Luminal breast cancer metastases and tumor arousal from dormancy are promoted by direct ...

14 Pages·2014·3.76 MB·English

Ogbaetal.BreastCancerResearch (2014) 16:489
DOI10.1186/s13058-014-0489-4
RESEARCH ARTICLE Open Access
Luminal breast cancer metastases and tumor
arousal from dormancy are promoted by direct
actions of estradiol and progesterone on the
malignant cells
Ndiya Ogba1*, Nicole G Manning1, Brian S Bliesner1, S Kelly Ambler1, James M Haughian1, Mauricio P Pinto1,
Paul Jedlicka2, Kristiina Joensuu3, Päivi Heikkilä3 and Kathryn B Horwitz1,2
Abstract
Introduction: Luminal, estrogen receptor-positive (ER+) breastcancers can metastasize but lie dormant for years
before recurrences prove lethal. Understanding the roles ofestrogen (E) or progestin (P) in development of luminal
metastases or in arousal from dormancy is hindered by few preclinicalmodels.We have developed such models.
Methods: Immunocompromised, ovariectomized (ovx’d) mice were intracardiac-injected withluminal or basal
human breast cancer cells. Four lines were tested: luminal ER+PR+cytokeratin5-negative(CK5−) E3and MCF-7 cells,
basal ER−PR−CK5+estrogenwithdrawn-line8 (EWD8) cells, and basal ER−PR−CK5− MDA-MB-231 cells. Development
ofmicrometastases or macrometastaseswas quantified in ovx’d mice and in mice supplementedwith E or Por
both. Metastatic deposits were analyzed byimmunohistochemistry for luminal, basal, and proliferation markers.
− −
Results: ER PR cellsgeneratedmacrometastases inmultiple organs in the absence or presence of hormones. By
contrast, ovx’d mice injected with ER+PR+ cells appeared to be metastases-free until they were supplemented with
Eor E+P. Furthermore, unlike parental ER+PR+CK5−cells, luminal metastases were heterogeneous, containing a
significant (6% to 30%) proportion ofnon-proliferative ER−PR−CK5+ cellsthat would be chemotherapy-resistant.
Additionally, because these cellslack receptors, they would also be endocrine therapy-resistant. With regard to
ovx’d control mice injected withER+PR+cells that appeared to be metastases-free, systematic pathologic analysis of
organs showed that some harbor a reservoir of dormant micrometastases that are ER+ but PR−.Such cells mayalso
be endocrine therapy- and chemotherapy-resistant. Their emergence as macrometastases can be triggered by E or
E+P restoration.
Conclusions: We conclude that hormonespromotedevelopment ofmulti-organ macrometastasesin luminal
− −
disease. The metastases display a disturbing heterogeneity, containing newly emergent ER PR subpopulations
that would be resistant to endocrinetherapy and chemotherapy. Similar cells are found in luminal metastases of
patients. Furthermore, lack ofhormonesis notprotective. While noovert metastases form inovx’d mice, luminal
tumor cellscan seed distant organs, where theyremain dormant as micrometastases and sheltered from therapies
butarousableby hormone repletion. This has implications for breast cancer survivorsor women with occult disease
who are prescribed hormones for contraception or replacement purposes.
*Correspondence:[email protected]
1DepartmentofMedicine,UniversityofColoradoAnschutzMedicalCampus,
12801E.7thAvenue,Aurora,CO80045,USA
Fulllistofauthorinformationisavailableattheendofthearticle
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Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,
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Ogbaetal.BreastCancerResearch (2014) 16:489 Page2of14
Introduction hormoneregulation.Oneinterestingnewmodelusesseri-
Luminal breast cancers represent over 70% of cases [1]. ally transplanted patient-derived luminal tumor xeno-
At least 1% of their cells express estrogen (ER+) or pro- grafts, three of which demonstrate E-dependent growth
gesterone (PR+) receptors or both [2], driving estrogen and retain luminal markers and gene expression profiles
(E)-dependent growth. Despite progress toward early [17]. The xenografts metastasize to lungs and lymph
diagnoses and advances in treatment, 20% to 30% of all nodes (LNs) [17], but the role of hormones, if any, in
patients with breast cancer and 40% to 50% of patients tumor-cell dispersal is unclear. Lacking efficient solid
with luminal breast cancer experience relapses that in- tumor models, a recent study used systemically injected
cludedistantmetastases[3,4].Thistendstooccurwithin ER+PR+MCF-7cellstoshowthattheycangenerateme-
− −
the first 5 years for patients with basal-like ER PR or tastases in an E-dependent manner but that the initial
HER2+ disease and later for patients with luminal dis- homing and seeding steps with development of micro-
ease [5]. In one study, median 15-year distant relapse metastases do not require E [18]. Additionally, two dor-
rates were 27.8% for luminal A and 42.9% for luminal B mancy models that included luminal cells were recently
[5]. Because molecular properties of primary tumors may described[19,20].However,theydonotaddresstherole
bepreserved inmetastases [6], adjuvant endocrine therap- ofhormonesinmetastasisorrecurrence[4].
