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Research Article

Estradiol Promotes Breast Cancer Cell Migrationvia Recruitment and Activation of NeutrophilsGabriela Vazquez Rodriguez1, Annelie Abrahamsson1, Lasse Dahl Ejby Jensen2, andCharlotta Dabrosin1

Abstract

Estradiol (E2) plays a key role in breast cancer progression.Mostbreast cancer recurrences express the estrogen receptor (ER), butnearly 50%of patients are resistant to antiestrogen therapy. Noveltherapeutic targets of ER-positive breast cancers are needed.Protumoral neutrophils expressing the lymphocyte function-associated antigen 1 (LFA-1) integrin may mediate cancer metas-tasis, andTGFb1 is themajor chemoattractant for neutrophils. Therole of E2 in neutrophil–ERþ breast cancer cell interactions isunknown. We studied this in vivo using murine breast cancers inimmunocompetentmice and humanbreast cancers in nudemice.Cell dissemination was evaluated in a zebrafish model, andmicrodialysis of breast cancer patients was performed. In vitrostudies were done with mammosphere cultures of breast cancercells and human neutrophils. We found that E2 increased the

number of LFA-1þ neutrophils recruited to the invasive edge ofmouse tumors, increased TGFb1 secretion and promoted neutro-phil infiltration inmammospheres, and inducedoverexpressionofLFA-1 in neutrophils. In zebrafish, in the presence of E2, neutro-phils increased dissemination of ERþ breast cancer cells via LFA-1and TGFb1, thus causing noninvasive cancer cells to be highlymetastatic. Time-lapse imaging in zebrafish revealed close inter-actions of neutrophils with cancer cells, which drove breast cancermetastasis. We also found that extracellular TGFb1 was overpro-duced in human breast cancer tissue compared with adjacentnormal breast tissue. Thus, E2 can regulate immune/cancer cellinteractions in tumormicroenvironments.Our results indicate thatextracellular TGFb1 is a relevant target in human breast cancer.Cancer Immunol Res; 5(3); 234–47. �2017 AACR.

IntroductionBreast cancer is the most common type of cancer affecting

women in the Western world, and metastasis is the main causeof death among breast cancer patients (1). Estrogen exposureplays a key role in breast cancer initiation and growth (2, 3). Twoof three breast cancers express the estrogen receptor (ER), andantiestrogen therapy is a cornerstone of the medical treatment ofthese patients (4). However, this long-term therapy only reducesthe risk of recurrence by30%to50%(5).Novel therapeutic targetsof estrogen-dependent breast cancer progression are thereforewarranted.

The importance of immune cells in tumor growth and metas-tasis has been described in numerous cancer types, includingbreast cancer (6, 7). Several types of immune cells express the ER,and estrogen affects the expression of inflammatory mediators inneutrophils and macrophages (8, 9). We have shown that estro-gen increases the influx of macrophages into breast cancers andinduces a protumorigenic phenotype (M2) in these cells (10).

Neutrophils also play an important role in tumor growth andmetastasis (7, 11). Neutrophils represent 40% to 75% of all whiteblood cells, and, similar to macrophages, neutrophils adopt aprotumorigenic phenotype (N2) or antitumorigenic phenotype(N1) in response to various cytokines (11–13).

TGFb1 is one of the most potent chemoattractants for neutro-phils (11). TGFb1 induces recruitment and N2 polarization ofneutrophils, and increases the number of circulating low-densityN2 neutrophils in several types of cancer, including breast cancer(11, 14, 15). TGFb1 is a multifunctional cytokine that has dualroles in tumorigenesis: in normal tissues and early stages ofcancer, it arrests cell proliferation and induces apoptotic path-ways, whereas in later stages, it promotes angiogenesis, growth,and tumor progression (16, 17). Cross-talk between TGFb1 andestradiol (E2) in breast cancer has been reported; for instance,TGFb1 and E2 together enrich cancer stem cell populations inbreast tumors, leading to increased migration and drug andradiation resistance (18). Estradiol also increases TGFb1 secretionin experimental breast cancers both in vitro and in vivo (19). Cell–cell interactions in tumormicroenvironments canbe enhanced byintegrins, which play a major role in cancer cell proliferation andmetastasis (20, 21). Expression of the lymphocyte function-asso-ciated antigen 1 integrin (LFA-1, aL/b2, CD11a/CD18) in neu-trophils promotes intra- and extravasation and transendothelialmigration (22, 23). As LFA-1 is involved in the first steps of celladhesion, and early steps of cancer cell dissemination would beefficient to target therapeutically, we focused our investigation onthis integrin (24).

It is clear that E2, TGFb1, and neutrophils are importantmediators of cancer progression and metastasis. However, therelationships among these three elements in the breast cancer

1Department of Oncology and Department of Clinical and Experimental Med-icine, Link€oping University, Link€oping, Sweden. 2Department of Medicine andHealth Sciences, Link€oping University, Link€oping, Sweden.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Charlotta Dabrosin, Link€oping University, UniversityHospital, SE-581 85 Link€oping, Sweden. Phone: 46-10-103-8595; Fax: 46-10-103-3090; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-16-0150

�2017 American Association for Cancer Research.

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metastasis process remain unaddressed. In the present study, E2increased the recruitment of neutrophils to the tumor-invasivemargin in vivo in two different mouse breast cancer models. Inaddition, E2 treatment promoted N2 polarization of neutro-phils by inducing overexpression of the integrin LFA-1. Theseresults were confirmed in vitro using mammosphere cultures. Ina zebrafish metastasis model, nonmetastatic ERþ breast cancercells became metastatic in the presence of neutrophils, and E2exposure further increased the dissemination of cancer cells viaTGFb1 and LFA-1 overexpression. Furthermore, in samplesfrom breast cancer patients, extracellular levels of TGFb1 weresignificantly increased, suggesting that this factor is a relevanttarget in human breast cancer.

Our data provide new insights into E2-dependent mechanismsof breast cancer progression. In addition, we also describe a novellow-cost protocol for paraffin embedding of mammospheres,which can be used to investigate molecular mechanisms andtherapeutic targets.

Materials and MethodsMicrodialysis of patients

The regional ethical review board of Link€oping approved thestudy,whichwas performed in accordancewith theDeclaration ofHelsinki. All subjects gave written informed consent. Womendiagnosed with breast cancer (n ¼ 12) underwent microdialysisbefore surgery. Characteristics of the cancers are shown in Sup-plementary Table S1. Before catheter insertion, 0.5 mL lidocaine(10 mg/mL) was administrated intracutaneously. One microdia-lysis catheter was inserted intratumorally into the breast cancerand another into adjacent normal breast tissue as previouslydescribed (M Dialysis; 71 cat. #P000127, 10-mm; refs. 25, 26).Catheters, connected to a pump (CMA 107; CMA MicrodialysisAB), were perfused with 154 mmol/liter NaCl and 60 g/literhydroxyethyl starch (Voluven; Fresenius Kabi AB; cat. #066287)at 0.5 mL/min. After 60-minute equilibration, the perfusate wasstored at �70�C.

