Tumor-Contacted Neutrophils Promote Metastasis by a CD90 ... · 2/3/2019 · tems), or mouse CD90 (1:50, catalog no.105312, BioLegend) overnightat4 C,followedbyasecondaryantibody(HRPrabbit

Translational Cancer Mechanisms and Therapy

Tumor-Contacted Neutrophils PromoteMetastasis by a CD90-TIMP-1 Juxtacrine–Paracrine LoopYing Wang1,2, Jianing Chen1,2, Linbin Yang1,2, Jiaqian Li1,2,Wei Wu1,2, Min Huang1,2,Ling Lin3, and Shicheng Su1,2

Abstract

Purpose: The different prognostic values of tumor-infiltrat-ing neutrophils (TIN) in different tissue compartments areunknown. In this study, we investigated their different prog-nostic roles and the underlying mechanism.

Experimental Design: We evaluated CD66bþ neutro-phils in primary tumors from 341 patients with breastcancer from Sun Yat-sen Memorial Hospital by IHC. Theassociation between stromal and parenchymal neutrophilcounts and clinical outcomes was assessed in a trainingset (170 samples), validated in an internal validation set(171 samples), and further confirmed in an external vali-dation set (105 samples). In addition, we isolated TINsfrom clinical samples and screened the cytokine profile byantibody microarray. The interaction between neutrophilsand tumor cells was investigated in transwell and 3DMatrigel coculture systems. The therapeutic potential ofindicated cytokines was evaluated in tumor-bearing immu-nocompetent mice.

Results: We observed that the neutrophils in tumor paren-chyma, rather than those in stroma, were an independent poorprognostic factor in the training [HR = 5.00, 95% confidenceinterval (CI): 2.88–8.68, P < 0.001], internal validation (HR ¼3.56, 95% CI: 2.07–6.14, P < 0.001), and external validation set(HR ¼ 5.07, 95% CI: 2.27–11.33, P < 0.001). The mechanisticstudy revealed that neutrophils induced breast cancer epithelial–mesenchymal transition (EMT) via tissue inhibitor of matrixmetalloprotease (TIMP-1). Reciprocally, breast cancer cellsundergoing EMT enhanced neutrophils' TIMP-1 secretion byCD90 in a cell-contact manner. In vivo, TIMP-1 neutralization orCD90 blockade significantly reduced metastasis. More impor-tantly, TIMP-1 and CD90 were positively correlated in breastcancer (r2 ¼ 0.6079; P < 0.001) and associated with poorprognosis of patients.

Conclusions: Our findings unravel a location-dictatedinteraction between tumor cells and neutrophils and pro-vide a rationale for new antimetastasis treatments.

IntroductionNeutrophils are themost prevalent white blood cells in human

blood and constitute a significant proportion of inflammatorycells in tumormicroenvironment of various types ofmalignancies(1–3). In mouse models, their role in cancer metastasis is con-troversial. It has been reported that neutrophils mediated byG-CSF suppress antitumor immunity and promote tumor colo-nization of distant organs (4, 5). In contrast, it has been shownthat tumor-entrained neutrophils inhibit lung metastasis (6)andmAb-induced tumor reduction is abolished in mice depletedof neutrophils (7). These contradictory observations highlight

the diversity of tumor-associated neutrophils and a pressing needto further evaluate their function in a more patient-relevantscenario (8, 9).

In patients with breast cancer, elevated neutrophil-to-lymphocyte ratio (NLR) in peripheral blood has been associ-ated with poor prognosis, particularly in triple-negative sub-type, which is most difficult to treat due to the lack of endocrineand target therapies (10, 11). However, the clinical significanceof neutrophils in tumors is less studied. Furthermore, thedifferent prognostic values of tumor-infiltrating neutrophils(TIN) in different tissue compartments are largely unknown.

Epithelial–mesenchymal transition (EMT) plays a crucial rolein tumor metastasis (12, 13). It has been well documented thatEMT endows tumor cells with enhancedmotility and invasiveness(14). In addition, we and others demonstrated that tumor cellsundergoing EMT foster a permissive microenvironment, whichreciprocally promotes EMT and metastasis by suppressing T-cellfunctions (15), recruiting monocytes (16), and skewing macro-phage polarization to protumor phenotypes (17–19). Therefore,the interaction between EMT and tumormicroenvironment exertsprofound effects on tumor dissemination. Understanding itscomplex cellular and molecular mechanisms is important fordesigning more effective antimetastasis treatments (20, 21).

Here, we investigated the clinical values of neutrophils asprognostic biomarkers in tumor parenchyma and stroma, respec-tively. In addition, we studied the functions and cytokine profilesof primary TINs isolated from clinical samples. Moreover, we

1Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics andGene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University,Guangzhou, China. 2Breast Tumor Center, Sun Yat-sen Memorial Hospital, SunYat-sen University, Guangzhou, China. 3Department of Internal Medicine, TheFirst Affiliated Hospital, Shantou University Medical College, Shantou, China.

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

Y. Wang, J. Chen, L. Yang, J. Li, and W. Wu contributed equally to this article.

Corresponding Author: Shicheng Su, Breast Tumor Center, Sun Yat-senMemorial Hospital, Sun Yat-sen University, 107 YanjiangWest Road, Guangzhou510120, China. Phone/Fax: 8620-8733-2022; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-2544

�2018 American Association for Cancer Research.

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explored the underlying mechanism in the Matrigel coculturesystem and in vivo models.

Materials and MethodsPatients and specimens

We obtained 446 tissue samples from patients with stage I–IIIprimary breast carcinomas for this study. The patients wereenrolled from two independent breast cancer centers. A total of341 patients enrolled between January 21, 2003 andDecember 8,2011 from Sun Yat-sen Memorial Hospital (Guangzhou, China)were randomized into the training cohort (n¼ 170) and internalvalidation cohort (n¼ 171). The independent cohort consisted of105 patients enrolled between January 20, 2008 and July 3, 2012from the First AffiliatedHospital of ShantouUniversity (Shantou,China). The last follow-up timewas February 10, 2017. This studywas approved by the Clinical Research Ethics Committee of bothhospitals. Written informed consents were obtained from allpatients before recruitment. All procedures performed in thisstudy were in accordance with the ethical standards of the insti-tutional research committee and the 1964 Helsinki Declarationand its later amendments or comparable ethical standards.