ies can improve initial survival rates even in patients with We previously demonstrated that in E-replete states,
advanced luminal disease [7]. Nevertheless, the survival ER+PR+ orthotopic xenografts metastasize to distant LNs
curve for luminal disease declines steadily after 5 years, andoccasionallytootherorgans[21].Detailedimmunohis-
overtaking more aggressive breast cancer subtypes after tochemistry (IHC) analyses of such tumors showed that
about 15 years [5,8]. Therefore, since they represent the during their expansion in mice, initially pure ER+PR+ cells
mostcommonformsofthedisease,luminaltumorsarere- develop cellular heterogeneity. At necropsy, presumptive
sponsible for most breast cancer deaths. Explanations for “luminal” tumors contain at least one cell subpopulation
prolongedluminaltumordormancyandtheirslowbutin- we call “luminobasal” that is ER−PR− and expresses cyto-
exorablerecurrenceandlethalityremainunclear,androles keratin5(CK5),aproteinusuallyassociatedwithbasal-like
of cellular heterogeneity and hormones in this process, if cancers [11,13,22]. In clinical samples of luminal disease,
any,arepoorlyunderstood. similar basal-like, ER−PR−CK5+ cells, whose numbers in-
The Women’s Health Initiative (WHI) report on post- crease with hormone therapies, can be found [23]. These
menopausal hormone replacement therapy (HRT) showed hormone-resistant,possiblychemo-resistantcellsarelikely
that the risks of combined E plus progestin (P), unlike to have a poor prognosis. The heterogeneity raises ques-
thoseofphysiologicalEalone,outweighedthebenefits[9]. tions about the identity of cell subpopulations in primary
Widespread acceptance of the WHI data led to a general luminal disease that are responsiblefor metastatic engraft-
decrease in HRT use. Concurrent reductions in the inci- mentandgrowth.
dence of invasive luminal cancers indirectly validated the In this study, we develop a luminal metastases model
WHIconclusions[10].However,explanationsforthedele- and assess the role of E and P in metastatic engraftment
terious effects on the breast of physiological E and P in and recurrence from dormancy. To short-circuit the
combinationHRTremainunclearpartlybecausehormonal cellular heterogeneity issue and study engraftment by
effectsoncarcinogenesisversusproliferationareoftencon- each cell population independently, T47D-derived solid
flated,andtheterm“risk”intimatesthatthehormonesare tumor xenografts were partitioned into their luminal
causative. P appears to have no effect on long-term tumor (called E3) and luminobasal (called EWD8) subpopula-
growth in vivo [11] but expands normal adult mammary tions [22]. These, plus established luminal (MCF-7) and
stem cells and cancer stem cells [12-14]. Regarding WHI, basal(MDA-MB-231)breastcancercelllines,weretagged
we therefore postulated that for E+P, the Pcomponent, in with luminescent and fluorescent markers for in vivo and
a non-proliferative step, reactivates cancer stem cells in ex vivo analyses. Cells were injected into the left ventricle
pre-existing but undiagnosed, perhaps dormant, disease of ovariectomized (ovx’d) immunocompromised mice,
[15].Thatsaid,littleisknownabouttherolesofEandPin and their colonization and proliferation in distant organs
metastasisandrecurrencefromdormancy. wasmonitoredintheabsenceofhormonesorfollowingE
Clinically,themajorsitesofluminalmetastasesarebone or E+P repletion. We found that luminobasal and basal
(>49%), followed by pleura/peritoneum, liver and lung cellsgeneratemetastasesregardlessofthehormonalstate.
(~20%), distant nodes (~14%), and brain (6%)[5]. The ER In contrast, luminal cells rarely form metastases unless E
and PR status of the primary tumor may be reflected in or E+P is restored. The organs colonized by luminal and
bone metastases [5,16], explaining the use of endocrine basal cells are similar and mimic the clinical pattern,
therapiestotreatdisseminateddiseasethatisrarelyreana- dominated by bone. Despite initial injection of in vitro
lyzed for biomarker expression. Few solid tumor models pureluminalcells,theirmetastasesinvivoexhibitcellu-
exist for detailed studies of luminal metastases and their lar heterogeneity, including outgrowth of ER−PR−CK5+
Ogbaetal.BreastCancerResearch (2014) 16:489 Page3of14
luminobasal subpopulations. These cells proliferate more guided by ultrasound imaging (Vevo770; VisualSonics,
slowly than surrounding luminal cells. Notably, although Toronto, ON, Canada). Mice (10 or more per group)
luminal cells seldom generate macrometastases in the ab- wereimplantedsubcutaneouslywithsilasticpelletscon-
sence of hormones, viable dormant micrometastases en- taining 10mgcellulose(C),2mg E+8mgcellulose(E),
graft at distant sites. If mice harboring such occult tumor 2 mg C+8 mg progesterone (P), or 2 mg E+8 mg pro-
cells are subsequently hormone supplemented, overt me- gesterone (E+P) that we previously showed release hor-
tastasesmaterialize. monesatphysiologicallevelsforpremenopausalwomen
[11]. Body weights were recorded weekly. To switch
Materials and methods from C to hormones after 8 weeks, mice were anesthe-
Celllines tized and cellulose pellets were excised and replaced
MCF-7 human breast cancer cells werefrom Sam Brooks with C, E, P, or E+P-releasing pellets. Development and
(MichiganCancerFoundation);T47DcellswerefromIafa progression of metastases were quantified weekly by
Keydar (Tel Aviv University, Israel). The T47Dco subline bioluminescent imaging with Xenogen in vivo imaging
describedinHorwitzetal.[24]haslowERandexception- systems (IVIS) 200 (Caliper Life Sciences, Hopkinton,
ally high PR that are not absolutely E-dependent. As de- MA,USA)tocapturephotonflux,afteranesthetizedmice
scribed [22], E3 and EWD8 are sublines of T47Dco. The wereintraperitoneallyinjectedwith75mg/kgD-Luciferin.
