Translational Cancer Mechanisms and Therapy

Reciprocal Network between Cancer Stem-LikeCells andMacrophages Facilitates theProgressionand Androgen Deprivation Therapy Resistanceof Prostate CancerHai Huang1, Chao Wang1, Fei Liu1, Hui-Zhen Li1, Guang Peng1, Xu Gao1, Ke-Qin Dong1,Hong-RuWang1, De-Pei Kong1, Min Qu1, Li-He Dai1, Kai-JianWang1, Zhe Zhou1, Jun Yang1,Ze-Yu Yang1, Yan-Qiong Cheng1, Qin-Qin Tian1, Dan Liu1, Chuan-Liang Xu1,Dan-Feng Xu2, Xin-Gang Cui3,4, and Ying-Hao Sun1

Abstract

Purpose: Cancer stem-like cells (CSC) contribute to the pro-gression and androgen deprivation therapy (ADT) resistanceof prostate cancer. As CSCs depend on their specific niche,including tumor-associated macrophages (TAM), elucidatingthe network between CSCs and TAMs may help to effectivelyinhibit the progression and ADT resistance of prostate cancer.

Experimental Design: The underlying intracellularmechanism that sustains the stem-like characteristics ofCSCs in prostate cancer was assessed via RNA sequencing,co-immunoprecipitation, chromatin immunoprecipitation,and other assays. A coculture system and cytokine antibodyarrays were used to examine the interaction network betweenCSCs and TAMs. In addition, an orthotopic prostate cancermodel was established to evaluate the in vivo effects of thecombined targeting of CSCs and their interaction with TAMson ADT resistance.

Results: Autophagy-related gene 7 (ATG7) facilitated thetranscription of OCT4 via b-catenin, which binds to the OCT4promoter, promoting CSC characteristics in prostate cancer,including self-renewal, tumor initiation, and drug resistance.In addition, CSCs remodeled their specific niche by educatingmonocytes/macrophages toward TAMs, and the CSC-educat-ed TAMs reciprocally promoted the stem-like properties ofCSCs, progression and ADT resistance of prostate cancer viaIL6/STAT3. Furthermore, the combined targeting of CSCs andtheir interactionwith TAMs by inhibiting ATG7/OCT4 and IL6receptor effectively ameliorated ADT resistance in an ortho-topic prostate cancer model.

Conclusions: Targeting CSCs and their niche may prove tobe a more powerful strategy than targeting CSCs alone, pro-viding a rational approach to ameliorating ADT resistance inprostate cancer. Clin Cancer Res; 24(18); 4612–26. �2018 AACR.

IntroductionIntratumor heterogeneity promotes tumor evolution and con-

tributes to disease progression, therapeutic failure, and patientsurvival (1). Cancer stem-like cells (CSC), a small subset of the

hierarchical organization of cells, offer an explanation for het-erogeneity among cancer cells (2). In different types of tumors,CSCs are functionally defined by their strong stem-like propertiesincluding self-renewal, chemoresistance, tumor initiation uponserial passages, and metastatic potential (3, 4). Thus, elucidatingthe molecular mechanisms responsible for CSCs would help todevelop new and promising therapies for advanced tumors inclinical practice.

Prostate cancer, with its strong heterogeneity, remains one ofthe most common causes of male cancer–related deaths world-wide (5). Androgen deprivation therapy (ADT), when applied toadvanced and recurrent prostate cancers, achieves short-termeffectiveness, but ultimately induces drug resistance, leading toincreased cancer-related deaths (6, 7). Increasing evidence hasindicated that ADT induces reprogrammingof prostate cancer andenriches a subpopulation of cells with CSC properties, which areresistant to ADT and drive prostate cancer progression (8, 9).Therefore, eliminating CSCs may be crucial for achieving a goodresponse of prostate cancer to ADT.

Intracellular programs including pluripotency transcriptionfactors (OCT4, Nanog, Sox2, etc.; refs. 10, 11) and aberrantsignaling pathways (Wnt/b-catenin, STAT3, NFkB, etc.; refs. 12–14) play a crucial role in maintaining the stem-like properties ofCSCs. Autophagy, a highly conserved catabolic process that func-tions as a cell survival mechanism under external stimuli such as

1Department of Urology, Changhai Hospital, Second Military Medical University,Shanghai, China. 2Department of Urinary Surgery, Ruijin Hospital, ShanghaiJiaotongUniversity School ofMedicine, Shanghai, China. 3Department of UrinarySurgery, The Third Affiliated Hospital of Second Military Medical University(Eastern Hepatobiliary Surgery Hospital), Shanghai, China. 4Department ofUrinary Surgery, Gongli Hospital, Second Military Medical University, Shanghai,China.

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

H. Huang, C. Wang, and F. Liu contributed equally to this article.

Corresponding Authors: Ying-Hao Sun, Department of Urology, ChanghaiHospital, Second Military Medical University, 168 Changhai Road, Shanghai200438, China. Phone/Fax: 8602-1350-30006; E-mail: sunyhsmmu@126.com;and Xin-Gang Cui, Department of Urinary Surgery, The Third Affiliated Hospitalof Second Military Medical University (Eastern Hepatobiliary Surgery Hospital),700 North Moyu Road, Shanghai 201805, China. Phone/Fax: 8602-1818-87661;E-mail: cuixingang@smmu.edu.cn

doi: 10.1158/1078-0432.CCR-18-0461

�2018 American Association for Cancer Research.

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chemotherapy or radiotherapy, has recently been shown to sup-port tumor cell survival, differentiation, and the self-renewal ofCSCs (15, 16). However, the related mechanisms need furtherstudy.

In addition to their internal characteristics, CSCs reside inniches that support their self-renewal (17). Recent studies indicatethat CSCs remodel their specific niche by recruiting monocytesand educating them to become tumor-associated macrophages(TAM; ref. 18). Furthermore, the CSC–TAM cross-talk facilitatestumor growth, metastasis, and chemoresistance (19, 20). Thus,jointly targeting CSCs and their niche components may prove toprevent tumor drug resistance and progression more effectivelythan targeting the CSCs alone. Although our studies and othershave shown that ADT-induced transdifferentiation attracts theinfiltration of TAMs and that their reciprocal network influencesADT resistance in prostate cancer (21–23), the molecular mech-anismunderlyingCSC–TAM interaction inADT resistance and theprogression of prostate cancer has not been elucidated.

In this study, we demonstrated that a subpopulation inprostatecancer harboring CSC characteristics could be identified by theepithelial marker OV6, which serves as a CSC marker in epithe-lium-derived malignant tumors and was associated with patientprognosis in our previous studies (24–27).We demonstrated thatan intracellular pathway, autophagy-related gene 7 (ATG7)/b-catenin/OCT4, sustained the self-renewal of OV6þ cells andfacilitated the progression and ADT resistance of prostate cancer.In addition, IL6/STAT3was found to be required for the reciprocalnetwork betweenOV6þ cells and TAMs. Finally, the joint targetingof OV6þ CSCs and their reciprocal network effectively amelio-rated ADT resistance in an orthotopic prostate cancer model.

