fibroblast subtypes regulate responsiveness of luminal...

Personalized Medicine and Imaging

Fibroblast Subtypes Regulate Responsiveness ofLuminal Breast Cancer to EstrogenHeather M. Brechbuhl1, Jessica Finlay-Schultz2, Tomomi M. Yamamoto1, Austin E. Gillen1,Diana M. Cittelly2, Aik-Choon Tan1, Sharon B. Sams2, Manoj M. Pillai3, Anthony D. Elias1,William A. Robinson1, Carol A. Sartorius2, and Peter Kabos1

Abstract

Purpose: Antiendocrine therapy remains the most effectivetreatment for estrogen receptor–positive (ERþ) breast cancer,but development of resistance is a major clinical complication.Effective targeting of mechanisms that control the loss of ERdependency in breast cancer remains elusive. We analyzed breastcancer–associated fibroblasts (CAF), the largest component of thetumor microenvironment, as a factor contributing to ER expres-sion levels and antiendocrine resistance.

Experimental Design: Tissues from patients with ERþ breastcancer were analyzed for the presence of CD146-positive(CD146pos) and CD146-negative (CD146neg) fibroblasts. ER-dependent proliferation and tamoxifen sensitivity were evalu-ated in ERþ tumor cells cocultured with CD146pos or CD146neg

fibroblasts. RNA sequencing was used to develop a high-confidence gene signature that predicts for disease recurrencein tamoxifen-treated patients with ERþ breast cancer.

Results: We demonstrate that ERþ breast cancers contain twoCAF subtypes defined by CD146 expression. CD146neg CAFssuppress ER expression in ERþ breast cancer cells, decrease tumorcell sensitivity to estrogen, and increase tumor cell resistance totamoxifen therapy. Conversely, the presence of CD146pos CAFsmaintains ER expression in ERþ breast cancer cells and sustainsestrogen-dependent proliferation and sensitivity to tamoxifen.Conditioned media from CD146pos CAFs with tamoxifen-resis-tant breast cancer cells are sufficient to restore tamoxifen sensi-tivity. Gene expression profiles of patient breast tumors withpredominantly CD146neg CAFs correlate with inferior clinicalresponse to tamoxifen and worse patient outcomes.

Conclusions: Our data suggest that CAF composition contri-butes to treatment response and patient outcomes in ERþ breastcancer and should be considered a target for drug development.Clin Cancer Res; 23(7); 1710–21. �2016 AACR.

IntroductionEstrogen receptor (ER) expression is the primary prognostic and

predictive biomarker for patients with breast cancer. Its presencedefines the luminal breast cancer subtypes (A and B) and deline-ates candidacy for antiendocrine therapy, which significantlyimproves survival outcomes (1, 2). Breast cancers, however,commonly display high heterogeneity of ER expression, whereindividual cells within a tumor vary in their level of ER expression.The fact that amajority of ERþ tumors contain a range of cells fromER� to ERþ led to the development of the Allred score for ERpositivity based on overall ER presence and intensity in anindividual tumor (3). Clinical presentationof only 1%ERþ tumorcells justifies the use of adjuvant antiendocrine therapy (3).

However, development of antiendocrine resistance remains amajor clinical problem that occurs in 40% of patients (4). Recur-rent tumors do not typically demonstrate complete loss of ERexpression (5); rather, they show a combination of both loss of ERexpression and loss of ER growth dependency.

To date, it remains unclear how individual tumors maintain abalance of ER-positive and -negative cells. Intrinsic cellular factorsdo not fully explain the range of ER expression within a singletumor; therefore, logic suggests the tumor microenvironment(TME) has a role in this phenomenon. In fact, expression patternsof proteins in the stromal/fibroblast component of breast cancer,such as platelet-derived growth factor receptor (PDGFRA andPDGFRB), CXCL1, CXCL14, CD10, and CD36, are prognostic ofpatient outcomes (6–12). Furthermore, Finak and colleaguesdescribe a stromal-derived profile consisting of seven stromalexpressed proteins that is predictive of breast cancer molecularsubtypes (13). Fibroblasts represent the most abundant cell typewithin the stroma (14), and we reasoned that in luminal breastcancer, the TME contains functionally and phenotypically distinctfibroblast subtypes that influence tumor cell ER expression andresponse to antiendocrine therapy.

The purpose of this studywas tofirst examinewhether subtypesof cancer-associated fibroblasts (CAFs) exist in luminal breastcancer and to then determine whether they have importantfunctional roles in dictating responsiveness of breast cancer cellsto estrogen. Intrinsic stromal fibroblasts are known to be hetero-geneous in both gene expression and function, which has madeit difficult to define functional subsets. CD146 (MCAM) was

1Department of Medicine, Division of Medical Oncology, University of ColoradoDenver, Aurora, Colorado. 2Department of Pathology, University of ColoradoDenver, Aurora, Colorado. 3Section of Hematology, Division of Hematology, YaleCancer Center and Yale University School of Medicine, New Haven, Connecticut.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Peter Kabos, University of Colorado Anschutz MedicalCampus, 12801 E 17th Ave MS 8117, Aurora, CO 80045. Phone: 303-724-3690;Fax: 303-724-3889; E-mail: [email protected]; and Heather Brech-buhl, [email protected]

doi: 10.1158/1078-0432.CCR-15-2851

�2016 American Association for Cancer Research.

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reported as a stromal cell-surface marker that defined fibroblastsubtypes in the hematopoietic stem cell niche (15). Given that alltissue stromal fibroblasts are mesenchymal in origin, we specu-lated that breast fibroblasts also contain both CD146-positive(CD146pos) andCD146-negative (CD146neg)fibroblasts and thatCD146 expressionwould define functional subsets of CAFs. Here,we describe a hierarchical organization in tumor-associated stro-ma, based upon CD146 expression, with implications for ther-apeutic sensitivity and disease progression.

