inorganoids treated with HGF�R5020 indicating that R5020also had an affect on MEC morphology in addition to in-creasing MEC proliferation.
It was not surprising that HGF stimulated proliferation inboth LECs and MECs because both cell types express c-Met,the receptor for HGF in intact mammary gland and in ourcultured mammary organoids. The increased proliferationobserved with R5020�HGF was not due to an increase inc-Met levels in either LECs or MECs because c-Met levelswere not changed by any of the treatment conditions and,more specifically, were not increased by the addition ofR5020.
We also observed that in the intact mammary gland, c-Metexpression in LECs was relatively constant from pubertythrough pregnancy and lactation, suggesting that c-Met ex-pression is not developmentally regulated in LECs. In contrast,we observed that during pregnancy and lactation c-Met ex-pression in ductal MECs was significantly decreased, comparedwith alveolar MECs. We speculate that this may be a mecha-nism to promote HGF-induced proliferation of alveolar MECsconcomitant with the specific expansion of alveoli number andsize during pregnancy and lactation, respectively.
PR and MEC proliferation
To determine the role of PR expression in MEC prolifer-ation, we analyzed PR isoform expression. We found thatPRA was the only isoform detected and localization of PRAwas confined to LECs under all treatment conditions. ER�was also detected in cultured mammary organoids and co-localized with PRA. Whereas the percentage of PRA-positivecells was not increased or decreased by any treatment con-dition, treatment with R5020 or HGF�R5020 significantlyreduced the overall intensity of PRA immunofluorescent
antibody staining, indicative of a decrease in PRA level. Thepercentage of PRA-positive LECs, the level of PRA expres-sion, colocalization of PRA with ER�, lack or PRB expression,and progestin-induced decrease in PRA levels in organoidswere similar to that reported for the adult intact mammarygland from which the organoids were derived (18). Takentogether, these results indicate that our mammary organoidculture model provides a relevant in vitro system for thestudy of progesterone action in normal mammary cells.
Interestingly, treatment of organoids with E2 did not in-crease the number of PRA� cells or intensity of PRA staining.This was not surprising because we previously reported thatE2 does not increase PR levels in primary monolayer cultureof mouse mammary epithelial cells. E2 up-regulation of PRrequires coculture with mammary fibroblasts (32). Becauseour organoid cultures were devoid of mammary stromalcells, the lack of E2 regulation of PR herein agrees with ourprevious findings of a requirement of mammary stroma forE2-induced PR up-regulation. The intact mammary gland isknown to contain ER� stromal cells, and it is plausible thatestrogen up-regulation of PR in vivo occurs through an in-direct mechanism involving estrogen action in ER� stromalcells (33). In this regard, preliminary studies of mouse PRpromoter regulation also suggest that E2 has no significantpositive effect on promoter activation (Haslam, S. Z., un-published observations).
RANKL and MEC proliferation
Previous observations of the lack of colocalization of PRwith proliferation markers in intact rodent and human mam-mary glands has formed the basis for the concept that pro-gestins can act on PR� cells to produce a paracrine factor(s)that acts on PR� cells to promote their proliferation (34, 35).Because MECs and a significant percentage of LECs do notcontain PR, we considered the possibility that R5020 induceda factor in PR� LECs that acted as a paracrine factor topromote proliferation in PR-negative (PR�) MECs. Embed-ded in this hypothesis is the concept that this paracrine factorwas not mitogenic by itself in either PR� MECs or PR� LECsbecause treatment with R5020 alone did not increase prolif-eration of LECs or MECs.
Based on previous reports, we considered RANKL a pos-sible candidate for the putative progestin-induced paracrinefactor (19). Both RANKL mRNA and protein were highlyup-regulated in R5020- and HGF�R5020-treated organoids.Because RANK expression was similar in all treatmentgroups, it was most likely that increased RANKL expressionwas responsible for increased MEC proliferation in responseto HGF�R5020 treatment. The addition of exogenousRANKL�HGF stimulated MEC proliferation to the samedegree observed after treatment with R5020�HGF, and co-treatment with HGF�R5020 plus a neutralizing antibody toRANKL produced a 70% inhibition of MEC proliferation.Thus, we concluded that progestin-induced RANKL syner-gizes with HGF to increase MEC proliferation and that ac-tivation of both RANKL/RANK and HGF/c-Met signalingpathways are required for the synergistic promotion of MECproliferation.