IHCFormalin-fixed and paraffin-embedded surgical specimens or

xenografts, liver, and lung tissues from animal experiments weresectioned at 4 mmand used for IHC staining. Antigen retrieval wasperformed using a pressure cooker for 9 minutes in antigenunmasking solution (ETDA, pH 8.0), followed by incubated in3% H2O2 for 10 minutes and blocked with PBS containing 5%BSA for 15 minutes. Afterward, the samples were incubated withprimary antibodies against human CD66b (1:100, catalog no.555723, BD Biosciences), mouse tissue inhibitor of matrix metal-loprotease (TIMP-1; 10 mg/mL, catalog no. AF-980, R&D Sys-tems), or mouse CD90 (1:50, catalog no.105312, BioLegend)overnight at 4�C, followed by a secondary antibody (HRP rabbit/mouse, GTVision III, GeneTech) for 1 hour at room temperature.Then, immunodetection was performed by using DAB (Dako)according to the manufacturer's instructions. Images wereobtained by microscopy (BX51WI, Olympus).

Isolation and culture of human neutrophilsPrimary TINs were isolated from fresh invasive ductal carcino-

ma samples obtained from surgery as described previously withslight modifications (17, 22). Briefly, fresh tissues were mincedinto small pieces on ice and digested by collagenase type I,collagenase type III, and hyaluronidase (1.5 mg/mL, SigmaAldrich) at 37�C with agitation for 30 minutes in DMEM with

10% FBS. The cell suspension was collected by filtering through a70mmcell strainer and the isolation of neutrophilswas performedby a magnetic-activated cell sorting using direct CD66b IsolationKit (catalog no. 130-104-913, Miltenyi Biotec) according to themanufacturer's instructions (23). In addition, autologous neu-trophils were isolated from EDTA-anticoagulated peripheralblood of the same patients using positive selection of CD66bþ

cellswithmagnetic beads. Remaining erythrocyteswere lysedwithAqua Braun (B. Braun). The isolated neutrophils were cultured inDMEM supplemented with 10% FBS at 37�C in 24-well cultureplates (Costar, Corning). In some experiments, neutrophils wereseeded at 2 � 105 cells/well. The condition media were collectedafter 24 hours and centrifuged at 3,000 rpm for 15minutes at 4�Cto remove the cell debris. All samples were collected from thedonors with informed consent, and all related procedures wereperformed with the approval of the internal review and ethicsboards of Sun Yat-sen Memorial Hospital.

Giemsa stainingThe morphology of neutrophils was evaluated by Giemsa

Staining. The isolated neutrophils were smeared onto a slide andstained in commercial Giemsa dye (catalog no.G4486,GBCBIO).After 30minutes, the dye was poured and smear was washed withwater. Images were obtained bymicroscopy (BX51WI,Olympus).

Culture of cancer cellsHuman breast cancer cell lines MCF-7, MDA-MB-231, and BT-

474 and mouse breast cancer cell line 4T1 were obtained fromATCC and cultured according to standard protocols. For theinduction of EMT of breast cancer cells, MCF-7 cells were culturedalone, treated with 30% condition media of primary TINs or 10ng/mL rhTIMP-1 (catalog no. 410-01-10, PeproTech) for fivedays. The culture media were changed every two days. In someexperiments, conditionmedia of TINswere pretreatedwith 10mg/mL neutralizing antibodies against TIMP-1 (catalog no. AF-970,R&D Systems), GM-CSF (catalog no. MAB215, R&D Systems),angiogenin (catalog no. AB-265-NA, R&D Systems), or CCL2(catalog no. MAB679, R&D Systems) for 30 minutes at 37�C.

Coculture of cancer cells and neutrophilsNoncontact transwell system. A total of 2 � 104 breast cancer cellswere seeded in the top chamber and 1 � 105 neutrophils wereseeded in the bottom chamber of 24-well Boyden chambers with0.4-mm pore size (Corning Incorporated). After 24 hours, the topchamberswere removed. Theneutrophils in the bottomchamberswere washed by PBS and cultured in fresh complete media foranother 12 hours. Afterward, the culture media of neutrophilswere collected for subsequent ELISA assay.

Mixed coculture system. A total of 2 � 104 breast cancer cells and1 � 105 neutrophils were mixed together and seeded in 24-wellculture plate. Neutralizing antibodies (20 mg/mL) againstFrizzled2 [catalog no. MOB-2608z-S(P), Creative Biolabs],TMPRSS4 (catalog no. 33R-5428, Fitzgerald Industries Interna-tional), CD90 (catalog no. GTX88569-PEP, GENETEX), EphA4(catalog no. LS-E9103, LifeSpan BioSciences), andMac-1 (catalogno.101201, BioLegend) were added in the coculture. After 24hours, neutrophils were isolated by CD66b magnetic-activatedcell sorting (catalog no. 130-104-913, Miltenyi Biotec) and cul-tured in fresh complete media for another 12 hours. Afterward,

Translational Relevance

We found parenchymal neutrophils, but not the stromalones, were an independent risk factor in breast cancer, espe-cially the triple-negative subtype with poor prognosis dueto the lack of targeted therapy. Furthermore, we uncoveredthat tumor-contacted neutrophils promote metastasis by aCD90-TIMP-1 juxtacrine–paracrine cycle. This study enhancesthe accurate prognosis prediction and provides new therapeu-tic strategies for patients with breast cancer.

Wang et al.

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the culture media of neutrophils were collected for subsequentELISA assay.

Three-dimensional coculture. Cells were seeded within the 8-wellchamber slides that were precoated with growth factor–reducedMatrigel (catalog no. 354230, BD Biosciences). A total of 2� 103

MCF-7 cells and neutrophils were mixed in a ratio of 1:5 andsuspended in the 400 mL DMEM þ 10% FBS þ 2% Matrigel.Afterward, the mixed cells were seeded in the chamber slides andcultured for 2 days.