Materials and MethodsCell culture

The human MCF-7 (p53 wild type, ERþ, luminal subtype)breast cancer cell lines were cultured in Modified Eagle Medium(MEM) supplemented with 5% FCS, nonessential amino acids(NEAAs), L-glutamine, and HEPES buffer at 37�C with a 5% CO2/95% atmospheric air. Human stromal cell lines HS5 and HS27Aand epithelial tumor cells UCD12 and T47D cells were grownin RPMI1640 supplemented with 5% FCS, NEAAs, penicillin(100 U/mL), and streptomycin (100 mg/mL). CD146 CAF sub-types are genetically and functionally akin to HS27a and HS5fibroblasts; however, unlike the HS27a and HS5 fibroblast celllines, our CAFs have a limited number of passages before theybecome senescent. Therefore, we used HS27a and HS5 fibroblastsin most of our studies and used our primary CAFs to verify ourfindings in a select set of studies. Unless otherwise indicated by thedesignation of CAF, described studies utilized HS27a and HS5fibroblasts. MCF-7, UCD12, and T47D were provided by thelaboratoryofC.A. Sartorius. The laboratoryofM.M.Pillai providedHS27a and HS5 cell lines. All cell lines used in this article wereauthenticated by STR profile testing in May 2016. For additionalmethods on cell culture drug line treatments and proliferationassays, please refer to the Supplementary Methods.

Generation of CAFsNormal and tumor tissue samples were collected from

patients at the University of Colorado Denver (Aurora, CO)

in accordance with an IRB-approved protocol. The tissue wascollected into ice-cold DMEM/F12 until ready for processing.Finely minced tissue was placed in collagenase digestion buffer(DMEM/F12 with 10 mmol/L HEPES, 2% BSA, 5 mg/mL insu-lin, 100 ng/mL hydrocortisone, 300 U/mL collagenase IV(1 mg/mL) and 100 U/mL hyaluronidase) overnight on arotator at 37�C. Digestion buffer was used at a volume of 10mL per 1 g of tissue. Following digestion, any oil layer wasgently aspirated off (common to normal breast tissue), and thesample was filtered through a 100-mm mesh into a 50-mLconical tube and centrifuged at 1,000 � g, at 4�C for 5 minutesto pellet the cells. The cell pellet was resuspended in 10 mL ofPBS and filtered through a 40-mm mesh into a new 50-mLconical tube. Differential centrifugation was used to enrich forstromal cell types. A slow speed, 80 � g, 4�C for 4 minutes, wasused to pellet epithelial cells. The supernatant was collected fora second centrifugation step, 100 � g, 4� C for 10 minutes. Theresulting pellet was enriched for stromal cells and was resus-pended in DMEM/F12, 5% FBS, insulin, NEAAs, and penicillin/streptomycin and cultured in standard cell culture flasks. Non-adherent and dead cells were washed out with PBS at 4 hours,24 hours, and twice weekly media changes until the culturesreached confluence in a 30-mm dish. Confluent cell cultureswere immortalized with E6E7 virus as described previously (16).Following selection of transduced cells with G418, limitingdilution and clonal selection was used to generate CD146pos

and CD146neg fibroblast subtypes from each patient sample.CD146 expression was verified by cytometry. For additionalmethods on flow cytometry, please refer to the SupplementaryMethods.

Animal experimentsAll animal experiments were conducted in an AAALAC-

accredited facility at the University of Colorado Denver underan IACUC-approved protocol. MCF-7 tumors labeled withZS-green were established by injecting 1 � 106 cells into themammary fat pad of NOD scid gamma (NSG) female mice.HS27a or HS5 cells were mixed with the tumor cells at a 1:1ratio (n ¼ 3–6 mice per stroma subtype). Tumors were allowedto grow for at least 5 weeks prior to removal. All tumorsreceived continuous estrogen supplementation throughout thestudy, as described previously (17). For the tamoxifen study,MCF-7 cells mixed with HS5 or HS27a cells were randomized toeither the right or left mammary fat pad of each mouse. Thetumors were established for 3 weeks and then the mice wererandomized into groups receiving peanut oil or 80 mg/kg4-hydroxytamoxifen. Treatments were given 3 times per weekby intraperitoneal injection for 8 weeks.

Human samplesHuman samples were collected under an approved COMIRB

protocol from a phase II clinical trial performed at University ofColorado consisting of 80 patients with stage II and III newlydiagnosed breast cancer (both ERþ and ER�). The trial wasdesigned to assess cellular heterogeneity in patients receivingneoadjuvant therapy. Available samples from patients with ERþ

disease were used. A board-certified pathologist reviewed histol-ogy. The tamoxifen outcome data come from the following GEOrecord number, GSE6532 (18–20). For additional gene expres-sion and immunocytochemistry methods, please refer to theSupplementary Methods.

Translational Relevance

Estrogen receptor (ER)–positive breast cancer is the mostcommon subtype. Targeting ER is an effective therapy, butdevelopment of antiendocrine resistance remains a majorcause of treatment failure. Attempts to uncover and therapeu-tically target mechanisms of antiendocrine resistance havefocused mainly on tumor-intrinsic traits. Here, we identifytwo subtypes of cancer-associated fibroblasts (CAF), based ontheir CD146 expression. We further show that CAF subtypesdifferentially contribute to tumoral ER expression and tamox-ifen sensitivity. CD146neg CAFs enforce ER-independentgrowth and mediate tamoxifen resistance by activating recep-tor tyrosine kinase pathways. Furthermore, the CAF subtypespredict treatment response and patient outcomes. We believethat these findings have clear clinical implications and supporta direct role for the tumor microenvironment in modulatingresponse to antiendocrine therapy. Insight into CAF–tumorinteractions and recognition of CAF subtypes in breast cancercould lead to further improvements in personalized care.

