Myeloid-Derived Suppressor Cells Endow Stem- like ... · progression, metastasis, and therapy resistance (6–8). In vivo, the stemness of cancer cells is not exclusively intrinsic

Microenvironment and Immunology

Myeloid-Derived Suppressor Cells Endow Stem-like Qualities to Breast Cancer Cells throughIL6/STAT3 and NO/NOTCH Cross-talk SignalingDongjun Peng1, Takashi Tanikawa1,Wei Li1, Lili Zhao2, Linda Vatan1,Wojciech Szeliga1,Shanshan Wan1, Shuang Wei1, Yin Wang1, Yan Liu1, Elzbieta Staroslawska3,Franciszek Szubstarski3, Jacek Rolinski4, Ewelina Grywalska4, Andrzej Stanisławek5,Wojciech Polkowski6, Andrzej Kurylcio6, Celina Kleer7,8, Alfred E. Chang1,8, Max Wicha8,9,Michael Sabel1,8,Weiping Zou1,8,10, and Ilona Kryczek1

Abstract

Myeloid-derived suppressor cells (MDSC) contribute toimmune suppression in cancer, but the mechanisms throughwhich they drive metastatic progression are not fully understood.In this study, we show how MDSC convey stem-like qualities tobreast cancer cells that coordinately help enable immune sup-pression and escape. We found that MDSC promoted tumorformation by enhancing breast cancer cell stem-like propertiesas well as by suppressing T-cell activation. Mechanistic investiga-tions indicated that these effects relied upon cross-talk betweenthe STAT3 and NOTCH pathways in cancer cells, with MDSCinducing IL6-dependent phosphorylation of STAT3 and activat-

ing NOTCH through nitric oxide leading to prolonged STAT3activation. In clinical specimens of breast cancer, the presence ofMDSC correlatedwith the presence of cancer stem-like cells (CSC)and independently predicted poor survival outcomes. Collective-ly, our work revealed an immune-associated mechanism thatextrinsically confers cancer cell stemness properties and affectspatient outcome. We suggest that targeting STAT3-NOTCH cross-talk between MDSC and CSC could offer a unique locus toimprove cancer treatment, by coordinately targeting a coupledmechanism that enables cancer stemness and immune escape.Cancer Res; 76(11); 3156–65. �2016 AACR.

IntroductionThe capacity of immunity to control and shape cancer progres-

sion (1, 2) has been the subject of intensive investigation. Activeprotective immunity contributes to tumor dormancy duringimmunoediting (3). Inhibitory immune elements formimmune-suppressive networks in the tumor microenvironmentand are the main obstacles for developing effective cancer immu-notherapies (4, 5). However, it is poorly understood whether the

immune-suppressive networks reshape cancer and cancer dor-mancy in specific human cancer, and in turn affect tumor pro-gression, metastasis, and therapeutic response in the immunecompetent host.

It is thought that dormant cancer cells may be stem-like cellswith high metastatic- and therapeutic-resistant potential. Thedefinition of cancer stem cells (CSC) remains largely operational.Nonetheless, CSCs are thought to be involved in tumor initiation,progression, metastasis, and therapy resistance (6–8). In vivo, thestemness of cancer cells is not exclusively intrinsic to CSCs.Extrinsic mechanisms (9) provided by the tumor microenviron-ment may be essential for forming the stem cell niche (10). Forexample, mesenchymal stem cells (10), myeloid cells (11–13),and T-cell subsets (14, 15) promote tumor metastasis. It is poorlyunderstood whether breast CSCs are the primary targets of thesecells (10, 14, 16, 17).

STAT3 activation is often observed in cancers, particularlymetastatic sites (18, 19). Persistently, activated STAT3 in tumorcells acts as a crucial oncogenic mediator and promotes tumor-igenesis (18, 20–23). Many factors can activate transiently STAT3.However, it is not well understood how STAT3 is persistentlyactivated in the cancer cells andwhat is the functional and clinicalconsequence of persistent STAT3 activation in human breastcancer.

In this report, we outline extensive studies on the interactionbetween myeloid-derived suppressor cells (MDSC) and CSCs inanimal models and patients with breast cancer, dissect cellular,and molecular mechanisms by which MDSCs reshape breastcancer stemness via STAT3 and NOTCH cross-talk, and reveal

1Department of Surgery, University of Michigan School of Medicine,AnnArbor,Michigan. 2DepartmentofBiostatistics,UniversityofMichi-gan School of Medicine, Ann Arbor, Michigan. 3St. John's CancerCenter, Lublin, Poland. 4Department of Clinical Immunology, MedicalUniversityofLublin, Lublin, Poland. 5DepartmentofOncology,MedicalUniversity of Lublin, Lublin, Poland. 6Department of Surgical Oncol-ogy, Medical University of Lublin, Lublin, Poland. 7Department ofPathology, University of Michigan School of Medicine, Ann Arbor,Michigan. 8University of Michigan Comprehensive Cancer Center,University of Michigan School of Medicine, Ann Arbor, Michigan.9Department of Medicine, University of Michigan School of Medicine,AnnArbor, Michigan. 10Graduate Programs in Immunology and TumorBiology, University of Michigan School of Medicine, Ann Arbor,Michigan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Weiping Zou, University of Michigan School of Med-icine, 109 Zina Pitcher Place, Ann Arbor, MI 48109. Phone: 734-615-5554; Fax:734-763-0143; E-mail: [email protected]; Ilona Kryczek, [email protected]

doi: 10.1158/0008-5472.CAN-15-2528

�2016 American Association for Cancer Research.