Murine breast cancer modelsMice were housed at Link€oping University. Animal care and

treatment conformed to regulatory standards. The institutionalanimal ethics committee at Link€oping University approved thisstudy. Female athymic mice (BALB/c-nu/nu) and FVB/N mice(6–8 weeks; Scanbur) were housed in ventilated cages with alight/dark cycle of 12/12 hours, with rodent chow and waterad libitum. Mice were anesthetized by intraperitoneal injection ofketamine/xylazine and oophorectomized prior to s.c. implanta-tionwith 3-mmpellets containing 17b-estradiol (0.18mg/60-dayrelease, cat. #NE-121; Innovative Research of America) that pro-duce serum concentrations of 150 to 250 pmol/L or placebo (27).

One week after surgery, MCF-7 cells, 5 � 106 in 200 mL PBS,were injected into the dorsal mammary fat pads. Because MCF-7cells require estrogen for tumor formation and growth, a non-estrogen control group was not possible in this in vivo model. Atsimilar tumor sizes, mice either continued with estradiol or ful-vestrant (5mg/mouse every3days, s.c.; AstraZeneca; cat. #099215),a pure antiestrogen, was added to estradiol treatment.

FVB/N mice were injected in the dorsal mammary fat pad withcells, 1 � 106 in 200 mL PBS, derived from a transgenic mousestrain expressing polyoma middle T (PyMT) antigen under thecontrol of the mouse mammary tumor virus long terminal repeat

(28). These mice developed spontaneous adenocarcinomas ofmammary epithelium by 8 to 10 weeks of age. Tumors from10-week-old mice were excised, dissociated in a collagenase/dispase solution (100 mL PBS) with 25 mg collagenase (Sigma;cat. #C7657) and 250 mg dispase (Roche; cat. #04 942 078 001),and cultured until confluence. As shown by us, and others, thesebreast tumors express the ER during the early stage of cancer anddecrease ER expression at later stages (29, 30). Harvesting tumortissue at early stages results in estrogen-dependent cancer growthin the syngeneic recipientmouse, as previously described (27, 30).Tumors were established in oophorectomizedmice with/withoutestrogen supplementation and with estrogen together with ful-vestrant as described above.

Cell lines and neutrophil primary culture conditionsERþ cell lines MCF-7 and ZR-75-1, and the ER� cell line MDA-

MB-231, were purchased in 2009, 2016, and 2009, respectively,from the American Type Culture Collection (ATCC). Cells,authenticated by the ATCC, were upon receipt immediatelyexpanded and stored in liquid nitrogen. A new aliquot wasresuscitated for each experiment and never used for more than6 months. MCF-7 and MDA-MB-231 were maintained in DMEMmedium (Gibco; cat. #11880) and ZR-75-1 cells in DMEM/F12(Gibco; cat. #11039) with 2 mmol/L glutamine (Gibco; cat.#25030), penicillin-G (50 IU/mL), streptomycin (50 mg/mL;Gibco; cat. #15070), and 10% and 5% FBS, respectively (Gibco;cat. #10270). Human venous neutrophils were freshly isolatedusing buffy coats from healthy female donor that were immedi-ately processed upon receipt. The buffy coat was diluted 1/3 incold DPBS 1X (Gibco; cat. #14200-067) with 0.1% heat-inacti-vated FBS (Invitrogen; cat. #16140-071) and 2 mmol/L EDTA(Invitrogen; cat. #AM9260G) and washed by centrifugation.Peripheral blood mononuclear cells were removed by densitygradient centrifugation with Ficoll-Paque (GE Healthcare; cat.#17-1440-02), and most of the red blood cells were removed bydensity gradient with sterile 3% dextran T-500 (Amersham Phar-macia Biotech AB; cat. # 17-0320-02) in 0.9 %NaCl. The residualred blood cells were completely eliminated by hypotonic lysis.Theneutrophilswere cultured at 37�Cand5%CO2 for 45minutesin DMEM/F12 with 0.02% of BSA (Merck; cat. #1.12018.0025),10 mg/mL apo-transferrin (Sigma; cat. #T2036), and 1 mg/mLinsulin (Sigma; cat. #I5500). For TGFb1, neutrophils were cul-tured for 12 hours in DMEM (Gibco) with 5% charcoal-filteredFBS (Gibco; cat. #12676-029) and 2 mmol/L glutamine (Gibco).Treatments were as follows: 10�9 mol/L E2 (Sigma; cat. #2758),10�6 mol/L fulvestrant (Sigma; cat. #I4409), recombinanthuman TGFb1 (200 pg/mL; rhTGFb1; Peprotech; cat. #100-21),and antibodies (5 mg/mL) to human TGFb1 (Acris Antibodies;cat. #DM1047), human LFA-1 (Biolegend; cat. #301213), or iso-type IgG (Biolegend; cat. #401408).

Mammosphere culture and neutrophil infiltration assayMDA-MB-231 cells were seeded at 3 � 103 cells/well in Ultra

Low Attachment 96-well plates (Corning Inc.; cat. #7007) inDMEM medium with 2% ECM gel (Sigma; cat. #E1270). MCF-7were seeded at 1 � 103 cells/well with 2.5% ECM gel in Mammo-cult medium (Stem Cell Technologies Inc.; cat. #05620). Plateswere centrifuged, 1,000 � g for 5 minutes, and incubated at 37�Cwith 5% CO2. Two days before neutrophil infiltration, mammo-spheres, sized 500 mm in diameter, culture mediumwas graduallychanged to serum-free DMEM/F12 supplemented as above. Note

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that 1 � 105 neutrophils and 1 � 104 neutrophils were added toMCF-7 andMDA-MB-231 mammospheres, respectively, and trea-ted for 5 days. Medium was collected and mammospheres werefixed in 4% paraformaldehyde (PFA). Fixed mammospheres werestained with Mayer's hematoxylin (Histolab Products AB; cat.#01820) during 5 minutes, washed with PBS, and rehydrated inethanol (70%2� 30minutes, 90%2� 30minutes, 99.5%3� 30minutes), tissue clear at 2 � 30 minutes (Histolab; cat. #14250),and liquid paraffin at 60�C 3 � 30 minutes in an eppendorf lid.Then, mammospheres were transferred to a warm metal mold at60�C for 30 minutes, centered with a warm spoon, and cooled atroom temperature for about 15minutes to keep them in place andthen placed at 60�C with more liquid paraffin and a plastic moldfor another 30 minutes. After incubation, the mold was carefullyplaced at room temperature for 10 minutes and then in 4�C tosolidify the paraffin.