Fibroblasts Regulate Estrogen Response in ERþ Breast Cancer

www.aacrjournals.org Clin Cancer Res; 23(7) April 1, 2017 1711




Statistical analysisStatistical analysis was completed using R-package software for

the gene expression datasets and with GraphPad Prism 6 analyt-ical software for all other experiments. For single comparisons, weused unpaired two-tailed t tests with assumptions of parametricdistribution Gaussian distribution and equal SDs. For multiplecomparisons, we used ordinary one-way ANOVA analysis withTukeymultiple comparisons tests. Significance was set at P < 0.05.All cell culture experiments consisted of at least n¼ 4 ormore andwere repeated at least once using the same ERþ breast cancer celltype, different ERþ subtype. Our in vivo experiment consisted ofn ¼ 3 to 6 animals per stromal subtype. Outliers were consid-ered to be two SDs from the mean, and data are presented asmean � SEM.

ResultsCD146 expression identifies subtypes of normal andcancer-associated stroma in breast tissue

To determine the prevalence of CD146pos/CD146neg cells inbreast cancer–associated stroma, we used dual immunohisto-chemical staining for CD146 and for the strongly associated

stromal marker vimentin. The stromal component of ERþ breastcancer–associated patient tissues contains mixed populations ofCD146pos and CD146neg cells (Fig. 1A). In fact, the stainingrevealed striking differences in the intensity and frequency ofCD146pos stroma between patient samples. We quantified thesedifferences by dual immunofluorescence (IF) staining for CD146and vimentin in a cohort of 17 patient samples previously scoredby a board-certified pathologist for ER expression.

In the clinical practice of pathology, cellular morphology isthe standard in identifying tumor cells and is the basis ofpathologic diagnosis. Stromal cells are long and spindly andbland in appearance with small nuclei and fine chromatin. Incontrast, breast tumor cells are easily identified as large pleo-morphic cells with anisonucleosis, characterized by coarse chro-matin and prominent nuclei. We stained our patient cohort fortumor cell marker cytokeratin 18 (CK18) and stromal markervimentin to verify that tumor cells were maintaining a distinctepithelial phenotype. All of our patient samples contained onlyCK18-positive, vimentin-negative tumor cells, and the stromalcompartment was characterized by spindle-shaped, vimentin-positive, CK18-negative cells (Supplementary Fig. S1). On thebasis of this combination of morphologic characteristics and

Figure 1.

Two subtypes of fibroblasts are presentin normal and breast cancer–associatedstroma based on CD146 expression, andincreased ratios of CD146pos fibroblastscorrelate with high ER expression. A,Immunohistochemical staining of patienttissue demonstrating the presence ofboth CD146pos (A; vector red stain, bluearrowhead) and CD146neg (A; DAB stain,yellow arrowhead) stroma. B,Quantification demonstrating thatpatients with Allred >6 have significantlyincreased CD146pos stroma compared toAllred � 6. C and D, IF staining of tissuefrom patients with ERþ breast cancerdemonstrates a decreased ratio ofCD146pos (C and D, red stain) to vimentin(C and D, green stain) expressing stromain patients with low Allred (C) comparedwith high Allred (D) scores. E,Representative histogramsdemonstrating the presence of bothCD146pos and CD146neg stroma inpatient-derived normal and breastcancer–associated tissue. n ¼ 14 patienttumor stroma samples and n ¼ 11matched normal breast tissue samples.Scale bars, 100 mm. T, tumor; V,endothelial vessels.

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supporting immunohistochemical pattern, we determined thatit was possible to distinguish tumor cells from stromal cells withhigh confidence.

On the basis of our cohort size, we used Allred 6 as our cut-offpoint for determining whether there was a potential relationshipbetween CD146 expression in the stromal component of breastcancer and the Allred score for ER expression. Ten samples hadAllred scores greater than 6 (high ER) and seven had scores lessthan or equal to 6 (lower ER). We quantified our IF staining bydetermining the percentage of vimentinþ/CD146pos andvimentinþ/CD146neg stroma. For this analysis, we manuallyexcluded tumor epithelial cells (vimentin negative, CK18 pos-itive) and obvious vessel structures (vimentin positive). Patientswho had an Allred score greater than 6 had a mean of 68%CD146pos stroma, whereas Allred scores equal to or less than 6had a mean of 18% CD146pos stroma (Fig. 1B). The range ofvimentinþ/CD146pos stroma cells in our 17 samples was 2% to87% (Fig. 1C and D). These data imply that threshold expres-sion for breast tumor ER is correlated to the CD146pos fibroblastsubtype.

We next tested normal breast tissue and cancer-associatedbreast tissue for the presence of CD146pos and CD146neg stromalsubtypes. Primary human cancer–associated and normal breaststroma cells were isolated from 11 ERþ breast cancer patientsamples, enriched for fibroblasts, and established as primary celllines using immortalization and clonal expansion. We deter-mined stromal cell enrichment by flow analysis and found thatour stromal isolation method resulted in a highly enrichedfibroblast fraction (95% VIMposþ, 92% FSP1þ) and was depletedof epithelial (3.9% CD136pos) and endothelial (CD31pos) cells,with minimal contribution of possible pericytes (0% CD31pos/NG2pos, 4.4% FSP1neg/NG2pos; Supplementary Figs. S2A, S2B,and S3A). We further validated that our cells expressed vimentinby gene expression and immunofluorescence staining (Supple-mentary Fig. S3B–S3D). Our method utilized several wash stepsto deplete the sample of poorly adherent hematopoietic cells.Finally, we flow sorted our clonal cell lines for expression ofCD146, and we identified two subtypes of normal fibroblasts(NBF) and CAFs in both normal breast and breast cancer–asso-ciated patient tissues (Fig. 1E). Taken together, these data dem-onstrate that the breast TME is composed of at least two CAFsubtypes, which can be identified according to CD146 expressionand are common to both normal breast stroma andbreast cancer–associated stroma. Patients with lower ER expression based onAllred scores (�6) have significantly decreased expression ofCD146pos CAF compared with patients with high ER (Allredscores > 6).