Materials and MethodsPatients and specimens

Clinical data, tissue specimen, and follow-up informationwerecollected from cohort one for 78 prostate cancer patients whowere diagnosed in Changhai Hospital (Shanghai, China) andcohort two for 67 prostate cancer patients who were diagnosed in

Changzheng Hospital (Shanghai, China) between 2012 and2013. Patients enrolled in these two cohorts were pathologicallydiagnosed as prostate cancer without distant metastasis andunderwent radical prostatectomy. Patients who received addi-tional treatment such as ADT, radiotherapy, or chemotherapywere not included. Follow-up timewas 42 (6–62)months. Tumorstage and Gleason Scores (GS) were assessed in terms of theAmerican JointCommittee onCancer (AJCC)2002 and theWorldHealthOrganization (WHO) classification guidelines. The time tobiochemical recurrence (BCR; cutoff: PSA ¼ 0.2 ng/mL) anddisease progression identified by MRI, CT, or ECT were selectedas the clinical endpoint of BCR-free survival and disease-freesurvival, respectively. Except for the two cohorts as above, thisstudy also included patients who were pathologically diagnosedcastration-resistant prostate cancer (CRPC; n ¼ 10) in which5 patients' samples were taken before and after ADT and NEPC(n ¼ 6) in Changhai Hospital (Shanghai, China). The sampleswere obtained after writing informed consent from patientsaccording to an established protocol approved by the EthicsCommittee of Second Military Medical University.

IHCThe IHC was done as reported previously (24). Primary anti-

bodies were used as follows: mouse anti-OV6 (MAB2020, R&DSystems), rat anti-F4/80 (ab6640, Dako), mouse anti-CD68(M0876, Dako), and rabbit anti-ATG7 (ab52472), mouse anti-b-catenin (ab22656), mouse anti-OCT4 (ab184665), and rabbitanti-STAT3 (ab68153) from Abcam, respectively. The proteinexpression was score by staining intensity and percentage ofpositively stained cells as reported previously (28). Hematoxylinand eosin (H&E)-stained sections of the prostate cancerspecimens were reevaluated by two experienced pathologists(Jun-hui Ge and Yong-wei Yu, Second Military Medical Univer-sity, Shanghai, China) to identify representative areas in doubleblind procedure. The percentage of positive cells (% of PPs) andthe staining intensity (SI value) were determined and multiplied(IRS value), and the score range is from aminimum score of 0 to amaximum score of 12. An IRS value more than one was consid-ered as positive (weak expression); an IRS value more than three,moderate expression; and an IRS value more than eight, strongexpression. The quantitative cutoff for each marker was defined:low expression (IRS value 0–3 including negative and weakexpression) and high expression (IRS value 4–12 including mod-erate and strong expression).

Immunofluorescence analysisThe immunofluorescence analysis was carried out as reported

previously (23). The primary antibodies were used as follows:mouse anti-OV6 (MAB2020, R&D Systems), rabbit anti-CD44(ab51037, Abcam), rabbit anti-CD133 (ab19898, Abcam), rabbitanti-STAT3 (ab68153, Abcam), rabbit anti-b-catenin (ab32572,Abcam).Nuclei were stained byDAPI (E607303, SangonBiotech)staining. Fluorescence images were observed and collected undera Leica DM5000B fluorescent microscope (Leica).

Cell cultureTHP-1, obtained from the Cell Bank of Type Culture Collection

of the Chinese Academy of Sciences (Shanghai, China), wasmaintained in RPMI1640 medium (C11875500CP, Gibco) sup-plemented with FBS (10%, 10099-141, Gibco) and 0.05 mmol/Lb-mercaptoethanol (07604, Sigma). C4-2B and C4-2 were kindly

Translational Relevance

Androgen deprivation therapy (ADT), when applied toadvanced and metastatic prostate cancers, is initially effectivebut inevitably induces drug resistance. Many studies haveindicated that ADT induces heterogeneity, including cancerstem-like cells (CSC), which are resistant to ADT and drive theprogression of prostate cancer. In fact, the specific microenvi-ronment should receive equal consideration in eradicatingCSCs and reversing the drug resistance of prostate cancer. Inthis study, we demonstrated that the combined targeting ofCSCs and their interaction with tumor-associated macro-phages (TAM) by inhibiting autophagy-related gene 7(ATG7)/OCT4 and IL6 receptor (IL6R) effectively amelioratedADT resistance in an orthotopic prostate cancer model. Ourstudy indicates that targetingCSCs jointlywith their nichemayprove to be a more powerful strategy than targeting CSCsalone, which provides a rational approach to amelioratingADT resistance in prostate cancer.

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provided and authenticated by Dr. Leland Chung (Cedars-SinaiMedical Center, Los Angeles, CA). C4-2B, C4-2, or C4-2B Cas9-ATG7-gRNA (ATG7KO) cells were cultured in RPMI1640medium(11835093, Gibco). C4-2B Cas9-gRNA NC cell line was main-tained in RPMI1640 medium (11835093, Gibco), 10 mg/mLblasticidin (S7419, SelleckChemicals), and0.5mg/mLpuromycin(A610593, Sangon Biotech). All cell lines were supplementedwith 1% penicillin/streptomycin (15140122, Gibco) and cul-tured at 37�C and 5% CO2.

The cell lines in this study were authenticated by short tandemrepeat (STR) profiling and tested for mycoplasma contaminationby Mycoplasma Detection Kit (B39032, Selleck Chemicals). Thelatest test was performed in January 2018. The cell lines used inthis study were within 40 passages. Permanent stocks of the celllines were prepared and stored in liquid nitrogen until use.

Spheroid formation assaySpheroid formation assay was carried out as described in our

previous study (24), Briefly, after magnetic sorting, single-cellsuspensions with 5,000 cells were seeded in 6-well ultra-lowattachment culture plates (Corning) cultured in serum-freeDMEM/F12 (Gibco), supplemented with B27 (1:50, Invitrogen),20 ng/mL EGF (Invitrogen), 10 ng/mL basic fibroblast growthfactor (Invitrogen), and ITS (1:100,Gibco) for 5days. The numberof spheroids formed was counted under a microscope and therepresentative pictures were taken.

Establishment of a prostate cancer cell line with knockout ofATG7 using CRISPR technology

The CRISPR-targeting sequence in this study, as shown inSupplementary Table S14, was designed on the basis of theOptimized CRISPR Design web tool (http://crispr.mit.edu/) forknockout of ATG7. C4-2B cells were transduced with sgATG7cloned into lenti Cas9-Blast (52962, Addgene), lentiGuide Purovector (52963, Addgene). Cells were selected with blasticidinpuromycin. First, C4-2B cells were transduced with lentivirusCas9 and were selected with 20 mg/mL blasticidin for fourdays. In each transduction, 100-mm dish was used to seed500 blasticidin-resistant cells and cultured until cell colonyformation. Individual colonies were shifted to 12-well plates asone colony per well and grown to confluence. Second, C4-2BCas9–stable cell line was transduced with lentiviruses sgRNAand were selected with 1 mg/mL puromycin. PCR or RT-PCR wasused to identify clones. A TOPO vector was used to packagegenomic PCR products to sequence alleles with indels ordeletions. Finally, Western blot analysis was used to validatethe ATG7 protein expression.

Gene knockdown, RNA interference, and plasmid transfectionGene knockdown, RNA interference, and plasmid trans-

fection were done as reported previously (23). The C4-2B orC4-2 was cultured in 6-well plates, inoculated at a density of5 � 104 cells/mL, and transfected with the shRNAs expressinglentivirus (sh-ATG7, shb-catenin, sh-OCT4, sh-STAT3) or con-trol lentivirus at a multiplicity of infection (MOI) of 45. After72-hour transfection, they were observed and photographedunder microscope. The sequences for shRNA were presented inSupplementary Table S14.

siRNA and plasmid transfection was carried out using Lipo-fectamine 3000 reagents (L3000015, Invitrogen) according to the

manufacturer's protocols. The sequences of si-Beclin1, si-ATG5,and ATG7 plasmid are shown in Supplementary Table S14.

Isolation of circulating monocytes and coculture assaysThe isolation of circulating monocytes was done as reported

previously (23), The C4-2B or C4-2 cells' coculture with circulat-ing monocytes or THP-1 was the same as in our previous study(23). After 5-day coculture in the 37�C, 5% CO2 condition, boththe supernatant and cells were collected for subsequent analysis.