Binding of RANKL to its receptor RANK initiates a sig-
Haslam et al. • Progestin and Proliferation of Mammary Cells Endocrinology, May 2008, 149(5):2098–2107 2105
naling cascade that activates nuclear factor-�B, resulting inp65 phosphorylation and nuclear translocation (21). How-ever, in vivo administration of exogenous RANKL to virginmice or RANKL treatment of primary epithelial cell culturesderived from virgin mammary gland fails to cause P65 nu-clear translocation, indicating a lack of epithelial cell re-sponse to RANKL (21). The same study showed that RANK,the receptor for RANKL, is expressed at low levels in basallylocated cells in ducts of the virgin gland. In that study it wasnot determined whether the cells expressing RANK wereLECs or MECs. RANK and RANKL expression is signifi-cantly increased at d 14–16 of pregnancy and is restricted tolobuloalveolar structures and coincides with the timing ofalveolar proliferation (21). The emerging picture is thatRANK and RANKL expression is temporally and spatiallyregulated, being most highly expressed during pregnancy topromote alveolar proliferation and survival. In agreementwith this interpretation is impaired alveologenesis inRANKL gene-deleted mice (25).
Although RANKL was highly up-regulated in LECs inR5020-treated organoids, R5020 did not cause proliferation ofLECs. RANKL was also up-regulated in LECs in HGF�R5020-treated organoids; however, proliferation of LECs wasnot increased above that of treatment with HGF alone.RANK, the receptor for RANKL was expressed in LECsunder all treatment conditions and was not an apparentlimiting factor for RANKL-induced LEC proliferation. Thereare several possibilities to explain the lack of an observedLEC proliferative response to RANKL in our studies. First,our organoids were derived from adult virgin mammarygland, and the alveologenic, proliferative response toRANKL occurs only in more differentiated LECs induced bypregnancy. In this regard, we previously reported that the Bisoform of PR is undetectable in the adult virgin mousemammary gland and is highly expressed only during preg-nancy (14). It is possible that RANKL stimulates proliferationonly in PRB-containing LECs. Another possibility is that aninteraction between the RANKL signaling pathway and an-other, as-yet-undefined, ligand and its signaling pathway arerequired for a proliferative response in LECs, much the sameway that RANKL and HGF coordinated signaling are re-quired to promote MEC proliferation. In this regard, HGF isa stroma-derived growth factor that acts in conjunction withR5020 to promote MEC proliferation. It is possible that an-other stroma-derived factor is required to act in conjunctionwith R5020 to promote LEC proliferation. Alternatively, it ispossible that RANKL is a paracrine factor that acts specifi-cally on MECs to promote their proliferation. Currently thedetailed mechanism(s) of RANKL action in the murine mam-mary gland have not been defined. Our results demonstratea novel role for RANKL acting in conjunction with HGF topromote MEC proliferation.
In summary, we have defined the morphological and pro-liferative responses of LECs and MECs during the processesof tubule or cyst formation in response to HGF and/orR5020-treatments, respectively, in 3-D, primary organoidculture system. The tubulogenesis response is initiated andcarried out in a stepwise progression by LECs. MECs do notappear to have an active role in this process. HGF by itselfmainly causes proliferation of LECs and to a lesser extent of
MECs. However, HGF�R5020 produce a synergistic increasein proliferation of MECs. This response appears to be me-diated by RANKL, a progestin-induced paracrine factor pro-duced in LECs that interacts with HGF to specifically in-crease proliferation of MECs.
In addition to their roles in normal mammary gland func-tion MECs also appear to play an important role in thedevelopment and progression of mammary cancers. In thisregard, it has been proposed the MEC may have tumorsuppressor properties (3, 36–38). Thus, understanding howcoordinated regulation of LECs and MECs occurs duringnormal mammary gland development and function mayprovide information relevant to understanding the basis ofthe aberrant organization observed in mammary cancers.
Acknowledgments
Received October 12, 2007. Accepted January 11, 2008.Address all correspondence and requests for reprints to: Sandra Z.
Haslam, Ph.D., Department of Physiology, 2201 Biomedical and PhysicalSciences Building, Michigan State University, East Lansing, Michigan48824. E-mail: [email protected].
This work was supported by National Institutes of Health Grant R01CA40104 (to S.Z.H.) and Department of Defense Breast Cancer ResearchProgram Fellowships DAMD17-03-1-0605 (to M.A.) and DAMD17-02-1-0488 (to K.S.).
Disclosure Statement: The authors have nothing to disclose.
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