Flow cytometryNeutrophils were resuspended in PBS containing 1% FBS and

stained with fluorescent-conjugated antibodies against CD66b(catalog no. 130-104-395, Miltenyi Biotec), CD11b (catalog no.11-0118-41, eBioscience), CD90 (catalog no. 328108, BioLe-gend) for 30 minutes at 4�C or propidium iodide solution(catalog no. BMS500PI, eBioscience) according to the manufac-turer's instructions. During the analysis of cytometry data, cellswere first gated on the basis of forward (FSC-A) and side (SSC-A)scatters to exclude cell debris.

Immunofluorescent stainingParaffin-embedded samples were sectioned at 4-mm thickness.

Antigen retrieval was performed by a pressure cooker for 15minutes in 0.01 mol/L citrate buffer (pH 6.0). Then, sectionswere blocked in PBS containing 10%donkey serumor 2%BSA for1 hour at room temperature. Cells for immunofluorescent stain-ing were fixed by 4% paraformaldehyde for 15 minutes at roomtemperature, washed with PBS, and permeabilized with 0.2%Triton X-100 in PBS for 15minutes. Thereafter, cells were blockedin PBS with 2% BSA for 1 hour at room temperature. Afterblocking, samples were incubated with primary antibodiesagainst E-cadherin (1:100, catalog no. Sc-7870, Santa CruzBiotechnology), Vimentin (1:100, catalog no. Sc-66002, SantaCruz Biotechnology), CD90 (1:50, catalog no. 328108, BioLe-gend), cytokeratin (1:100, catalog no. ZM-0069, Zsbio),CD66b (1:100, catalog no.555723, BD Biosciences), and TIMP-1(10 mg/mL, catalog no. ZAa0429, Zsbio) overnight at 4�C. Theslides were then incubated in Alexa Fluor–conjugated secondaryantibodies (Invitrogen) for 1hour at room temperature.DAPIwasused for counterstaining the nuclei. The images were obtained bylaser scanning confocal microscopy (LSM780, Zeiss).

Migration and invasion assayMigration and invasion assay were performed in 8-mm 24-well

Boyden chambers (Corning). For invasion assay, Matrigel (cata-log no. 354230, BD Biosciences) diluted with DMEM in 1:3 ratiowas precoated in the chambers and solidified for 30 minutes in37�C. A total of 5 � 104 breast cancer cells suspended in 100 mL0.2%BSADMEMwere added in the top chamber,whereas 2�105

primary TINs or autologous peripheral blood neutrophils in600 mL 5% FBS DMEM were added to the bottom chambers. Thecancer cells migrated and invaded for 10 hours and 24 hours,respectively. Afterward, cancer cells in the top chamber wereremoved by swab and the cells attached in the underside ofchambers were fixed by 4% paraformaldehyde for 15 minutesand then stained by 0.5% crystal violet. Quantification wasperformed by the mean number of cells in five microscopic fieldsper chamber. To offset the influence of proliferation, we pre-treated tumor cells with 10 mg/mL mitomycin C (catalog no.

S8146, Selleck Chemicals) to inhibit cell proliferation. In someexperiments, TINs in the bottom chamberwere pretreatedwith 10mg/mL neutralizing antibody against TIMP-1 (catalog no. AF-970,R&D Systems), GM-CSF (catalog no. MAB215, R&D Systems),angiogenin (catalog no. AB-265-NA, R&D Systems), or CCL2(catalog no. MAB-679, R&D Systems) for 30 minutes at 37�C.

Western blot analysisProtein was extracted from the cells using RIPA buffer, resolved

by SDS–polyacrylamide gels and then transferred to polyvinyli-dene difluoride membranes. Primary antibodies againstE-cadherin (catalog no. Sc-7870, Santa Cruz Biotechnology),Vimentin (cat no. Sc-66002, Santa Cruz Biotechnology), Snail(Cell Signaling Technology, catalog no. 9782T), Twist (Abcam,catalogno. ab49254), Slug (Cell Signaling Technology, catalogno.9782T), ZEB-1 (Cell Signaling Technology, catalog no. 9782T),CD90 (Abcam, catalogno. ab133350), andGAPDH(ProteinTech,catalog no. HRP-60004) were used. Peroxidase-conjugated sec-ondary antibodies (Cell Signaling Technology) were used and theantigen–antibody reaction was visualized by enhanced chemilu-minescence assay (ECL, Thermo Fisher Scientific).

Animal experiments4T1 cells-luc (5 � 105 cells/mouse) were inoculated into the

mammary fat pads of 6-week-old female BALB/c mice. For neu-trophil depletion, rat anti-Ly6G antibody (12.5 mg/mouse, cata-log no. BE0075-1, BioXcell) or rat IgG isotype control (12.5 mg/mouse, catalog no. BE0089, BioXcell) was administered daily viaintraperitoneal injection after the grafts were palpable. For neu-tralization of TIMP-1, TIMP-1 mAb (0.4 mg/kg, catalog no. AF-980, R&D Systems) or the IgG control (0.4 mg/kg, catalog no.ab37373, Abcam)was administrated via intraperitoneal injectiononce a week after the grafts were palpable. For neutralization ofCD90,CD90mAb (250mg/mouse, catalog no. BE0066, BioXCell)or the IgG control (250 mg/mouse, catalog no. BE0089, BioXcell)was administrated every 3 days via intraperitoneal injection afterthe grafts were palpable. We examined the metastasis using PET/CT imaging (Siemens) and IVIS Lumina Imaging System (Xeno-gen). The tumors, lung, and liver tissues were harvested forhematoxylin and eosin (H&E) and IHC staining when the tumorsreached 1.5 cm in diameter.

Cytokine antibody arraysWe used Human Cytokine Antibody Arrays V Kit (catalog no.