CD146pos CAFs are functionally and phenotypically akin toCD146pos fibroblasts found in normal bone marrow

We next used the cobblestone area assay (CSA) to determinewhether the CAF subtypes we derived from human ERþ breastcancer tissue had similar functionality to the CD146pos andCD146neg bone marrow–derived HS27a and HS5 fibroblasts. Adescription of this assay can be found in the SupplementaryMethods. Mononuclear cell (MNC)/HS27a cocultures had 5.8-fold more CSA than MNC/HS5 cocultures (Supplementary Fig.S4A and S4B). Similar to the results with normal fibroblasts,MNCs cocultured with CD146pos compared with CD146neg CAFcells had significantly more CSA (threefold greater; Supplemen-tary Fig. S4A and S4B). These results show that our CAFs promote

equivalentMNCbehavior as normal bonemarrow–derived fibro-blasts when stratified by CD146 expression.

To determine whether CAF cell isolates were phenotypicallyakin to HS27a and HS5 normal fibroblasts, we used gene expres-sion signatures and hierarchical clustering analysis. The geneexpression signatures of CD146neg CAFs demonstrated significantsimilarity by clustering in the same family with normal HS5fibroblasts (Supplementary Fig. S4C). Likewise, CD146pos CAFsclustered with HS27 fibroblasts (Supplementary Fig. S4C). OurCAF cell lines have similar expression levels for genes associatedwith activated fibroblasts, including procollagen type I alpha,smooth muscle actin, and fibroblast activation protein alpha(Supplementary Table S1). These data support the assertion thatour human cancer–derived stromal subtypes have a fibroblastgene signature that is similar to the human bonemarrow–derivedHS27a and HS5 normal fibroblasts.

CAF subtypes differentially influence ER expression in breastcancer cells

To pursue a functional role for CAFs in distinguishing tumorcharacteristics, we compared the phenotype and growth of ERþ

breast cancer cells (BCC) grown in conjunction with the twofibroblast subtypes. ERþ MCF-7 BCCs were cocultured withCD146pos and CD146neg fibroblasts in estrogen-depleted mediafor 5 days and stained for ER using IF (Fig. 2A). Cytokeratin 18(CK18) was used to positively identify tumor cells. ER expressionwas significantly higher inMCF-7 cells when they were coculturedwith CD146pos fibroblasts (74% vs. 37% ERþ cells, Fig. 2B).Similar results were observed when we cocultured BCCs with ourprimary CAF subtypes (Supplementary Fig. S5A).

To assess whether CAF subtypes would similarly affect ER intumors, we grew ERþMCF-7 cells coimplanted with the fibroblastsubtypes as xenografts. A 1:1 mixture of MCF-7 cells was injectedwith CD146pos or CD146neg fibroblasts. Tumors were establishedand allowed to grow with estrogen supplementation for 4 weeksprior to collection. Tumor sizes at 4 weeks were not significantlydifferent between fibroblast subtypes. We costained the tumorswith ER plus CK18 to identify tumor cells. MCF-7 xenografttumors mixed with CD146pos fibroblasts expressed higher levelsof ER (Fig. 2C) compared with MCF-7/CD146neg mixed tumors(38% vs. 24%, Fig. 2D). These data show that CD146neg fibro-blasts drive decreased ER expression in BCCs and suggest onepossible mechanism for stroma-induced development of anti-endocrine resistance.

ERþ breast cancer cells use ER growth-dependent pathwayswhen stimulated by CD146pos fibroblasts

To determine whether CD146-positive or -negative fibroblastsinfluence estrogen-dependent proliferation in ERþ breast cancercells, we used coculture and conditioned media (CM) experi-ments. We analyzed proliferation rates using a total protein assayor live cell imaging using Incucyte Zoom.MCF-7 cell cultures weregrown in estrogen-starved conditions for 72 hours prior to treat-ment with CM. RT-PCR analysis–verified ER expression, in theabsence of estrogen, was decreased simply by treatingMCF-7 cellswith CM from CD146neg fibroblasts (Fig. 3A). In vehicle-treatedsamples after 72 hours, CM from both CD146pos and CD146neg

fibroblasts increased MCF-7 BCC proliferation 5-fold (Fig. 3B).Treatment with estrogen (17b-estradiol, E2) significantlyincreased proliferation of BCCs grown in unconditioned orCD146pos CM (2.9-fold and 1.3-fold, respectively; Fig. 3B).






Estrogen did not alter proliferation of BCCs treated withCD146neg CM (Fig. 3B). Tamoxifen significantly inhibited estro-gen-induced proliferation of BCCs in unconditioned or CD146pos

fibroblasts CM by more than sevenfold (Fig. 3B). However,proliferation of BCCs with CD146neg fibroblast CM was not

significantly changed with tamoxifen (Fig. 3B). Similar resultswere obtained using cocultures of MCF-7 with fibroblasts insteadof CM, and with two other ERþ BCCs, UCD12 (SupplementaryFig. S5B) and T47D (Supplementary Fig. S5C and S5D). Thesedata demonstrate that ERþ BCCs influenced by CD146pos

Figure 2.