Optimization for neutrophils/mammospheres concentrationwas performed ranging from 2 � 104 to 1 � 105 per mammo-sphere. For MCF-7 mammospheres, in E2 group, the variousconcentration of infiltrating neutrophils per mammosphere wasnot significantly different. The experiments were set up using 1 �105 neutrophils/MCF-7mammospheres/well because there was alower variability for this concentration (Supplementary Fig. S2A).However, for MDA-MB-231 mammospheres, 1 � 105 neutro-phils/mammospheres/well resulted in structural damaged andreduced growth (Supplementary Fig. S2B); therefore, a concen-tration of 1 � 104 neutrophils/mammospheres/well was used.

Quantification of TGFb1 and soluble ICAM-1TGFb1 and intercellular adhesion molecule 1 (ICAM-1) were

analyzed with ELISAs (Quantikine TGFb1 ELISA kit; R&D Sys-tems; cat. #DB100B and ICAM-1 ELISA; BioVision; cat. #K7161-100) according to the manufacturer's instructions.

Invasion assayMDA-MB-231 mammospheres were infiltrated with neutro-

phils during 3 days. Invasion assay was performed by the provi-der's instructions (R&D Systems; cat. #3500-096-K). Images weretaken using the inverted microscope Axio Vert.A1 with AxioCamICm1 CCD camera (Zeiss) at days 0 and 5. Invaded area (mm2)was calculated by subtracting the area at day 0 to the total areacalculated at day 5 by using ImageJ 1.50i software.

Immunocytochemistry, immunohistochemistry,immunofluorescence, and Giemsa staining

Neutrophils, fixed in 4% PFA, were exposed to anti–humanLFA-1 (Biolegend; cat. #301213) and Alexa-Fluor 488 (Abcam;cat. #ab150113).Mouse tumor sectionswere stainedwith rat anti-mouse lymphocyte antigen 6 complex locus G6D (Ly6G) 1:400(BD Pharmingen; cat. #551459), using rat on a mouse HRPPolymer kit (BioCare Medical; #RT517G), counterstained withMayer's hematoxylin. Negative controls did not stain. For immu-nofluorescence, sections were stained with rat anti-mouse Ly6G1:400 (BD Pharmingen), rabbit anti-mouse LFA-1 1:100 (Abcam;cat. #ab203336), anti-rabbit Alexa 546 1:200 (Life Technologies;cat. #A11010), and anti-rat Alexa 488 1:200 (Life Technologies;cat. #A21210), and mounted using SlowFade Gold antifadereagent withDAPI (Life Technologies; cat. #S36938). Images wereacquired byOlympus BX43microscope light/fluorescencemicro-scope, excitation filters BP460-495 and BP530-550, using an

Olympus DP72 CCD camera and analyzed using Olympus Cell-Sens Imaging software.Mammospheres,fixed for 30minuteswith4% PFA at room temperature, were stained with anti-humanCD45 1:200 (Biolegend; cat. #9624-01) and anti-mouse Alexa-Fluor 488 1:500 (Abcam; cat. #ab150113), and mounted inSlowFade Gold antifade reagent with DAPI. Images were takenusing Zeiss Axio Imager with LSM 700 upright confocal micro-scope, and the infiltration of neutrophils was analyzed usingthe ImageJ 1.50i software. Images of MCF-7 mammosphereswere deconvolved for better visualization where specified withHuygens compute engine 15.10.1p6 using classic maximumlikelihood estimation.

Cytospins of freshly isolated neutrophils were dried and stain-ed with a SNABB-DIFF kit (LABEX Instrument AB; cat. #2115)following the manufacturer's instructions. For immunocyto-chemistry, cytospins of freshly isolated neutrophils were dried,fixed with cold acetone for 10 minutes at �20�C, and incubatedwith anti–human LFA-1 at 1:100 (Biolegend; cat. #301213) at4�C overnight. The MACH Universal HRP-polymer detectionsystem (Histolab; cat. #BC-BRI4012L) and the betazoid DABKit (Histolab; cat. #BC-BDB2004H) were used. Slides weremounted with Glycergel (Dako; cat. #C0563), and negative con-trol did not show stain. For immunohistochemistry, MCF-7 andMDA-MB-231 mammospheres were stained with mouse anti-human ICAM-1/CD54 1:50 (Biolegend; cat. #353101), mouseanti-human ICAM-2/CD102 1:100 (Novus Biological;cat. #NBP2-00320), and rabbit anti-human ICAM-3/CD501:100 (Sino Biological; cat. #10333-R002-50), and counter-stained with Mayer's hematoxylin (Histolab). For immunofluo-rescence, sections of PyMT mouse tumors were double stainedwith rat anti-mouse Ly6G 1:400 (BD Pharmingen; cat. #551459)and rabbit anti-mouse F4/80 1:25 (Abcam; cat. #ab111101) at 4�Covernight. Anti-rabbit Alexa 488 1:200 (Abcam; cat. #ab150077)and anti-rat Alexa 546 1:200 (Invitrogen; cat. #A11081) were usedas secondary antibodies. Slides were mounted using SlowFadeGoldantifade reagentwithDAPI (Life Technologies; cat. #S36938).Images were acquired by Olympus BX43 microscope light/fluorescence microscope with excitation filters BP460-495 andBP530-550, using an Olympus DP72 CCD camera, and ana-lyzed using Olympus CellSens Imaging software.

Migration, survival, and retention assaysMCF-7 cells were cultivated during 3 days in DMEM (Gibco;

cat. #11880) with 10% charcoal-filtered FBS (Gibco; cat. #12676-029), 50 IU/mL Penicillin-G, 50 mg/mL streptomycin (Gibco;cat. #15070), and 2mmol/L glutamine (Gibco; cat. #25030)� E210�9mol/L (Sigma; cat. #2758). Conditionedmedia fromMCF-7cells were used for migration and survival assays. Human neu-trophils were freshly isolated as described above and re-sus-pended in DMEM/F12 with 0.02% of BSA (Merck; cat.#1.12018.0025), 10 mg/mL apo-transferrin (Sigma; cat. #T2036),and 1 mg/mL insulin (Sigma; cat. #I5500). Migration assay wasperformed with a CytoSelect 96-well cell migration assay kit (CellBiolabs; cat. #CBA-105) according to the manufacturer�s instruc-tions. Note that 5� 104 neutrophils/well were placed in the upperchamber � E2 10�9 mol/L and conditioned media in the lowerchamber, and migrated cells were quantified after 24-hour incu-bation at 37�C by using the Spark 10M multimode microplatereader (TecanGroupLtd.). For survival assay, 5�105neutrophils/well were cultured in MCF-7 cell–conditioned media � E2 in a96-well plate. Living cells were quantified at 24 and 48 hours of

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Figure 1.