AAH-CYT-5-4, RayBiotech) to evaluate the cytokine profiles.The arrays were blocked, incubated with 1 mL of supernatantsfrom neutrophils overnight at 4�C, and then probed withbiotin-conjugated antibodies (1/250) for 2 hours. Afterward,the array membranes were incubated with horseradish perox-idase–linked secondary antibody (1/1,000) for 1 hour at roomtemperature. The membranes were then incubated with chemi-luminescent substrate and exposed to X-ray films. The signals asrelative units of spot color density were evaluated by ImageJsoftware. The Excel-based analysis software tools provided byRayBiotech company (https://www.raybiotech.com/analysis-tools) were used for the automatic data sorting, averaging,background subtraction, positive control normalization, anddata comparison. The formula used for data normalization is asfollows: X(nY) ¼ X(Y) x P1/P(Y), where P1 ¼ the average signaldensity of the positive control spots on the reference array,P(Y) ¼ the average signal density of the positive control spots

Neutrophils Induce EMT by a CD90-TIMP-1 Loop

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on Array Y, X(Y) ¼ the signal density for a particular spot onArray for sample, and "Y", X(nY) ¼ the normalized value forthat particular spot "X" on Array for sample "Y".

ELISATIMP-1 (catalog no. E-EL-H0184), GM-CSF (catalog no. E-CL-

H0081), angiogenin (catalog no. E-CL-H0006), CCL2 (catalogno. E-EL-H0020), CCL5 (catalog no. E-EL-H0023), VEGF (catalogno. E-EL-H0111), TNFa (catalog no. 88-7346-88), and TGF-b1(catalog no. 88-8350-22) ELISA kits were purchased fromElabscience or Thermo Fisher Scientific. All experiments wereperformed according to the manufacturer's instructions.

siRNA transfectionTo knock down specific target genes, cells were plated at 5 �

105 cells/mL and transfected with specific siRNA duplexes usingLipofectamine 3000 Transfection Reagent (Invitrogen, catalog no.L3000015) according to the manufacturer's instructions. siRNAswere provided by GenePharma Inc. Oligonucleotide sequence ofsiRNAs are as following: Twist siRNA-1, 50-GCAAGAUUCAGACC-CUCAATT-30 (sense), Twist siRNA-2, 50-CCUGAGCAACAGC-GAGGAATT-30 (sense).

Statistical analysisWe identified the optimumcutoff for high and lowneutrophils'

infiltration, TIMP1þ neutrophils, or CD90þ tumor cells in tumorsusing X-tile plots based on the association with the patients'survival by X-tile software version 3.6.1 (Yale University Schoolof Medicine, New Haven, CT) as we reported previously (24, 25).Statistical analyses were performed using SPSS version 19.0 (SPSSInc). AssociationbetweenTINs and clinicopathologic featureswasassessed by c2 test. DFS and OS was estimated using the Kaplan–Meier method and log-rank test. The univariate and multivariateCox proportional hazards models were used to identify indepen-dent prognostic factors. We used receiver operating characteristics(ROC) curves to investigate the prognostic sensitivity and spec-ificity of TINs. All experiments for cell cultures were performedindependently at least three times and in triplicate for each time.P values less than 0.05 were considered statistically significant.

ResultsNeutrophils are present in both tumor parenchyma and stromaof breast cancer

To investigate the presence of TINs in breast cancer, weobtained clinical breast cancer samples from two independentinstitutes and performed IHC staining for CD66b as a marker ofneutrophils (26). A total of 341 patients from Sun Yat-senMemorial Hospital were randomized into the training set(170) and internal validation set (171). The independent setcomprising 105 patients was enrolled from the First AffiliatedHospital of Shantou University (Shantou, China). Patient char-acteristics are listed in Supplementary Table S1.

CD66bþ neutrophils were absent in normal breast tissues. Incontrast, neutrophils can be observed in both parenchyma andstroma of breast cancer tissues (Fig. 1A). Among the training,validation and independent sets, TINs were identified in 78 of170 (45.88%), 74 of 171(43.3%), and 51 of 105 (48.57%) casesin tumor parenchyma, respectively, and60of 170 (35.29%), 63of171 (36.84%), and 39 of 105 (37.14%) cases in tumor stroma,respectively.

Parenchymal TINs, but not stromal ones, are associated withpoor prognosis

To investigate the clinical significance of TINs in breast cancer,we used X-tile plots to generate the optimum cut-off value (11.0cells/HPF) for the CD66bþ TINs in tumor parenchyma based onassociation with disease-free survival (DFS) in the training set(Supplementary Fig. S1A). The univariate analysis showed signif-icant association between TINs in tumor parenchyma and DFS(Fig. 1B; Supplementary Table S2).More importantly, the patientswith high TINs in parenchyma exhibited worse overall survival(OS) compared with the ones with low TINs in tumor parenchy-ma (Fig. 1C; Supplementary Table S3). Cox multivariate analysisdemonstrated that TINs in parenchyma was an independentprognostic factor for both DFS (Table 1) and OS (SupplementaryTable S4). In addition, high TINs in tumor parenchyma wassignificantly associated with advanced histologic grade, tumorsize, lymph nodes metastasis, high TNM stage, triple-negativebreast cancer subtype, and distant metastasis (SupplementaryTable S5). To confirm these findings, we applied the same cut-off setting to the validation group and independent one. In bothcohorts, TINs in parenchyma was an independent prognosticfactor (Table 1; Supplementary Table S4) and correlated withsimilar clinicopathologic features (Supplementary Table S5).

To assess the sensitivity and specificity of parenchymal TINs asa prognostic biomarker, we calculated the area under the receiveroperating characteristic curve (AUC). TINs in tumor parenchymashowed a good prognostic value of DFS in the training [AUC ¼0.72; 95% confidence interval (CI): 0.63–0.83], validation(AUC ¼ 0.77; 95% CI: 0.69–0.85), and independent set (AUC¼ 0.84; 95% CI: 0.76–0.92), respectively (Fig. 1D). Similarly,parenchymal TINs also showed a good prognostic value of OS inthe training (AUC¼0.79; 95%CI: 0.67–0.91), validation (AUC¼0.78; 95% CI: 0.69–0.88), and independent set (AUC ¼ 0.84;95% CI: 0.75–0.93), respectively (Supplementary Fig. S1B).

Similarly, we also used X-tile plots to generate the optimumcut-off value (5.0 cells/HPF) for the TINs in stroma in thetraining set (Supplementary Fig. S1C). We found that TINs atstroma was not significantly associated with DFS or OS in allthree cohorts (Supplementary Fig. S1D). High TINs in stromawas significantly associated with tumor size in the training setand lymphovascular invasion in internal validation set, whichwere not validated by other cohorts (Supplementary Table S6).Collectively, our data indicated that TINs in parenchyma, butnot ones in stroma, are associated with poor prognosis ofpatients with breast cancer.