CD146pos CAFs sustain ERexpression in ERþbreast cancer cells, whereasCD146neg CAFs promote decreasedER expression.A,Representative IF staining for ER (red)and the tumor cell maker CK18 (green) in cocultures of MCF-7 cells with HS27a (CD146pos) or HS5 (CD146neg) fibroblasts showing decreased ER expression inCD146neg cocultures compared with CD146pos cocultures. B, Analysis of four replicates imaged in three positions and quantified for the percentage of ERþ

tumor cells demonstrates a significant reduction in ER expression in cocultures with CD146neg fibroblasts. C, Mixed MCF-7/fibroblast (HS27a or HS5) tumors wereestablished in NSG mice and harvested for IF staining for ER (red) and CK18 (green) showing decreased ER expression in tumors mixed with CD146neg fibroblasts.D, Analysis of five animals per group, imaged in three positions and quantified for the percentage of ERþ tumor cells demonstrates a significant reduction in ERexpression in tumors mixed with CD146neg fibroblasts. Percent ERþ cells ¼ (triple-positive ERþ/CK18þ/DAPIþ)/(all CK18þ/DAPIþ). Outliers were consideredto be two SDs from the mean and excluded. Scale bars, 20 mm.

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fibroblasts remain estrogen responsive and antiestrogen sensitive;however, influence from CD146neg fibroblasts renders ERþ BCCsestrogen unresponsive and tamoxifen insensitive.

As our data indicated that CD146 fibroblast subtypes influenceBCC response to tamoxifen, we next tested the response oftamoxifen-resistant MCF-7/TAMR-1 (TAMR-1) cells to tamoxifenwhen grown under the influence of CD146pos or CD146neg

fibroblasts. TAMR-1 cells are a tamoxifen-resistant derivative ofMCF-7 BCCs (21).We cultured TAMR-1 cells in unconditioned orconditioned media from CD146pos or CD146neg fibroblasts andtreated with 10 nmol/L E2 alone or with 100 nmol/L 4-hydroxy-tamoxifen (tamoxifen). Tamoxifen treatment had no effect onproliferation of BCCs in unconditioned or CD146neg fibroblastsCM (Fig. 3C). However, TAMR-1 cells cultured in CD146pos

fibroblast CM had significantly decreased proliferation withtamoxifen (29% compared with control; Fig. 3C), suggestingTAMR-1 cells gained sensitivity to tamoxifen.

To substantiate our in vitro results, we establishedMCF7 tumorsmixed with CD146-positive or -negative fibroblasts in the mam-mary fat pad of NSG mice and then treated the mice withtamoxifen. Animals were given a 1 mg subcutaneous estrogenpellet that remained in place throughout the experiment. Tumorswere established for 3 weeks and then randomized into groupsreceiving tamoxifen or vehicle (average tumor size¼ 54mm3; Fig.4A). Two weeks after tamoxifen treatment, MCF-7/CD146neg

tumors were significantly larger than MCF-7/CD146pos fibro-blasts (P < 0.001; 326 � 67 mm3 vs. 73� 39 mm3, respectively).

Vehicle-treated MCF-7/CD146neg fibroblasts were significantlylarger than MCF-7/CD146pos tumors by 7 weeks (P < 0.001;151�84mm3vs. 151�84mm3, respectively).MCF-7/CD146neg

mixed tumors did not respond to tamoxifen treatment (Fig. 4Aand B). However, tamoxifen-treated MCF-7/CD146pos tumorswere significantly smaller than vehicle-treated MCF-7/CD146pos

tumors (P <0.03; 0.4mm3 vs. 0.17mm3; Fig. 4A and B). AlthoughMCF-7/CD146pos tumors were significantly smaller than MCF-7/CD146neg tumors, all of the tumors contained dense regions ofCK18-positive tumors with vimentin-positive stroma presentthroughout (Supplementary Fig. S6A). These data support thehypothesis that fibroblast subtypes can influence tamoxifen sen-sitivity of ERþ BCCs.

ERþ breast cancer cells activate receptor tyrosine kinasepathways when stimulated by CD146neg fibroblasts

Evidence suggests that early development of endocrine resis-tance involves cross-talk between ER, EGFR/HER2, and IGF1Rpathways (22). Therefore, we next determined whether growth ofERþ BCCs influenced by CD146neg fibroblasts was sensitive toEGFR inhibition. We cocultured GFP-labeled MCF-7 BCCs withCD146pos orCD146negfibroblasts in serum-reducedmedia (2.5%FBS) for 48 hours and then treated the cells with the EGFR-specificinhibitor gefitinib (10 mmol/L). Proliferation was unchanged forMCF-7 cells cocultured with CD146pos fibroblasts and treatedwith gefitinib, whereas it was reduced by 60% in gefitinib-treatedBCCs cocultured with CD146neg fibroblasts (Fig. 5A).

Figure 3.

Influence of CD146neg CAFs programsERþ breast cancer cells to bypassestrogen-dependent proliferation anddecrease tamoxifen (Tam) sensitivity.A,MCF-7 cells cultured for 5 days in CMfrom CD146pos (HS27a) fibroblasts havesignificantly more ER gene expressionthan MCF-7 cells grown in CM fromCD146neg (HS5) fibroblasts. B, SRB totalprotein analysis of MCF-7 cells culturedinCM fromCD146pos (HS27a)fibroblastshave significantly increasedproliferation in response to estrogentreatment and significantly decreasedproliferation in response to treatmentwith 4-OH-tamoxifen. In contrast, CMfrom CD146neg fibroblasts rendersMCF-7 cells unresponsive to estrogenand 4-OH-tamoxifen treatment. Dataare normalized against an estrogen-withdrawn (EWD) untreated control(Cont) collected at the time oftreatment. C, SRB total protein analysisof tamoxifen-resistant TAMR-1 cellscultured in CM from CD146pos (HS27a)fibroblasts have significantly decreasedproliferation when treated with 4-OH-tamoxifen. Veh., vehicle. Data arenormalized against an estrogen-withdrawn (EWD) untreated controlcollected at the time of treatment.�� , P < 0.001; �� , P < 0.0001.