celllineswerederivedfromsolidtumorxenograftsgrown Signal intensity was quantified with Living Image 2.60.1
in ovx’d NSG (non-obese diabetic/severe combined im- software (Caliper Life Sciences). Mice were euthanized if
munodeficientgamma)miceeitherwithout(Ewithdrawn, imaging showed extensive metastases or at signs of
EWD)orwith17β-estradiol(E)supplementation.Bygene morbidity. At necropsy, organs with ZsG fluorescent me-
profiling, E3 cells cluster with luminal cell lines and tastases were found by using UV light (Illumatool 9900;
EWD8 cells cluster with basal-like triple-negative (TN) Lightools Research, Encinitas, CA, USA) coupled to a
cell lines [22]. Their T47Dco origin was confirmed by dissecting microscope (SZ-61; Olympus, Tokyo, Japan)
shorttandemrepeat(STR)andkaryotypeanalyses;their and photographed with a digital camera (Olympus C-
luminal versus basal-like classification and isogenicity 5050). Organs with ZsG fluorescence were collected
were demonstrated by gene expression profiling [22]. and processed for IHC [21]. Additionally, apparently
BT-474 and MDA-MB-231 cells were from the ATCC metastasis-free non-fluorescent organs, including lungs,
(Manassas, VA, USA). These cells were also authenti- liver, adrenals, LNs, and long bones, were harvested
catedbySTRanalysis.Allcellsaremycoplasma-free. frommiceintheCgroup.
To track metastases at necropsy, cells were tagged
withZsGreen(ZsG)byretroviralinfection[21].Toquan-
tifymetastaticburdeninlivingmice,cellswerealsomodi- Three-dimensionalcoloniesandhistology
fied to express luciferase. For this, pMSCV-Luciferase Briefly [25], for three-dimensional (3D) colonies, 4×104
PGK-hygro (gift of Heide Ford, University of Colorado) E3 or MCF-7 cells were plated into 8-well chambered
was transfected into PT-67 packaging cells. ZsG-tagged slides (BD Biosciences, East Rutherford, NJ, USA) pre-
cells were incubated in filtered, virus-containing superna- coated with 60 μL growth factor-reduced phenol red-free
tants and selected for hygromycin resistance. Luciferase Matrigel. Colonies were grown 1 week in phenol red-free
expression was confirmed witha kit (Promega, Fitchburg, MEM with 5% charcoal-stripped FBS, without hormones
WI, USA). EWD8 cells were cultured in phenol red-free (C/ethanol,1:1,000vol/vol)orwithE(10nM),orEandP
minimum essential medium (MEM) and 5% fetal bovine (100nM),inmediarefreshedevery2days.Matrigelblocks
serum (FBS) depleted of hormones by incubation with wereembedded in Histogel(Richard-Allen Scientific, part
dextran-coatedcharcoal pellets followed by filtration[22]. of Thermo Fisher Scientific, Waltham, MA, USA), fixed
E3cellswereculturedinthesamemediumsupplemented overnight in 4% paraformaldehyde, switched to 70% (vol/
with 10 nM E. MCF-7 and MDA-MB-231 cells were vol) ethanol, and paraffin-embedded. Mouse organs were
grownasdescribed[22]. alsofixedin4%paraformaldehydeandprocessedasabove.
Bone samples were decalcified in 10% EDTA for 1 week
Intracardiacinjectionsandmetastasesquantification prior to fixation. Paraffin-embedded samples were sec-
Animal protocols were approved by the University of tioned (4 μm) serially, deparaffinized in xylene, and
Colorado Institutional Animal Care and Use Committee. rehydrated in graded alcohol. To locate micro- or
Female NSG mice were ovx’d at 3 to 4 weeks (The macrometastases, every 5th or 10th section was stained
Jackson Laboratory, Bar Harbor, ME, USA). Six-week- withhematoxylin and eosin (H&E) until tumor cells ap-
old mice were anesthetized with isoflurane and injected peared microscopically, after which continuous sections
in the left ventricle with 105 tumor cells suspended in were saved for IHC. In some studies, CK8/18 IHC re-
0.1mLphosphate-bufferedsalinebyusinga26Gneedle placedH&E.
Ogbaetal.BreastCancerResearch (2014) 16:489 Page4of14
Immunohistochemistry betweenE3and EWD8 areespeciallyuseful since theyare
For antigen retrieval, slides were heated in citrate buffer isogenic “twins”, having been derived from the same lu-
and sections were blocked 1 hour in 10% normal goat minalparentalcells[22].Allcellsweremodifiedtoexpress
serum. Primary antibodies, including clones and concen- luciferase and ZsG. To confirm cell subtype assignment
trations or dilutions, are listed in Table S1 (Additional priorto IC injections,the cells weregrown as3D colonies
file 1). These were detected either with fluorescent sec- in phenol red-free Matrigel and analyzed for luminal and
ondary antibodies conjugated to AlexaFluor-488 or -555 basal markers (Additional file 2). E3 cells are ER+PR+;
(1:200, Invitrogen, Waltham, MA, USA) and counter- MCF-7 cells are ER+PR− in phenol red-free medium (but
stained with 4′,6-diamino-2-phenylindole (DAPI) or with PR can be induced by E [29]). Both are CK5− and vimen-
horseradish peroxidase-labeled secondary antibodies and tin−. EWD8 cells lack luminal markers and are CK5+
−
DAB+ chromogen/substrate (Dako, Glostrup, Denmark). vimentin ; MDA-MB-231 cells lack luminal markers and
SlideswereimagedwithaNikonEclipseE600microscope areCK5−vimentin+.
(Nikon,Tokyo, Japan) equipped with a CoolSnap fx cam- Progressionofmetastasesinhormone-freeorhormone-
era (Photometrics,Tucson, AZ, USA) operatedby Image- replete mice was quantified weekly by whole-body
−
Pro software (Media Cybernetics, Rockville, MD, USA) bioluminescent luciferase imaging (BLI). Basal-like ER
−
[22]. For CK5, phosphor-histone H3 (pHH3) and nuclear PR EWD8 cells were highly aggressive, producing exten-
proliferationmarker(Ki67) quantification, photographs of sivemetastaseswithin20daysintheabsenceofhormones
atleastfiverandom40×fields(3Dcolonies)or100×fields (C)(Figure1A,B).AlthoughEandE+Pappeartoacceler-
(xenografts)perconditionwereusedtocalculatevalues. ate EWD8 metastasis, the effects are not statistically sig-
nificant (C versus E, P=0.857; C versus E+P, P=0.819)
Clinical (Figure1A).Micehadtobesacrificedafterabout30days.