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the pathologic, clinical, and therapeutic relevance of the interac-tion between MDSCs and CSCs in breast cancer.

Materials and MethodsHuman subjects, tissues, and cells

Patients diagnosed with breast cancer were recruited in thestudies. All use of human subjects in this study was approved bythe local Institutional Review Boards. We collected fresh breastcancer tissues from the University of Michigan Surgery Clinic.Fresh tumors were processed into single cell suspension andimmediately used for MDSC and tumor (stem) cell analysis(13, 15, 24, 25). Specifically, tumor cell suspensions were pre-pared from solid tumors by enzymatic digestion in 50 mL ofHank's Balanced Salt Solution (Life Technologies) containing 40mg of collagenase, 4 mg of DNase I, and 100 units of hyaluron-idase (Sigma Co.) for 2 hours. Cells were washed twice in HBSS.MDSCs were enriched by depleting tumor cells, B- and T-cellsubsets (PE-selection kit; StemCell Technology) and 7-AAD exclu-sion, and sorted with high-speed sorter FACSaria (Becton Dick-inson) to high purity (>97%). We studied formalin-fixed, paraf-fin-embedded breast cancer tissues from 104Her-2/neuþ patientsin Poland (cohort 1; refs. 26, 27), and from 84 breast cancerpatients from the University of Michigan School of Medicine(cohort 2) and 90 breast cancer patients from Poland (cohort3) for this study (Supplementary Table S1). Cohort 1 was initiallyrecruited for clinical trials with Herceptin treatment (26, 27).Cohorts 2 and 3 were randomly recruited regardless of Her-2expression status. After pathologic review, a tissue microarray(TMA) was constructed from the most representative area ofparaffin-embedded breast cancer tissues. The TMAs were used forspecific immunohistochemistry staining. Fivehundred andninetythree patients with breast cancer were evaluated in The CancerGenome Atlas (TCGA) breast datasets in Oncomine.org

Immunohistochemistry analysisImmunohistochemistry staining was performed as previously

described (28–30). Tissues were stained with polyclonal rabbitanti-human-CD33 (1/10 dilution, DAKO; ref. 13), ormouse anti-human ALDH1 (Becton Dickinson; ref. 24). We used horseradishperoxidase–based detection system to detect positive cells (EnVi-sion, DAKO). The specimens were digitalized with an automatedplatform (Aperio Technologies) and ScanScope XT and SpectrumPlus using TMA software version 9.1 scanning system. Cores weremanually scored in high resolution of �40. A mean score ofduplicate cores from each individual tissue was calculated. Anydiscrepancies were resolved by subsequent consultation withdiagnostic pathologist. The cores were quantified and analyzedfor the expression of ALDH1 and CD33 with an Aperio imagingsystem (Genetix). Cytoplasmic expression of ALDH1 was evalu-ated, whereas nuclear staining alone was considered nonspecificand was not included in the analysis. Intensity of staining wasscored as 0 (no expression), 1þ (less than1%of positive cells), 2þ(1%–5%ofpositive cells), 3þ (5%–20%ofpositive cells), and4þ(more than 20% positive cells). The intratumoral CD33þ cellswere quantified and expressed as the numbers of CD33þ cells per0.6mm2 of tumor section. The tissues were divided into high andlow CD33þ cell infiltration based on the median value. Theoptimal cutoff points were calculated using the ROC curve.Human vimentin and E-cadherin were detected in paraffin-embedded MCF-7 cells with the PathScan EMT Duplex IF Kit(Cell Signaling Technology).

Cell linesMCF-7 andMDA-MB-231 cell lines (ATCC) were characterized

and authenticated by the vendor using short tandem repeat DNAprofiling. The cell lines were passaged in our laboratory for fewerthan 6 months. All cell lines were maintained in DMEM supple-mented with 10% FBS plus 100 U/mL penicillin and 100 mg/mLstreptomycin (P/S).

Human xenograft tumor modelHuman xenograft tumor model was established in female

NOD-scid IL2Rgnull (NSG) mice with modifications (13, 24,25). Briefly, MCF-7 breast cancer cells plus MDSCs were mixedwith anti-IL6 (200 mg, mouse IgG2b; R&D Systems) and iNOSinhibitor (L-NMMA, 50 mmol; EMD Millipore), and were subse-quently inoculated subcutaneously into NSGmice supplied withestradiol-17b pellet. Tumor development was monitored andtumor size was measured.