NanoLC-ESI-MS/MS analysisThe nano-scale liquid chromatography tandem electrospray

ionizationmass spectrometry (NanoLC-ESI-MS/MS) analysis wasperformed as reported previously (23). For the identified proteinsreported here, the certainty should be >98% if the identification isbased on LC/MS-MS sequencing of one peptide and >99.9% ifbased on the sequencing of two or more peptides.

Magnetic cell sorting and flow cytometry assayThe magnetic-activated cell sorting (MACS) assay was per-

formed with a MiniMACS Cell Sorter (Miltenyi Biotec) asreported in previous study (24). Flow cytometric assay wasperformed with a Cyan ADP Sorter (Beckman Coulter). Pros-tate cancer cell lines were labeled with APC-conjugated-OV6antibody (FAB2020A, R&D Systems), FITC-conjugated CD44antibody (130-095-195, Miltenyi Biotec), PE-conjugated-CD133 (130-098-826, Miltenyi Biotec), PE-conjugated-CD14antibody (REA599, Miltenyi Biotec).

Assessment of apoptosisApoptotic cells were evaluated by ANXA5 and PI staining

(Invitrogen) according to the manufacturer's instructions, andanalyzed by flow cytometry with a Cyan ADP Sorter (BeckmanCoulter)

RNA-seq and analysisRNA was isolated from OV6�, OV6þ without and with ATG7-

knockdown C4-2B cells with TRIzol reagent (Invitrogen), Thetotal RNA was purified by the Qiagen RNeasy Mini Kit (Qiagen)and then the purified RNAwas checked to determine the quantity.Single and double stranded cDNA were synthesized from mRNAsamples. The double-strand cDNA was then purified for dAtailing, end repair, adaptors ligation, and DNA fragment enrich-ment. Quantify the libraries using Qubit (Invitrogen) based onthe Qubit user Guide. The constructed library was sequenced onIllumina Hiseq 4000 sequencer.

The TopHat version 1.2.0 was used to align the paired-end rawreads that allows two mismatches in the alignment. Cufflinksversion 2.0.0 was used to assemble the aligned reads into tran-scripts using. The distribution of the reads and the alignmentquality were estimated using SAM tools. From the aligned reads,the gene and transcript expression was performed using cufflinksversion 1.3.036. The differential transcripts were analyzed viacuffdiff. Finally, GO and pathway functional analyses were per-formed for the differentially expressed transcripts.

RT-PCRThe RT-PCR was done as reported previously (24). Briefly,

RNAiso Plus (9109, Takara) and PrimeScript One Step RT reagentKit (RR037B, Takara) were used to extraction and reverse tran-scription of RNA. The results were normalized by the expression

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of the b-actin gene and each measurement was performed intriplicate. Fold change relative to control group was determinedby2�DDCt. Theprimer sequenceswere presented in SupplementaryTable S14.

Western blot analysisTheWestern blot analysiswas done as described inour previous

study (24) and the following primary antibodies were used rabbitanti-Beclin1 (3495), rabbit anti-ATG5 (12994), rabbit anti-LC3B(3868), rabbit anti-b-actin (4970) from Cell Signaling Technol-ogy, and rabbit anti-STAT3 (ab68153), mouse anti-OCT4(ab184665), rabbit anti-P-STAT3 (ab76315), mouse anti-b-cate-nin (ab22656), rabbit anti-b-TrCP (ab71753) from Abcam, over-night in the blocking solution at 4�C. Then, the membranes wereincubated with anti-rabbit IgG-HRP–linked antibody (7074S) oranti-mouse IgG-HRP–linked antibody (7076S) from Cell Signal-ing Technology at room temperature for 1–2 hours (1: 5,000),respectively. To analyze ATG7 and b-catenin protein interactions,coimmunoprecipitation experiments were performed usingOV6þ C4-2B cells according to previously published protocols(24) and the following primary antibodies were used: rabbitanti-ATG7 (1:100, ab52472) and mouse anti-b-catenin(ab22656) from Abcam. b-Actin protein level was used as aninternal control.

Cell migration assay and cell proliferation assayThe cell migration ability of sorted monocytes and THP-1 was

evaluated by cell migration assay as we reported in previous study(23). The proliferation of C4-2B and C4-2 cells in indicatedcondition was evaluated using a CCK-8 kit (CK-04, Dojindo) aswe reported in previous study (23). The proliferation rates werepresented as a proportion of the control value which was detectedat the first time point.

Autophagy analysis and mRFP-GFP-LC3B systemThe number of autophagosomes in OV6� and OV6þ prostate

cancer cells was analyzed by electronmicroscopy (Hitachi-7650).The mRFP-GFP-LC3B adenovirus construct was provided byHanbio Inc. (autophagy-adv-GFP-RFP-LC3B-1000). This con-struct fluorescence in terms of the difference in pH between theneutral autophagosome and the acidic autolysosome, and exhib-ited redfluorescence or the red/green (yellow)makes it possible tomonitor progression of autophagic flux. Confocal fluorescencemicroscopy was used to scan and assess the fluorescence.

Chromatin immunoprecipitation analysis and Duolink PLATo determine the association of transcription factor b-catenin

and OCT4 promoter, ChIP assays were performed according topreviously published protocols (23). Primers complementaryto the promoter region of OCT4 (forward: 50- GCCCATT-CAAGGGTTGAGCACT-30; reverse: 50- GGTTCAAAGAAGCCTGG-GAGGG -30)were used to detectOCT4 genomicDNA andprimersspecific to human GAPDH promoter was used as control (kitsupplied). Enrichment of the targets was calculated as follows:fold enrichment ¼ 2(Ct[OCT4�ChIP] �Ct[IgG]).

We detected the interaction between b-catenin and ATG7using the Duolink PLA according to previously publishedprotocols (23).

Luciferase reporter assayThe interaction between b-catenin and theOCT4 promoter was

evaluated by using a luciferase reporter assay as we reported inprevious study (23). The b-catenin–binding sites of the OCT4promoter (sequence: CTTTGAA, �1302 to �1308 relative to theOCT4 transcription site) or its deletion mutant was cloned intothe pGL3-basic luciferase reporter vector (Promega). Cells werecollected 48 hours after transfection and OCT4 transcriptionactivity was evaluated by measuring luminescence with theDual-Luciferase Assay Kit (E1910, Promega). Fold induction wasderived relative to normalized reporter activity.

Antibody–microarray experiment and ELISAThe antibody–microarray experiment was done as we

previously reported (23). Cytokine profiles were measuredby Quantibody Human Inflammatory Array 3 (QAH-INF-G3,RayBiotech) which permitted detection of 40 inflammation-associated cytokines.

The IL6 or PSA levels in cell culture medium or serum weremeasured using ELISA Kit for IL6 (SEA079Hu, Cloud-CloneCorp), PSA Quantikine ELISA Kit (DKK300, R&D Systems),respectively, according to the manufacturer's instructions.

Animal experiments and in vivo imagingAll experimental animal procedures were approved by the

Animal Care and Use Committee of the Second MilitaryMedical University, Shanghai, China. NOD-SCID mice (male,7 weeks old) were purchased from the Shanghai LaboratoryAnimal Center (SLAC) and housed under specific pathogen-freeconditions. The orthotopic prostate cancer xenograft assaywas done as previously reported (23). A total of 5 � 105 OV6þ

C4-2B cells or C4-2 cells mixed with Matrigel (1:1) wereorthotopically injected into both anterior prostates ofNOD-SCID mice through an abdominal incision. Three weeksafter the cell injections, the animals were grouped so that theaverage bioluminescence index was similar in the indicatedgroups. These mice in treatment group were treated withcastration/enzalutamide (30 mg/kg, twice a week) by intraper-itoneal injection, combined with or without tocilizumab (5 mg/mL) and ATG7KO, while the control group was treated withvehicle. Bioluminescence data were quantified using IVIS Lumi-na II imaging system (PerkinElmer).