Seventy-two primary tumors and matching metastases In contrast, mice injected with isogenic luminal ER+PR+
−
were collected from the archives of the Department of CK5 E3 cells appeared to be metastasis-free in the ab-
Pathology, Helsinki University Hospital, Finland, with sence of hormones (C) up to the study termination of
approval from the ethics committee of the Helsinki 80 days (Figure 1A). However, if E3-injected mice were
University Central Hospital and with patient consent. supplementedwithEorE+Pstartingonday0,metastases
Clinicopathological and other data have been published begantodevelopaftermorethan28daysandthetumor
[26,27].Brain metastasesfromfourpatients were cutinto burden increased weekly thereafter (Figure 1A, B). E-
4-micron sections and stained by dual colorimetric IHC supplementationsignificantly(P<0.05) increasedtumor
(Envision G/2 Doublestain; Dako) for PR (NeoMarkers, burden relative to C, and addition of P to E exacerbated
Freemont, CA, USA) and CK5 (Novocastra, Leica this(P<0.005).
Biosystems, Nussloch, Germany) [22]. As surrogates for overall health, mouse body weights
werealsomonitored(Additionalfile3A).Control(C)mice
Statisticalanalysis withEWD8metastaseslostweightrapidly,appearedsickly
Data are reported as mean±standard error of the mean after 20 days, and were sacrificed early. C mice injected
(SEM) of three or more independent experiments and with E3 cells gained weight; ones receiving E or E+P
were analyzed statistically with Prism v6.0 (GraphPad maintained starting weights for up to 80 days. These data
Software,LaJolla,CA,USA)byStudent’sttest.Differences are reflected in metastasis-free survival as estimated by
in survival curves among treatment groups were per- Kaplan-Meier plots (Figure 1C) with median survival of
formed by using Kaplan-Meier semi-parametric method 30 days for EWD8 mice regardless of hormonal state,
withthelog-ranktest.P valuesoflessthan0.05werecon- which is significantly (P=0.003) less than the 65- to 70-
sideredstatisticallysignificant. day median survival for E3 mice on E or E+P, and the in-
definitesurvivalofcontrolE3mice.
Results To test a second luminal cell line, MCF-7 cells were IC
HormonespromotemetastasisofER+PR+breastcancer injectedintoovx’dmicewithout(C)orwithEorE+Psup-
cells plementation(Figure1D).LikeE3cells,MCF-7cellsfailed
To analyze effects of ovarian hormones on metastases, to produce metastases in the absence of hormones, but
ovx’dNSGmiceimplantedwithcontrolcellulose(C),E,or metastaseswereevidentafter20daysinE-orE+P-supple-
E+P-releasing pellets [11] were intracardiac (IC)-injected mented mice. Compared with E3 cells, MCF-7 cells are
into the arterial circulation with 105 tumor cells. Two more sensitive to E. In the presence of hormones, the
luminal ER+PR+CK5− (E3 and MCF-7) and two basal-like MCF-7metastaticburdenincreasedprogressivelyuntilthe
(EWD8 and MDA-MB-231) cell lines were compared. study was terminated at 45 days. Body weights remained
EWD8 are TN ER−PR−HER2− but CK5+EGFR+ [22]; relativelystable under all conditions (Additional file 3B).
MDA-MB-231areTNandCK5−EGFR+[28].Comparisons In sum, two different luminal breast cancer cell lines
Ogbaetal.BreastCancerResearch (2014) 16:489 Page5of14
Figure1Hormonesarenecessaryforluminaltumormetastases.(A)Ovariectomized(Ovx’d)non-obesediabetic/severecombined
immunodeficientgamma(NSG)micewereintracardiac(IC)-injectedwith105luciferaseandZsGreen-taggedbasal-likeestrogenwithdrawn-line8
(EWD8)orluminalE3cells,andimplantedwithcellulosecontrol(C)orwithestrogen(E)orestrogen+progestin(E+P)-releasingpellets.Weekly
quantitationusedinvivobioluminescentimaging(BLI).Dataarepresentedasmean±standarderrorofthemean(SEM)ofBLIsignal(n=20per
treatmentgroup).*P<0.05,**P<0.005,Student’sttest.(B)RepresentativeluciferaseBLIwhole-bodyimages.(C)Kaplan-Meiersurvivalcurves
(n=20pertreatmentgroup);P=0.003,log-ranktest.(D)Ovx’dNSGmicewereICinjectedwith5×105luciferaseandZsGreen-taggedluminal
MCF-7cellsandimplantedwithC,E,orE+Ppellets.WeeklyinvivoluciferaseBLIquantitation.Dataarepresentedasmean±SEMofBLIsignal
(n=20pertreatmentgroup).*P<0.05,Student’sttest.
introduced into the circulation of immune compro- hormone biosynthesis [31]. Basal-like EWD8 (Figure 2C)
mised mice demonstrate that metastasis is promoted by orMDA-MB-231cells(Additionalfile4B)arehighlyag-
ovarian hormones (Figure 1). In contrast, formation of gressive and do not require hormones to form multi-
metastases from basal-like cells is largely hormone- organ metastases. In the case of bones, for example,
independent. Taken together, these data suggest that basal cells formed metastases at 80% or greater fre-
major effects of E or E+P are on the malignant luminal quency in bothC andhormone-treated mice (Figure2C
cells.Thismaybefurthermodifiedbyeffectsofhormones andAdditionalfile4B).