Primary TINs promote metastasis of breast cancer cellsGiven TINs in tumor parenchyma were a prognostic factor

predicting poor prognosis, we further investigated its underlyingmechanism. We isolated primary TINs from freshly resectedhuman breast cancer tissues as reported previously (17, 22). Thepurity of isolated TINs determined by CD11bþCD66bþ cells weremore than 90% (Fig. 2A). The isolated CD11bþCD66bþ cellsfrom tumors exhibited deeply lobed nucleus (Fig. 2B and C). Theexpression of CD66b remained unchanged after magnetic sortingand up to 48 hours of culture (Supplementary Fig. S2A). Theapoptosis of neutrophils increased after 48 hours of culture alone.However, coculture with tumor cells significantly reducedthe apoptosis of neutrophils (Supplementary Fig. S2B). Weobserved that primary TINs, rather than untreated autologousneutrophils isolated from peripheral blood, significantly

Wang et al.





Figure 1.

TINs in parenchyma, but not the ones in stroma, are associated with poor prognosis. A, Representative images for IHC staining of CD66b in normal breasttissue, breast cancer tissue, and appendicitis tissue. The neutrophils in appendicitis tissue served as positive controls for CD66b staining. Boxes indicate the areawithhigher magnification. Scale bars, 50 mm. B and C, Kaplan–Meier survival curves for patients with breast cancer with high and low infiltration of CD66bþ TINsin tumor parenchyma in the training (n ¼ 170), internal (n ¼ 171), and independent (n ¼ 105) cohorts, respectively. The optimal survival cutoff point forthe CD66bþ TINs infiltrationwas determined by X-Tile based on the associationwith disease-free survival (B) or overall survival (C) in the training cohort and appliedto the other two cohorts. D, ROC analysis and AUC were used to assess the prognostic capacity of DFS according to the parenchymal CD66bþ TINs.






promoted migration and invasion of multiple breast cancer celllines (Fig. 2D and E). In BALB/c mice, depletion of neutrophilsby anti-Ly6G inhibited the metastasis of synergic 4T1 cells toboth lung and liver (Fig. 2F and G).

EMT plays a crucial role in breast cancer metastasis. There-fore, we investigated whether TINs induce EMT of breast cancercells. We cultured epithelial-like breast cancer cell MCF-7 alone,or with 30% conditioned media from untreated neutrophils orautologous primary TINs. We found that MCF-7 cells treatedwith the conditioned media of TINs, but not the ones culturedalone or treated with the conditioned media of control neu-trophils became stretched and elongated (Fig. 2H), downregu-lated in epithelial biomarker E-cadherin expression, whileincreased in mesenchymal biomarker vimentin expression(Fig. 2H and I). In addition, stimulation with the conditionedmedia of TINs increased levels of EMT transcriptional factorTwist, but not Snail, Slug, or ZEB1 in tumor cells (Fig. 2I).Together, these results indicated that TINs induce EMT andmetastasis of breast cancer cells.

Primary TINs promote metastasis of breast cancer cellsby TIMP-1

To identify the cytokines secreted by TINs that induce EMT, weused antibody array to compare the cytokine production profile ofprimary TINs to the one of autologous peripheral neutrophils.Three cytokines, TIMP-1, GM-CSF, and angiogenin, increased inTINs (Fig. 3A; Supplementary Table S7), which was confirmed byELISA (Fig. 3B).

To further explore which of these cytokines is responsible forEMT induced by TINs, we added neutralizing antibodies againstthese cytokines and observed that neutralization of TIMP-1, butnot GM-CSF or angiogenin, significantly reduced the migrationand invasion of tumor cells induced by TINs (Fig. 3C). Moreover,blockade of TIMP-1, rather thanGM-CSFor angiogenin,markedlyinhibited EMT of tumor cells treated with the conditioned mediaof TINs (Fig. 3D and E).

It has been reported that TGFb within the tumor microenvi-ronment induces a population of TINs with N2 phenotype (27).To investigate the difference of N1 and N2 cytokines betweenneutrophils and TINs, we evaluated the levels of TGFb1, N1cytokine (TNFa), and N2 cytokines (VEGF, CCL5, and CCL2) inTINs and paired peripheral neutrophils by ELISA. Our datashowed that CCL2, rather than TNFa, significantly increased inTINs compared with peripheral neutrophils (SupplementaryFig. S3A). VEGF and CCL5 exhibited a trend of elevation inTINs. However, the difference was not statistically significant(Supplementary Fig. S3A). Neutralization of CCL2 did notsignificantly reduce the migration and invasion of tumor cellsinduced by TINs (Supplementary Fig. S3B). The TGFb1 secreted

by TINs was very low (Supplementary Fig. S3A), suggestingTGFb1 may not be mainly derived from neutrophils in thetumor microenvironment.

Consistently, three-dimensional culture demonstrated that theTINs with increased TIMP-1 production compared with controlneutrophils induced EMT of cocultured tumor cells (Fig. 3F). Inaddition, treatment of rhTIMP-1 upregulated vimentin anddownregulated E-cadherin of tumor cells. rhTIMP-1 stimulationincreased the level of Twist, rather than other EMT factors such asSnail and ZEB1 (Fig. 3G). Moreover, rhTIMP-1 enhanced migra-tion and invasion of tumor cells (Supplementary Fig. S3C).Silencing Twist abrogated the EMT (Fig. 3G) and migration andinvasion (Supplementary Fig. S3C) induced by rhTIMP-1. In vivo,administration of TIMP-1 neutralization antibody intraperitone-ally markedly inhibited tumor metastasis (Fig. 3H). Collectively,these data demonstrated that TINs promote metastasis of breastcancer cells via TIMP-1.