For in vivo limiting dilution assay, sorted OV6þ, OV6�,OV6þCD44þ, OV6þCD44�, OV6�CD44þ tumor cells were dilut-ed in appropriate cell dose and injected in NOD/SCID mice, thenumber of tumors formed fromeach cell dose injectedwas scored.According to the ELDA software (http://bioinf.wehi.edu.au/software/elda/index.html; the Walter and Eliza Hall Institute). Thefrequency of CSCs was calculated.

Statistical analysisCategorical data were presented as number (%) and continu-

ous data were presented as median (range). Numerical data wereexpressed as the mean � SD. Statistical differences betweenvariables were analyzed by two-tailed Student t test, categori-cal/binary measures were assessed by c2 test or Fisher exact testand ANOVA for continuous measures. Survival curve was plottedby the Kaplan–Meier method and compared using the log-rankanalysis. Difference was considered significant at P < 0.05. Allexperiments for cell cultures were performed independently atleast three times and in triplicate each time. Data analysis was

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performed by the GraphPad Prism 5 (GraphPad Software, Inc.)and SPSS 22.0 (IBM Corporation).

ResultsOV6 serves as an indicator for tumor heterogeneity and for theprognosis of patients with prostate cancer

On the basis of our previous studies (24–27), we postulatedthat OV6 was associated with the heterogeneity and progressionof prostate cancer. By IHC, the positive staining forOV6 in normalprostate basal cells suggests that OV6 is an epithelial marker (Fig.1A), which is consistent with the results of our previous studies(24–27). Samples from locally advanced prostate cancer (LAPC),CRPC, and neuroendocrine prostate cancer (NEPC) exhibitedhigher OV6 expression than localized prostate cancers (Fig.1A). In prostate cancerswith differentGleason Scores (GS), higherOV6 expressionwas found in sampleswithGS>7 than in sampleswith GS < 7 (Fig. 1B). Even in the same sample, staining showedstronger OV6 expression in worse differentiated areas (Fig. 1B). Inaddition, a positive correlation between the percentage of OV6and GS was observed in fresh clinical samples assessed by immu-nofluorescence analysis (Fig. 1C; Supplementary Fig. S1A). Theseresults indicate that the differential expression ofOV6 predicts thedegree of differentiation and the progression of prostate cancer.

We next examined the relationship between the clinical out-comes of prostate cancer patients and OV6 expression. Patientsfrom two independent clinical centers were respectively dividedinto OV6high and OV6low groups according to OV6 expression insamples of prostate cancer (Supplementary Fig. S1B; Supplemen-tary Table S1 and S2). The OV6high group exhibited worse BCRand disease-free survival (DFS; Fig. 1D and E; SupplementaryTable S1 and S2). In addition, we investigated whether OV6 wasassociatedwith androgen deprivation therapy (ADT) resistance inprostate cancer. As expected, higher OV6 expression was observedin samples from the same patient with metastatic prostate cancercomplicated by ADT failure than in the corresponding tissuesbefore ADT (Fig. 1F; Supplementary Fig. S1C). Accordingly, OV6expression was higher in ADT-treated xenografts than in na€�veones from a mouse model of orthotopic prostate cancer (Fig. 1G;Supplementary Fig. S1D–S1F), which was described in our pre-vious study (23).

To summarize, our findings indicate that OV6 is a potentmarker for poor differentiation of prostate cancer and effectivelyevaluates the prognosis and disease progression of prostate cancerpatients.

OV6-positive prostate cancer cells harbor cancer stem-likecharacteristics

Given that CSCs offer an explanation for tumor heterogeneityand progression, we next examined whether OV6 served as aputative CSCmarker in prostate cancer. First, higher expression ofOV6 was observed in sphere-forming prostate cancer cells (withCSC properties; ref. 29) than in adherent cells, whichwas assessedby flow cytometry and immunofluorescence analysis (Fig. 2A;Supplementary Fig. S2A and S2B). Second, multimarker analysesrevealed that OV6þ cells in prostate cancer cell lines C4-2B alsoexpressed CD44 or CD133, and the percentage of double positivecells (OV6þCD44þ or OV6þCD133þ) was increased in spheres(Supplementary Fig. S2C). Third, the colocalization of OV6 withCD44 or CD133 was observed in spheres from C4-2B (Supple-mentary Fig. S2D). These data suggest that the pattern of OV6

expression in prostate CSCs may be similar to that of CD44 orCD133.

Then,we compared the stem-like properties ofOV6þ andOV6�

cells separated from prostate cancer cell lines via MACS (Supple-mentary Fig. S2E). First, higher expression of stem-associatedgenes was observed in OV6þ cells than in OV6� cells (Fig. 2B;Supplementary Fig. S2F). Second, OV6þ cells formed morespheres in serial passages than OV6� cells (Fig. 2C; Supplemen-tary Fig. S2G). Third, OV6þ cells initiated tumors with a higherincidence and frequency in NOD/SCID mice in two serial gen-erations (Fig. 2D; Supplementary Fig. S2H and S2I; Supplemen-tary Table S3). In addition, OV6þCD44þ C4-2B cells exhibitedhigher incidence and frequency of tumor initiation thanOV6�CD44þ cells in NOD/SCID mice, but there is no statisticaldifference in tumorigenicity between OV6þCD44þ andOV6þCD44� cells (Supplementary Fig. S2J; Supplementary TableS4). The results indicate that OV6 may play a stronger role thanCD44 in the tumor initiation of CSCs. Furthermore, we examinedwhether OV6þ cells exhibited heightened resistance to chemo-therapy or ADT. After the administration of docetaxel (a chemo-therapy drug) or enzalutamide (an androgen receptor antago-nist), the ratio of OV6þ cells in prostate cancer was elevated(Supplementary Fig. S2K). We also observed that OV6þ cellspresented heightened cell survival or decreased apoptosis duringdocetaxel or enzalutamide treatment, compared with OV6� cells(Fig. 2E–H; Supplementary Fig. S2L–S2O). Collectively, thesedata demonstrate thatOV6þ cells exhibit strong stem-like abilitiesin prostate cancer.

To examine themechanismsunderlying the regulationofOV6þ

cells, RNA-seq was employed. Among the differentially expressedgenes, we noticed that many stem or autophagy-related genesexhibited increased expression in OV6þ cells, which possessedrelative quiescence (Fig. 2I; Supplementary Fig. S2P–S2T; Sup-plementary Table S5–S7). As autophagy has been reported to playcrucial roles in CSCs, we examined whether autophagy mediatedthe stem-like properties of OV6þCSCs. First, electronmicroscopyand Western blot analysis demonstrated that more autophago-somes and increased LC3BII was observed in OV6þ CSCs than inOV6� cells (Fig. 2J and K). In addition, an autophagosomal–lysosomal fusion process was activated to a greater extent inOV6þ

CSCs, suggesting a heightened autophagic flux (Fig. 2L). Second,after the treatment of autophagy inhibitor 3-methyladenine (3-MA), OV6þ CSCs exhibited decreased stem-associated genes andself-renewal compared with the control na€�ve OV6þ CSCs (Fig.2MandN; Supplementary Fig. S2U and S2V). Furthermore,OV6þ

CSCs subjected to 3-MA treatment initiated tumors inNOD/SCIDmice with a lower efficiency and lower percentage of OV6 intwo serial generations than OV6þ CSCs without 3-MA treatment(Fig. 2O–P; Supplementary Fig. S2W).

