notch signaling via wnt regulates the proliferation of … · carcinoma. introduction macrophages...

Tumor Biology and Immunology

NOTCH Signaling via WNT Regulates theProliferation of Alternative, CCR2-IndependentTumor-AssociatedMacrophages inHepatocellularCarcinomaYu-Chen Ye1, Jun-Long Zhao2,Yi-Tong Lu2, Chun-ChenGao2,Yang Yang2, Shi-Qian Liang2,Ying-Ying Lu2, Lin Wang1, Shu-Qiang Yue1, Ke-Feng Dou1, Hong-Yan Qin2, and Hua Han1,2

Abstract

Tumor-associated macrophages (TAM) play pivotal roles intumor progression and metastasis, but the contribution andregulation of different macrophage populations remainunclear. Here we show that Notch signaling plays distinctroles in regulating different TAM subsets in hepatocellularcarcinoma (HCC). Myeloid-specific NOTCH blockade byconditional disruption of recombination signal bindingprotein Jk (RBPj cKO) significantly delayed the growth ofsubcutaneously inoculated Lewis lung carcinoma (LLC), butaccelerated orthotopically inoculated hepatic Hepa1-6tumors in mice. In contrast to subcutaneous LLC, RBPj cKOsignificantly increased the number of TAMs in hepaticHepa1-6 tumors despite impeded differentiation of mono-cyte-derived TAMs (moTAM). The dominating TAMs inorthotopic HCC manifested properties of Kupffer cells (KC)and hence are tentatively named KC-like TAMs (kclTAM).The increased proliferation of RBPj cKO kclTAMs was main-tained even in Ccr2�/� mice, in which moTAMs were genet-ically blocked. NOTCH signaling blockade accelerated pro-

liferation of kclTAMs via enhanced b-catenin–dependentWNT signaling, which also downregulated IL12 and upre-gulated IL10 expression by kclTAMs likely throughc-MYC. In addition, myeloid-specific RBPj cKO facilitatedhepatic metastasis of colorectal cancer but suppressed lungmetastasis in mice, suggesting that the phenotype of RBPjcKO in promoting tumor growth was liver-specific. Inpatient-derived HCC biopsies, NOTCH signaling negativelycorrelated with WNT activation in CD68þ macrophages,which positively correlated with advanced HCC stages.Therefore, NOTCH blockade impedes the differentiation ofmoTAMs, but upregulates Wnt/b-catenin signaling to pro-mote the proliferation and protumor cytokine productionof kclTAMs, facilitating HCC progression and hepatic metas-tasis of colorectal cancer.

Significance: These findings highlight the role of NOTCHand WNT signaling in regulating TAMs in hepatocellularcarcinoma.

IntroductionMacrophages infiltrating tumors, known as tumor-associated

macrophages (TAM), are pivotally involved in tumor initiation,progression, and metastasis (1, 2). TAMs are broadly classifiedinto the M2 macrophage activation subtype, attributing to theirupregulated expression of M2 markers including IL10, mannosereceptor, and arginase, as well as their enhanced activities

in suppressing antitumor immunity and promoting tumorneovascularization (1–3). Compelling evidence has demonstrat-ed that the majority of TAMs are derived from CCR2þ inflamma-torymonocytes in bonemarrow (BM), therefore targeting CCR2þ

monocytes to block TAM input has been recognized as a prom-ising therapy (2–4). However, it is increasingly clear that TAMsconstitute heterogeneous populations (5, 6). Inmousemammarycancer models and human breast cancers, as well as in trans-planted murine Lewis lung carcinoma (LLC), spontaneous lungcancer, and glioma, TAMs originate frommonocyte input but canmaintain their population by local proliferation (5–10). On theother hand, embryonic hematopoiesis–derived tissue-residentmacrophages, which are maintained by local mitogenic signalssuch as colony-stimulating factor (CSF)-1 and IL4 (11–14), alsoparticipate in tumor progression and metastasis as shown inpancreatic ductal adenocarcinoma (15) and certain lung metas-tasis events (16). It is important to dissect themechanisms for theregulation of these distinct monocyte and macrophage popula-tions in tumors for more efficient intervention.

Hepatocellular carcinoma (HCC) is a leading cause of can-cer-related death worldwide (17). In liver, resident macro-phages or Kupffer cells (KC) are primarily generated duringembryogenesis and sustained by self-renewal, but recent evi-dence has demonstrated their input from BM also (18–20). KCs

1Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military MedicalUniversity, Xi'an, China. 2State Key Laboratory of Cancer Biology, Department ofMedical Genetics and Developmental Biology, Fourth Military Medical Univer-sity, Xi'an, China.

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

Y.-C. Ye, J.-L. Zhao, and Y.-T. Lu contributed equally to this article.

Corresponding Authors: Hong-Yan Qin, State Key Laboratory of Cancer Biol-ogy, Department of Medical Genetics and Developmental Biology, FourthMilitary Medical University, Chang-Le Xi Street #169, Xi'an 710032, China.Phone: 8629-8477-4487; Fax: 8629-8324-6270; E-mail: [email protected];and Ke-Feng Dou, [email protected]

Cancer Res 2019;79:4160–72

doi: 10.1158/0008-5472.CAN-18-1691

�2019 American Association for Cancer Research.

CancerResearch

Cancer Res; 79(16) August 15, 20194160

on February 18, 2021. © 2019 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 2, 2019; DOI: 10.1158/0008-5472.CAN-18-1691

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-18-1691&domain=pdf&date_stamp=2019-7-29

[email protected]

[email protected]

http://cancerres.aacrjournals.org/

produce IL6 to promote diethylnitrosamine-induced HCC andare responsible for the gender disparity in HCC (21–23). KCsalso play a key role in cholangiocellular carcinogenesis (24).Monocyte-derived macrophages, on the other hand, are respon-sible for the clearance of senescent premalignant hepato-cytes (25). However, precise contributions of different macro-phage populations and their regulation in hepatic cancers havenot been fully understood.

The recombination signal binding protein-Jk (RBPj)-mediatedNOTCH signaling regulates cell differentiation and plasticity incooperation with other pathways (26, 27). NOTCH signalingplays a critical role in differentiation and functional plasticity ofTAMs (7, 28, 29). NOTCH signaling is required for monocyte-derived TAM differentiation as shown in myeloid-specific Rbpjknockoutmediated byCD11c-Cre inmice (7).On the other hand,forced Notch activation by myeloid-specific overexpression ofNotch intracellular domain (NICD)mediated by LyzM-Cre resultsin attenuated TAM phenotypes in mice (29). In this study, weshow that myeloid-specific Rbpj knockout blocked TAM differen-tiation from monocytes in HCC as reported previously in breastcancermodels (7), but a TAMpopulationwith KC-like phenotypeexpanded via proliferation and constituted an alternative sourceof TAMs to facilitate tumor growth and hepatic metastasis. Ourevidence also supported that NOTCH-WNT signaling governedthe expansion of these KC-like TAMs in HCCs.

Materials and MethodsPatients and biopsies

Human HCC biopsies were obtained from patients hospital-ized in the Department of Hepatobiliary Surgery, Xijing Hospital,Fourth Military Medical University (Xi'an, China), and stagedaccording to the AJCC Cancer Staging Manual (8th ed.; Supple-mentary Table S1). The use of human samples was approved bythe Ethics Committee of Xijing Hospital and conformed toDeclaration of Helsinki. Written informed consent was obtainedfrom all patients involved.

Mice and tumor modelsLyzM-Cre (stock #019096, Jackson Laboratory), Rbpj-floxed

(Rbpjf; ref. 30), and Ccr2 knockout (stock #004999, JacksonLaboratory) mice were brought about on the C57BL/6J back-ground in a specific pathogen-free facility. Mice were mated andgenotyped by PCR. Male littermates of 8- to 10-weeks old wereused in experiments. All animal experiments were reviewed andapproved by the Animal Experiment Administration Committeeof the Fourth Military Medical University to ensure ethnical andhumane treatment of animals.