onthemetastaticniche. Among the four cell lines, minor organ-specific differ-
ences were observed: Liver metastases were lower for E3
Hormonespromoteluminalmetastasisinmultipleorgans than MCF-7 cells, and abdominal/intestinal, mammary
Clinically, major sites of metastases are bones, liver, and gland LN, renal, and uterine metastases were higher for
lungs[5,30].ZsGreenfluorescenceatnecropsy(Figure2A) E3 than MCF-7 cells (Figure 2B and Additional file 4A).
was used to track the organ specificity of metastatic cells. Also, brain metastases were lower for EWD8 than for
Inhormone-replete(butnothormone-deprived)mice,lu- MDA-MB-231 cells (Figure 2C and Additional file 4B).
minalE3(Figure2B)andMCF-7cells(Additionalfile4A) Nonetheless, the number of metastatic sites observed for
colonize multiple organs, including bones, liver, lungs, each cell line demonstrates the critical role of hormones
brain, adrenals, and LNs. Quantitation of the organ sites (especially E) for metastases by luminal E3 and MCF-7
colonizedindicatethatEenhancestheincidenceofmetas- cells and the hormone independence for metastases by
tases (blue bars), which is not significantly altered by P basal-likeEWD8andMDA-MB-231cells.
(red bars) (Figure 2B and Additional file 4A). Overall, in
theabsenceofhormones(greenbars),thefrequencyofE3 Cellularheterogeneityinluminalandbasalmetastases
andMCF-7bonemetastasesinCmiceislessthan2%but Over70%ofpatientswithbreastcancersuccumbtolytic
averages65%inE-orE+P-repletemice.Luminalmetasta- bone lesions that cause fractures, pain, and associated
ses to some organs (adrenals, for instance) exhibit some complications [32]. Since clinically, metastases can con-
independence from exogenous hormones (Figure 2B and tain cells that differ from those at the primary site [33],
Additionalfile4A)possiblybecauseofendogenoussteroid experimental bones were sectioned and analyzed by
Ogbaetal.BreastCancerResearch (2014) 16:489 Page6of14
Figure2Hormonescontrolmetastasesofluminalbutnotbasal-liketumorcells.(A)Ovariectomized(Ovx’d)non-obesediabetic/severe
combinedimmunodeficientgamma(NSG)micewereintracardiac-injectedwith105luciferaseandZsGreen-taggedbasal-likeEWD8orluminalE3
cellsandimplantedwithcellulose(C)orwithestrogen(E)orestrogen+progestin(E+P)-releasingpellets.Representativeimagesatnecropsy
ofZsGfluorescentmetastasesindifferentorgansareshown.BF,brightfieldimage(n=20pertreatmentgroup).(B)Bargraphshowsthe
percentageofmiceineachtreatmentgroup—C(green),E(blue),andE+P(red)—withE3metastasestodistantorgans.Dataarepresentedas
meanpercentagespergroup(n=20pertreatmentgroup).(C)Bargraphshowsthepercentageofmiceineachtreatmentgroup—C(green),E
(blue),andE+P(red)—withEWD8metastasestodistantorgans.Dataarepresentedasmeanpercentagespergroup(n=20pertreatmentgroup).
(D)NumberofZsG+metastaticsitespermouseatnecropsyforE3andEWD8cellsunderC(green),E(blue),orE+P(red)conditions.Dataare
presentedasmean±standarderrorofthemean(SEM)(n=20pertreatmentgroup).**P<0.005,Student’sttest.
H&E and IHC. Multiple bone sections of ovx’d C mice CK5+ cells in E+P. MCF-7 colonies contain about 3%
injected with luminal E3 or MCF-7 yielded no tumor CK5+ cells in E or E+P. Differences between colony as-
cells (Figure 3A)confirmingthenecropsydata. However, says and in vivo metastases may be due to signals con-
morethan50%of bonesinE-orE+P-supplementedmice tributed bythemetastatic niche.
contained tumor cells (Figure 3B). These were generally Detailed marker analyses of luminal E3 and MCF-7
foundinlongbonesand spine,and werecharacterized by bone metastases are shown in Additional file 6A and B.
− −
islands of malignant cells infiltrating the marrow space, The ER PR subpopulation loses the luminal markers
−
associatedwithvariablebonedestruction. CK8/18andclaudin3(CLD3)andisHER2 (unlikeBT-
Unlike the homogeneous ER+PR+CK5− luminal state of 474 controls). Luminal bone metastases also lack the
E3 and MCF-7 cells at the time of injection (Additional mesenchymal marker vimentin, unlike basal-like MDA-
file 2), bone metastases are heterogeneous (Figure 3B, MB-231 metastases that retain vimentin (Additional
middle and lower panels), consisting not only of the ex- file6AandB).
pected ER+PR+ luminal cells (green nuclei) but also of Basal-like EWD8 and MDA-MB-231 cells also colonize
multiple basal-like ER−PR−CK5+ luminobasal cells (red bone but do so even in the absence of hormones
cytoplasm) [22]. E3 metastases contained about 8% lumi- (Additional file 7A). Histopathology again shows malig-
nobasalcellsinthepresenceofE,whichweresignificantly nant epithelial cells infiltrating the marrow with bone
(P=0.037) increased to about 18% by addition of P to E destruction;EWD8infiltratesalsoshowsignsofdesmo-
(Figure 3C, top panel). MCF-7 metastases contained plasia (Additional file 7A, top H&E panel). Receptor
high levels of luminobasal cells (25% to 30%) in both E conversion in clinical cases has been reported [34,35].
andE+P(Figure3C,bottompanel). Because EWD8 cells have luminal parentage [22], we
Although hormone-dependent acquisition of hetero- looked for reversions by using ER and PR as markers.