Autophagy-related gene 7 maintains the expansion and self-renewal of OV6þ stem-like cells via OCT4 in prostate cancer

In addition to above results that autophagy was required inOV6þ CSCs, RNA-seq and Western blot analysis both exhibitedthat autophagy-related gene 7 (ATG7)was elevated inOV6þCSCs(Fig. 2I and K). Thus, we established ATG7-deficient cell lines(C4-2B-ATG7KO) using CRISPR/Cas9 technology to examinewhether ATG7 regulated OV6þ CSCs (Supplementary Fig.S3A). Notably, silencing ATG7 reduced the expression of stem-associated genes and the self-renewal and tumorigenicity ofOV6þ

CSCs (Fig. 2M–O; Supplementary Fig. S2U–S2W). In contrast, the

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Figure 1.

OV6 serves as an indicator for tumor heterogeneity and for prognosis in patients with prostate cancer. A, Representative hematoxylin and eosin (H&E)staining and IHC staining for OV6 in normal prostate and prostate cancer tissues, ranked by different disease stages: localized, LAPC, CRPC, andneuroendocrine prostate cancer (NEPC; scale bar ¼ 50 mm). The IHC scores for OV6 in the corresponding groups are presented (right). B, RepresentativeH&E staining and IHC staining for OV6 in regions with different Gleason scores (GS) in the same sample from patients with prostate cancer. The percentagesof OV6 expression distributed in regions with different GSs are shown (right; scale bar ¼ 50 mm). C, Representative immunofluorescence staining of OV6 insamples with different GSs. Enlarged images are presented in the top. The nucleus is stained with DAPI dye. Red staining indicates OV6-positive cells inprostate cancer (scale bar ¼ 50 mm). D and E, Kaplan–Meier curves for BCR and disease-free survival (DFS) in prostate cancer patients were analyzed accordingtoOV6expression. (D, cohort 1,n¼78;E, cohort 2,n¼67). RepresentativeH&E staining and IHC staining forOV6 in the followinggroups:F,Pre-ADT versus post-ADTtissues from the same patient with metastatic prostate cancer (n ¼ 5; scale bar ¼ 50 mm). G, Na€�ve versus post-ADT tissues from C4-2B (n ¼ 5) orC4-2–derived orthotopic prostate cancers (n ¼ 5; scale bar ¼ 50 mm). All data represent the means � SD (� , P < 0.05; �� , P < 0.01; and ��� , P < 0.001).ADT, androgen deprivation therapy; PCa, prostate cancer.

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upregulation of ATG7 enhanced the stem-like properties ofOV6þ CSCs (Fig. 3A–C; Supplementary Fig. S3B–S3F). Inaddition, after knockdown of other autophagy-related genesBeclin1 or ATG5 in prostate cancer, the expression of stem-associated genes and the self-renewal of OV6þ CSCs were notinhibited (Supplementary Fig. S3G–S3I). Therefore, ATG7 wasrequired for sustaining the expansion and self-renewal of OV6þ

CSCs in prostate cancer.Next, to elucidate the mechanisms underlying ATG7-mediated

OV6þ CSCs, RNA-seq was applied to ATG7-knockdown andcontrol OV6þ CSCs (Fig. 3D; Supplementary Fig. S3J–S3L; Sup-plementary Table S8–S10). We noticed that the expression levelsof stem-associated genes and the pathways they participate inwere reduced in ATG7-knockdown CSCs, and in particular, OCT4was significantly downregulated (Fig. 3D; Supplementary Fig.S3L). In addition, OCT4 knockdown alleviated the ATG7-pro-moting stem-like characteristics of OV6þ CSCs (Fig. 3A–C; Sup-plementary Fig. S3B–S3E and S3M). Furthermore, ATG7 upregu-lation enhanced OCT4 expression and transcriptional activity(Fig. 3A, E, and F). Thus, OCT4 was required for the regulationof the stem-like characteristics of OV6þ CSCs by ATG7.

Furthermore, we next examined how ATG7 regulated OCT4expression and transcription in OV6þ CSCs. Knowing that ATG7was not a transcription factor, we assumed that OCT4 transcrip-tion was mediated by an indirect mechanism. Then, we immu-noprecipitated the ATG7 protein with an anti-ATG7 antibody andanalyzed the results by Nano LC-ESI-MS/MS (Fig. 3G; Supple-mentary Table S11). Among the ATG7-interacting proteins (Fig.3G; Supplementary Table S11), b-catenin had been reported topromote OCT4 transcription (30). Co-immunoprecipitation (co-IP) andDuolink proximity ligation assays also demonstrated thatthe direct interaction between ATG7 and b-catenin occurred inOV6þ CSCs (Fig. 3H and I). But ATG7 knockout interrupted theirinteraction (Fig. 3I). These results prompted us to examine howATG7 facilitated OCT4 transcription via b-catenin in OV6þ CSCs.First, b-catenin knockdown alleviated the ATG7-promoting stem-like characteristics of OV6þ CSCs (Fig. 3A–C; Supplementary Fig.S3B–S3E, S3N). Second, b-catenin knockdown alleviated theATG7-induced increase inOCT4 expression (Fig. 3A andE). Third,aChIP assay showed thatb-cateninbound toOCT4promoter (thesequence was reported in previous studies; ref. 30) in OV6þ cells,and this binding was enhanced by ATG7 overexpression (Fig. 3J).In addition, although ATG7 enhanced OCT4 transcriptional

activity, mutated variants of the b-catenin–binding sites onOCT4promoter abolished this effect (Fig. 3F). These results indicate thatATG7mediates OCT4 expression and transcription via b-catenin.

Given that b-catenin is phosphorylated by a cytoplasmicdestruction complex, then ubiquitinated by b-transducinrepeat-containing E3 ubiquitin protein ligase (b-TrCP) andfinally degraded by proteasomes, and given that stabilizedb-catenin enters the nucleus to perform its biological function(31), we next determined how ATG7 mediated b-catenin sta-bilization in the cytoplasm. As shown in Supplementary Fig.S3O, ATG7 promoted the interaction between b-catenin andAxin1 or b-TrCP, while ATG7 silencing achieved the oppositeeffect. In addition, ATG7 facilitated b-catenin to enter thenucleus, but ATG7 knockdown decreased b-catenin expressionin the nucleus of OV6þ cells, as shown in Fig. 3K. The resultsindicate that ATG7 facilitates stem-like properties and OCT4transcription via b-catenin stabilization.

OV6þ CSCs educate monocytes/macrophages toward tumor-associated macrophages, which reciprocally facilitate the self-renewal of OV6þ CSCs

On the basis of the above results, we next examined whetherATG7 or OCT4 inhibition could reverse the ADT resistance ofOV6þ CSCs in prostate cancer. As expected, in vitro experimentsshowed that ATG7 or OCT4 inhibition enhanced the effects ofenzalutamide or abiraterone (another ADT drug) on OV6þ CSCs(Fig. 4A and B).However, the same interventionmeasures did noteffectively inhibit ADT resistance of OV6þ CSCs in an orthotopicprostate cancer model although enzalutamide induced ATG7expression (Fig. 4C–E; Supplementary Fig. S4A–S4D). The differ-ence between in vivo and in vitro experiments might ultimately liein the role of the microenvironment, which has been reported tosupport the self-renewal of CSCs (17). In addition, our studiesand others have demonstrated that tumor-associated macro-phages (TAM) facilitate enzalutamide resistance inprostate cancer(21, 23) Accordingly, samples from ADT-treated OV6þ CSCs-derived orthotopic prostate cancers without or with ATG7 (orOCT4) inhibition still presented highOV6 expression and F4/80þ