LLC,Hepa1-6 (HCC), andCMT93 (colorectal cancer) cellswereobtained from the ATCC repository in 2015. These cells wereauthenticated by both morphologic profiling and short tandemrepeat profiling and tested by PCR to exclude Mycoplasma con-tamination. Cells were maintained in DMEM supplemented with10% FCS and 2 mmol/L L-glutamine (Invitrogen). Both LLC andCMT93 cells were derived from the C57BL strain (H-2b). Hepa1-6cells were derived fromC57Lmice (H-2b) but bear the sameMHCgenotype as that of C57BL strain. So we tentatively used Hepa1-6cells to establish orthotopic HCC models in mice on C57BLbackground. For lung cancer models, LLC cells (5 � 106 cells/200 mL) were inoculated subcutaneously on the right side of theback of recipient mice. For orthotopic HCC models, mice were

anesthetized by intraperitoneal injection of 0.6% pentobarbitalsodium (10 mL/g, Sigma-Aldrich). Hepa1-6 cells (5 � 106 cells in30 mLMatrigel, Sigma) were injected into liver parenchyma of leftlobe. Mice were sacrificed 3 weeks after inoculation. Tumors wereweighed and tumor volume was estimated as (L � S2) � 0.52 (L,long diameter; S, short diameter). In some experiments, Hepa1-6cells, CMT93, or LLC cells were transduced with a lentivirusexpressing firefly luciferase (Genechem) following the manufac-turer's protocol, and hepatic tumor growth was monitored usingan in vivo imaging system (IVIS; Xenogen, Perkin-Elmer). For livermetastasis, CMT93 cells (1 � 106) were injected into spleenparenchyma of mice. For lung metastasis, LLC cells (1 � 106)were injected via tail vein. Both liver and lung metastasis wasmonitored using IVIS. ICG-001 (MedChem Express) was admin-istered by intraperitoneal injection (5 mg/kg in saline; Supple-mentary Fig. S1A–S1D for tumor models).

HistologyMice were perfused with PBS before tumors were collected and

fixed in cold 4% paraformaldehyde and treated with 30% sucroseat 4 �C.Hematoxylin and eosin staining was performed routinely,and pictures were taken under a microscope (BX51, Olympus)equipped with a CCD camera (DP70, Olympus). For immuno-fluorescence, samples were embedded in optimum cutting tem-perature compound and cryosectioned at 5 or 60-mm thickness,followed by immunostaining according to standard protocolswith antibodies listed in Supplementary Table S2. Nuclei werecounterstained with Hoechst 33258 (Sigma). Images were col-lected under a laser scanning confocal microscope (FV1000,Olympus) or a fluorescence microscope (BX51, Olympus).Three-dimensional reconstruction of images was performed withthe NIS-Elements Viewer 4.20 software (Nikon) after CD31staining.

Cell culture and transfectionMurine primary KCs were isolated by density gradient centri-

fugation and cultured as described previously (30). For transfec-tionwith siRNA, KCs (5� 105) were seeded in 24-well plates, andtransfected with synthetic siRNA or negative control (NC; 50nmol/L, RiboBio) using Lipofectamine 2000 (Invitrogen) follow-ing the manufacturer's protocol. Medium was replaced withnormal medium 4 hours after adding the liposome–nucleotidemixture, and cells were assayed 24 hours later. In some experi-ments, KCswere treatedwithHepa1-6 conditionalmedium (CM)or Wnt3a (100 ng/mL) for 24 hours. Monocytes were isolated byFACS according to the markers described previously (31) andstimulatedwithHepa1-6CM for 24 hours before FACS analysis ofintracellular Ki67.

For reporter assay, the murine b-catenin (b-Ctnn) promoterfragment (�1,237–þ131, Genebank accession # NC_000075.6)was cloned by PCR to construct pCtnnb1-pro-luc using pGL3-basic (Invitrogen; ref. 32). KCs (1 � 106) were transfectedwith 1 mg pCtnnb1-pro-luc and 50 ng pRL-TK by Nucleofector(Lonza) according to the recommended protocol, and culturedfurther with Hepa1-6 CM and a g-secretase inhibitor (GSI;2.5, 5.0, 10 mmol/L; DAPT, Sigma) for 24 hours. Luciferaseactivity was detected using a Dual-Luciferase Reporter Assay Kit(Promega) and a chemiluminometer (Luminoskan Ascent,Labsystems).

Short hairpin RNA (shRNA) targetingb-catenin (Shb-Ctnn) andthe NC were inserted into GV493 lentivirus vector that expresses

Notch Regulates TAM Subsets in HCC

www.aacrjournals.org Cancer Res; 79(16) August 15, 2019 4161




GFP (Genechem). HEK293T cells were transfected to prepare viralparticles. KCs from RBPj cKO and control mice were stablytransduced with Shb-Ctnn or NC shRNA using the lentiviralparticles. The infected GFP-labeled KCs (2.5 � 105) were mixedwith Hepa1-6 cells (5 � 106) in 30 mL Matrigel, and wereorthotopically inoculated in liver of wild-type mice. Mice weremaintained for 3 weeks after the inoculation. In addition, ade-novirus-expressing NICD (Genechem)was applied to infect wild-type KCs according to the manufacturer's protocol.

FACS assayMice were perfused with PBS and tumors were dissected and

finely minced, followed by incubation with 1mg/mL collagenaseV and 4 mg/mL DNase I (Roche) at 37 �C for 1 hour. Single-cellsuspensions were obtained by passing through a 70-mm cellstrainer. After erythrolysis, cells were resuspended in FACS buffer,and incubated with antibodies listed in Supplementary Table S2for 20minutes in dark on ice. Intracellular stainingwas performedwith a permeabilization buffer (eBioscience) and stained withantibodies. Cells were analyzedwith a FACSCalibur or a FACSAriaII flow cytometer (BD Immunocytometry Systems), and datawereanalyzed using the FlowJo 7.6.1 Software (TreeStar). Cell viabilitywas evaluated with 7-amino-actinomycin D (BD Biosciences).Cell sorting was performed using the FACSAria II Flow Cytometer(BD Biosciences).

ELISASerum IL10 in mice was determined with an ELISA Kit

(eBioscience) according to the manufacturer's instructions.Wnt3a level was also determined with a kit (MyBioSource).

RT-PCRTotal RNA was extracted from tissue or cell samples using the

TRizol Reagent (Invitrogen), and reverse transcribed into cDNAwith the PrimeScript RT Reagent Kit (Takara Biotechnology).qPCR was performed using the SYBR Premix ExTaq II (TakaraBiotechnology) and Applied Biosystems 7500 Real-time PCRSystem, with b-actin as a reference control. Primers are listed inSupplementary Table S3.

Western blottingCells were lysed with the RIPA Lysis Buffer (Beyotime Biotech-

nology) containing 10 mmol/L phenylmethanesulfonyl fluoride.Samples were separated by SDS-PAGE and electro-transferredonto polyvinylidene fluoride membranes. Membranes wereblocked with 5% skim milk in PBS-0.1% Tween 20, and thenincubated at 4 �C overnight with primary antibodies, followed bywashing and incubation with secondary antibodies at roomtemperature for 1 hour. After washing, blots were developed withenhanced chemiluminescence (Pierce, Thermo Fisher Scientific)and detected using ChemiScope Imaging System (Clinx ScienceInstruments). b-ACTIN was used as an internal reference. Anti-bodies are listed in Supplementary Table S2.

Statistical analysisQuantification of histologic image was conducted with Image

Pro Plus 6.0 Software (Media Cybernetics). Statistical analyseswere performed with GraphPad Prism 5 software. The compar-isons between groupswere undertaken using paired and unpairedStudent t tests or one-way ANOVA with Tukey multiple compar-ison test. Results were expressed as means � SD. P <0.05 wasconsidered as significant.