geneity is not observed for cells grown on plastic, it can However, EWD8 metastases uniformly retain the basal
− − −
bestudiedin3Dcolonyassays(Additionalfile5).Forin- markerCK5 and are ER , PR , and vimentin (Additional
stance, E3 colonies contain 0% CK5+ cells in E and 14% file 7A). A “double negative” ER−PR−CK5− subpopulation
Ogbaetal.BreastCancerResearch (2014) 16:489 Page7of14
In previous studies of primary luminal cancers, we
showed that the ER+PR+ population suppresses mitosis
− −
of the ER PR subpopulation [23]. The latter would
therefore be both hormone-resistant because of a lack of
receptors and chemotherapy-resistant because of quies-
cence [23]. To assess proliferation of cell subpopulations
in our models, sections of E3, MCF-7, and EWD8 bone
metastases from E- or E+P-treated mice were dual-stained
for CK5 and the mitosis marker pHH3 [36]. We find that
regardless of the hormone state, in both E3 and MCF-7
bonemetastases(Figure3D),theER−PR−CK5+luminobasal
(LB) subpopulation is significantly (P <0.005) growth-
suppressedcomparedwithitsER+PR+CK5−luminal(LUM)
neighbors. In EWD8 bone metastases (Additional file 7B),
growth of the pre-existing CK5+ population is significantly
(P <0.005) reduced compared with the newly formed
− − −
double-negativeER PR CK5 subpopulation.Insum,in
addition to marker heterogeneity, cell growth is hetero-
geneousinmetastases,makingtherapydifficult.
Additional file 8A shows brain metastases caused by
E3andMCF-7cells.Likebone,brainmetastasesarehet-
erogeneous, containing both the injected ER+PR+CK5−
− −
luminal population (green) and the newly arisen ER PR
CK5+ luminobasal subpopulation (red). Therefore, this
phenotypicconversionisnotorgan-specific.Similarcellu-
lar heterogeneity marks luminal metastases of patients as
showninaclinicalsampleof brainmetastases(Additional
file8B)[27].Overall,thesedatademonstratethatinjection
of apparently homogeneous tumor cells generates metas-
tases with heterogeneous cell populations, partly in re-
sponse to hormones. Furthermore, these subpopulations
Figure3HomogeneousER+PR+CK5−luminalE3andMCF-7cells
canexhibitdivergentproliferationrates.
generateheterogeneousbonemetastasescontainingER−PR−
CK5+luminobasalcells.(A)Representativehematoxylinandeosin
(H&E)andimmunohistochemistry(IHC)ofcontrol(C)boneslacking Luminaltumordormancyandreactivationbyhormones
metastasesdespiteintracardiac(IC)injectionofE3orMCF-7cells. Our models demonstrate that IC-injected malignant
H&E:(B)bone,(BM)marrow.Scalebars:50μm.IHC:estrogen luminal cells form macrometastases in hormone-
receptor(ER)orprogesteronereceptor(PR)(green),cytokeratin5 supplemented but not in hormone-free ovx’d mice.
(CK5)(red),and4′,6-diamino-2-phenylindole(DAPI)(blue)by
immunofluorescent(IF)staining(n=5pergroup).Scalebars:20μm. What happened to the disseminated tumor cells injected
(B)H&EandIHCofE-orE+P-treatedmicewithbonescontaining into hormone-free mice? To address this, hormone-free
metastasesafterICinjectionofE3orMCF-7cells.H&E:bone(B)and ovx’d C mice were IC-injected with 105 Luc/ZsG-E3 or
tumor(T)cells(n=5pergroup).Scalebars:50μm.IHC:ERorPR Luc/ZsG-MCF-7cellsandmonitoredfor 8weeksbyIVIS
(green),CK5(red)andDAPI(blue)byIFstaining(n=5pergroup).
(Figure 4A). Few metastases were imaged by IVIS despite
Scalebars:20μm.(C)PercentageofCK5+cellsinE-orE+P-treated
the fact that 8 weeks is more than sufficient for these to
E3andMCF-7bonemetastases.Mean±standarderrorofthemean
(SEM)valuesareshown(n=5pergroup).*P<0.05,Student’sttest. be visible in hormone-supplemented mice (Figure 1A,
(D)ProliferationratesofCK5+andCK5−cellsmeasuredby 1D).At8weeks,theCpelletswereremovedandreplaced
phosphor-histoneH3(pHH3)inE-andE+P-treatedE3andMCF-7 with C, E, P, or E+P-releasing pellets, and mice were
bonemetastases.Mean±SEMvaluesareshown(n=5pergroup).
monitored for another 8 weeks. As shown for E3 injected
**P<0.005,Student’sttest.
mice (Figure 4A), under C or P-restored conditions, mice
remained metastases-free. However, metastases emerged
was also detected (Additional file 7A). Double-negative in E- or E+P-restored mice. Results from IVIS imaging
cells resemble MDA-MB-231 metastases with regard to were confirmed at necropsy (Figure 4B), with few E3
CK5 loss, but unlike MDA-231 cells, they lack vimentin ZsG+ fluorescent deposits after 16 weeks in organs of C
(Additionalfile7A).Theseanalysesdemonstrateinteresting (1/11;9%)orP-treatedmice(1/15;6%)butrobustZsG+
cellularheterogeneityevenwithinbasal-likemetastases. metastases in one or more organs of E (8/16; 50%) or
Ogbaetal.BreastCancerResearch (2014) 16:489 Page8of14
Figure4(Seelegendonnextpage.)
Ogbaetal.BreastCancerResearch (2014) 16:489 Page9of14
(Seefigureonpreviouspage.)