To further examine this effect, we performed a dose–responseexperiment using CM from MCF-7 cells or CM from fibroblastsand treatment with 0 to 25 mmol/L gefitinib (EGFR inhibitor) or 0to 50 mg/mL trastuzumab (HER2-specific inhibitor). Proliferationwas measured with a total protein assay. At the maximum con-centration of gefitinib (2.5 mmol/L), MCF-7 cells had a growthreduction of 17%, 25%, and 46%when grown inCM fromMCF-7cells, CD146pos fibroblasts, or CD146neg fibroblasts, respectively(Fig. 5B). Trastuzumab treatment causedMCF-7 growth reductionbetween 3% to 12% in MCF-7 CM, 15% in CD146pos CM, and24% to 31% in CD146neg CM (Fig. 5C). Western blot analysis ofMCF7 cells treated with CM fromMCF-7, CD146pos, or CD146neg

fibroblasts demonstrates that MCF7 cells grown in all conditionsexpress a small amount of EGFR (Supplementary Fig. S6B) andthat only CM from the fibroblasts result in detectable phosphor-ylated EGFR protein (Fig. 5D). These data suggest that CD146pos

fibroblasts stimulate EGFR and CD146neg fibroblasts stimulateboth EGFR and HER2 in ERþ BCCs.

To further examine which tyrosine kinase pathways might beactive inCMcultures, we used a humanphospho-RTK array panel.As previously demonstrated byWestern blot analysis, MCF-7 cellsplus CD146pos or CD146neg CMwere positive for phospho-EGFR,but not phospho-ERBB2/HER2 (Fig. 5D). Interestingly, the cul-tures grown with CD146neg fibroblasts were also positive forphosphorylated IGF1R, which has been implicated in promotingtamoxifen resistance (23–25). Our data suggest that BCCs influ-enced by CD146neg fibroblasts escape estrogen-dependent pro-liferation and exhibit tamoxifen resistance through activation ofEGFR, HER2, and IGF1R.

CD146 expression in fibroblasts correlates with patientoutcomes

Previous analysis by Finak and colleagues (13) demonstratedthat the gene signature from the total stromal component of

patients with breast cancer accurately identifies normal versuscancer tissue and predicts patient outcomes (13). We comparedthe gene signatures ofHS27a,HS5, CD146pos CAFs andCD146neg

CAFs to the published gene expression data of normal and breastcancer–associated stroma. We verified considerable overlap ofexpressed annotated genes in all four cell types (HS27a, HS5,CD146pos, and CD146neg), with the gene set used to identifybreast stromal origin and to generate predictions of breast cancerpatient outcomes (128/163 stromal genes identified by Finak andcolleagues; ref. 13).We then used the 128 identified stromal genesin our dataset to determine whether CD146pos or CD146neg

fibroblasts clustered with normal breast stroma or breast can-cer–associated stroma in the published dataset.

Because HS5 cells and our CD146neg CAFs cluster in a singlefamily, and HS27a cells cluster with our CD146pos CAFs, wepooled the genes from each subtype together for our comparison.The gene expression profile fromHS5 (CD146neg) fibroblasts andourbreast cancer patient–derivedCD146negCAFs alignedwith theFinak and colleagues' gene profile pattern for breast cancer asso-ciated stroma, whereas a CD146pos gene profile from HS27a orour CD146pos CAFs alignedwith normal breast-associated stroma(Fig. 6A). Furthermore, CD146neg CAFs predicted poor/mixedclinical outcomes for patients with ERþ breast cancer comparedwith CD146pos CAFs, which were aligned with better clinicaloutcomes (Fig. 6B). These data demonstrate that CD146 expres-sion is a distinguishing characteristic of stromal fibroblasts in thenormal and diseased breast that mimics the fibroblast hierarchypresent in the hematopoietic system, demarcates normal versustumor-associated stroma, and is predictive of disease outcomes.

CAF-induced gene expression signature predicts breast cancerrecurrence in patients treated with tamoxifen

We cocultured MCF-7 cells with CD146pos or CD146neg fibro-blasts and used RNA sequencing to determine gene expression

Figure 4.

CD146neg CAFs promote tamoxifenresistance in ERþ breast cancer tumors.Mixed MCF-7/fibroblast (HS27a or HS5)tumors were established in NSG mice for3 weeks and then treated with 4-hydroxy-tamoxifen (4-OH-TAM) or peanut oil(vehicle) for 8 weeks.A, Tumors establishedwith CD146pos (HS27a) fibroblasts hadsignificantly smaller tumors by 6weeks aftertreatment with tamoxifen. In comparison,tumors established with CD146neg (HS5)fibroblasts did not respond to tamoxifentreatment. � , P < 0.05; �� , P < 0.01. B, Tumorsestablished with CD146pos (HS27a)fibroblasts and treated with tamoxifenweighed significantly less at excisioncompared with vehicle. In comparison,tumor established with CD146neg (HS5)fibroblasts did not respond to 4-OH-TAM.n ¼ 6 mice per group.

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changes in the BCCs. Analysis revealed that MCF-7 cells cocul-tured with CD146neg fibroblasts had increased expression oftranscripts from 21 genes identified in the literature as upregu-

lated in tamoxifen resistance (Supplementary Table S2; refs. 26–32). Conversely, MCF-7 cells cocultured with CD146pos fibro-blasts had increased expression of 15 genes identified in the

Figure 5.