Immunosuppressive assayCD45þCD33þCD14þCD15�/dim and CD45þCD33þCD14�

CD15bright MDSC subsets were sorted from breast tumor tissues.MDSCs were cultured with T cells (4� 104) at different ratio in thepresence of 2.5 mg/mL anti-human CD3 and 1.25 mg/mL anti-human CD28 for 3 days. T-cell proliferation was determined byKi67 expression and CFSE dilution. T-cell cytokine expression wasevaluated by intracellular staining. The analysis was gated onCD3þ

T cells.

Lentiviral vector constructionMCF7 cells were transfected with GFP-expressing lentiviral

vectors encoding shSTAT3 (Supplementary Table S2) or nonfunc-tional scrambled control. After transfection, the transfected cellswere selected and cultured for further experiments.

Flow-cytometry analysis and cell sortingSingle cell suspensions were made from different organs and

tumor tissues. Cells were labeled with fluorescence-conjugatedantibodies to CD3, CD4, CD5, CD11b, CD14, CD15, CD16,CD19, CD33, CD45, HLA-DR, ALDH, and STAT3 (BD Pharmin-gen). The cells were analyzed by LSR II (BD Biosciences). Theprimary cells were sorted from fresh tissues by high speed sorterARIA (BD Biosciences).

Western blot analysisCell lysates were prepared with SDS lysis buffer and clarified

by centrifugation, and protein concentration were determined bythe BCA Protein Assay Kit (Thermo Scientific). Equivalentamounts of total cellular proteins were separated by SDS-PAGE,and transferred onto polyvinylidene difluoride membranes. Pro-teins are detected with the antibodies against STAT3 and p-STAT3(Y705; Cell Signaling Technology), NOTCH1 (Abcam), NICD(Cleaved NOTCH1 Val1744; Cell Signaling Technology), andGAPDH (6C5; Santa Cruz Biotechnology). Epithelial-mesenchy-mal transition (EMT)-associated proteins were detected with theEpithelial–Mesenchymal Transition (EMT) proteins AntibodySampler Kit (Cell Signaling Technology).

Real-time RT-PCR and ELISA assayReal-time PCR was performed as we described previously (24,

31). All the primers were included in the Supplementary Infor-mation (Supplementary Table S2). Cytokine production was

MDSC and Cancer Stem Cell

www.aacrjournals.org Cancer Res; 76(11) June 1, 2016 3157




measured by ELISA as described by the manufacturer's protocol(R&D Systems; DY206). Nitric oxide (NO) was detected in cul-tured supernatant according to themanufacturer's protocol (TotalNO and Nitrate/Nitrite Parameter Assay Kit; R&D Systems,KGE001) and normalized to the medium control.

Tumor sphere formationMDSCs were sorted from human breast tumor tissues by high-

speed sorter (FACSAria; BD Biosciences) as described previously(13). Sphere assay was performed as described previously (24,

32). Briefly, tumor cells or sorted tumor cell subsets were plated inultra-low attachment plates (Corning) in serum-free EBM-2 or X-VIVO medium (Lonza) supplemented with 5 mg/mL insulin(Sigma), 20 ng/mL human recombinant EGF (Invitrogen), at adensity of 1,000 to 10,000 viable cells per well. Spheres (>50 mm)were counted after 1 to 6 weeks.

MDSC and tumor cocultureHuman breast cancer cells (106/mL) were cocultured with

MDSCs in Transwell system with 3 mmol/L Notch inhibitor,

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Figure 1.MDSCs are functionally relevant in human breast cancer. A–D, phenotype of MDSCs. Fresh breast cancer tissueswere separated into single cell suspension. The cellswere stained with isotype control antibodies and relevant antibodies. Polychromatic flow cytometry analysis was performed on these cells by gating onsingle cells in different gates (G1–G3). A, the relationship among CD45þ, CD5þ, CD19þ, and CD33þ cells. Top, FSC, SSC, and CD45 gates; middle, isotype controls ingates 1�2�3; bottom, CD19, CD5 and CD33 in gates 1�2�3. B, the phenotype of MDSCs. The expression of CD11b, CD14, CD15, CD16, HLA-DR, and 7-AAD wasanalyzed in CD5�CD19�CD33þCD45þ cells. C, the percentage of different antigen expressing cells in CD5�CD19�CD33þCD45þ cells. D, the percentage of CD33expressing cells in CD45þ cells. One of 10 representative patients is shown (A and B). Columns represent the mean � SEM (C and D; n ¼ 10). E–G, MDSCssuppressed T-cell activation. CD3þT cellswere activatedwith anti-CD3 in the presence ofMDSCs. E, T cellswere culturedwithMDSCs at different ratio (MDSC: T-cell)for 48 hours. Ki67þCD3þ T cells were measured by flow cytometry; n ¼ 3. F, T cells were labeled with CFSE and subsequently cultured with MDSCs. TheCD3þ T-cell divisions were analyzed by FACS on day 10. G, effector molecules were detected in CD3þ T cells by intracellular staining and analyzed by FACS.MDSC: T-cell ratio was 0.5:1 (F and G). n ¼ 4, � , P < 0.03, Wilcoxon pair test.

Peng et al.

Cancer Res; 76(11) June 1, 2016 Cancer Research3158




g-secretase inhibitor I (Z-LLNLe-CHO; Calbiochem) or/and500 nmol/L STAT3 inhibitor (Cucurbitacin I, Calbiochem).Tumor cells were subjected to genetic and functional analyses.