ResultsMyeloid-specific Rbpj knockout promoted the growth oforthotopic HCC

NOTCH signaling appeared activated in TAMs, likely byligands expressed by tumor or microenvironmental cells (Sup-plementary Fig. S1E). Myeloid-specific Rbpj disruption withLyzM-Cre (RBPj cKO) resulted in retarded growth of subcuta-neous LLC tumors as compared with the control (LyzM-Cre-RBPjf/þ; Supplementary Figs. S1A and S2A–S2C). FACS analy-ses showed that RBPj cKO reduced TAMs (F4/80hiCD11bhi) andincreased F4/80intCD11bhiLy6Chi monocytes (SupplementaryFig. S2D), likely due to blocked differentiation of monocyte-derived TAMs (moTAM) as demonstrated by CD11c-Cre–mediated Rbpj knockout in murine breast cancer (7). Consis-tently, immunofluorescence showed that accompanied withreduced F4/80þ TAMs, CD31þ vascular staining decreasedsignificantly in subcutaneous LLC tumors from RBPj cKO mice(Supplementary Fig. S2E). Therefore, consistent with previousreports (7), myeloid NOTCH blockade by LyzM-Cre–mediatedRbpj knockout reduced TAMs and suppressed the growth ofsubcutaneous LLC tumors.

In liver, macrophage development appeared normal in staticRBPj cKO mice (Supplementary Fig. S3A and S3B). We foundthat, unexpectedly, orthotopic HCC tumors established byinoculation of Hepa1-6 cells in the liver (Supplementary Fig.S1B) grew significantly larger in the Rbpj cKO mice than in thecontrol (Fig. 1A and B; Supplementary Fig. S3C–S3E). Tumor-infiltrating macrophages and vasculature increased as shownby immunofluorescence (Fig. 1C). FACS showed that CD8þ

T cells decreased (Fig. 1D and E). LyzM-Cre specifically acti-vates LoxP-mediated DNA recombination in macrophages andneutrophils (33). In RBPj cKO mice, while total CD11bþ

myeloid cells (Fig. 1D and F) and MHCIIhiCD11chi dendriticcells (Supplementary Fig. S4A) in hepatic Hep1-6 tumorsappeared unchanged, and F4/80�CD11bþLy6Ghi tumor-associated neutrophils (TAN) showed a tendency of decrease,but not statistically significant, the F4/80hiCD11bþLy6G� TAMpopulation increased significantly (Fig. 1D and G). TumorIL10 mRNA and serum IL10 increased in RBPj cKO micebearing orthotopic Hepa1-6 tumors (Fig. 1H). These dataindicated that despite repressing subcutaneous LLC tumors,blocking NOTCH signaling by RBPj cKO promoted orthotopicHepa1-6 tumor growth with increased TAMs.

RBPj cKO increased TAMs despite impeded differentiation inhepatic Hepa1-6 tumors

To determine the identity of increased TAMs in hepatic Hepa1-6 tumors of RBPj cKO mice, single-cell suspensions from ortho-topic Hepa1-6 tumors of perfused mice were analyzed by FACSafter staining with CD11b and F4/80 (Fig. 2A; refs. 34, 35). TheF4/80hiCD11blo macrophage population (G1), which normallyrepresents KCs in quiescence but may also include monocyte-derived macrophages (20), increased significantly in both cellpercentage andnumber (Fig. 2B). These TAMswere Ly6G-negativeand Ly6C-low/negative, and expressed mature TAM markers(VCAM1 and MHCII) and KC markers (MARCO and TIM4) andnegative for the eosinophil marker SiglecF (Fig. 2C). Moreover,they expressed higher IL10 in RBPj cKO mice (Fig. 2D). In FACS-sorted F4/80hiCD11blo TAMs, qRT-PCR confirmed reducedexpression of NOTCH downstream genes Hes1 and Hey1

Ye et al.

Cancer Res; 79(16) August 15, 2019 Cancer Research4162




(Supplementary Fig. S4B), and increased IL10, and PD-L1 andPD-L2 mRNA accompanied by decreased IL12 and IL1b expres-sion (Fig. 2E). FACS with Ly6C staining further showed that theF4/80þ population contained low percentages of Ly6Chi mono-cytes and Ly6CintmoTAMs, but themajority (around 90%) of thispopulation was Ly6Clo/� macrophages that resembled KCs (36).In this F4/80þ population from hepatic Hepa1-6 tumors in RBPjcKO mice, monocytes increased, moTAMs decreased, andLy6Clo/� TAMs increased mildly but significantly (Fig. 2F). TheseF4/80þCD11bloLy6Clo/� TAMs were then tentatively named asKC-like TAMs (kclTAM), although their ontogeny might be com-plex (35, 36). Further analysis revealed that the F4/80hiCD11blo

population (Fig. 2A) and the F4/80þCD11bloLy6Clo/� popula-tion (Fig. 2F) were the same population of kclTAMs cells (Sup-plementary Fig. S4C and S4D). AlthoughmoTAMs decreased, thetotal TAMnumber still accumulated significantlywith the increaseof kclTAMs in RBPj cKOmice (Supplementary Fig. S4E). Notably,kclTAMs dominated the total TAMs both in RBPj cKO andCtrl mice (>90%), with higher proportion in the RBPj cKO

ones (Supplementary Fig. S4E). Coinoculation of Hepa1-6 withKCs indeed promoted HCC tumor growth, suggesting a pro-tumor role of kclTAMs (Supplementary Fig. S4F). In subcuta-neous LLC tumors, consistent with previous findings in breastcancer models (7), staining with anti-CD11b, F4/80, and Ly6Cshowed that monocytes (F4/80þCD11bhiLy6Chi) increased andmoTAMs (F4/80þCD11bhiLy6Cint) decreased upon NOTCHblockade, respectively (Supplementary Fig. S4G and S4H).These results suggested that although NOTCH blockade dis-rupted moTAMs differentiation from inflammatory monocytes,kclTAMs increased alternatively, participating in acceleratedgrowth of hepatic Hepa1-6 tumors.

RBPj cKO promoted the proliferation of kclTAMs in hepaticHepa1-6 tumors

Next, we analyzed mechanisms underlying increased kclTAMsin RBPj cKO mice. Apoptosis did not change in F4/80hiCD11blo

TAMs from RBPj cKOmice (Supplementary Fig. S5A and S5B). Incontrast, cytoplasmic staining of Ki67 indicated that Ki67þ cells

Figure 1.

Myeloid-specific Rbpj knockout promoted the growth of orthotopic hepatic Hepa1-6 tumors with increased TAMs. A, Hepa1-6 cells were orthotopically inoculatedin liver of RBPj cKO or control (Ctrl) mice. Tumors were dissected 3 weeks after the inoculation and photographed. Tumor weights were compared (n¼ 13).B, tumor growth was monitored with in vivo imaging (n¼ 11). C, Tumor sections were stained with anti-F4/80 or anti-CD31 (60 mm) immunofluorescence andcounterstained with Hoechst. F4/80þ cells or CD31þ pixels were compared (n¼ 5). D, Single-cell suspensions from tumors were stained as indicated andanalyzed by FACS. The percentage and/or number of CD4þand CD8þ T cells (E), CD11bþmyeloid cells (F), and TAMs (F4/80þCD11bþ Ly6G�) and TANs(F4/80�CD11bþLy6Gþ; G) were quantitatively compared (n¼ 7). H, IL10 mRNA in tumors and serum IL10 of the mice were determined by qRT-PCR and ELISA(n¼ 5), respectively. Bars, mean� SD. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; n.s, not significant.






increased remarkably in the F4/80hiCD11blo TAM population inhepatic Hepa1-6 tumors from RBPj cKO mice, suggestingincreased proliferation (Fig. 3A and B). However, no significantincrease in Ki67þ cells was detected in the monocyte/moTAM(F4/80intCD11bint) or neutrophil (F4/80�CD11bhi) compart-ment (Supplementary Fig. S5C and S5D). Immunofluorescencestaining confirmed that F4/80þKi67þ TAMs increased in hepaticHepa1-6 tumors from RBPj cKO mice (Fig. 3C). Next, KCs wereisolated from RBPj cKO and control mice (SupplementaryFig. S5E) and cultured with Hepa1-6 CM, which may providemitogenic stimulation for KCs. EdU incorporation assay showedmore EdUþ cells in Rbpj-deficient KCs than that of the control(Fig. 3D and E). Cell-cycle analysis showed that the G2–M pro-portion increased in Rbpj-deficient KCs as compared with thecontrol (Fig. 3F). In normal KCs treated with Hepa1-6 CM andGSI, aNOTCHsignal inhibitor,more proliferative EdUþ cellsweredetected compared with that of the control (Supplementary Fig.S5F andS5G).However, cytoplasmicKi67 stainingof FACS-sortedBM monocytes stimulated with Hepa1-6 CM showed that thepercentage of Ki67þ RBPj cKOmonocytes displayed a tendency ofdecrease but not statistical significance, whose phenotype was stilldifferent from that of RBPj cKO kclTAMs (Supplementary Fig.S6A–S6C). Together, these data suggested that RBPj cKO increasedkclTAMs by promoting their proliferation in hepatic tumors.