TINs promote EMT by a CD90-TIMP-1 juxtacrine–paracrineloop

Given that TINs induced EMT by TIMP-1, we then askedwhether cancer cells undergoing EMT induce TIMP-1 overexpres-sion in neutrophils in turn. We induced EMT of MCF7 cells(MCF7EMT) by TIN conditioned media for 5 days. Then, wecoculturedMCF7EMT cells with unstimulated neutrophils isolatedfrom peripheral blood in the transwell system. We observed thatalthough TIMP-1 in cocultured neutrophils slightly increased, itslevel was much less than the one in TINs (Fig. 4A). Therefore, wemixed neutrophils with MCF7EMT cells for 24 hours and retrievedthe neutrophils by CD66b magnetic beads. We found that thesecretion of TIMP1 in neutrophils mixed with MCF7EMT cellssignificantly increased, which was comparable with the level inTINs (Fig. 4A). Thus, these data suggest that cancer cells under-going EMT induce TIMP-1 overexpression in neutrophils in a cell-contact manner.

A panel of membrane proteins was elevated in cancer cells afterEMT, including Frizzled2, TMPRSS4, CD90, and EphA4 (19, 28,29). Among them, neutralization of CD90 abrogated TIMP-1overexpression in neutrophils mixed with MCF7EMT cells(Fig. 4B). Consistently, neutralization of Mac-1, the neutrophilreceptor of CD90, in both a cis- and trans- manner (19, 30), alsoexerted similarly effects (Fig. 4C).

To investigate whether TIMP-1 induced upregulation of CD90in tumor cells, we induced EMT of MCF7 cells by treatment ofrhTIMP-1 and evaluated CD90 by flow cytometry, Western blot-ting, and immunostaining. We observed that CD90 level wasmarkedly elevated in rhTIMP-1–treated tumor cells comparedwith untreated ones (Supplementary Fig. S4A–S4C). Of note,treatment of TGFb1, another EMT inducer, also upregulated

Table 1. Multivariate Cox regression analyses of DFS in the training, validation, and independent cohorts

DFSTraining cohort (n ¼ 170) Validation cohort (n ¼ 171) Independent cohort (n ¼ 105)

Factor HR (95% CI) P HR (95% CI) P HR (95% CI) P

Tumor grade (III vs. I–II) 1.282 (0.718–2.289) 0.401 1.123 (0.644–1.959) 0.682 2.228 (0.935–5.307) 0.070Tumor size (>2 cm) 2.168 (1.152–4.079) 0.016� 1.976 (1.054–3.704) 0.034� 4.424 (1.274–15.368) 0.019�

LN (positive vs. negative) 3.098 (1.535–6.251) 0.002�� 2.970 (1.525–5.782) 0.001�� 4.947 (1.619–15.116) 0.005��

ER (positive vs. negative) 0.673 (0.340–1.335) 0.257 0.676 (0.337–1.356) 0.271 0.471 (0.180–1.229) 0.124PR (positive vs. negative) 0.875 (0.402–1.907) 0.737 0.924 (0.465–1.836) 0.822 1.800 (0.557–5.822) 0.326CD66bþ TINs in parenchyma (high vs. low) 3.331 (1.827–6.073) <0.001

��2.055 (1.132–3.731) 0.018� 4.546 (1.869–11.056) 0.001��

NOTE: P value was calculated by two-sided log-rank test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.Abbreviations: ER, estrogen receptor; LN, lymph node; PR, progesterone receptor; TNM, tumor–node–metastasis.

Wang et al.





Figure 2.

Primary human TINs promote EMT and metastasis. Fresh carcinoma tissues were digested by collagenase. The TINs and autologous neutrophils fromperipheral bloodwere isolated by CD66bmagnetic beads.A–C, The neutrophil purity was determined by flow cytometry (A) and immunofluorescent staining (B) forCD66b and CD11b. Arrows indicate the area ofmagnification. Scale bars, 10 mm.C, The Giemsa staining of isolated neutrophils. Scale bars, 200 mm.D and E,Migrationand invasion assays of cancer cells with indicated treatments were performed in the transwell system. Cancer cells were added in the top chamber, whereasprimary TINs or autologous peripheral blood neutrophils were added to the bottom chambers. Three independent experiments were performed for theneutrophils isolated from each of the 5 patients. D, Representative images. Scale bars, 100 mm. E, Statistical analysis, mean� SEM. �� , P < 0.001 by Student t test.F, Neutrophils in 4T1 tumor-bearing mice were depleted by anti-Ly6G intraperitoneal injection. Representative images of lung and liver metastasis evaluated byPET-CT. SUV, standardized uptake value. G, Statistical analysis of metastasis calculated by the standardized uptake value (SUV). n¼ 8 for each group, mean� SEM.�� , P <0.01 comparedwith untreated group by Student t test.H and I,MCF-7 cellswere cultured alone (�), or treatedwith conditionedmedia (CM) of primary TINs orautologous blood neutrophils. Three independent experiments were performed for the neutrophils isolated from each of the three patients. H, Representativeimages for morphology, E-cadherin, and vimentin expression. Scale bars, 20 mm. I, Representative Western blotting images for EMT markers (n ¼ 3).






Figure 3.

TINs promote metastasis by TIMP-1. A, Representative cytokine arrays for the conditioned media of TINs and autologous neutrophils from peripheral blood.Boxes indicate the cytokines with significant changes and table on the right summarizes the relative signal intensity of indicated cytokines. Mean � SEM,n¼ 5 patients. B, Indicated cytokines levels were validated by ELISA. Mean� SEM, � , P < 0.05, �� , P < 0.001 compared with peripheral neutrophils by Student t test.Three independent experiments were performed for each of the 6 patients. C, Neutralizing antibodies against GM-CSF, angiogenin, or TIMP-1 were addedin the transwell cocultured system of MCF-7 cells and primary TINs. Representative images and quantification of migration and invasion assays of MCF-7 cellswith indicated treatment are shown. Scale bars, 100 mm. Mean � SEM. �� , P < 0.001 by Student t test. Three independent experiments were performedfor the neutrophils isolated from each of the 3 patients. D and E, Conditioned media of TINs were preincubated with neutralizing antibodies against GM-CSF,angiogenin, or TIMP-1. D, Representative immunofluorescent images for E-cadherin and vimentin (E-cad/Vim) expression in MCF-7 cells with indicated treatments.Scale bars, 20 mm (n ¼ 3). E, The representative images of Western blotting analysis for the expression of EMT markers in MCF-7 cells with indicated treatments(n ¼ 3). F, MCF-7 cells and primary TINs or autologous peripheral neutrophils were cocultured in a Matrigel three-dimensional system. The representativeimages of immunofluorescent staining for E-cadherin, vimentin, CD66b, and TIMP-1 are shown. Scale bars, 20 mm (n ¼ 3). G, Representative images of Westernblotting analysis for EMT markers in MCF-7 cells with indicated treatments (n ¼ 3). H, 4T1 cells were injected into the mammary fat pads of BALB/c mice andTIMP-1–neutralizing antibodies were administrated via intraperioneal route once per week at 0.4 mg/kg after the syngeneic grafts were palpable. The metastasiswas evaluated by IVIS. Representative bioluminescence images (left) and the quantification of bioluminescence signal (right). n ¼ 8 for each group,mean � SEM (�� , P < 0.01 by Student t test).