Statistical analysisThe Wilcoxon signed-rank test was used to determine pair-

wise differences and the Mann–Whitney U test was used todetermine differences between groups. P < 0.05 was consideredas significant. All statistical analysis was done on Statisticasoftware (StatSoft Inc.). Overall patient survival was measuredfrom the date of diagnosis to tumor-related death. Data werecensored at the last follow-up for patients who were alive at thetime of analysis. Spearman correlation coefficients were com-puted to assess relationships between MDSCs and ALDH.Survival curves were constructed using the method ofKaplan–Meier and survival differences were assessed using thelog-rank test. The Cox proportional hazards model was used toassess the effect of MDSC infiltration after adjusting importantprognostic factors, including cohort, histotype, tumor type,stage, grade, and treatment. Statistical significance was definedas a P value of <0.05. All analyses were performed using SAS 9.3software.

ResultsMDSCS are functionally relevant in human breast cancer

We isolated myeloid cells from human breast cancer forthe phenotypic, molecular, functional, and clinical studies.Polychromatic flow-cytometry analysis demonstrated thatthere existed substantial CD45þ immune cell subsets,including CD19þCD45þ cells, CD5þCD45þ cells and CD5�

CD19�CD33þCD45þ cells in fresh breast cancer tissues(Fig. 1A). CD5�CD19�CD33þCD45þ cells expressed highlevels of CD11b and low to medium levels of CD14, CD15,CD16, and HLA-DR (Fig. 1B and C). There were 22% of lin�

CD33þCD11bþCD45þ cells in total CD45þ immune cellsin fresh breast cancer tissues (Fig. 1D). The lin�

CD33þCD11bþCD45þ cells were sorted by high speed sorterto high purity (>97%) and were subjected to a suppressionassay. These cells suppressed T-cell proliferation as shownby reduced Ki67 expression in T cells (Fig. 1E), decreasedCFSE-labeled T-cell divisions (Fig. 1F) and effector T cells(Fig. 1G). The percentages of CD14þCD15�/dim cells werehigher than that of CD14�CD15high cells (Fig. 1C) and the twosubsets were capable of inhibiting T-cell proliferation (Supple-mentary Fig. S1B). On the basis of the phenotype and immune

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Figure 2.MDSCs are clinically relevant in human breast cancer. Immunohistochemistry was performed in human breast tumor tissues. CD33þ cells were quantified in 0.6mm2.A, five representative cases with different levels of CD33þ cell infiltration are shown. Top, �40; bottom, �20. B–D, relationship between MDSCs and patientoutcome. Breast cancer patients were divided into high and low CD33þ cell infiltration based on the median value of CD33 expression (see Materials and Methods).Kaplan–Meier curves were drawn based on the OS of all the three cohorts (n ¼ 278, P < 0.01, log-rank test; B), or the cohort 1 (N ¼ 104, P ¼ 0.01, log-ranktest; C), or the combined cohorts 2 and 3 (N ¼ 174, P < 0.01, log-rank test; D).






suppressive capacity, lin�CD33þCD11bþCD45þ cells are referredas MDSCs.

MDSCS are clinically relevant in human breast cancerGiven the high levels of CD33 expression on MDSCs, we

attempted to quantify MDSCs with CD33 in the paraffin-fixedbreast cancer tissues. When we stained single cells from freshbreast cancer tissue cells with anti-CD33 (Supplementary Fig.S1A), we found that CD33high cells were basically confinedtoCD3�CD19�CD45þ cells (Supplementary Fig. S1A). Thus,CD33 may be an operational marker to phenotypically defineMDSCs in paraffin-fixed breast cancer tissues. We quantifiedCD33þ cells with immunohistochemical staining (IHC) in threepatient cohorts (Supplementary Table S1). The European patientcohort 1 (cohort 1) included 104 treatment-na€�ve Her-2/neuþ

primary breast cancer patients. The Michigan patient cohort(cohort 2) and the Poland patient cohort 3 (cohort 3) included84 and 90 breast cancer patients, respectively, regardless of theirHer-2/neu status (Supplementary Table 1S).We observed that thelevels of CD33þ cells were variable from patient to patient (Fig.2A). However, the levels of CD33þ cells were comparable among

three cohorts (Supplementary Fig. S2). For survival analyses,similar to our tumor-associated regulatory T-cell analysis (29),we summed the three cohorts and divided the patients into highand low groups based on the median levels of CD33þ cells.

Kaplan–Meier analyses indicated that high levels of CD33þ

cells correlated with reduced overall survival (OS) comparedwith low levels of CD33þ cells in the total patient population inunivariate analysis (Fig. 2B; Supplementary Table S1). Aspatients in cohort 1 were exclusively Her2þ, to avoid potentialbias due to patient distribution, we independently analyzed thecohort 1 and the combined cohorts 2 and 3. We found that highlevels of CD33þ cells were associated with reduced OS com-pared with low levels of CD33þ cells in the cohort 1 (Fig. 2C)and the combined cohorts 2 and 3 (Fig. 2D). In a multivariateanalysis, including covariates of cohorts, histology, tumor type,stage, treatment, and grade, high MDSC infiltration was againassociated with shorter survival in all the three cohorts (Sup-plementary Table S3), the cohort 1 (Supplementary Table S4)and the combined cohorts 2 and 3 (Supplementary Table S5).Thus, MDSCs are functionally and clinically important inpatients with breast cancer.