The increased proliferation of Rbpj cKO kclTAMs wasmaintained in Ccr2�/� mice

To further investigate the macrophage subpopulationsin HCC in RBPj cKO mice, we bred mice to obtain RBPj cKOon Ccr2�/� background. Ccr2 knockout reduced the growth ofhepatic Hepa1-6 tumors accompanied by a 34.1% (7.81 � 0.86in control vs. 5.14 � 0.46 in CCR2 knockout) reduction in theF4/80hiCD11blo population (Fig. 4A and B; SupplementaryFig. S7A). F4/80intCD11bint monocytes/moTAMs decreasedmore significantly (Fig. 4A and B). However, when NOTCHsignaling was interrupted by RBPj cKO on the Ccr2�/� back-ground, the growth of hepatic Hepa1-6 tumors increased ascompared with the Ccr2�/� control (Fig. 4C and D; Supple-mentary Fig. S7B–S7D). Flow cytometry showed that, com-pared with the Ccr2�/� control, the F4/80hiCD11blo populationincreased by 28.3% (5.41 � 0.30 in Ccr2�/� control to 6.94 �0.13 in Ccr2�/� RBPj cKO) in hepatic Hepa1-6 tumors fromRBPj cKOmice (Fig. 4E and F). Anti-Ki67 and anti-IL10 stainingshowed that cells in the F4/80hiCD11blo population weremore proliferative and expressed more IL10 (Fig. 4E and F).Staining of Ly6C indicated that while monocytes andmoTAMs were almost absent likely due to Ccr2 deficiency,F4/80þCD11bloLy6Clo/� kclTAMs increased (Fig. 4G and H).These data further suggested that NOTCH blockade in myeloid

Figure 2.

Myeloid-specific Rbpj knockout increased kclTAMs in orthotopic hepatic Hepa1-6 tumors. A, Hepa1-6 cells were orthotopically inoculated in livers of Rbpj cKO orcontrol (Ctrl) mice for 3 weeks. Single-cell suspensions from tumors were stained with anti-CD11b and -F4/80, followed by FACS. B, The percentage and numberof F4/80hiCD11blo TAMs (G1) were determined (n¼ 7). C, F4/80hiCD11blo TAMswere further displayed for TAM, KC, and eosinophil markers. D, F4/80hiCD11blo

TAMswere further analyzed for cytoplasmic IL12 and IL10 by FACS and quantitatively compared (n¼ 7). E, The expression of IL1b, IL12, IL10, PD-L1, and PD-L2 inFACS-sorted F4/80hiCD11blo TAMs from G1 was determined by qRT-PCR (n¼ 6). F, Single-cell suspensions from the hepatic Hepa1-6 tumors were analyzed byFACS after staining with anti-F4/80, -CD11b, and -Ly6C. Percentages of monocytes (F4/80þCD11bintLy6Chi), moTAMs (F4/80þCD11bhiLy6Cint), and kclTAMs(F4/80þCD11bloLy6Clo/�) were determined (n¼ 7). Bars, mean� SD. � , P < 0.05; �� , P < 0.01; n.s, not significant.

Ye et al.





cells promoted kclTAM proliferation and IL10 expression inde-pendent of CCR2 in HCC.

RBPj cKO led to enhanced activation of WNT signaling inkclTAMs

Next, we investigated the molecular mechanisms ofenhanced proliferation in RBPj cKO kclTAMs. The expressionof CSF-1R, CSF-2Rb2, and IL4R in F4/80hiCD11blo TAMsdid not change in RBPj cKO as detected by qRT-PCR (Supple-mentary Fig. S8A). We then examined the activity of WNTsignaling, which is repressed by NOTCH activation (27),by immunofluorescence staining with anti-F4/80 and anti-b-catenin. In hepatic tumors from RBPj cKO mice, the meanfluorescence intensity of both total and nuclear b-catenin inF4/80þ TAMs increased significantly (Fig. 5A and B). Theexpression of WNT downstream molecules Axin2, b-catenin,c-Myc, and cyclin D1 increased significantly in FACS-sortedF4/80hiCD11blo TAMs from RBPj cKO mice, as determinedby qRT-PCR (Fig. 5C). The protein level of b-CATENIN, acti-vated b-CATENIN (a-b-ctnn), and c-MYC also increased inthese cells (Fig. 5D). Rbpj-knockout KCs cultured withHepa1-6–derived CM displayed upregulated mRNA expressionof c-Myc, b-catenin, cyclin D1, and Axin2 (Fig. 5E), as well asprotein expression of b-CATENIN, a-b-CTNN, and c-MYC(Fig. 5F). We also carried out a reporter assay by transfectingKCs with pCtnnb1-pro-luc and cultured in the presence of

Hepa1-6 CM and GSI. The result showed that blockingNOTCH signaling upregulated b-catenin mRNA, and increasedluciferase expression driven by the b-catenin promoter (Fig. 5Gand H). ELISA showed that Hepa1-6 secreted WNT3a, whichcould stimulate the proliferation of KCs (SupplementaryFig. S8B and S8C). Surprisingly, compared with controlKCs, RBPj cKO KCs cultured in vitro appeared to exhibitupregulation of WNT signaling independent of exogenousNotch ligands. Thus, we supposed that KCs could expressNotch ligands themselves and signal to each other. Indeed,Hepa1-6 CM stimulated KCs to express JAG1 and elevatedthe expression of NICD with more translocation into thenucleus (Supplementary Fig. S8D and S8E), suggesting acti-vation of NOTCH signaling. To further confirm whether theNOTCH activation of KCs was dependent on the JAG1 ligandexpressed, we treated KCs with Jag1 siRNA, which efficientlyknocked down the expression of JAG1, as well as that of NICD(Supplementary Fig. S8F and S8G). Next, we used the adeno-virus to overexpress NICD in Hepa1-6 CM-treated KCs andfound that the protein level of b-CATENIN and a-b-CTNN, andthe mRNA level of b-catenin and Axin2 were downregulatedwith reduced proliferative capacity of KCs as measured byEdU staining (Supplementary Fig. S8H and S8J). These resultssuggested that NOTCH signaling could negatively regulate theactivation of WNT/b-CATENIN signaling and the proliferationof kclTAMs.

Figure 3.

RBPj cKO increased kclTAM proliferation. A, Single-cell suspensions from the hepatic Hepa1-6 tumors were stained for F4/80, CD11b, and Ki67, followed by FACS.B, The percentage of Ki67þ F4/80hiCD11blo TAMs (or kclTAMs) was determined (n¼ 7). C, Hepatic Hepa1-6 tumors from RBPj cKO and control (Ctrl) mice weresectioned and stained with anti-F4/80 and Ki67 immunofluorescence. The number of F4/80þKi67þ TAMswas determined (n¼ 5). D, KCs from RBPj cKO andcontrol mice were treated with Hepa1-6–derived CM for 24 hours and were labeled with EdU and observed under a fluorescence microscope. E, The number andpercentage of EdUþ cells were quantitatively compared (n¼ 4). F, KCs from RBPj cKO and control mice were treated with Hepa1-6–derived CM for 24 hours.Cell-cycle progressionwas determined by FACS after propidium iodide staining. The proportion of cells in each phase was compared between the two groups(n¼ 3). Bars, mean� SD. � , P < 0.05; �� , P < 0.01.