TAM infiltration (Supplementary Fig. S4A–S4C).Thus, we examined the interaction between OV6þ CSCs and

TAMs in prostate cancer. First, a positive correlation betweenOV6and CD68 was observed in prostate cancer patients (Fig. 4F;Supplementary Fig. S4E). Furthermore, patients with high

Figure 2.OV6-positive prostate cancer cells harbor cancer stem-like characteristics. A, Flow cytometric analysis was performed to detect the percentage of OV6 inspheres and adherent cells fromdifferent prostate cancer cell lines.B,RT-PCRanalysis of the expression of stem-like–associated genes (CD133, CD44, SOX2, OCT4, MYC,KLF4) in OV6� and OV6þ C4-2B cells. Data are expressed as the fold change relative to OV6� C4-2B cells. C, Comparison of representative images and numbersofOV6þ andOV6� cell-derived spheres fromserial passages (scale bar¼ 100mm).D,MaleNOD/SCIDmice (n¼ 6/group)were subcutaneously injectedwith 5� 103, 1�104, and 2 � 104 OV6� or OV6þ cells. Bioluminescent imaging for subcutaneous tumors from mice injected with OV6� or OV6þ C4-2B cells after transplantationfrom the primary and secondary generation are shown, and the tumorigenic cell frequency estimate and P values were calculated by the limiting dilution analysis tool(http://bioinf.wehi.edu.au/software/elda/). E–H, OV6� or OV6þ C4-2B cells were treated with docetaxel (E) or enzalutamide (ENZ, G) for 72 hours, and apoptosiswas evaluated by Annexin V-FITC/PI staining. Cell viability was measured by CCK-8 assays, and the data are presented as the fold change relative to treatment-freegroups: (F, docetaxel; H, ENZ). I, RNA-seq was applied, and a heatmap depicting the significantly differentially expressed genes in OV6þ and OV6� C4-2B cellsfromthree independent experiments is shown.J,Electronmicroscopy imagesshowingautophagosomes inC4-2BorC4-2cells groupedbyOV6�andOV6þ (scalebar¼ 2mm; the arrow indicates autophagosome). K,Western blot analysis of ATG7, Beclin1, ATG5, LC3B-I/II in OV6� andOV6þ groups of C4-2B or C4-2 cells. b-Actin served asthe internal control. L, Representative images of LC3B staining in C4-2B or C4-2 cells infected with adenovirus-delivering mRFP-GFP-LC3B: OV6� and OV6þ.Quantificationofautophagosomeandautolysosome formation representingpuncta staining sitespercell infive independent images(scalebar¼ 15mm).M,Expressionofstem-like-associated genes in OV6þ C4-2B cells: control, 3-MA, and ATG7-knock out (KO) cells. N, Numbers of spheres were respectively compared between control,3-MA–treated, and ATG7KO OV6þ C4-2B cells. O, Subcutaneous tumors from mice injected with 2 � 104 OV6þ C4-2B cells: control, 3-MA, and ATG7KO (6/group). P,Representative H&E staining and IHC staining of OV6 and ATG7 in subcutaneous tumor tissues from OV6þ C4-2B cells: control, 3-MA, and ATG7KO in two generations(scale bar ¼ 50 mm). All data represent the means � SD (� , P < 0.05; �� , P < 0.01; and ��� , P < 0.001).

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expression levels of bothOV6 and CD68 exhibited the worst BCRand DFS (Fig. 4G and H; Supplementary Table S12). Second,freshly isolated healthy donor circulating monocytes (Supple-mentary Fig. S4F) and the humanmonocytic cell line THP-1 wereexposed to conditionedmedium (CM) fromOV6� orOV6þ cells,and their migration properties were tested. A higher number ofmonocytes migrating toward OV6þ CM than toward OV6� CM

was recorded (Fig. 4I). Fourth, in the presence of OV6þ CM, themonocytes expressed a higher percentage of M2 markers anddecreasedM1 phenotype genes thanmonocytes exposed toOV6�

CM (Fig. 4J). These results indicate that OV6þ cells can recruitmonocytes and educate them toward TAMs.

To determine whether OV6þ cell-educated TAMs facilitated thestem-like properties of OV6þ CSCs, a coculture system was

Figure 3.

Autophagy-related gene 7 (ATG7) maintains the expansion and self-renewal of OV6þ stem-like cells via OCT4 in prostate cancer. A, qRT-PCR analysis ofstem-like–associated genes in OV6þ C4-2B cells: control, ATG7OE, ATG7OE þ shb-catenin3#, and ATG7OE þ shOCT43# (OE, overexpression). B, Number of OV6þ

C4-2B spheres were compared among the control, ATG7OE, ATG7OE þ shb-catenin3#, and ATG7OE þ shOCT43# groups. C, First and second generation ofsubcutaneous tumors frommice injected with 2� 104 OV6þ C4-2B cells: control, ATG7OE, ATG7OEþ shb-catenin3#, and ATG7OEþ shOCT43# (6/group).D,Heatmapof the significantly differentially expressed genes (DEG) found by RNA-seq for shATG7 compared with the control OV6þ C4-2B cells. E, Western blot analysisof OCT4 and b-catenin in OV6þ C4-2B cells: control, ATG7 overexpression (ATG7OE), ATG7OE þ shb-catenin2#, and ATG7OE þ shb-catenin3#. F, The binding ofb-catenin to the OCT4 promoter was validated via luciferase assays in OV6þ C4-2B cells: control and ATG7OE. b-Catenin–binding sites were inhibited inreporter constructs harboring mutated variants (Mut). G, ATG7-interacting proteins identified via Nano LC-ESI-MS/MS and the STRING protein–protein interactionnetwork are shown.H,Western blot assaywas used to detect endogenous ATG7 coimmunoprecipitatedwith endogenous b-catenin in OV6þC4-2B cells. IgG servedas a control for immunoprecipitation. I, Representative images of the interaction between ATG7 and b-catenin in OV6þ C4-2B cells without or with ATG7KO,confirmed via Duolink staining assays (scale bars ¼ 5 mm). J, ChIP-PCR analysis was performed to examine the binding of b-catenin to the OCT4 promoterin OV6þ and OV6þ/ATG7OE C4-2B cells. K, Representative immunofluorescence staining of b-catenin in OV6þ C4-2B cells grouped as control, ATG7OE, andATG7KO. The nucleus is stained with DAPI dye (scale bar ¼ 25 mm). All data represent the means � SD (� , P < 0.05; �� , P < 0.01; and ��� , P < 0.001).

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Figure 4.

OV6þ CSCs educate monocytes/macrophages toward tumor-associated macrophages, which reciprocally facilitate the self-renewal of OV6þ CSCs. CCK8 experimentswere used to assess the proliferation at the indicated times in the following groups: A, OV6þ C4-2B or C4-2 cells were exposed to normal conditions, enzalutamide(ENZ, 10 nmol/L) with knockdown of ATG7 or OCT4. B, OV6þ C4-2B or C4-2 cells were exposed to normal conditions, abiraterone (Abi, 8 mmol/L) with knockdownof ATG7 or OCT4. C, OV6þ C4-2B cells stably expressing luciferase were injected into the prostate glands of NOD/SCID mice without or with enzalutamide (ENZ,30 mg/kg) treatment alone or combined with ATG7KO or shOCT43#. D and E, At the third week after injection, photon flux and serum PSA levels were examinedin thedifferentgroupsofmice.The resultsarepresentedas the fold increase in tumorgrowthover timeat the thirdweekafter injection.F,RepresentativeH&EstainingandIHC staining of OV6 and CD68 in prostate cancer patients (scale bar ¼ 50 mm). G and H, Kaplan–Meier curves for BCR and DFS in prostate cancer patients (n ¼ 145)were analyzed according to the combined OV6 and CD68 expression. I, Representative images and counts of migrated cells from sorted monocytes or THP-1cocultured with OV6� or OV6þ C4-2B cells (scale bar ¼ 50 mm). J, Expression of M1- and M2-related genes evaluated by qRT-PCR analysis in sorted monocytesor THP-1 cocultured with OV6� or OV6þ C4-2B cells. K, Real-time quantitative PCR analysis of stem-like–associated genes in OV6þ C4-2B cells cocultured withsortedmonocytes or THP-1 comparedwith cells cultured alone. L,Number of sphereswas compared amongOV6þC4-2B cells cultured alone andwith sortedmonocytesor THP-1. M, OV6þ C4-2B cells were exposed to normal conditions or ENZ (10 nmol/L) and cocultured without or with sorted monocytes or THP-1. CCK8 experimentswere used to assess the proliferation at the indicated times. All data represent the means � SD (� , P < 0.05; �� , P < 0.01; and ��� , P < 0.001).