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Figure 3.MDSCs promote and correlate human breast CSCs. A, human MDSCs increased breast cancer sphere formation. Human MCF-7 breast cancer cells were culturedwith MDSCs in sphere forming condition (24). Numbers of spheres are expressed as mean � SEM, n ¼ 4, MDSCs derived from three different patients; � , P < 0.05,Wilcoxon pair test. B, human MDSCs increased ALDHþ breast CSCs. MCF-7 breast cancer cells and MDSCs were cocultured in sphere-forming condition(24). ALDHþ cells were determined by FACS gated on CD33� tumor cells. Results are expressed asmean� SEM, triplicates with MDSCs derived from three differentpatients; � , P < 0.05, Wilcoxon pair test. C, human MDSCs stimulated human breast CSC core gene transcripts. MCF-7 cells were cocultured with primaryMDSCs in Transwell for 48 hours. Stem cell core genes were quantified by real-time PCR. Results are expressed as the mean relative values� SD. Experiments weretriplicates with MDSCs from three different patients. D, relationship between MDSCs and ALDH-1þ CSCs in breast cancer tissues. CD33þ cells and ALDH-1þ

tumor cells were evaluated by immunohistochemistry staining in tissues obtained from patients of cohorts 1 and 2. CD33þ cells were quantified as the numbersof CD33þ cells/0.6mm2 in TMAs. ALDH-1þ cells were scored from 0 to 4 based on the intensity of ALDH-1 expression (see Materials and Methods). Theircorrelation was analyzed by Pearson correlation. N¼ 178; P < 0.00000001, R¼ 0.4. E, effects of MDSCs on human MCF-7 breast tumor growth in NSGmice. Humanbreast cancer MCF7 cells were mixed with MDSCs and inoculated subcutaneously into NSG mice supplied with E2 (estradiol-17b pellet). Tumor growth wasmonitored. N¼ 5/group; � , P < 0.05 (Wilcoxon pair test). F, effects of MDSCs on human MCF-7 incidences in NSG mice. 105 MCF7 cells were mixed with MDSCs andinoculated subcutaneously into NSG mice supplied with E2 (estradiol-17b pellet). Tumor incidence was monitored for 16 days. N ¼ 5/group.

Peng et al.





MDSCs induce human breast CSCsNext, we investigated the mechanisms by which MDSCs are

associated with poor patient outcome. CSCs contribute to tumorprogression and therapeutic resistance (7, 8, 33). We reasonedthat MDSCs might affect CSC biologic behavior. We showed thathuman breast cancer–associated MDSCs promoted MCF-7 breastcancer sphere formation (Fig. 3A). ALDH-1þ cells are enrichedwithCSCs in breast and ovarian cancer cells (24, 34).Weobservedthat MDSCs enhanced human breast ALDHþ cells (Fig. 3B),stimulated multiple core stem cell gene expression (Fig. 3C), buthave no effect on cancer cell proliferation (Supplementary Fig.S3A). To test whether MDSCs were directly associated with CSCsin patients with breast cancer, we quantified ALDHþ CSCs inhuman breast cancer tissues. Breast cancer cells expressed a varietyof ALDH levels (Supplementary Fig. S3B). The median levels ofCD33þ cells (MDSCs) positively correlated with that of ALDHþ

CSCs (Fig. 3D). Similar results were observed in the cohort 1(Supplementary Fig. S3C) and cohort 2 (Supplementary Fig. S3B),respectively. Finally, we evaluated the relevance of the interactionbetween MDSCs and tumor cells in the human xenograft model.Human cancer MDSCs were co-injected with MCF7 breast cancercells into in female NOD-scid IL2Rg null (NSG) mice. We foundthat MDSCs accelerated tumor progression (Fig. 3E). Further-more, MDSCs increased the incidence of breast cancer tumorformation in NSG model (Fig. 3F). Cancer stemness is oftenassociated with epithelial–mesenchymal transition (EMT). Wefound that MDSCs increased EMT-related gene expression asshown byWestern blotting (Supplementary Fig. S3E) and immu-nofluorescence staining (Supplementary Fig. S3F). The data sup-port a role ofMDSCs in human breast cancer in vivo. Thus,MDSCsare biologically and clinically linked to breast cancer stemness.