Rbpj deficiency enhanced proliferation of kclTAMs viaWNT/b-CATENIN signaling

We next accessed whether blockingWNT signaling in vivo couldinhibit kclTAM proliferation in hepatic Hepa1-6 tumors in RBPjcKO mice with ICG-001, an antagonist of WNT/b-CATENINpathway (37). Injection of ICG-001 significantly suppressedgrowth of orthotopic Hepa1-6 tumors in both control and RBPjcKO mice (Supplementary Fig. S9A). FACS analyses showed thattheF4/80hiCD11blopopulationdecreased significantlyupon ICG-001 injection (Fig. 6A and B). In this population, Ki67þ prolifer-ative cells and IL10-expressing cells, both of which increased inhepatic tumors of RBPj cKOmice, decreased significantly (Fig. 6Aand B). CD8þ T cells increased, whereas regulatory T (Treg) cellsdecreased (Fig. 6C; Supplementary Fig. S9B and S9C). These datasuggested that WNT signaling was required for enhanced prolif-eration and IL10 expression in Rbpj-deficient kclTAMs. However,reduced tumor growthmight not be solely attributed to decreasedkclTAMs in the presence of ICG-001, because Wnt signaling playsmultiple roles in different cell types in tumor.

In cultured RBPj cKO KCs, b-catenin siRNA repressedb-CATENIN and also AXIN2 expression, and c-MYC siRNArepressed c-MYC expression efficiently (SupplementaryFig. S10A–S10D). EdU staining indicated that knockdownof b-catenin abrogated the enhanced proliferation of Rbpj-deficient KCs (Fig. 6D; Supplementary Fig. S10E). Moreover,

knockdown of b-catenin or c-Myc in Rbpj-deficient KCs withsiRNAs reversed the downregulated IL12 and IL1b and upre-gulated IL10 as shown by qRT-PCR (Fig. 6E and F). These datasuggested that WNT/b-CATENIN signaling was required fornot only accelerated proliferation, but also likely for theirprotumor cytokine secretion of Rbpj-deficient kclTAMs, likelythrough upregulated c-MYC (38).

To further show that WNT signaling mediated kclTAM pro-liferation under RBPj cKO, we transfected KCs from RBPj cKOand control mice with b-catenin shRNA or NC shRNA(both labeled with GFP), and coinoculated these KCs withHepa1-6 cells in liver of wild-type mice. KCs were transfectedwith high efficiency, and b-catenin was effectively knockeddown with downregulated Axin2 expression (SupplementaryFig. S10F–S10H). The result showed that Rbpj-deficient KCssignificantly promoted tumor growth, but this effect was abro-gated by b-catenin shRNA transfection (SupplementaryFig. S11A). FACS analysis showed that kclTAMs, which con-tained the GFPþ KCs, increased in the Rbpj-deficient group, butthis increase was canceled by b-catenin shRNA transfection(Supplementary Fig. S11B and S11C). Moreover, GFPþ RBPjcKO KCs exhibited stronger proliferation as assessed by Ki67staining, which was also canceled by b-catenin shRNA transfec-tion (Supplementary Fig. S11B, S11D, and S11E). These datasupport the conclusion that increased kclTAM proliferation in

Figure 4.

NOTCH blockade in macrophages promoted the growth of hepatic Hepa1-6 tumors independent on CCR2.A, Hepa1-6 cells were inoculated in the liver of Ccr2knockout or control (Ctrl) mice. Tumors were dissected 3 weeks after the inoculation andmyeloid cells were analyzed by FACS. B, F4/80intCD11bint

monocytes/moTAMs and F4/80hiCD11blo TAMs in tumors were determined. Hepa1-6 cells were inoculated in liver of CCR2�/�LyzM-Cre-RBPjf/f or control(CCR2�/�LyzM-Cre-RBPjf/þ) mice for 3 weeks. Tumor growth was monitored by in vivo imaging (C) and tumor weight (on day 21; D). E, Single-cell suspensionsfrom the tumors were stained with anti-CD11b and -F4/80, together with cytoplasmic staining for Ki67 or IL10, and analyzed by FACS. F, The percentage ofkclTAMs (CCR2-independent F4/80hiCD11blo, G1), Ki67

þ, or IL10þ kclTAMs, was determined. G, Cells in Ewere stained with anti-F4/80, -CD11b, and -Ly6C andanalyzed by FACS. Percentages of kclTAMs (F4/80þCD11bloLy6Clo/�) were determined. Bars, mean� SD, n¼ 11. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

Ye et al.





orthotopic Hepa1-6 tumors of RBPj cKO mice was mediated byWNT signaling pathway.

Myeloid RBPj cKO promoted hepatic metastasis of colorectalcancer cells but suppressed lung metastasis

To test the effect of myeloid NOTCH blockade on hepaticmetastasis, we inoculated luciferaseþ CMT93 colorectal cancercells into spleen of RBPj cKO and control mice (SupplementaryFig. S1C). Live imaging indicated that hepatic metastatictumors were significantly larger in RBPj cKO mice than that inthe control (Fig. 7A; Supplementary Fig. S12A). Histologic stain-

ing showed that the hepatic replacement areas in RBPj cKO micewere significantly larger (Fig. 7B). Flow cytometry indicated thatF4/80hiCD11blo TAMs increased significantly in metastatictumors from RBPj cKO mice (Fig. 7C and D). These cells weremore proliferative and expressed higher IL10 (Fig. 7C and D).Taken together, these results indicated that NOTCH blockade inmyeloid cells promoted liver metastasis of colorectal cancer.

To confirm the liver specificity of this observation, we estab-lished lung metastasis model in control and RBPj cKO miceby injection of luciferase-expressing LLC cells via tail vein (Sup-plementary Fig. S1D).Different from livermetastasis, live imaging

Figure 5.

RBPj cKO promotedWNT signaling in kclTAM by upregulating b-CATENIN. A, Sections of hepatic Hepa1-6 tumors were stained with F4/80 and b-CATENINimmunofluorescence, and observed under a confocal fluorescence microscope. White boxes indicate the representative macrophages with nuclear b-CATENIN,with magnified images on the right. B, Themean fluorescence intensity of total or nuclear b-CATENIN staining in F4/80þ cells was determined (n¼ 5). C, F4/80hiCD11blo TAMswere FACS sorted. ThemRNA level of Axin2, b-catenin, c-Myc, and cyclin D1was determined with qRT-PCR.D, The protein level of b-CATENIN,a-b-CTNN, and c-MYC was determined byWestern blotting and quantitatively compared (n¼ 6). E, The mRNA levels of Axin2, b-catenin, c-Myc, and cyclin D1 inRBPj cKO and control (Ctrl) KCs cultured with Hepa1-6 CMwere determined with qRT-PCR. F, The protein level of b-CATENIN, a-b-CTNN, and c-MYC wasdetermined byWestern blotting and quantitatively compared (n¼ 4). G, KCs from normal mice were nucleofected with pCtnnb1-pro-luc and pRL-TK, andtreated with Hepa1-6 CM and GSI (2.5, 5.0, 10 mmol/L) or DMSO for 24 hours (n¼ 5). The protein level of NICD and HES1 was detected byWestern blotting.H, ThemRNA level of b-cateninwas determined by qRT-PCR. Reporter assay was performed to determine the activity of Ctnnb1 promoter. Bars, mean� SD.� , P < 0.05; �� , P < 0.01; �� , P < 0.001.






showed that lung metastatic tumors were significantly smaller inRBPj cKOmice than that in the control (Supplementary Fig. S12Band S12C). Macroscopic tumor counting indicated that thelungs of RBPj cKO mice possessed less metastatic foci (>2 mm;Supplementary Fig. S12D and S12E). Flow cytometry indicatedthat F4/80hiCD11bhi TAMs decreased significantly in metastatictumors from RBPj cKO mice with more F4/80intCD11bintLy6Chi

monocytes (Supplementary Fig. S12F–S12H), which suggestedblocked differentiation of moTAMs. However, the proliferativecapacity of these TAMs (F4/80hiCD11bhi) appeared unaffectedas determined by Ki67 intracellular staining (SupplementaryFig. S12F and S12I). Taken together, these results indicated thatNOTCH blockade in myeloid cells suppressed lung metastasis,which suggested that the phenotype that RBPj cKO promotedtumor growth was liver specific.