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CSC-Mø Facilitates the Progression and ADT Resistance of PCa

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employed. After the coculture of TAMs and OV6þ CSCs, stem-associated gene expression and sphere-forming capacity wereenhanced significantly (Fig. 4K–L). In addition, TAMs enhancedthe enzalutamide resistance of OV6þCSCs (Fig. 4M). Therefore, areciprocal interaction between OV6þ CSCs and TAMs occurs inprostate cancer and influences ADT resistance.

IL6 signaling mediates the reciprocity between OV6þ CSCs andTAMs

To search for the inflammatory factors facilitating the interactionbetween OV6þ CSCs and TAMs, the cytokine profiles in the CMfrom monocytes, OV6þ CSCs alone, or cocultured monocytes/OV6þCSCswere analyzedby aRayBioHumanCytokineAntibodyArray (Fig. 5A–E; Supplementary Fig. S5A and S5B; SupplementaryTable S13). Compared with the CM from monocytes or OV6þ

CSCs cultured alone, the CM from cocultured monocytes/OV6þ

CSCs presented upregulated cytokines (Fig. 5A and B; Supplemen-tary Table S13). We then compared the upregulated proteins inboth comparable groups and, by using a Venn diagram, found that13 proteins were shared (Fig. 5C). To select the important upre-gulatedproteins,weusedmetrics, combiningbothP value and foldchange, to evaluate the protein relevance rank. The distribution ofthe rank score for each candidate upregulated protein is shownin Fig. 5D. In addition, we also used rank scores as filteringmetricsto analyze the shared upregulated proteins. We used a rank scoregreater than the 75% quantile as the upregulation cutoff, and thenthe shared upregulated proteins were obtained by the intersectionof the proteins via P value or rank scores (Fig. 5E). Finally, weobtained 8 shared upregulated proteins as candidates (Fig. 5E) andperformed pathway analysis, which also indicated that IL6 signal-ing pathway was activated (Supplementary Fig. S5A and S5B). Inconclusion, IL6 is identified as the crucial factor in the interactionbetween OV6þ CSCs and TAMs.

On the basis of the ELISA results, IL6 secretion was elevated inthe CM from cocultured TAMs/OV6þ CSCs compared with theCM from monocytes or OV6þ CSCs alone (Fig. 5F). In addition,the addition of a neutralizing antibody against IL6 to the CM ofcocultured TAMs/OV6þ CSCs suppressed the recruitment andeducation of TAMs by OV6þ CSCs (Fig. 5G–J). Furthermore,treatment with IL6 antibody also inhibited the ability of TAMsto facilitate the stem-like properties ofOV6þ cells, including stem-associated gene expression, self-renewal, and tumorigenicity (Fig.5K–M). IL6 exerts its biological effects by binding to IL6 receptor(IL6R) and triggers STAT3 activation (32). As expected, the FDA-approved drug tocilizumab (an IL6R inhibitor) or STAT3 knock-down also alleviated the promotion of the stem-like properties ofOV6þC4-2B cells by TAMs (Fig. 5K–M; Supplementary Fig. S5C).In addition, tocilizumab (an IL6R inhibitor) abated the increasedprotein level of p-STAT3 and nuclear STAT3 expression in OV6þ

C4-2B cells by TAMs (Supplementary Fig. S5D and S5E). Thus,IL6/STAT3 is required for the reciprocal interaction betweenOV6þ CSCs and TAMs in prostate cancer.

Joint targeting of OV6þ CSCs and their interaction with TAMsameliorates ADT resistance in an orthotopic prostate cancermodel

On the basis of above results, we examined whether a strategytargeting OV6þ CSCs and their network with TAMs reversed theADT resistance of OV6þ CSCs in vivo. OV6þ C4-2B or C4-2 cellswere injected into the prostates of mice, and all mice wererandomly divided into five groups (Fig. 6A; Supplementary Fig.

S6A). As shown in Fig. 6A–C and Supplementary Fig. S6A–S6C,enzalutamide-treated mice became resistant to ADT, and ATG7silencing or IL6R inhibition alone achieved limited suppression oftumor growth and the enzalutamide resistance of OV6þ CSCs.However, jointly targeting OV6þ CSCs and their interaction withTAMs by ATG7 silencing and tocilizumab effectively inhibitedtumor growth and improved the enzalutamide resistance ofOV6þ

CSCs in vivo (Fig. 6A–C; Supplementary Fig. S6A–S6C). In addi-tion, the levels of OV6 and F4/80 were elevated in the enzaluta-mide-treated xenografts and targeting only ATG7 silencing oronly IL6R inhibition partly attenuated the expression of OV6and F4/80 (Fig. 6A; Supplementary Fig. S6D–S6H). However,the combination of the two treatments exerted better therapeuticeffects on enzalutamide resistance of OV6þ CSCs in vivo (Fig. 6A;Supplementary Fig. S6A, S6D–S6E).

DiscussionTumor heterogeneity presents a great challenge in cancer treat-

ment, as tumor cell populations evolve andadapt to the stress of theexternal environment (1, 2). CSCs, an important example ofheterogeneity, are well known to survive many therapies includingchemotherapy, radiotherapy and endocrine therapy (33, 34). Thus,the therapeutic eradication of existing CSCs may be a promisingpolicy to overcome treatment resistance. Although many studieshave examined the mechanisms driving the expansion and self-renewal of CSCs, the effect of scavenging CSC populations in vivo isnot as ideal as in vitro. In our study, we found that an intracellularpathway, ATG7/b-catenin/OCT4, sustained the self-renewal ofOV6þ CSCs and facilitated ADT resistance in prostate cancer. Asexpected, blocking the pathway by ATG7 or OCT4 inhibitionimpaired the stemness of CSCs and reversed the ADT resistanceof OV6þ cells in vitro. However, experiments revealed that ATG7 orOCT4 knockdown treatment failed to inhibit ADT resistance in anorthotopic prostate cancer model.

In fact, the specific microenvironment or niche should receiveequal consideration in eradicating CSCs and reversing the drugresistance of tumors (17). CSCs reside in the microenvironmentand remodel their specific niche by recruiting mesenchymal stemcells, inflammatory cells, or immune cells (17). In addition,educated cells within the CSC niche produce factors that recip-rocally facilitate the expansion and self-renewal of CSCs andpromote tumor cell growth, invasion, andmetastasis (35). There-fore, a better understanding of the biology and niche componentsof CSCs as well as their network signaling is beneficial foreffectively eliminating CSCs and improving the therapeutic effecton tumors in clinical practice. Among the niche components,TAMs, which are derived from monocytes/macrophages, play aprotumor role and correlate with poor prognosis in patients withvarious cancers (36, 37). Although the interaction between CSCsand TAMs has been reported to promote the growth, progression,and stem-like characteristics of tumors, understanding of the in-depth mechanisms and a combined CSC-TAM targeting policy inprostate cancer remain lacking and need further study.