MDSCs induce humanbreast CSCs through STAT3 andNOTCHsignaling

STAT3 (18, 20–23) and NOTCH (20, 35, 36) activation isobserved in a variety of cancers and may control cancer progres-

sion and metastasis. We found that MDSCs strongly inducedSTAT3 phosphorylation in MCF-7 and MDA-MB-231 breast can-cer cells cocultured with MDSCs (Fig. 4A). Interestingly, NOTCHwas also activated in MCF-7 breast cancer cells by MDSCs asshownbyhigh expression ofNOTCH2,NOTCH3, CHERP,HEY1,and HEY2 transcripts (Fig. 4B) and of intracellular domain ofNOTCH (NICD) expression (Fig. 4C). MDSCs promoted breastcancer stemness and were associated with the levels of ALDHþ

breast CSCs in breast cancer tissues (Fig. 3). We hypothesized thatSTAT3 and NOTCH activation was involved inMDSC-stimulatedbreast cancer stemness. To test this hypothesis, we blockedNOTCH and STAT3 signaling in human MDSC and breast cancercell coculture. The Notch inhibitor or the STAT3 inhibitor par-tially, and their combination completely, reduced ALDHþ breastCSCs induced byMDSCs (Fig. 4D). The data indicate that MDSCsactivate STAT3 and NOTCH, and induce human breast CSCs.

MDSC-derived IL6 and NO mediate CSC inductionAs MDSCs induced CSCs partially via STAT3 activation, we

examined howMDSCs activated STAT3. IL6 has been reported toactivate STAT3 and was associated with tumorigenesis (18, 20–23). Tumor-associated CD14þCD15�/dim MDSCs expressed highlevels of IL6 (Fig. 5A; Supplementary Fig. S4A). Breast cancer cellsexpressed IL6 receptor (Supplementary Fig. S4B) and IL6-activat-ed STAT3 (Supplementary Fig. S4D). STAT3 inhibition partiallysuppressed breast CSCs induced by MDSCs (Fig. 4C). It suggeststhat MDSCs may promote CSCs via IL6-mediated STAT3activation.

As MDSCs induced CSCs partially through NOTCH activa-tion, we further examined how MDSCs activated NOTCH.MDSCs may release reactive nitrogen intermediates to suppressimmune responses (37). We detected high levels of NO in theculture supernatants of CD14þCD15�/dim and CD14�CD15þ

MDSCs (Fig. 5B; Supplementary Fig. S4C). GSNO-stimulatedNOTCH activation (Fig. 5C) and NOTCH gene expression(Fig. 5D).

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+MDA-MB-231Figure 4.

MDSCs induce CSCs through STAT3 andNOTCH signaling pathways. A, effects ofMDSCs on STAT3 activation in MDA-MB-231and MCF-7 tumor cells. Tumor cells werecocultured with MDSCs in Transwell system,and STAT3 phosphorylation in tumor cellswas detected by Western blot. One of threeexperiments is shown. B and C, effects ofMDSCs on NOTCH signaling activation inMCF-7. Tumor cells were cocultured withMDSCs in Transwell system. Expression oftumor Notch signaling transcripts wasquantified by real-time PCR on day 1 (B) andcleaved NOTCH (NICD) was detected byWestern blot on day 3. C and D, effects ofbiochemical inhibition of STAT3 and NOTCHon MDSC-induced human ALDHþ breast cells.Human breast cancer cells were coculturedwith MDSCs in the presence of Notch inhibitoror STAT3 inhibitor. Human ALDHþ breastCSCs were analyzed by FACS. Results areexpressed as the percentage ofALDHþ cells�SD; n ¼ 4; � , P < 0.05 (Wilcoxon pair test) incomparison with the control (MDSCs).






Next, we evaluated the relative impact of MDSC-derived IL6and NO on CSC induction. In the coculture of breast cancer cellsand MDSCs, anti-IL6 mAb or iNOS inhibitor partially, and theircombination completely, reduced sphere formation stimulatedby MDSCs (Fig. 5E). Thus, our results suggest that MDSC-derivedIL6 andNOmay collaboratively activate STAT3 andNOTCH, andinduce breast CSCs.

STAT3 and NOTCH cooperatively support cancer stemnessFinally, we investigated howMDSC-derived IL6 and NO coop-

eratively activate STAT3 and NOTCH, and support cancer stem-ness. We first evaluated whether STAT3 and NOTCH signalingreciprocally affected their counterpart's expression in the cocul-ture of MDSCs and breast cancer cells. Genetic knockdown ofSTAT3 with specific shSTAT3 abrogated the NOTCH activationmediated byMDSCs, as shown by reduced NICD expression (Fig.6A), and whereas the inhibition of NOTCH with NOTCH inhib-itor reduced STAT3 activation mediated by MDSCs (Fig. 6B).STAT3 inhibitor was included as a positive control (Fig. 6B).

We further examined the roles of NO and IL6 in MDSC-mediated STAT3 activation and the kinetics of STAT3 phosphor-ylation. MDSCs mediated potent tumor STAT3 activation (Fig.4A; Fig. 6A and B). This activation was partially reduced by iNOSinhibitors and anti-IL6 mAb, and completely blocked by bothiNOS inhibitor and anti-IL6 mAb (Fig. 6C). The data raise thepossibility that MDSCs induce potent STAT3 activation via IL6and NO signaling collaboration in human breast cancers.