E2 increased tumor growth and neutrophil recruitment and polarization in mouse breast cancer models. A, Oophorectomized FVB/N mice supplemented withphysiologic concentrations of estradiol (E2; n ¼ 10), placebo (n ¼ 5), or E2 þ fulvestrant (Fulv) treatment (5 mg/mouse, every third day s.c.; n ¼ 8) were injectedwith PyMT tumor cells in themammary fat pad, and tumor growthwasmonitored using a caliper. Tumor sectionswere stained for neutrophils (Ly6G) and quantified asdescribed in Materials and Methods. ��, P < 0.01 and �� , P < 0.001 compared with control and þþ, P < 0.01. þþþ, P < 0.001 compared with E2. Scale bars, 20 mm.B,Tumor sections from tumors treatedasdescribed inAwere stained for neutrophils (Ly6G)andLFA-1 expressionandquantifiedasdescribed inMaterials andMethods.�� , P < 0.01, compared with control; þþ, P < 0.01 compared with E2. Scale bars, 50 mm. C, Oophorectomized BALB/c-nu/nu mice were supplemented withphysiologic levels of E2 and injected with MCF-7 cells in the mammary fat pad. At similar tumor sizes, one group continued with E2 treatment (n ¼ 8), and the othergroup received an additional Fulv treatment (5 mg/mouse every third day s.c.; n ¼ 11). Tumor sections were stained for neutrophils (Ly6G) and quantified asdescribed in Materials andMethods. �� , P < 0.01 and ��, P < 0.001. Scale bars, 50 mm.D, Tumor sectionswere stained for neutrophils (Ly6G) and LFA-1 expression andquantified as described in Materials and Methods. �� , P < 0.0001. Scale bars, 50 mm. Data are representative of at least two seperate experiments.






Figure 2.

E2 increased the infiltration of human neutrophils via TGFb1 in MCF-7 mammospheres. A, MCF-7 cells were cultured in monolayer or as mammospheres.Western blot was performed for detection of ERa expression; lane 1 represents cells from monolayer culture and lane 2 cells from mammosphere culture.Mammosphere size increased in the presence of E2 (n ¼ 12 in each group). M, molecular weight marker; �� , P < 0.0001. (Continued on the following page.)






culture with trypan blue viability exclusion and reported aspercentage of survival. For retention study, 7 � 105 neutro-phils/mL were added to a monolayer culture of MCF-7 cellsgrown inDMEM(Gibco)with 10%charcoal-filtered FBS (Gibco),Penicillin-G (50 IU/mL), streptomycin (50 mg/mL; Gibco), and2 mmol/L glutamine (Gibco) � E2 10

�9 mol/L (Sigma). Cocul-tures were incubated at 37�C, and concentration of neutrophilsin culture supernatant was quantified at 24 and 48 hours ofculture in a B€urker chamber. Number of retained neutrophilswas calculated by subtracting the concentration of neutrophils inculture supernatant to the initial concentration of neutrophilsadded to MCF-7 cell cultures.

Zebrafish experimentsThe institutional animal ethics committee at Link€oping Uni-

versity approved all zebrafish experiments. Cells were treatedwith/without E2 before injections. Cancer cells were labeled withFast DiI oil red dye (ThermoFisher Scientific; cat. #1635639), aspreviously described (31). Human neutrophils were labeled with6mg/mLofDiBblue dye (Biotium; cat. #60036) and re-suspendedin medium with/without E2. Neutrophils were diluted 1:2 (50%)with cancer cells, and anti–human TGFb1, LFA-1, and isotypecontrol antibodies were added at 0.1 mg/mL with/without E2immediately before injection.

Transgenic Tg(fli1:EGFP)y1 zebrafish embryos, with green fluo-rescent vessels, were raised in E3 medium with 1-phenyl-2-thio-urea (PTU). Cells were implanted into the perivitelline space of2-day old zebrafish embryos, as previously described (31), andincubated at 28�C in E3 medium with 0.2 mmol/L PTU. Cellmigration to the tail region was evaluated 24 hours after injec-tion, the embryos were anesthetized with 0.02% MS-222(Sigma-Aldrich; cat. #E10521), and pictures were acquired usingan Olympus BX43 light/fluorescence microscope (10�/0.30magnification), excitation filters BP360-370, BP460-495, andBP530-550, using an Olympus DP72 CCD camera. Images wereacquired with the Olympus CellSens.

For time-lapse experiments, zebrafish embryoswere embeddedin 0.5% agarose low gelling temperature (Sigma; cat. #A9045)with 0.02% MS-222 and placed in 35 mm glass bottom petridishes (MatTek Corporation; cat. #P35G-1.5-10-C) with E3embryo medium containing 0.2 mmol/L PTU and with/withoutE2. Z-stack images were acquired every 6 and 15 minutes for tailregion and injection site, respectively. A Zeiss Observer withLSM 700 inverted microscope equipped with incubator chamberwas used for Supplementary Videos S1 to S3. A Nikon EclipseTE2000-U inverted confocal microscope equipped with BioRadRadiance 2100MP laser scanning system and 2-photon excitationMaiTai laser was used for Supplementary Video S4. Time-lapseimages were acquired with the Zeiss ZEN software.

Equipment and settingsMammosphere confocal imaging was performed as follows:

image size (pixels) 1024 � 1024, 16-bit depth, averaging 4, ECPlan-Neofluar 10�/0.3 M27 objective, laser wavelength 488and 405 nm, Alexa-fluor 488 excitation/emission (nm):488/518, DAPI excitation/emission (nm): 405/435, binningmode 1 � 1, and PMT detector. Time-lapse Video 3 was acquiredwith LaserSharp2000 software, image size 1024 � 768, 8-bitdepth, 10� objective magnification, 1.2 objective zoom, PMTdetector, detection filters HQ450/80, E570LP, and HQ515/30.Time-lapse Supplementary Videos S1, S2, and S4 were acquiredwith Zeiss ZEN software, at 512 � 512 resolution, 8-bit depth,Plan-Apochromat 10�/0.45 M27 objective magnification, PMTdetector, binning mode 1 � 1.