Wang et al.




Figure 4.

TINs promote EMT by a CD90-TIMP-1 juxtacrine–paracrine loop. A, Neutrophils from peripheral blood were cocultured with untreated MCF-7 cells or MCF-7 cellsunderwent EMT (MCF7EMT) by pretreatment of TIN-conditioned media in transwell system or directly. After 24 hours, neutrophils were isolated by CD66b magneticbead isolationand their level ofTIMP-1wasassessedbyELISA.Mean� SEM.n.s., not statistically significant; �� ,P<0.01; �� ,P<0.001byStudent t test. Three independentexperimentswereperformed for theneutrophils isolated fromeachof the4patients.BandC,Peripheral neutrophilsweremixedwithMCF7EMT in thepresenceof indicatedneutralizing antibody. After 24 hours, neutrophils were isolated by CD66b magnetic bead isolation and their level of TIMP-1 was assessed by ELISA. Mean � SEM.�� ,P <0.01 byStudent t test. Three independent experimentswere performed for the neutrophils isolated fromeachof the 3 patients.D andE,4T1 cellswere injected intothe fat pads of BALB/c mice and neutralizing antibodies against TIMP-1 (D) or CD90 (E) were administrated after the syngeneic grafts were palpable. Representativeimages of IHC staining for CD90 (D) and TIMP-1 (E) in xenograft (left) and the quantification (right) are shown. Scale bars, 100 mm. n¼ 8 for each group, mean� SEM.�� , P < 0.001 by Student t test. F, 4T1 cells were injected into the mammary fat pads of BALB/c mice. The indicated antibodies were administrated after the syngeneicgrafts were palpable. The lung and liver metastases were evaluated by IVIS. Representative bioluminescence images (left) and the quantification of bioluminescencesignal (right) demonstrated the lung and liver metastasis. n¼ 6 for each group, mean� SEM (n.s., not statistically significant; � , P < 0.05; �� , P < 0.01 by Student t test).






CD90 expression (Supplementary Fig. S4A–S4C). Moreover,we found that addition of TIN conditioned media, rather thanperipheral neutrophil one, significantly increased CD90 in MCF7cells (Supplementary Fig. S4D).More importantly, TIMP-1 block-ade abolished the CD90 induction in MCF7 cells by the condi-tioned media of TINs (Supplementary Fig. S4E).

In vivo, CD90 staining was predominantly observed in tumorcells, whereas TIMP-1 blockade significantly reduced CD90expression (Fig. 4D). Reciprocally, administration of CD90 neu-tralization antibody intraperitoneally markedly reduced TIMP-1levels in tumors (Fig. 4E) and distant metastasis (Fig. 4F). Incontrast, no difference in metastasis was observed in the micetreated with IgG and the ones treated with anti-CD90 antibodieswhen anti-TIMP-1 neutralizing antibody was coinjected (Fig. 4F).Collectively, these data suggested that tumor-contacted neutro-phils promote EMTby aCD90-TIMP-1 juxtacrine–paracrine loop.

Both TIMP-1 and CD90 are associated with poor prognosis ofpatients with breast cancer

To investigate the clinical significance of the CD90–TIMP-1loop in human breast cancer, we investigate the expression ofCD90, TIMP-1, EMT marker E-cadherin, and neutrophil markerCD66b in human breast cancer. We observed that CD90 is over-expressed in tumor cells with downregulation of E-cadherincompared with the ones with abundant E-cadherin expression(Fig. 5A). In addition, TIMP-1 is predominantly expressed in TINsin tumor parenchyma, rather than the ones in stroma (Fig. 5A).Consistent with the ex vivo data, TIMP-1 levels were stronglycorrelated with CD90 levels (Fig. 5B). More importantly, bothCD90 and TIMP-1were associatedwith long-termpoor prognosis(Fig. 5C).

DiscussionNeutrophils are the first responders to tissue damage. It has

been well documented that they also play a critical role inmultiple types of cancer, the "unhealed wound" (31, 32).Evidences for both pro- and antitumor roles have been reported(4, 5, 33, 34), highlighting our lack of detailed knowledgeabout their diversity in tumors. Therefore, careful dissections ofinteraction between neutrophils and cancer cells by moreclinically relevant approaches are of paramount importancefor developing neutrophil-targeted oncology treatment (3, 8).By analyzing the clinical samples from three cohorts of patientswith breast cancer, we found that neutrophils in tumor paren-chyma, rather than the ones in stroma, were an independentrisk factor, especially in the triple-negative subtype, which isnotoriously poor prognostic due to the lack of targeted therapy.By isolating primary TINs from clinical samples and cytokinemicroarray screening, we uncovered that tumor-contacted neu-trophils induced EMT by a CD90-TIMP-1 juxtacrine-paracrinecycle (Fig. 5D). More importantly, we highlighted the thera-peutic opportunities by showing blockade of either TIMP-1 orCD90 inhibited breast cancer metastasis in vivo.