To further confirm negative regulation of WNT signalingby Notch activation in TAMs in patient samples, we collectedbiopsies from patients with HCC with different diseasegrades (Supplementary Table S1). Immunofluorescence stain-ing of tumor sections showed that in CD68þ macrophages,the level of nuclear NICD was negatively correlated with thatof b-CATENIN (P < 0.0001; Fig. 7E and F), which positivelycorrelated with higher, more malignant HCC grades (Fig. 7G;

Supplementary Fig. S12J). These data suggested that inhibition ofNOTCHsignaling likely led to enhancedWNTactivation in TAMs,which marks higher disease grades in patients with HCC.

DiscussionAlteration in NOTCH signaling is frequently found when

macrophages are challenged by stress-derived damage-associatedmolecular pattern and infection-derived pathogen-associatedmolecular pattern signals, which are enriched in liver sinusoids.As for our experimental system, NOTCH signaling was activatedin TAMs of orthotopic Hepa1-6 HCC tumors (SupplementaryFig. S1E), possibly by ligands expressed by tumor or microenvi-ronmental cells. Previous reports have shown that NOTCH sig-naling is of prominent importance to both differentiation andfunctional plasticity of TAMs in various tumormodels (7, 28, 29).In this study, we attempted to access the role of NOTCH signalingin HCC TAMs subsets.

Distinct roles of NOTCH signaling in different TAMpopulations in HCC

In this study, we disrupted canonical NOTCH signaling inmyeloid cells by knockout of Rbpj with LyzM-Cre (33). NOTCH

Figure 6.

RBPj cKO promoted kclTAM proliferation and hepatic tumor growth through upregulatedWNT signaling. A, RBPj cKO and control (Ctrl) mice bearing hepaticHepa1-6 tumors were treated with ICG-001 (5 mg/kg) or saline (n¼ 11). Single-cell suspensions from tumors were analyzed by FACS after staining withanti-F4/80 and CD11b, and cytoplasmic staining of Ki67 and IL10. B, The percentage and number of F4/80hiCD11blo TAMs (or kclTAMs, G1), and the percentage ofKi67þ or IL10þ TAMs in kclTAMs were determined. C, The number of CD8þ T cells and Treg cells (CD4þCD25þFoxp3þ) were determined by FACS (n¼ 6).D, RBPj cKO and control KCs were transfected with b-catenin siRNA or NC for 24 hours. Cells were stimulated with Hepa1-6 CM for 24 hours and analyzed by EdUincorporation. The percentage and number of EdUþ cells per field were compared (n¼ 4). RBPj cKO and control KCs were transfected with b-catenin siRNA (E),c-Myc siRNA (F), or NC for 24 hours and stimulated with Hepa1-6 CM for 24 hours. The mRNA level of IL1b, IL10, and IL12was analyzed by qRT-PCR (n¼ 5). Bars,mean� SD. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

Ye et al.





blockade mediated by this Cre transgene resulted in reducedtumor growth in the subcutaneous lung cancer model, likelyattributed to reduced monocyte-to-TAM differentiation andhence TAM input, as demonstrated previously (7). However, thescenario appears different in liver, which is the largest reservoir ofimmunosuppressive-resident macrophages (35). When NOTCHwas blocked by myeloid-specific Rbpj ablation, growth of hepaticHepa1-6 tumors was accelerated accompanied by increasedTAMs. The dominating TAMs exhibited certain surface markersof KCs, including F4/80hiCD11bloLy6Clo/�TIM4þMARCOþ, andwere tentatively named as kclTAMs in this study. Similar to manyother cancers, monocytes are recruited and differentiate intoTAMs in CCR2- and NOTCH-dependent ways to promote HCCgrowth. However, our data have shown that despite impeded

monocyte-to-TAM differentiation, disruption of NOTCH signal-ing increased kclTAMs in HCC instead through enhanced in situproliferation (Supplementary Fig. S13). It has been documentedthat KCs are with heterogeneous origins, namely embryonichematopoiesis–generated KCs and BM monocyte–derivedKCs (20, 35). The results reported here could not clarify whetherthese kclTAMs originate from bona fideKCs, or differentiated fromBM-derived or extramedullary-derived monocytes, or even trans-formed from moTAMs. The possibility existed that in the totalTAMs pool of HCC, KCs might account for only a small fraction.More experiments are required to answer this question by usingfate-tracing strategies and/or gene expression profiling. In a gen-eral way, our data suggests that irrespective of origins, kclTAMsas a whole expanded under the control of NOTCH signaling.

Figure 7.

RBPj cKO promoted hepatic metastasis.A, RBPj cKO and control (Ctrl) mice were inoculated in spleen with CMT93 colorectal cancer cells expressing luciferaseand photographed in 3 weeks under an Xenogen IVIS after intraperitoneal injection with luciferin. Average radiance was compared (n¼ 11). B, Livers of the micewere photographed, and liver sections were stained with hematoxylin and eosin. The metastatic areas were quantitatively compared. C, Single-cell suspensionsfrom livers were analyzed by FACS after staining for CD11b, F4/80, and cytoplasmic Ki67 or IL10. D, The percentage of F4/80hiCD11blo TAMs (or kclTAMs, G1) andKi67þ or IL10þ F4/80hiCD11blo TAMswas determined. E, Human HCC biopsies (T1, 11; T2, 11; T3, 12; T4, 10) were stained with anti-CD68 (pink), anti-NICD (red), andanti-b-catenin (green) immunofluorescence, counterstained with Hoechst, and observed under a laser scanning confocal microscope. F, Representative imagesof cells are shown belowwith high magnitude. The expression of nuclear NICD and nuclear b-CATENIN was quantified in CD68þ cells (more than 200 CD68þ cellsin randomly selected fields for each sample) usingmean density (IOD/area), and the correlation between nuclear NICD and nuclear b-CATENIN in CD68þ cellswas analyzed.G, The number of CD68þ cells with nuclear b-CATENIN per field (40�) was correlated with disease stages of HCC (also see SupplementaryFig. S12J). Bars, mean� SD. � , P < 0.05; �� , P < 0.001.






NOTCH signaling appears to repress their proliferation, so thatwhen NOTCH was blocked by RBPj cKO, this inhibitory role wascanceled. Of note, NOTCH blockade resulted in a 2- to 3-folddecrease of tumor size in breast cancers (7) and subcutaneous LLC(Supplementary Fig. S2), but an approximately 2.5-fold increasein orthotopic HCC, suggesting that kclTAMs compensated func-tionally the loss of moTAMs under NOTCH deficiency.

Ablation or pharmaceutical inhibition of CCR2 suppressesHCC growth, highlighting the role of CCR2-mediated monocyterecruitment inHCC (4). In our experimental systemof orthotopicHCC tumors, however, the phenotype of increased proliferatingkclTAMs and accelerated tumor growth in RBPj cKO mice stillexisted under Ccr2 knockout background. Therefore, althoughtargeting CCR2þ monocyte–derived TAMs via CCL2–CCR2 axishas been suggested as a promising therapy for HCC (4), thistreatment might not be as effective in the absence of potentialNOTCH signaling–mediated suppression of kclTAMs.