Western blotLysates were loaded on a 4% to 15% polyacrylamide gel

(BioRad; cat. #456-1083), transferred to PVDF membrane(BioRad; cat. #170-4156), and incubated with anti-human ERa(Dako; cat. #M7047) overnight at 4�C and then with anti-mouseimmunoglobulins HRP (Dako; cat. #P0447). ECL detection kit(GE Healthcare; cat. #RPN2232) following the manufacturer'sinstructions was used.

Statistical analysisData are presented as mean � SEM. Two-tailed Student t tests

for paired and unpaired analyses, where appropriate, were used. AP value < 0.05 was considered statistically significant. Statisticswere performed with Prism 6.0 (GraphPad software).

ResultsE2 increased tumor growth and chemotaxis of protumoralneutrophils in breast cancer in vivo

To elucidate whether E2 affects the recruitment and polarizationof neutrophils in ERþ breast cancer, we set up two different mousetumor models of breast cancer. Murine PyMT tumors displayed E2-dependent growth in immune-competentmouse thatwas inhibitedby fulvestrant treatment (Fig. 1A). In PyMT tumors, E2 induced asignificant increase of neutrophils in the invasive margin comparedwith tumors grown without E2, and this was reversed by fulvestrant(Fig. 1A). Furthermore, E2-recruited neutrophils in the tumor siteshowed increased expression of LFA-1 integrin, whereas fulvestrant-treated tumors exhibited similar LFA-1 expression as control tumors(Fig. 1B). Inhumanestrogen-dependent experimental breast cancers(MCF-7) established in nudemice, the pure antiestrogen fulvestrantsignificantly decreased E2-induced tumor growth (Fig. 1C). Also,fulvestrant significantly reduced the E2-induced recruitment ofneutrophils (Fig. 1C) and significantly decreased the percentage of

(Continued.) B, Freshly isolated human neutrophils at 1 � 105 were added to MCF-7 mammospheres and treated for 5 days with E2 and the antiestrogenfulvestrant (Fulv). Infiltrated neutrophils were counted (n¼ 5 in each group); �, P < 0.05 compared with control; þþ, P < 0.01 compared with E2. Scale bar, 200 mm.C, Orthogonal projection of MCF-7 mammosphere in E2 treatment showing the localization of neutrophils within the mammospheres. Scale bar, 200 mm.D, Culture medium from neutrophil-infiltrated MCF-7 mammospheres treated � E2 and fulvestrant (Fulv) for 5 days was analyzed for TGFb1 with ELISA (n ¼ 6 ineach group); �� , P < 0.001 compared with control; þþþþ, P < 0.0001 compared with E2. E, TGFb1 quantification with ELISA in culture medium from MCF-7mammospheres� E2 andmammosphereswith or without neutrophils and treatedwith E2 for 5 days (n¼ 6 in each group); �� ,P <0.01 comparedwithmammospherewithout E2. F, For inhibition experiments, neutrophil-infiltrated MCF-7 mammospheres were treated for 5 days with anti–human TGFb1, and infiltratedneutrophils were counted (n ¼ 8 in each group); � , P < 0.05 compared with control; þþ, P < 0.01 compared with E2. Scale bar, 200 mm. G, Neutrophils wereadded toMCF-7mammosphereswith orwithout human recombinant TGFb1 200pg/mLduring 5days, and infiltrated neutrophilswere counted (n¼6 in each group);� , P < 0.05 compared with control. Scale bar, 200 mm. Data are representative of at least two seperate experiments.






LFA-1þ neutrophils (Fig. 1D). The results were similar in tumors ofcomparable size from the various treatment groups as well as intumors of different sizes, i.e., large control tumors versus smallertumors from the treatment groups. However, it cannot be ruled outthat in the larger tumors, other factor may contribute to the recruit-ment and polarization of the neutrophils. In Supplementary Fig.S1A, we show that the neutrophil marker Ly6G specifically stainsmurine neutrophils and not macrophages.

E2 increased neutrophil infiltration via TGFb1 in vitroTo analyze the mechanism underlying neutrophil recruitment

in response to E2, we set up aMCF-7mammosphere breast cancermodel in vitro. First, we developed a protocol for paraffin embed-ding of mammospheres to avoid agarose or clot embedding, asdescribed inMaterials andMethods. The isolated cells frombloodthat were used for all experiments indeed were neutrophils withtheir characteristic multi-lobulated nuclei and with variousexpression of LFA-1 (Supplementary Fig. S1B and S1C).

Some studies suggest that ERþ cells lose ER expression whencultured in mammospheres, but mammospheres maintainedERa expression and their growth increased significantly inresponse to E2 (Fig. 2A). Whenmammospheres were established,human neutrophils and treatments were added. Infiltration ofhuman neutrophils was significantly increased by E2, which wasreduced in the presence of fulvestrant (Fig. 2B). This was notdependent on the number of neutrophils that were added to themammospheres, as similar results were found at three differentconcentrations of neutrophils (Supplementary Fig. S2A). Orthog-onal projection in Fig. 2C reveals that infiltrated neutrophilsremained in the outer layers of mammospheres, resembling whatwas observed in vivo. The E2-inducedmigrationof neutrophilswasconfirmed using the Boyden chamber; 37,863 � 1,687 neutro-phils migrated in the control versus 45,606� 977 in the E2 group(P < 0.01). E2 exposure and coculture increased the survival ofneutrophils; after 48 hours, 18% � 2% survived in single cultureversus 35% � 4% in coculture (P < 0.05). In the presence of E2,32% � 1% survived in single culture versus 53% � 1% incoculture (P < 0.01). In line with previous data of neutrophilsurvival in human in tissue in vivo (32), neutrophils were detectedup to 5 days in three-dimensional (3D) mammosphere culture.The retention of neutrophils to cancer cells increased duringE2 exposure; 1.9 � 105 � 0.1 versus 3.1 � 105 � 0.09 in theE2 group (P < 0.01).

TGFb1 secretion in neutrophil-infiltrated MCF-7 mammo-spheres was significantly increased by E2, but was significantlyreduced by fulvestrant (Fig. 2D). The cancer cells were the mainsource of TGFb1 (Fig. 2E). To evaluate whether neutrophilinfiltration in response to E2 was mediated by TGFb1, we addedanti–human TGFb1 to the mammospheres. Significantlydecreased E2-induced infiltration of neutrophils was detected(Fig. 2F). Treatment with hrTGFb1 induced infiltration ofneutrophils similar to E2 (Fig. 2G). The presence of neutrophilsdid not affect the proliferation of the mammospheres (datanot shown). This was supported by mammosphere size, whichwas unchanged after addition of TGFb1 antibody (778 � 7 and769 � 7 mm, respectively).