TINs can be found in almost every type of malignancies.Although their presence is undeniable, their prognostic value iscontroversial (2, 3). Positive (35), negative (36), or no (37)association between TINs and patient prognosis have beenreported, highlighting their heterogeneity and diversity. Intrigu-ingly, we observed that neutrophils in tumor parenchyma, ratherthan the ones in stroma, were significantly correlated with

worse long-term survival of patients with breast cancer. To ourknowledge, this is the first study to identify the different prog-nostic values of TINs in different tissue compartments. It has beenreported that TINs can be classified as N1 (anti-) and N2 (pro-)subtypes, highlighting the heterogeneity of TINs (27). Our reportprovided the clinical evidence to support the hypothesis thatthe plasticity of neutrophils is constantly mediated by differentsignals within the tumor microenvironment (38, 39).

Despite the progress in the treatment of early-stage cancer,metastatic diseases are still incurable. EMT has been identifiedas a critical regulator of metastasis (12). We and others havepreviously revealed a crosstalk between cancer cells with EMTtraits and inflammatory cells, including monocytes, macro-phages, and fibroblasts (16, 17, 19). In this study, we discoveredthat neutrophils, another major cellular component of the tumormicroenvironment, induced EMTof breast cancer cells by TIMP-1.In turn, breast cancer cells undergoing EMT maintained andreinforced TIMP-1 production from neutrophils by CD90 in acell-contact fashion. The cell–cell contact–dependent interactionsidentified here provided the mechanistic insight to the clinicalobservation that neutrophils in tumor parenchyma, but not theones in stroma, were strongly associated with worse clinicaloutcomes in patients with breast cancer. Indeed, TIMP-1 andCD90were positively correlated in human breast cancer and bothassociated with long-term poor prognosis. Our data here providenew evidences to support the emerging notion that varioussignaling molecules secreted extracellularly or located on themembrane ofmultiple stromal cell subsets in themilieu facilitate,perhaps in a coordinated manner, EMT induction and metastasisof cancer cells.

Neutrophils are a major source of matrix metalloproteinase 9(MMP9), which facilitates tumor cell invasion in the extracellularmatrix (9). Unexpectedly, we found that neutrophils producedTIMP-1, the inhibitor of MMP9, which directly enhanced tumorcell motility by inducing EMT. More broadly, the crosstalkbetween neutrophils and cancer cells with EMT traits observedhere may represent a general mechanism that also operates inother carcinoma types. Elevated TIMP-1 has been correlated withpoor prognosis of multiple cancers, including breast (40), lung(41), and lymphoma (42). In addition, inhibition of TIMP-1reduces liver metastasis of colorectal cancer (43, 44). Therefore,our and others' data suggested that tumor cells utilize the bilateralproperties of TIMP-1 for cancer metastasis.

CD90 (Thy-1) is a membrane-anchoring protein that faces theextracellular matrix. It can bind to Mac1 (CD11b/CD18)expressed in neutrophils and macrophages, and initiate signalingcascades in both cis- and trans-manner (30). CD90 is mainlyexpressed in endothelial cells and mediates extravasation ofmyeloid cells in inflammation (45). Despite the fact that itsfunction in tumor metastasis remains unclear, accumulatingevidences indicate that CD90 is overexpressed in cancer stemcells of various tumor types (19, 46, 47), which are closely linkedto EMT. We extend the knowledge of this emerging field byshowing (i) CD90 in cancer cells with EMT traits inducesTIMP-1 production in neutrophils, which reciprocally reinforcedEMT and (ii) blockade of CD90 inhibited breast cancermetastasisin vivo. How CD90 induces TIMP-1 expression in neutrophils andthe therapeutic value of CD90 blockade in other cancer typeswarrant further studies.

Collectively, our study demonstrated a juxtacrine–paracrineloop between neutrophils and tumor cells with EMT traits, which

Wang et al.





Figure 5.

Both TIMP-1 andCD90are associatedwith poor prognosis.A,Representative images of H&E staining and immunofluorescence staining of CD66b, TIMP-1, E-cadherin,CD90, and cytokeratin (CK) in serial sections of breast cancer samples. Scale bars, 50 mm.B, The correlation between the number of TIMP-1þ TINs (TIMP-1þ CD66bþ)and the number of CD90þ tumor cells (CD90þCKþ) in human breast cancer (n¼ 341). CK, cytokeratin. C,Kaplan–Meier curves of patientswith low and high numbersof TIMP-1þ TINs andCD90þ tumor cells (n¼ 341).D, Schematics highlighting themajor findings of this study. Neutrophils induced EMTvia TIMP-1. Reciprocally, tumorcells undergoing EMT enhanced neutrophils' TIMP-1 by CD90 in a cell-contact manner. Blocking this juxtacrine–paracrine loop reduces metastasis in vivo.






is important for breast cancer metastasis and a potential thera-peutic target for cancer treatment.

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

Authors' ContributionsConception and design: S. SuDevelopment of methodology: Y. Wang, J. Chen, J. Li, W. Wu, M. Huang, S. SuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Wang, J. Chen, L. Yang, J. Li, W. Wu, M. Huang,L. LinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Wang, J. Chen, J. Li, M. HuangWriting, review, and/or revision of the manuscript: J. Chen, M. Huang, S. SuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Wang, J. Li, W. Wu, L. Lin, S. SuStudy supervision: S. Su

Acknowledgments

This work was supported by grants from the National Key Researchand Development Program of China (2017YFA0106300), the NaturalScience Foundation of China (81622036, 81472468, 81672614,81802656, 81802645, and 81672620), Science Foundation of GuangdongProvince (2016A030306023, 2017A030313878, and 201710010083),the Tip-top Scientific and Technical Innovative Youth Talents of Guang-dong special support program (2016TQ03R553), and the GuangzhouScience Technology and Innovation Commission (201508020008 and201508020249).

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 August 6, 2018; revised November 8, 2018; accepted November 21,2018; published first November 27, 2018.

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Published OnlineFirst November 27, 2018.Clin Cancer Res Ying Wang, Jianing Chen, Linbin Yang, et al.

Paracrine Loop−CD90-TIMP-1 Juxtacrine Tumor-Contacted Neutrophils Promote Metastasis by a

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Tumor-Contacted Neutrophils Promote Metastasis by a CD90 ... · 2/3/2019 · tems), or mouse CD90 (1:50, catalog no.105312, BioLegend) overnightat4 C,followedbyasecondaryantibody(HRPrabbit