Influence of CD146neg CAFs programs ERþ breast cancer to express tyrosine kinase receptor pathways shown to promote tamoxifen resistance. A, Live cell imagingand quantification of MCF-7 proliferation in response to the specific EGFR inhibitor gefitinib demonstrates that CD146neg fibroblasts (HS5) cause significantlydecreased proliferation in MCF-7 cells, whereas proliferation of MCF-7 cells cocultured with CD146pos (HS27a) fibroblasts is not significantly changed. Veh., vehicle.B, SRB total protein analysis of MCF-7 cells cultured in CM with 2.5% reduced charcoal-stripped serum from MCF-7 cells or either fibroblast subtype (HS27aand HS5) have significantly decreased proliferation in response to gefitinib treatment. C, SRB total protein analysis of MCF-7 cells cultured in CM with 2.5% reducedcharcoal stripped serum from CD146neg (HS5) fibroblasts significantly decreased proliferation in response to trastuzumab treatment. D, Western blot analysisofMCF-7 cells demonstrates that onlyMCF-7 cells grown in CM fromCD146pos (HS27) or CD146neg (HS5) fibroblasts express pEGFRprotein. Receptor tyrosine kinasearrays demonstrate phosphorylated EGFR protein (pEGFR) in MCF-7 cells influenced by both fibroblast subtypes (HS27a and HS5). Phospho-IGF1R is presentonly in MCF-7 cells influenced by CD146neg fibroblasts (HS5). �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.






literature as downregulated in tamoxifen resistance (Supplemen-tary Table S3). From these lists, we identified a set of nine genesdirectly influenced by fibroblast cocultures (five upregulatedgenes in MCF-7 cells cocultured with CD146neg fibroblasts andfour genes upregulated in MCF-7 cells cocultured with CD146pos

fibroblasts) that produced a high-confidence gene signature that

reliably predicts recurrence-free survival in patients treated withtamoxifen (training set: 181patients,P¼3�10�5; and validationset: 87 patients, P ¼ 0.00147) (Fig. 6C). Importantly, the nine-gene set was not predictive of recurrence-free survival in a set of125 patients that were not treated with tamoxifen, suggesting it islinked to tamoxifen resistance and not a mere association with

Figure 6.

CD146neg CAF gene signature predicts poorer clinical outcomes and produces an epithelial signature that is predictive of decreased recurrence-free survival intamoxifen-treated patients. A, Hierarchical clustering of Affymetrix gene expression data for normal fibroblasts (HS27a and HS5) and our primary CAFs comparedwith Finak and colleagues' data demonstrates that CD146pos fibroblasts cluster with normal breast stroma, whereas CD146neg fibroblasts cluster with tumor-associated stroma. B,Hierarchical clustering of Affymetrix gene expression data for normal fibroblasts (HS27a and HS5) and our primary CAFs compared with Finakand colleagues' data demonstrates that CD146pos fibroblasts predict for better patient outcomes and CD146neg fibroblasts for poorer patient outcomes.Triplicate samples were used for gene expression array analysis. C, Patient training and validation sets for a gene signature consisting of five genes (PRKCA,MACROD2, SMARCA4, BNIP3, and MYO1B) predicted to be up and four genes (RPLP1, CDC42EP4, MAP2K4, and SIAH2) predicted to be down in the epithelialcomponent of ERþ breast cancer was generated from RNA sequencing data in MCF-7/CAF cocultures. The gene signature demonstrates significant predictivepower of increased recurrence-free survival in patients with the CD146pos signature. �� , P < 0.001; �� , P < 0.0001.

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poor patient prognosis (Supplementary Fig. S6C). Furthermore, asignature composed of all 36 genes is not predictive of recurrence-free survival in the tamoxifen-treated groups (Supplementary Fig.S6D). Because CD146 correlates with Allred score, we askedwhether our gene set was predictive of recurrence-free survivalsimply due to the fact that ER expression correlates with betterpatient outcomes in patients treated with tamoxifen. Consistentwith this idea, we split the same data cohorts, along the median,into high and low ER expression and assessed recurrence-freesurvival. If our CD146-based gene signature and, by extension,the stromal composition were only a surrogate for ER expression,then a significant survival advantagewould be expected in patientswith higher ER expression. However, high ER gene expression wasnot predictive of recurrence-free survival in tamoxifen-treated or-untreated patients (Supplementary Fig. S6E–S6G), and we con-clude that the stromal composition significantly influences tamox-ifen response in a novel way that does not resolve simply on ERexpression. These data show that influence from CD146pos fibro-blasts is predictive of improved recurrence-free survival aftertamoxifen treatment,whereas influence fromCD146negfibroblastsproduces a tumor gene signature predictive of poorer prognosis inpatients with breast cancer treated with tamoxifen.

DiscussionThis study demonstrates how functionally distinct subtypes of

CAFs can directly affect ER expression and growth dependency inluminal breast cancers. We identified two subtypes of CAFs thatcontribute to tumor ERheterogeneity influence tumor response toantiendocrine therapy. Development of tamoxifen-resistant celllines, such as TAMR-1 cells, requires escalating doses and long-termagent exposure (up toone year; refs. 33, 34); strikingly, in thisstudywe show that a similar phenotype can be achieved by a shortcoculture (5 days) with CD146neg CAFs. Equally important, wecan reverse the tamoxifen-resistant phenotype by a short coculturewith CD146pos CAFs. These data strongly support the need toconsider CAFs to further elucidate mechanisms of antiendocrineresistance. As proof of principle, we were able to identify anepithelial gene signature enforced byCAF subtypes that accuratelypredicted recurrence-free survival in tamoxifen-treated patients.