LFA-1 integrin overexpression in human neutrophils inducedby E2

Next, we investigated whether E2 affects LFA-1 expression inneutrophils. Primary cultures of human neutrophils were

exposed to E2, which significantly increased LFA-1 expression,and this effect was reduced in the presence of fulvestrant (Fig. 3A).Human recombinant TGFb1 treatment increased LFA-1 expres-sion in a similar fashion to E2 treatment (Fig. 3A). As furtherconfirmation, anti–human TGFb1 decreased LFA-1 expression(Fig. 3B), and significantly higher concentrations of secretedTGFb1 were observed in E2-exposed neutrophils (Fig. 3C). Fur-thermore, anti–human LFA-1 significantly decreased E2-inducedneutrophil infiltration into mammospheres (Fig. 3D), underscor-ing the key role of LFA-1 expression in neutrophil migration.

E2-induced LFA-1 promoted neutrophil-dependentdissemination of ERþ breast cancer cells

PyMT transgenic mice develop metastases, especially to thelungs, at late carcinoma stages at approximately 14 to 18 weeks ofage, and at this stage, the tumors have lost their ER expression(28, 29, 33). Here, we harvested tumors from 10-week-old miceand injected the cells into the mammary fat pad in recipientsyngeneicmice to achieve anER-expressing breast cancermodel inan immune competentmouse. However, these tumors would notmetastasize and therefore could not be used for studies ofmechanisms of E2-dependent metastatic growth. For these rea-sons, we used a zebrafish model to evaluate whether theE2-induced overexpression of LFA-1 in neutrophils, via TGFb1,increased the dissemination of otherwise nonmetastatic ERþ

breast cancer cells. As previously shown, E2 exposure alone didnot affect the dissemination of MCF-7 cells (10). However, thepresence of neutrophils significantly increased dissemination ofMCF-7 cells (Fig. 4A). The presence of E2 further increased cancercell dissemination (Fig. 4A). In addition, the number offishwherecancer cells were disseminated increased from 33% � 3.7%to 56% � 3.2% in the presence of neutrophils, P < 0.05, and to88%� 6.1% in the presence of neutrophils and E2, P < 0.01. Anti–human TGFb1 and anti–human LFA-1 significantly reducedthe dissemination of MCF-7 cells, confirming the importanceof TGFb1 and LFA-1 for cancer cell dissemination capacity(Fig. 4B and C).

To corroborate the results obtained with MCF-7 cells, weevaluated another ERþ breast cancer cell line in the presence ofneutrophils and E2. Similar to the MCF-7 cells, anti–humanTGFb1 and anti–human LFA-1 antibodies inhibited the dissem-ination of ZR-75-1 cell injected together with neutrophils in thepresence of E2 (Fig. 4D). These data suggest that E2-induceddissemination via LFA-1 might be conserved among ERþ breastcancer cells.

Neutrophils mediate the intra- and extravasation of breastcancer cells

In the zebrafish dissemination experiments, we observed that ahigh proportion ofmigrated breast cancer cells were accompaniedby neutrophils, especially in the presence of E2. Time-lapse imag-ing in zebrafish showed that neutrophils increased the migrationand invasion of ERþ breast cancer cells at the tumor injection site,and that neutrophils comigrated with cancer cells and promotedcancer cell intravasation (Fig. 5A and B; Supplementary Videos S1and S2). Neutrophils intravasated together with cancer cells fromthe tumor primary site, helping to establish newmetastatic nichesand to extravasate circulatory cancer cells in distant sites of the fish(Fig. 5B; Supplementary Video S3). Anti–human LFA-1 blockedthe neutrophil-induced migration and intravasation of cancercells (Supplementary Video S4).






ER� cancer cell invasion anddisseminationwas independent ofE2 and neutrophils

To evaluate whether E2-induced breast cancer progressionand metastasis via LFA-1 plays a role in ER� breast cancer, ER�

MDA-MB-231 was investigated. Growth of MDA-MB-231tumors in nude mice was independent on E2 exposure. Tumorvolume 3 weeks after cancer cell injection in mice was 18 5 �45 mm3 versus 173 � 55 mm3, P ¼ 0.87, � E2, respectively. No

differences in neutrophil count in the invasive margin intumors grown were detected � E2; 552 � 101 versus 654 �64, respectively, P ¼ 0.4.

Exposure to E2 did not affect the invasive capability of thesecells in the presence or absence of neutrophils, and no significantdifference in the invasion of neutrophils into MDA-MB-231mammospheres was detected (Fig. 6A and B). Estradiol didnot affect TGFb1 secretion or infiltration of neutrophils in the

Figure 3.

E2 induced LFA-1 expression in human neutrophils via TGFb1. A and B, Freshly isolated human neutrophils were cultured with E2, fulvestrant (Fulv), humanrecombinant TGFb1 (200 pg/mL), anti–human TGFb1, or isotype control at 5 mg/mL for 45 minutes. Cells were fixed and stained with anti–human LFA-1 (green), andnumber of LFA-1 positive cells were counted (n ¼ 8–9 in each group); �� , P < 0.01, compared with control; þ, P < 0.05; þþ, P < 0.01 compared with E2. Scale bar,50 mm. C, TGFb1 quantification with ELISA in culture medium of neutrophils cultured with or without E2 for 12 hours (n ¼ 5 in each group); � , P < 0.05 comparedwith control. D, For inhibition experiments, neutrophil-infiltrated MCF-7 mammospheres were treated for 5 days with anti–human LFA-1 at 5 mg/mL, andnumber of infiltrated neutrophils were counted. MCF-7 mammosphere images were deconvolved with Huygens software for better visualization (n ¼ 7 ineach group); �� , P < 0.001 compared with control; þ, P < 0.05 compared with E2. Scale bar, 200 mm. Data are representative of at least two seperateexperiments.






Figure 4.

Anti–LFA-1 and anti-TGFb1 antibodies inhibited E2-induced neutrophil/MCF-7 cancer cell dissemination. A, Transgenic zebrafish embryos (green blood vessels)were injected with MCF-7 (red) � E2 � neutrophils (blue) as described in Materials and Methods. Before injection, MCF-7 cells were cultured either with orwithout 10�9mol/L E2. Disseminated cells were counted (n¼ 18–22 in each group); � , P <0.05 and �� , P <0.01. Arrowheads show comigratedMCF-7/neutrophils cells(violet color). Scale bar, 100 mm. B, Zebrafish embryos were injected with MCF-7 (red) and neutrophils (blue) in the presence of E2. Anti–human LFA-1 or isotypecontrol were added and disseminated cells were counted (n ¼ 24–44 in each group); � , P < 0.05 compared with MCF-7; þ, P < 0.05 compared with MCF-7 þneutrophils þ isotype control. Arrowheads show comigrated MCF-7/neutrophils cells (violet color). Scale bar, 100 mm. C, Zebrafish embryos were injectedwith MCF-7 (red) and neutrophils (blue) þ E2. Anti–human TGFb1 or isotype control antibodies were added, and the amount of disseminated cells was counted(n ¼ 20–44 in each group); �� , P < 0.01 compared with MCF-7; þ,P < 0.05 compared with MCF-7 þ neutrophils þ isotype control. Arrowheads show comigratedMCF-7/neutrophils cells (violet color). Scale bar, 100 mm. D, Zebrafish embryos were injected with ZR-75-1 and neutrophils þ E2. Anti–human LFA-1, TGFb1, orisotype control were added, and the amount of disseminated cells was counted (n ¼ 20–30 in each group); � , P < 0.05 compared with ZR-75-1; þþ, P < 0.01compared with ZR-75-1 þ neutrophils þ isotype control. Data are representative of at least two seperate experiments.