In addition, KCs are physiologically immune-suppressivethrough secretion of IL10 and expression of PD-L1/2 to main-tain liver tolerance (35, 39). Notch deficiency increased theexpression of IL10 and PD-L1/2 and reduced the expression ofIL12 and IL1b in kclTAMs of orthotopic HCC, likely leading toexacerbated protumor activity. Indeed, our data indicated thatCD8þ cytotoxic T cells decreased in cKO HCC tumors (Fig 1Dand E). However, more functional experiments are still neededto investigate the downstream cellular and molecular regula-tory mechanisms of RBPj cKO kclTAMs. Therefore, myeloid-specific NOTCH deficiency could not only compensate thedecreased TAM input from monocytes with increased localkclTAM proliferation, but could probably also elicit strongerprotumor activity of kclTAMs, participating in accelerated HCCgrowth. Importantly, however, the phenomenon discussedabove was only based on the orthotopic Hepa1-6 HCC model,and many other HCC cell lines are still needed to confirm theconclusion.

Mitogenic signal for kclTAM proliferation in HCCTheWNT/b-CATENIN signaling pathway plays a critical role in

liver development, physiology, and pathology (40). WNTligands are produced by different hepatic cell populations suchas liver sinusoidal endothelial cells, and support the replenish-ment of hepatocytes and regulate their metabolism (41). WNT/b-CATENIN pathway is frequently upregulated in HCC andparticipates in maintenance of tumor-initiating cells, drug resis-tance, angiogenesis, and metastasis (42). Upregulated WNTligands are detected in human HCC and HCC cell lines (43).Our data showed that Hepa1-6 cells secreted WNT3a, a typicalligand activating canonical WNT/b-CATENIN pathway, whichcould serve as a mitogen for kclTAMs. However, we could notexclude that Hepa1-6 produced other WNT ligands or other typesof mitogens regulating the proliferation of kclTAMs. Our dataindicate that NOTCH deficiency can result in expansion ofkclTAMs by upregulating b-CATENIN, the key mediator of WNTpathway. In addition, WNT signaling also regulates macrophagepolarization under different disease contexts (44, 45), and c-MYChas been reported to promote M2-like phenotype of TAMs (38).Consistently, our findings indicated that the strengthened protu-mor secretory phenotypeofRbpj-deficient kclTAMs appeared tobedependent on upregulated c-Myc downstream to b-catenin (Fig 6Eand F). In human samples, increased CD68þ TAMswith activatedb-CATENIN is associated with advanced tumor stages of HCC

patients. These findings add further mechanistic evidence fortargeting WNT signaling in patients with HCC.

The mutual regulation of NOTCH and WNT signaling path-ways are critically involved in numerous developmental process-es, including hematopoiesis, by influencing cell proliferationand differentiation (27). A NOTCH-on/WNT-off or WNT-on/NOTCH-off pattern has been noticed in various signaling settings,in which NOTCH pathway and WNT pathway regulate eachother at different signaling levels (27). In this study, NOTCHactivation in KCs appeared to suppress WNT signaling by down-regulating b-catenin expression at the mRNA level. But detailedeffect of NICD/RBPj complex or its downstream HES familytranscription regulators onb-catenin promoter and the underlyingmechanisms require further clarification. Moreover, we couldnot exclude that NOTCH may inhibit the transcription mediatedby b-catenin through protein–protein interactions, which hasbeen revealed in several models (46). It would be interesting toask whether the NOTCH–WNT axis functions as a hepatic nicheregulating KC homeostasis under physiologic and/or pathologicconditions, as suggested in recent studies (20).

NOTCH signal also regulated kclTAM population in hepaticmetastatic growth

Myeloid-specific Rbpj deficiency promoted the hepatic meta-static growth of CMT93 colorectal cancer with more proliferativekclTAMs, which expressed higher IL10. This phenotype was con-sistent with that in orthotopic Hepa1-6 HCC. Myeloid-specificRbpj deficiency in liver seemed to provide a prometastatic nichefor hepaticmetastasis of colorectal cancer. KCs play bimodal rolesin colorectal cancer liver metastasis (47, 48). At early stage, KCsprevent livermetastasis by efficiently killingmetastatic cancer cellsthrough phagocytosis. However, the activities of KCs are modu-lated by other cells including cancer cells and cytokines in liversinusoids during liver metastasis, often leading to increasedcolorectal cancer cell arrest and colonization in liver (47). Thevariation in tumoricidal role of KCs could be somewhat excludedbecause we did not observe a significant difference in tumorgrowth between control and RBPj cKO group at early stage ofthis metastasis model (Supplementary Fig. S12A). Our resultssuggested that the variation of prometastatic role of KCs might bemore important with Rbpj-deficient kclTAMs expressing higherIL10. However, the precise regulatory mechanism of this immu-nosuppressive cytokine still needs further investigation. Besides, itremains to be elucidated whether RBPj cKO facilitated coloniza-tion of colorectal cancer cells in liver at advanced stage andwhether activated WNT signaling participate in this metastaticmodel after myeloid NOTCH blockade. It would also be inter-esting to determine the corresponding activation status ofNOTCH and WNT signals in human biopsies of liver metastasis.Moreover,APC deficiency is a highly frequent drivingmutation ofcolorectal cancer (49). Whether APC mutation, which leads toenhanced WNT signaling, in KCs could facilitate liver metastasiswould be worthy of further investigation.

Myeloid NOTCH blockade exerted distinct effects on differentmetastatic models. Compared with the control mice, the growthof subcutaneous LLC and lungmetastasis were both inhibited butthat of orthotopicHepa1-6HCCandCMT93 livermetastasis bothpromoted in the RBPj cKOmice. This probably suggested that thephenotype that RBPj cKO promoted tumor growth was a liver-specific phenomenon. Of note, from the metastatic model alone,RBPj cKO promoted liver metastasis but repressed lung

Ye et al.





metastasis. We considered the contribution of different macro-phage subsets as a possiblemechanism. As for the livermetastasis,the injected CMT93 cells via spleen arrived at the liver sinusoidsanddirectly interactedwithKCs, andNOTCHblockade promotednot only the proliferation but also the TAM phenotype (such asthe IL10 secretion) of KCs to facilitate liver metastasis (see Fig. 7Cand D). As for the lung metastasis model via tail vein injection,Qian and colleagues reported previously that in the metastasisprocess of breast cancer cells to the lung,monocyteswere recruitedand differentiated into TAMs or metastasis-associated macro-phages, which facilitated lung metastasis by promoting cancercell extravasation and survival (50, 51). We supposed that RBPjcKO also impeded the differentiation of moTAMs, and thus lungmetastasis was repressed.

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

Authors' ContributionsConception and design: H.-Y. Qin, H. HanDevelopment of methodology: J.-L. Zhao, K.-F. Dou, H.-Y. Qin

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y.-C. Ye, J.-L. Zhao, C.-C. Gao, Y. Yang,S.-Q. Liang, Y.-Y. Lu, L. Wang, S.-Q. Yue, K.-F. Dou, H.-Y. QinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y.-C. Ye, J.-L. Zhao, C.-C. Gao, Y.-Y. Lu, L. Wang,S.-Q. Yue, K.-F. Dou, H.-Y. Qin, H. HanWriting, review, and/or revision of the manuscript: K.-F. Dou, H.-Y. Qin,H. HanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y.-T. Lu, K.-F. Dou, H.-Y. QinStudy supervision: K.-F. Dou, H.-Y. Qin

AcknowledgmentsThis study was supported by National Natural Science Foundation of

China (81530018, 31371474, 31570878, 31730041, and 31130019) and theMinistry of Science and Technology of China (2015CB553702).

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 June 4, 2018; revised October 23, 2018; accepted June 25, 2019;published first July 2, 2019.

References1. Qian BZ, Pollard JW. Macrophage diversity enhances tumor progression

and metastasis. Cell 2010;141:39–51.2. Mantovani A, Marchesi F, Malesci A, Laghi L, Allavena P. Tumour-

associated macrophages as treatment targets in oncology. Nat Rev ClinOncol 2017;14:399–416.

3. Noy R, Pollard JW. Tumor-associated macrophages: from mechanisms totherapy. Immunity 2014;41:49–61.

4. Li X, YaoW, Yuan Y, Chen P, Li B, Li J, et al. Targeting of tumour-infiltratingmacrophages via CCL2/CCR2 signalling as a therapeutic strategy againsthepatocellular carcinoma. Gut 2017;66:157–67.