We further investigated the kinetics and persistence of STAT3activation via IL6 and NO interaction. We observed that STAT3

activation induced by IL6 was faint within 1 hour (Fig. 6D). Incontrast, addition of GSNO sustained and prolonged STAT3activation (Fig. 6D). Interestingly, GSNO potently activatedNOTCH (Fig. 5C and D) and weakly stimulated STAT3 activation(Fig. 6D). Thus, the data suggest that MDSC-derived NO activatesNOTCH to facilitate and sustain persistent cancer STAT3 phos-phorylation stimulated byMDSC-derived IL6 and IL6may not besolely responsible for long-lasting STAT3activation in tumor cells.

We tested the relevance of the interaction between MDSCs andtumor cells in the human xenograftmodel.Human cancerMDSCswere coinjected with MCF7 breast cancer cells into in femaleNOD-scid IL2Rg null (NSG) mice. We found that MDSCs accel-erated tumor progression. The effect was blocked by the treatmentwith anti-IL6 and iNOS inhibitor (Fig. 6E). In line with this, wefound that MDSCs increased ALDH1 expression in tumor cellsand blockade of iNOS and IL6 abolished this effect (Fig. 6F andG). Furthermore, we analyzed the correlations among ALDH1A1,IL6, andCD33 transcripts in TCGAbreast cancer data (Oncomine.org.).We observed strong correlations amongALDH1A1, IL6, andCD33 transcripts (Supplementary Fig. S5). These data support arole of MDSCs and MDSC-derived IL6 and NO in human breastcancer progression in vivo.

Altogether, we have demonstrated that MDSC-derived IL6initiates STAT3 phosphorylation, MDSC-derived NO activatesNOTCH, and NOTCH subsequently and collaboratively actswith IL6 to promote prolonged STAT3 activation. Thus, MDSCsmay play a role in stimulating and maintaining CSC poolthrough the interaction between IL6/STAT3 and NO/NOTCH(Fig. 6H).

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Figure 5.MDSC-derived IL6 andNOmediateCSC induction. A andB, IL6 andNO released byMDSCs. Breast cancer–associatedMDSCswere cultured for 2 to 3days. IL6 (A) andNO (B) were detected in the supernatants and normalized to medium. Results are expressed as the mean values � SEM. Two independent experiments(MDSC 1 and 2) with triplicates; � , P < 0.05. C and D, effects of GSNO onNOTCH signaling. MCF-7 tumor cells were cultured with GSNO for 3 days. NICDwas detectedby Western blot analysis (C), and NOTCH associated gene expression was analyzed by real-time PCR (D). Results are expressed as the mean � SEM; N ¼ 4;� , P < 0.05. E, effects of IL6 and NO on human MDSC-induced breast cancer sphere formation. Human MCF-7 breast cancer cell sphere assay was performed for10 to 15 days with MDSCs in the presence of anti-IL6 mAb and iNOS inhibitors. One of four experiments is shown.

Peng et al.





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Figure 6.Activation of STAT3 and NOTCH supports cancer stemness. A, effects of shSTAT3 on MDSC-mediated NOTCH activation in MCF-7 cells. MCF-7 cells were culturedwith MDSCs for 24 hours in Transwell system. Active domain of NOTCH (NICD) was detected by Western blot analysis. B, effects of biochemical inhibitionofNOTCH andSTAT3 onMDSC-mediatedSTAT3 activation in breast cancer cells. MDA-MB-231 cellswere culturedwithMDSCs for 24 hours in Transwell system in thepresence of STAT3 inhibitor (Cucurbitacin I) or NOTCH inhibitor (g-secretase inhibitor I). STAT3 activation was detected by Western blot analysis. C, effectsof IL6 and NO on human MDSC-induced breast cancer STAT3 phosphorylation. Human breast cancer MDA-MB-231 cells were cultured with MDSCs in Transwellsystem in the presence of anti-IL6 mAb and iNOS inhibitor for 6 hours. STAT3 phosphorylation was detected with Western blot analysis. D, effects of IL6and NO on breast cancer STAT3 phosphorylation. MCF-7 cells were treated with IL6 and GSNO for different time periods. STAT3 phosphorylation was detectedwithWestern blot analysis. One of three experiments is shown. E–G, effects of MDSCs on MCF-7 breast tumor growth in NSG mice. MCF7 cells were coculturedwith MDSCs in the presence of anti-IL6 mAb and iNOS inhibitor, or isotype antibody for 1 hour, and inoculated subcutaneously into NSG mice suppliedwith E2 (estradiol-17bpellet). E, tumor growthwasmonitored.N¼ 5/group; � ,P<0.05 (Mann–WhitneyU test). F andG,ALDH1 cells in PAN-Keratinþ tumor cellswereevaluated by immunofluorescence staining of paraffin-embedded tissues. F, results are expressed as the mean relative values � SEM (Mann–WhitneyU test, P < 0.05). G, one of 5 representative tissues is shown. H, scheme of MDSC and CSC cross-talk. MDSCs activate STAT3 and NOTCH via IL6 and NO,respectively. NOTCH activation facilitates and sustains persistent STAT3 activation via IL6, and the cross-talk between IL6/STAT3 and NO/NOTCH promotes breastcancer stemness.