Androgen deprivation therapy (ADT) is a crucial treatment foradvanced or metastatic prostate cancer. Despite the effectivenessof ADT in the initial stage, drug resistance is inevitable due to theinduced heterogeneity and the microenvironment. A recent studyindicates that targeting colony-stimulating factor 1 receptor(CSF1R) can reverse TAM-mediated resistance to ADT in prostatecancer (21). In addition, our study demonstrates that blockade of

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Figure 5.

IL6 signaling mediates the reciprocity between OV6þ CSCs and TAMs. A, Cytokine profiles were analyzed by using a RayBio Human Cytokine AntibodyArray. Heatmap of significantly expressed cytokines in CM from monocytes (mono) cocultured with OV6þ C4-2B cells and CM from monocytes alone.B, Heatmap of significantly expressed cytokines in CM frommonocytes cocultured with OV6þ C4-2B cells and CM from OV6þ C4-2B cells alone. C, Venn diagram ofthe significantly expressed cytokines of the indicated groups. D, Rank score of the significantly differentially expressed cytokines. E, Venn diagram of thesignificantly differentially expressed cytokines of the indicated groups is shown. F, ELISA was conducted to detect the IL6 concentration (pg/mL) in CM from OV6þ

C4-2B cells cultured alone or with monocytes of healthy donors or THP-1 cells. G, The number of migrated cells among monocytes of healthy donors culturedalone or with OV6þ C4-2B cells in the presence or absence of IL6 antibody (20 mg/mL). H, The number of migrated cells among THP-1 cells cultured aloneor with OV6þ C4-2B cells in the presence or absence of IL6 antibody (20 mg/mL). I, Real-time quantitative PCR analysis of M1- and M2-related genes in sortedmonocytes cultured alone or with OV6þ C4-2B cells in the presence or absence of IL6 antibody (20 mg/mL). J, qRT-PCR analysis of M1- and M2-related genesin THP-1 cells cultured alone or with OV6þ C4-2B cells in the presence or absence of IL6 antibody (20 mg/mL). K, qRT-PCR analysis of stem-like–associated genes inOV6þ C4-2B cells cultured alone or with sorted monocytes in the presence or absence of IL6 antibody (20 mg/mL), tocilizumab (To; 5 mg/mL), shSTAT32#, orshSTAT33#. L, First- and second-generation numbers of sphereswere compared amongOV6þC4-2B cells cultured alone orwith sortedmonocytes in the presence orabsence of IL6 antibody (20 mg/mL), To (5 mg/mL), shSTAT32#, or shSTAT33#. M, Subcutaneous tumors from mice injected with 2 � 104 OV6þ C4-2B cellsunder different conditions, cultured alone or with sorted monocytes in the presence or absence of IL6 antibody (20 mg/mL), To (5 mg/mL), shSTAT32#, orshSTAT33# (6/group). All data represent the means � SD (� , P < 0.05; �� , P < 0.01; and ��� , P < 0.001).

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Figure 6.

Joint targeting of OV6þ CSCs and their interaction with TAMs ameliorates ADT resistance in an orthotopic prostate cancer model. A, C4-2B OV6þ cells culturedin the absence or presence of tocilizumab (To, 5 mg/mL) and/or ATG7 knockout cells were injected into the prostate glands of NOD/SCID mice subjected tocastration/enzalutamide treatment or control group. Representative hematoxylin and eosin (H&E) staining and IHC staining for OV6, F4/80, ATG7, OCT4,and STAT3 was performed in orthotopic xenografts from the different mouse groups (scale bar ¼ 50 mm). B and C, At the third week after injection,photon flux and serum PSA levels were examined in the different groups of mice. The results are presented as the fold increase in tumor growth over timeof the third week after injection. D, Schematic representation of the mechanism and the preclinical significance of our study. All data represent the means � SD(�� , P < 0.01).

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the association between ADT-induced transdifferentiation andTAMs inhibited enzalutamide resistance in an orthotopic prostatecancer model (23). In this study, CSCs and their interaction withTAMs in the niche were both considered. Combined targeting ofOV6þ cells and the network signaling between OV6þ cells andTAMs amelioratedADT resistance in anorthotopic prostate cancermodel. In conclusion, our study indicates that targeting CSCs andtheir niche may prove to be a more powerful strategy thantargeting the CSCs alone, which provides a rational approach toameliorating ADT resistance in prostate cancer (Fig. 6D). Ourfuture studies will further elucidate the interaction and networksignaling between the heterogenicity of prostate cancer and itsmicroenvironment, which will assist in searching for the criticaltarget, overcomingADT resistance and improving theprognosis ofpatients with advanced prostate cancer.

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

Authors' ContributionsConception and design: C. Wang, X.-G. Cui, Y.-H. SunDevelopment of methodology: H. Huang, C. Wang, F. Liu, G. Peng,Y.-Q. Cheng, D. LiuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Huang, C. Wang, F. Liu, H.-Z. Li, G. Peng, X. Gao,K.-Q. Dong, H.-R. Wang, D.-P. Kong, K.-J. Wang, Z. Zhou, X.-G. Cui, Y.-H. SunAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Huang, C. Wang, F. Liu, K.-Q. Dong, L.-H. Dai,Z. Zhou, J. Yang, Z.-Y. YangWriting, review, and/or revision of the manuscript: H. Huang, C. Wang

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.Wang, F. Liu, M. Qu, Y.-Q. Cheng, Q.-Q. TianStudy supervision: X. Gao, M. Qu, C.-L. Xu, D.-F. Xu, X.-G. Cui, Y.-H. Sun

AcknowledgmentsThe authors thank Dr. Leland Chung (Cedars-Sinai Medical Center, Los

Angeles, CA) for providing prostate cancer cell line C4-2B and C4-2. They alsothank Dr. Zhe-wei Wang at Translational Medicine Center, Changzheng Hos-pital, the Second Military Medical University (Shanghai, China) for flowcytometry analysis. The authors are grateful for technical support from theDepartment of Biophysics of Second Military Medical University (Shanghai,China) for electron microscopy. This work was supported by the NationalNatural Science Foundation of China (no. 81472397, 81773154, 81772747,81772720, and81301861),National KeyBasic ResearchProgramofChina (973Program, no. 2012CB518306), Research Program of Science and TechnologyCommission of Shanghai Municipality (no. 14411950100), the Program forShanghai Municipal Health and Family Planning Commission ImportantDiseases Joint Research Project (no. 2013ZYJB0101), Shanghai Natural ScienceFoundation of China (no. 13ZR1450700), Innovation Program of ShanghaiMunicipal Education Commission (no. 2017-01-07-00-07-E00014), theShanghai Medical Guidance (Chinese and Western Medicine) Science andTechnology Support Project (no. 17411960200), Key Construction Project ofZhangjiang National Innovation Demonstration Zone the National New DrugInnovation Program (no. 2017ZX09304030), and Shanghai Clinical MedicalCenter for Urinary System Diseases (no. 2017ZZ01005).

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 February 12, 2018; revised April 3, 2018; accepted April 20, 2018;published first April 24, 2018.

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Huang et al.

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2018;24:4612-4626. Published OnlineFirst April 24, 2018.Clin Cancer Res   Hai Huang, Chao Wang, Fei Liu, et al.   Deprivation Therapy Resistance of Prostate CancerMacrophages Facilitates the Progression and Androgen Reciprocal Network between Cancer Stem-Like Cells and

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