presence or absence of anti–human LFA-1 (Fig. 6C and D). Inaddition, the dissemination of MDA-MB-231 cells was not affect-ed by the presence of neutrophils or E2 (Fig. 6E). Higher con-centrations of neutrophils resulted in structural damage to themammospheres (Supplementary Fig. S2B).

LFA-1 ligand expression differs among ERþ and ER� breastcancers

Immunostaining of paraffin-embedded mammospheresshowed differential expression of LFA-1 ligands. For instance, weobserved high expression of ICAM-3 and no or very low expres-sion of ICAM-1 and ICAM-2 in the nonmetastatic ERþ MCF-7mammospheres (Supplementary Fig. S3A), indicating that neu-trophil infiltration could be carried out in this model through cellinteractions via LFA-1 and ICAM-3 binding. On the other hand,the metastatic ER� MDA-MB-231 mammospheres showed highexpression of ICAM-1 and ICAM-3 and weak expression ofICAM-2 (Supplementary Fig. S3A). In addition, ER� MDA-MB-231mammospheres produced higher quantities of soluble ICAM-1 thanERþ MCF-7 mammospheres (Supplementary Fig. S3B).

Extracellular TGFb1 was increased in human breast cancersGiven the key role of TGFb1 in mediating E2-induced breast

cancer progression and dissemination in vitro and in vivo, it isimportant to knowwhether extracellular concentrations of TGFb1are affected in breast cancer patients. Therefore, we performedmicrodialysis for in vivo detection of TGFb1 in women with ERþ

breast cancer. We found that TGFb1 in breast cancers was signif-

icantly higher compared with adjacent normal breast tissue,suggesting TGFb1 as a possible target for human breast cancertreatments (Fig. 7).

DiscussionIn this article, we showed that estrogen exposure increased the

secretion of TGFb1, leading to increased accumulation of neutro-phils in the invasive margin of ERþ breast cancers. In addition toincreased neutrophil numbers, exposure to E2 increased the expres-sion of LFA-1 in neutrophils. Neutrophils increased the ability ofnonmetastatic ERþ breast cancer cells to disseminate, and E2exposure further enhanced this process via increased expression ofTGFb1 and LFA-1. Our observation of higher extracellular levels ofTGFb1 in human breast cancers compared with normal adjacentbreast tissue suggests that TGFb-1maybe exploited therapeutically.

Exposure to E2 plays a key role in breast cancer growth andmetastasis (34). During the first 5 first years after diagnosis, ERþ

breast cancer patients have a better prognosis than ER� breastcancer patients. However, the prognoses of these two groupsconverge over time; five to ten years after diagnosis, there is nodifference in recurrence rates, and beyond 10 years, the risk ofrecurrence and death is higher in the ERþ group (35, 36). Abouthalf of ERþ breast cancers respond to hormone therapy; however,25% of patients who receive hormone therapy will relapse (5).Because more than two thirds of breast cancers express the ER,investigating mechanisms of ERþ breast cancer dissemination iskey for designing novel therapeutics for these cancers.

Figure 5.

Human neutrophils comigrated withbreast cancer cells to promotedissemination and extravasation. A,ZR-75-1 cells (red), cultured with E2,were coinjected 1:2 with humanneutrophils (blue) into zebrafishembryos. Arrows show thecomigrated cells in blood circulationand in different stages of extravasationin the zebrafish tail. CC, cancer cells.Scale bar, 100 mm.B,MCF-7 cells (red),cultured with E2, were coinjected 1:2with human neutrophils (blue) intozebrafish embryos. Arrows show thecomigrated neutrophils with MCF-7 atthe injection site (violet color). Scalebar, 50 mm. Data are representative ofat least two seperate experiments.






Figure 6.

Triple-negative MDA-MB-231 breast cancer cell invasion and metastasis were independent of human neutrophils. A, MDA-MB-231 mammospheres were appliedto 3D culture for invasion assay � neutrophils � E2. Images were taken at days 0 and 5, and invaded area was calculated (n ¼ 4 in each group). Scale bar,200 mm. B, Freshly isolated human neutrophils at 1� 104 were added to MDA-MB-231 mammospheres and treated for 5 days� E2, and infiltrated neutrophils werecounted (n ¼ 6). Scale bar, 200 mm. C, TGFb1 quantification with ELISA in culture medium from MDAMB231 mammospheres � E2 for 5 days (n ¼ 6 in eachgroup). D, MDA-MB-231 mammospheres infiltrated with human neutrophils at 1 � 104 and treated for 5 days � E2 and � anti–human LFA-1 at 5 mg/mL, infiltratedneutrophils were counted (n ¼ 6 in each group). E, Zebrafish embryos were injected with MDA-MB-231 (red) and neutrophils (blue) � E2, and the amount ofdisseminated cells was counted (n ¼ 18–21). Scale bar, 100 mm. Data are representative of at least two seperate experiments.






Although the mechanisms of estrogen-mediated primarycancer growth are well studied, mechanisms behind estro-gen-dependent breast cancer dissemination need to be eluci-dated. One major obstacle for this research is that murinemouse models of breast cancer that spontaneously metastasizelose ER expression during the dissemination process; the metas-tases are then ER�. There has been a general belief that this isalso a natural cause of breast cancer progression in humans.Evidence shows that this is not the case; the majority of ERþ

primary breast cancers maintain ER expression in metastases,and nearly a third gain ER expression in metastatic lesions (37).This underscores the importance of understanding estrogen-dependent mechanisms of breast cancer progression as a pre-requisite for finding novel therapeutic targets.