Development of antiendocrine resistance has been linked toactivation of signaling pathways that converge on PI3K/AKT/mTOR signaling, including EGFR, HER2, and IGF1R (24, 35,36). Although there is reasonable controversy regarding the roleof IGF1R signaling in conditions of sustained antiendocrineresistance (37), several studies demonstrate IGF1R as amodifyingfactor (23–25, 35). Our data focus on short-term cultures anddemonstrate phosphorylation of EGFR and IGF1R as well asdecreased proliferation in response to the HER2 inhibitor trastu-zumab in ERþ BCCs influenced by CD146neg fibroblasts. Discov-eries of RTK activation in tamoxifen resistance prompted severalclinical trials utilizing single receptor tyrosine kinase inhibitors(RTKI) in combination with antiendocrine therapy as a way todelay endocrine resistance (38–42). Unfortunately, the variableoutcome in many of these trials led to conclusions that targetingRTKs was largely ineffective for ERþ breast cancer (42, 43).

Our data demonstrate that fibroblast subtypes, present in theTME, can dictate which RTKs are active in ERþ BCCs. For example,both CD146pos and CD146neg fibroblasts promote EGFR phos-phorylation in ERþ BCCs, but CD146neg fibroblasts also promoteIGF1R phosphorylation and sensitize ERþ BCCs to the HER2

inhibitor trastuzumab. These data would suggest that a singleEGFR RTKI combined with antiendocrine therapy might work fortumors with a high percentage of CD146pos fibroblasts in theTME, but a use of a broadRTKI that can target EGFR,HER2, and/orIGF1R simultaneously may be a better choice for tumors expres-singmostly CD146neg fibroblasts in the TME. In fact, recent effortssuggest that combining multiple RTKIs, or using a broad-spec-trum RTK inhibition approach with AKT/mTOR inhibitors incombination with antiendocrine therapy is a more effectiveapproach (44–50). Our data suggest that the examination of TMEand fibroblast subtypes may lead to improvements in personal-ization of care.

Specifically in luminal breast cancers, ER serves as both aprognostic and predictive marker in patients and forms the basisof clinical decision-making (1, 2). However, efficacy of treatmentis limited by development of antiendocrine resistance that leadsto treatment failure and disease recurrence and progression.Although ER expression is correlated with better patient out-comes, it does not predict for which patients will have recurrentdisease after tamoxifen treatment. Here, we have identified anepithelial gene signature based on stromal influence that ispredictive of recurrent disease after tamoxifen treatment. Thissignature could be used to guide aggressive treatment from thetime of diagnosis in patients with the CD146neg signature. Min-imally, our data present a new paradigm for considering CAFs as aheterogeneous population that has significant impact on endo-crine response. However, whether the ratio of CD146pos/CD146neg cells is host dependent remains to be answered. Priorstudies have shown recruitment of stromal components totumors, and it is therefore conceivable that some tumors aremore apt at recruiting distant stroma into the microenvironmentthan others (51–53). Furthermore, it is unclear whether recruitedCAFshave a givenCD146phenotype/signature prior to arriving orwhether this is a programmable state.

In summary, we have shown that CAFs do not represent ahomogeneous cell population but contain at least two distinctcellular subtypes that differentially influence breast cancer cellswith respect to their molecular characteristics, phenotypic behav-ior in disease progression, and markers of therapeutic response.Our data support the hypothesis that tumors hijack normalstromal components of the tissue microenvironment and use itto their advantage. The generation of CAF cell lines and their studywith a broader range of metastatic transplant models will alsoprovide a model system to functionally define the breast cancermicroenvironment. We believe that studies of CAF biology andimproved targeting of their interactions with tumor cells willenhance our ability to deliver personalized therapy.

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

Authors' ContributionsConception and design: H.M. Brechbuhl, T.M. Yamamoto, M.M. Pillai,C.A. Sartorius, P. KabosDevelopment of methodology: H.M. Brechbuhl, T.M. Yamamoto, A.-C. Tan,P. KabosAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H.M. Brechbuhl, J. Finlay-Schultz, T.M. Yamamoto,D.M. Cittelly, A.-C. Tan, P. KabosAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H.M. Brechbuhl, J. Finlay-Schultz, T.M. Yamamoto,A.E. Gillen, A.-C. Tan, S.B. Sams, P. Kabos






Writing, review, and/or revision of themanuscript:H.M. Brechbuhl, J. Finlay-Schultz, T.M. Yamamoto, D.M. Cittelly, A.-C. Tan, M.M. Pillai, A.D. Elias, W.A.Robinson, C.A. Sartorius, P. KabosAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H.M. Brechbuhl, P. KabosStudy supervision: H.M. Brechbuhl, P. KabosOther (pathology/histology support): S.B. Sams

AcknowledgmentsWe would like to acknowledge Veronica Wessells for assistance with tissue

processing, the animal core facility, and the Biochemistry and MolecularGenetics high-throughput sequencing core (School of Medicine) at the Uni-versity of Colorado, Denver.

Grant SupportThis work was supported in part by NIH (NCI CA164048 and CA205044 to

P. Kabos, HL104070 to M. Pillai, and CA140985 to C. Sartorius), Grohne Fundfor StemCell Research for Breast Cancer (to P. Kabos), and the Cancer League ofColorado (to P. Kabos). Additional support was provided in part by theUniversity of Colorado Cancer Center's Flow Cytometry Shared Resourcefunded by NCI grant P30CA046934.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 23, 2015; revised September 12, 2016; acceptedSeptember 19, 2016; published OnlineFirst October 4, 2016.

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2017;23:1710-1721. Published OnlineFirst October 4, 2016.Clin Cancer Res Heather M. Brechbuhl, Jessica Finlay-Schultz, Tomomi M. Yamamoto, et al. Cancer to EstrogenFibroblast Subtypes Regulate Responsiveness of Luminal Breast

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