Figure4Hormonesreactivatedormantluminalmicrometastases.(A)Ovariectomized(Ovx’d)non-obesediabetic/severecombined
immunodeficientgamma(NSG)micewereintracardiac(IC)-injectedwith105luciferase-andZsG-taggedE3cellswithouthormonesupplementation
(cellulose,orC)andmonitoredfor8weeks.Atweek8,theCpelletwasremovedandreplacedwithC(n=11),estrogen(E)(n=16),progestin
(P)(n=15),orestrogen+progestin(E+P)(n=17)-releasingpelletsforanother7to8weeks.Weeklybioluminescentluciferaseimaging(BLI)
imaginginlivemicebyinvivoimagingsystems(IVIS).Dataarepresentedasmean±standarderrorofthemean(SEM)ofBLIsignal.*P<0.05,
**P<0.005,Student’sttest.(B)Bargraphshowsthepercentageofmiceineachtreatmentgroup—C(green),E(blue),E+P(red),andP(gray)—
withE3metastasesindistantorganswithout(C)orwithE,P,orE+Prestoration.Dataarepresentedasmeanpercentagespergroup.(C)
Immunohistochemistry(IHC)forCK8/18,ER,PR,andCK5inorganswithE3micrometastases(C)andmacrometastases(EorE+P).Forpercentage
ofCK5+cells,mean±SEMvaluesareshown(n=3pergroup).Scalebars:50μm.(D)IHCforCK8/18andnuclearproliferationmarker(Ki67)in
organswithE3orMCF-7micrometastases(C)andmacrometastases(E+P).ForpercentageofKi67+cells,mean±SEMvaluesareshown
(n=3pergroup).Scalebars:50μm.
E+P (10/17; about 59%) mice. Guided by ZsG fluores- (Additional file 9B). As described for bone metastases
cence, organs were sectioned and stained with H&E. (Figure3D),theCK5+luminobasalsubpopulationismark-
−
This confirmed the presence of macrometastases in edly growth-suppressed compared with its CK5 neigh-
multiple organs of E- or E+P-replete mice, most com- bors(Additionalfile9C).
monly in lungs, brain, adrenals, LNs, abdomen, kidney, To contrast proliferation rates between macro- and
and uterus (Figure 4B). (Organs with macrometastases micrometastases, Ki67 was quantified (Figure 4D); 35%
often had micrometastases in adjacent regions.) C or P to 37% of adrenal or mammary gland LN E+P-treated E3
mice showed few if any metastases in random H&E- macrometastatic cells were proliferating, compared with
stainedsectionsofsimilarorgans(Figure4B). 0% to 4% of similar micrometastatic cells (Figure 4D).
To explain how luminal metastases could arise in C Thus, E3 micrometastases are functionally dormant as
mice that appeared to be metastases-free prior to hor- definedclinically[37,38].MCF-7cellsaremoreprolifer-
mone resumption, we reasoned that one or more organs ative even as micrometastases with about 20% Ki67+
must have harbored micrometastases that had escaped cells (Figure 4D), which may explain their more aggres-
detection because of random sectioning or fluorescence sivebehavior(compareFigure1AwithFigure1D).
below the limits of detection. We therefore returned to Overall, these data demonstrate that, even in the ab-
E3-injected C mice necropsied at 16 weeks, and system- sence of hormones, luminal tumor cells disseminated
atically serial-sectioned select entire organs, immuno- through the circulation can, and do, implant and survive
staining every 10th 4 μm section for the luminal marker at metastatic sites; that entry into “dormancy” varies
CK8/18. This marker detects micrometastases—defined among tumors (as it does here for T47D-derived versus
as fewer than 100 cells—more effectively than H&E. We MCF-7 cells)makingsome less perilousthanothers; and
found CK8/18+ micrometastases in adrenal glands, uteri, that some cells enter a state of proliferative stasis from
and mammary gland LNs of C mice (Figure 4C). For whichtheycanescapeifEorE+Pisrestored.Theclinical
comparison, CK8/18+ E3 macrometastases in mammary implications are grave with regard to hormone supple-
gland LNs of E- and E+P-treated mice are also shown mentation for women who are breast cancer survivors or
(Figure4C).Apparently metastases-freeC miceinjected haveoccultdisease.