The zebrafish metastasis model allows for such investigation(10, 31). By using this model, we showed here that neutrophilsincreased ERþ breast cancer cell dissemination, which is in agree-ment with recent published data (7). We also showed that non-metastatic ERþ breast cancer cells became highly metastatic in thepresence of neutrophils and that estrogen treatment may furtherenhance this dissemination capability. Aside from the effects ofcell–cell interactions between neutrophils and breast cancer cells,neutrophils may also pave the way for distant metastases byinfiltrating and creating premetastatic niches before cancer cellsarrive (7). Neutrophils did not increase the dissemination abilityof ER� metastatic breast cancer cells in the presence or absence ofE2, suggesting that thismechanismmay primarily be important inlow-grade cancers, such as ERþ breast cancers.

Neutrophils possess dual roles in cancer progressiondependingon their phenotype or number in the tumor microenvironment(38, 39). Massive infiltration of neutrophils may elicit a directcytotoxic effect by releasing hydrogen peroxide, leading to tumorregression, whereas a low-grade neutrophil gradient is tumorprogressive (38, 39). This is in line with our present data showingthat addition of a high number of neutrophils caused a disinte-gration of mammospheres in vitro.

According to previous reports, high expression of ICAM mole-cules in cancer tissues is related to poor prognosis and increased

cancer malignancy (40, 41). These data support our results forhighly metastatic and invasive ER� MDA-MB-231 mammo-spheres, which exhibited high expression of ICAM-1 and -3, weakexpression of ICAM-2, and secretion of ICAM-1 molecules. Thenonmetastatic ERþ MCF-7 mammospheres showed high expres-sion of ICAM-3 only. ICAM-1 molecules interact with the actin-containing cytoskeleton anda-actinin, inducing cytoskeletal rear-rangement (42, 43). This activity could explain the neutrophil-independent invasion and dissemination ofMDA-MB-231 cancercells because their high expression of ICAM molecules promotestheir increased motility and invasion.

TGFb1 exerts a potent chemotactic effect in neutrophils(44, 45). Many cell types, including immune cells, produceTGFb1, which is secreted in a latent form bound to variousproteins (45). Events in the microenvironment, such as proteaseactivities, release active TGFb1 into the extracellular fluid where itbecomes available for cell–cell interactions (19). A correlationbetween TGFb1 and increased metastasis has previously beenshown only by immunostaining human breast cancers. However,previous studies did not determinewhether TGFb1 in its bioactiveform is elevated in human breast cancer. Here, we provideevidence that, indeed, extracellular concentrations of TGFb1 areincreased in human breast cancers, suggesting that TGFb1 is aviable target for treatment.

In addition to its chemotactic effects, TGFb1 may decreaseneutrophil cytotoxicity and mediate a polarization from antitu-mor N1 neutrophils to protumor N2 neutrophils (11). In ourstudy, LFA-1 integrin, which is associated with N2 polarizationand prolonged survival of neutrophils (46), increased upon E2exposure via elevated TGFb1. This LFA-1 overexpression in neu-trophils also increased cell–cell interactions, which led toincreased ERþ breast cancer cell dissemination. We confirmedthe roles of LFA-1 and TGFb1 in mediating E2 effects by showingthat inhibition of these proteins by antibodies successfullyblocked cancer cell dissemination in the presence of E2.

Wehavepreviously shown that E2 does not affect the number ofneutrophils in central tumor tissues and that the numbers ofneutrophils in the centers of tumors are very low (10).Our presentdata suggest that neutrophils are preferentially localized in theinvasive margin and that E2 significantly affects the number ofthese neutrophils both in immune-competent and immune-defi-cient mousemodels of breast cancer. Also, the presence of LFA-1–expressing neutrophils in the invasive edges of tumors signifi-cantly increased with E2 treatment. This suggests that combinedtherapies that target cell–cell interactions in tumormicroenviron-ments could be feasible in ERþ breast cancer.

Our data were in part generated using mammosphere cultures,and we described here a protocol for paraffin embedding of thesespheres. 3D models such as tumor spheres or microtissue moreclosely mimic the tumor microenvironment than do monolayersof cancer cells. Previous groups have attempted diverse paraffin-embedding techniques with several disadvantages, includingdifficulties in obtaining accurate sections of spheres due to aga-rose/clot embedding (47–49). When agarose/clot embedding ofmammospheres is performed, mammospheres do not stay in asingle plane but sink to different deep layers. In order to analyzeall mammospheres, multiple slices must be collected. Our pro-tocol provides a new and improved method for paraffin embed-ding. With the method described herein, it is possible to analyzeall paraffin-embeddedmammospheres simultaneously in a singleslice, reducing slide number and staining procedures.

Figure 7.

Extracellular in vivo TGFb1 levels in human breast cancers. Twelvepostmenopausal breast cancer patients underwent microdialysis beforesurgery. One catheter was inserted into the breast cancer and another intoadjacent normal breast tissue, and extracellular TGFb1 in the microdialysateswas analyzed. �� , P < 0.01.






In summary, neutrophils may respond tomicroenvironmentalcues and acquire a protumorigenic phenotype. Our data provideinsight into these mechanisms in breast cancer, showing that E2promotes breast cancer metastasis by enhancing cell–cell inter-actions between neutrophils and ERþ breast cancer cells. E2increased the expression of LFA-1 via TGFb1, leading to cancercellmigration from the tumor primary site to distant sites, creatingnew metastatic niches. These interactions caused nonmetastaticcells to become highly metastatic. Our data are clinically relevantbecause we detected increased TGFb1 in breast cancers of women.Our work contributes to the understanding of the immune-modulatory effects of E2 in ERþ breast cancer that drive breastcancer metastasis and provides insights into potential therapeutictargets for disseminated hormone-dependent breast cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. DabrosinDevelopment of methodology: G. Vazquez Rodriguez, A. Abrahamsson,C. Dabrosin

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):G.VazquezRodriguez, A.Abrahamsson, L.D.E. Jensen,C. DabrosinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):G. Vazquez Rodriguez, A. Abrahamsson, C. DabrosinWriting, review, and/or revision of the manuscript: G. Vazquez Rodriguez,A. Abrahamsson, C. DabrosinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Vazquez Rodriguez, A. Abrahamsson,C. DabrosinStudy supervision: C. Dabrosin

Grant SupportThis work was supported by grants to C. Dabrosin from the Swedish Cancer

Society (2015/309), the Swedish Research Council (2013-2457), LiU-Cancer,and Research Funds of Link€oping University Hospital.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 1, 2016; revised January 18, 2017; accepted January 20, 2017;published OnlineFirst February 3, 2017.

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2017;5:234-247. Published OnlineFirst February 3, 2017.Cancer Immunol Res Gabriela Vazquez Rodriguez, Annelie Abrahamsson, Lasse Dahl Ejby Jensen, et al. and Activation of NeutrophilsEstradiol Promotes Breast Cancer Cell Migration via Recruitment

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