DiscussionIn this study, we have generated important novel insights into

MDSC and CSC immunobiology and pathology in the context ofhuman breast cancer. (i) MDSCs provide extrinsic signals for CSCrenewal and promote tumor metastatic and tumorigenic poten-tial. (ii) MDSCs affect CSC biology through IL6/STAT3 and NO/NOTCH signaling pathways. (iii) NO/NOTCH signaling enforcesand sustains persistent and potent IL6/STAT3 activation, andaffects cancer stemness. (iv) The interaction between MDSCs andCSCs is biologically and clinically relevant in patients with breastcancer.

Immune-suppressive effects of MDSCs are relatively well stud-ied in tumor-bearing mouse models (38). Myeloid cells, includ-ing MDSCs and macrophages, have been linked with cancerstemness (13, 39, 40). However, the non-immunologic effectsof MDSCs are poorly understood in human breast cancer. It hasbeen reported that peripheral bloodMDSCs correlatewith clinicalcancer stage, metastatic tumor burden, and doxorubicin–cyclo-phosphamide chemotherapy (41). In line with this, we havefound high numbers of MDSCs in breast cancer tissues. To oursurprise, MDSCs directly promote and maintain the CSC poolthrough two integrated signaling pathways: IL6/STAT3 and NO/NOTCH signaling pathways.

The link between IL6 and STAT3 has been reported in severaltypes of cancer (18, 20–23). Interestingly, IL6 alone inducestransient STAT3 phosphorylation, whereas MDSCs inducelong-lasting STAT3 activation. MDSC-derived NO activatesNOTCH and contributes to sustained STAT3 phosphorylationthrough IL6 andNO collaborative action. In support of this, it hasbeen demonstrated that NO stimulates NOTCH signaling anddelivers a survival signal to glioma cells (42) and drosophilablood cells (43). Thus, although many factors can regulateNOTCH and STAT3 signaling pathways in cancer, our worksupport the notion that MDSCs integrate the signaling networksbetween NO/NOTCH and IL6/STAT3 in breast cancer. We pro-pose that MDSCs contribute to persistent and potent STAT3activation in breast cancer, which promotes and maintains theCSC pool. Given the role of CSCs in cancer metastasis, our workalso supports the notion that STAT3 signaling is crucial formyeloid cell colonization at future metastatic sites (19).

After deciphering the molecular and cellular importance of thecross-talk between MDSCs and tumor cells in CSCs, we havefurther addressed the biological and clinical relevance of thiscross-talk in patients with breast cancer. MDSCs correlate withCSCs content in the human breast cancer microenvironment, andare adversely associatedwith patient survival. It has been reported

that response to Herceptin (44) and chemotherapy (45) is in partregulated by immune components in tumor-bearing mousemodels. Given the relevance of CSCs in tumor relapse and therapyresistance (7, 8, 33), our data point toward a possibility thatimmune-suppressive element, MDSCs, directly target the cancerstemness signaling pathway and may potentially affect cancertherapy. Altogether, our results suggest that anticancer therapyshould simultaneously target hostMDSCs and cancer (stem) cellsto improve therapeutic efficiency and abrogate therapy resistance.We have shown that CD33 is an operational marker for humantumor–associated MDSCs (13). Targeting CD33 is considered astrategy to treat patients with acute promyelocytic leukemia (46).Therefore, targeting CD33 signaling may be an optional regimento treat breast cancer patients.

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

Authors' ContributionsConception and design: T. Tanikawa, W. Zou, I. KryczekDevelopment of methodology: T. Tanikawa, S. WanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Peng, T. Tanikawa, W. Li, S. Wei, Y. Wang, Y. Liu,E. Staroslawska, F. Szubstarski, J. Rolinski, E. Grywalska, A. Stanisławek,W. Polkowski, A. Kurylcio, C. Kleer, A.E. Chang, M. Sabel, I. KryczekAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Peng, T. Tanikawa, L. Zhao, M. Sabel, I. KryczekWriting, review, and/or revision of the manuscript: D. Peng, M. Wicha,M. Sabel, W. Zou, I. KryczekAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Vatan, W. Szeliga, E. Staroslawska,F. Szubstarski, J. Rolinski, E. Grywalska, A. Stanisławek, W. PolkowskiStudy supervision: M. Wicha, I. Kryczek

AcknowledgmentsThe authors thank Daniel Hayes for fruitful discussion and intellectual

support, and Deborah Postiff, Michelle Vinco, Jackline Barikdar, and RonaldCraig in the Pathology Department for their technical assistance.

Grant SupportThis work was supported in part by research grants from the NIH/NCI R01

grants (W. Zou; CA123088, CA099985, CA193136, and CA152470) and theNIH through the University of Michigan's Cancer Center Support Grant(CA46592).

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 September 16, 2015; revised March 2, 2016; accepted March 17,2016; published OnlineFirst April 6, 2016.

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2016;76:3156-3165. Published OnlineFirst April 6, 2016.Cancer Res Dongjun Peng, Takashi Tanikawa, Wei Li, et al. SignalingBreast Cancer Cells through IL6/STAT3 and NO/NOTCH Cross-talk Myeloid-Derived Suppressor Cells Endow Stem-like Qualities to

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