with MCF-7 cells also harbored CK8/18+ micrometas-
tases in adrenal glands, which are compared with CK8/ Discussion
18+ MCF-7 macrometastases in adrenal glands of E- Models
andE+P-treatedmice(Additionalfile9A). Thoughconsidered a stringent“intrinsic”molecularsub-
To contrast molecular markers of micro- versus type definable by presence of steroid receptors, luminal
macrometastases, sections adjacent to CK8/18+ ones breast cancer is a complex disease. Among other vari-
were immunostained for ER, PR, and CK5. Control E3 ables, tumors that are ER+PR+, ER+PR− or ER−PR+,
micrometastases were surprisingly PR− and also CK5− HER2− or HER2+, or low- or high-grade are all classified
−
(Figure 4C). MCF-7 micrometastases were PR with as luminal [39,40]. Tumors that may have initiated years
about 9% CK5+ cells (Additional file 9A). E3 macrome- or decades earlier are diagnosed in the pre-, peri-, and
tastases, derived from hormone-treated mice, were ER+, post-menopausal years, each with the attendant hormonal
strongly PR+ with about 14 (E) or 27% (E+P) CK5+ cells changes that differ from womanto woman.Untilrecently,
(Figure 4C). Similarly, E- or E+P-treated MCF-7 macro- clinicalassays,includinggeneexpressionprofiling,assessed
metastases were ER+, PR+, and CK5+ (Additional file 9A). the bulk of a breast cancer to assign its subtype. However,
Of note, PR+ and CK5+ heterogeneity are restored or such assays cannot define intratumoral cell heterogeneity
enhanced in dormant tumors reactivated by hormones [41].IHCanalysesclearlydemonstratethatinvariably,indi-
in both E3 and MCF-7 adrenal and LN metastases vidualluminaltumorsconsistofcellmixtures,onlyoneof
Ogbaetal.BreastCancerResearch (2014) 16:489 Page10of14
whichistheclassicER+PR+hormone-responsivecell.Here, other hand, the present studies show for the first time
weuseIHCtoexaminetheroleofovarianhormonesinlu- that for receptor-positive luminal cells, the later steps
minal breast cancer metastasis by using four experimental are hormone-dependent. Compared with basal-like MDA-
cells: two luminal and two basal. Like their clinical coun- MB-231 and EWD8 cells, which metastasize efficiently in
terparts,themodelsare variable:MCF-7areER+butneed ovx’d mice with or without hormones, luminal E3 and
estradiol to induce PR; T47Dco and their E3 derivatives MCF-7cellsdosomainlyif hormonesarerestored.Estro-
are ER+ with partially constitutive elevated PR. Both are gens appear to be key. Effects of P are variable, amplifying
− − −
CK5 . EWD8, also derived from T47Dco, is ER PR and theE-dependentmetastasisrateofE3cells(Figure1A)but
CK5+; MDA-MB-231 is basal-like and CK5−. We describe failing to do so for MCF-7 cells (Figure 1D). This is an-
aroleforhormonesindevelopmentofluminalmetastases, otherexampleofluminaltumorvariability.Thesedataval-
the cellular heterogeneity of micro- and macrometastases idate the clinical practice of using adjuvant therapies that
−
arising from apparently homogeneous cell models, a PR suppress E production or ER signaling to slow develop-
dormantstatethatmaybeasanctuaryfromtherapies,and ment of luminal metastases [7]. However, such preventive
aroleforhormonesinescapefromdormancy.Tothebest strategieswouldfailfortheER−PR−CK5+luminobasalsub-
ofourknowledge,theseobservationsarenovel. populations commonly found in luminal disease [22,23].
The cellular heterogeneity is clearly demonstrable with Thismayexplainwhy,comparedwithER+PR+primarytu-
just two markers—PR and CK5—that theoretically gener- mors, ER tumors are lost in about 25% of metastases and
atefourpossiblecelltypes:luminalPR+CK5−,luminobasal PRtumorsarelostatanevengreaterrate[44,45]andsug-
PR−CK5+, double-negative PR−CK5−, and double-positive gests that adjuvant combination therapies that target both
PR+CK5+. To various degrees, we observeall fourin clin- cell types might be extremely effective. However, chemo-
ical and model luminal disease. It is not unreasonable to therapies are unlikely to work since luminobasal cells are
postulate that, if more markers were analyzed, even quiescent when mixed with luminal cells (Figure 3D and
greater heterogeneity would be observed, ultimately Additionalfile9C)[23].
demonstratingthateverysingleluminaltumorisunique
in its cellular composition and therapy vulnerability. Hormonespromoteluminalmetastasesatmultiple
Thishasclearimplicationsfordiseasemanagement. organs
Luminal and basal cells tend to colonize the same or-
HormonespromotemetastasisofER+PR+cells gans: bones, liver, lungs, brain, and adrenals (Figure 2
As has been elegantly demonstrated, metastasis requires and Additional file 4). The same sites are colonized in
completion of a complex series of steps, including escape patients regardless of breast tumor subtype [46]. How-
of tumor cells from the primary site into surrounding tis- ever, this tends to be hormone-dependent for ER+PR+
− −
sues,theirsurvivalandtransportvialymphaticsandblood luminalcells and hormone-independent for ER PR basal
vessels, and their capture andexpansionindistant organs cells, suggesting that the exogenous hormones target the
[33].Wepreviouslyshowedthatxenograftedsolidluminal malignant cells directly. Of course, the metastatic niche
tumors efficiently metastasize to local and distant LNs may also be targeted by hormones [47]. Additionally,
[21].Ontheotherhand,comparedwithbasaltumors,lu- some organs, like adrenals and brain and tissues like fat,
minal tumor metastasis to distant organs is highly ineffi- arecapableofendogenousEandPbiosynthesis[31].Such
cient, requiring months of observation [42] and risking extragonadalhormoneproductionincreaseswithage[48],
prematuremouseattrition.Xenografttumor volumesthat which might drive the slow but eventual development of
are achieved in 20 to 30 days with basal cells require 150 experimental luminal metastases from solid tumor xeno-
to200dayswithluminalcells.Therefore,todesignstudies grafts in rodents or hormone-dependent metastases in
in which hormone supplementation is the major variable postmenopausal women. Significantly, in women, no life
and in which different cell subpopulations can be ana- stageisentirelydepletedofovarianhormones.
lyzed, we used IC injection, which circumvents the first
steps in the metastasis cascade. Under these conditions, Heterogeneityofmetastasesfrompureluminalprogenitors
basal metastases were quantifiable in 20 to 30 days; lu- The origins of intratumoral heterogeneity remain under
minal metastases in 40 to 80 days (Figure1). Parenthetic- intense debate. Explanations include differentiation from
ally, these numbers suggest that a major impediment to cancer stem cells, clonal expansion of intrinsic subpopu-
experimental studies of luminal metastases from solid tu- lations, microenvironmental selection, random muta-
mors is the early local invasion and intravasation steps tions creating genetic diversity, and epigenetic switches
[33] but that once tumor cells are in the blood, later [33,41]. It is likely that at one time or other all of these
eventsunfoldrapidlyregardlessoftumorsubtype. comeintoplay.Thepresentdata focusonbonemetasta-
Based on our experience and that of others [43], these ses, although heterogeneity is observed in other sites as
early steps cannot be hastened by hormones. On the well (Additional file 8; data not shown). In our studies,