5. FranklinRA, LiMO.Theontogenyof tumor-associatedmacrophages: a newunderstanding of cancer-elicited inflammation. Oncoimmunology 2014;3:e955346.

6. Lahmar Q, Keirsse J, Laoui D, Movahedi K, Van Overmeire E, VanGinderachter JA. Tissue-resident versus monocyte-derived macrophagesin the tumor microenvironment. Biochim Biophys Acta 2016;1865:23–34.

7. Franklin RA, LiaoW, Sarkar A, KimMV, BivonaMR, Liu K, et al. The cellularandmolecular origin of tumor-associatedmacrophages. Science 2014;344:921–5.

8. Campbell MJ, Tonlaar NY, Garwood ER, Huo D, Moore DH, KhramtsovAI, et al. Proliferating macrophages associated with high grade, hor-mone receptor negative breast cancer and poor clinical outcome.Breast Cancer Res Treat 2011;128:703–11.

9. Strachan DC, Ruffell B, Oei Y, Bissell MJ, Coussens LM, Pryer N, et al.CSF1R inhibition delays cervical and mammary tumor growth inmurine models by attenuating the turnover of tumor-associated macro-phages and enhancing infiltration by CD8þ T cells. Oncoimmunology2013;2:e26968.

10. Tymoszuk P, EvensH,Marzola V,Wachowicz K,WasmerMH,Datta S, et al.In situ proliferation contributes to accumulation of tumor-associatedmacrophages in spontaneous mammary tumors. Eur J Immunol 2014;44:2247–62.

11. Ginhoux F, Guilliams M. Tissue-resident macrophage ontogeny andhomeostasis. Immunity 2016;44:439–49.

12. Epelman S, Lavine KJ, Randolph GJ. Origin and functions of tissuemacrophages. Immunity 2014;41:21–35.

13. Jenkins SJ, Ruckerl D, Thomas GD, Hewitson JP, Duncan S, BrombacherF, et al. IL-4 directly signals tissue-resident macrophages to proliferatebeyond homeostatic levels controlled by CSF-1. J Exp Med 2013;210:2477–91.

14. Ensan S, Li A, Besla R,DegouseeN,Cosme J, RoufaielM, et al. Self-renewingresident arterial macrophages arise from embryonic CX3CR1(þ) precur-

sors and circulating monocytes immediately after birth. Nat Immunol2016;17:159–68.

15. Zhu Y, Herndon JM, Sojka DK, Kim KW, Knolhoff BL, Zuo C, et al. Tissue-residentmacrophages in pancreatic ductal adenocarcinoma originate fromembryonic hematopoiesis and promote tumor progression. Immunity2017;47:323–38.

16. Sharma SK, ChintalaNK, Vadrevu SK, Patel J, KarbowniczekM,MarkiewskiMM. Pulmonary alveolar macrophages contribute to the premetastaticniche by suppressing antitumor T cell responses in the lungs. J Immunol2015;194:5529–38.

17. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancerstatistics, 2012. CA Cancer J Clin 2015;65:87–108.

18. Hoeffel G, Chen J, Lavin Y, Low D, Almeida FF, See P, et al. C-Myb(þ)erythro-myeloid progenitor-derived fetal monocytes give rise to adulttissue-resident macrophages. Immunity 2015;42:665–78.

19. Gomez Perdiguero E, Klapproth K, Schulz C, Busch K, Azzoni E, Crozet L,et al. Tissue-residentmacrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature 2015;518:547–51.

20. Scott CL, Zheng F,DeBaetselier P,Martens L, Saeys Y,DePrijck S, et al. Bonemarrow-derived monocytes give rise to self-renewing and fully differenti-ated Kupffer cells. Nat Commun 2016;7:10321.

21. Wan S, Zhao E, Kryczek I, Vatan L, Sadovskaya A, Ludema G, et al. Tumor-associated macrophages produce interleukin 6 and signal via STAT3to promote expansion of human hepatocellular carcinoma stem cells.Gastroenterology 2014;147:1393–404.

22. Naugler WE, Sakurai T, Kim S, Maeda S, Kim K, Elsharkawy AM, et al.Gender disparity in liver cancer due to sex differences inMyD88-dependentIL-6 production. Science 2007;317:121–4.

23. Wu J, Li J, Salcedo R, Mivechi NF, Trinchieri G, Horuzsko A. The proin-flammatory myeloid cell receptor TREM-1 controls Kupffer cell activationand development of hepatocellular carcinoma. Cancer Res 2012;72:3977–86.

24. YuanD,Huang S, Berger E, Liu L, Gross N, Heinzmann F, et al. Kupffer cell-derived Tnf triggers cholangiocellular tumorigenesis through JNK due tochronic mitochondrial dysfunction and ROS. Cancer Cell 2017;31:771–89.

25. Kang TW, Yevsa T, Woller N, Hoenicke L, Wuestefeld T, Dauch D, et al.Senescence surveillance of pre-malignant hepatocytes limits liver cancerdevelopment. Nature 2011;479:547–51.

26. KopanR, IlaganMX. The canonicalNotch signalingpathway: unfolding theactivation mechanism. Cell 2009;137:216–33.

27. Hayward P, Kalmar T, Arias AM. Wnt/Notch signalling and informationprocessing during development. Development 2008;135:411–24.






28. Wang YC, He F, Feng F, Liu XW, Dong GY, Qin HY, et al. Notch signalingdetermines the M1 versus M2 polarization of macrophages in antitumorimmune responses. Cancer Res 2010;70:4840–9.

29. Zhao JL,Huang F,He F, GaoCC, Liang SQ,MaPF, et al. Forced activationofnotch in macrophages represses tumor growth by upregulating miR-125aand disabling tumor-associated macrophages. Cancer Res 2016;76:1403–15.

30. He F, Guo FC, Li Z, Yu HC, Ma PF, Zhao JL, et al. Myeloid-specificdisruption of recombination signal binding protein J ameliorates hepaticfibrosis by attenuating inflammation through cylindromatosis in mice.Hepatology 2015;61:303–14.

31. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regulation ofmyeloid cells by tumours. Nat Rev Immunol 2012;12:253–68.

32. Li Q, Dashwood W-M, Zhong X, Al-Fageeh M, Dashwood RH. Cloning ofthe rat b-catenin gene (Ctnnb1) promoter and its functional analysiscompared with the Catnb and CTNNB1 promoters. Genomics 2004;83:231–42.

33. Clausen BE, Burkhardt C, ReithW, Renkawitz R, Forster I. Conditional genetargeting in macrophages and granulocytes using LysMcre mice.Transgenic Res 1999;8:265–77.

34. Beattie L, Sawtell A, Mann J, Frame TC, Teal B, de Labastida Rivera F, et al.Bone marrow-derived and resident liver macrophages display uniquetranscriptomic signatures but similar biological functions. J Hepatol2016;65:758–68.

35. Krenkel O, Tacke F. Liver macrophages in tissue homeostasis and disease.Nat Rev Immunol 2017;17:306–21.

36. Bleriot C, Dupuis T, Jouvion G, Eberl G, Disson O, Lecuit M. Liver-residentmacrophage necroptosis orchestrates type 1 microbicidal inflammationand type-2-mediated tissue repair during bacterial infection. Immunity2015;42:145–58.

37. Henderson WR Jr, Chi EY, Ye X, Nguyen C, Tien YT, Zhou B, et al.Inhibition of Wnt/beta-catenin/CREB binding protein (CBP) signalingreverses pulmonary fibrosis. Proc Natl Acad Sci U S A 2010;107:14309–14.

38. PelloOM,DePizzolM,MiroloM, Soucek L, Zammataro L, Amabile A, et al.Role of c-MYC in alternative activation of humanmacrophages and tumor-associated macrophage biology. Blood 2012;119:411–21.

39. Wu K, Kryczek I, Chen LP, Zou WP, Welling TH. Kupffer cell suppres-sion of CD8þT cells in human hepatocellular carcinoma is mediated by

B7-H1/Programmed Death-1 interactions. Cancer Res 2009;69:8067–75.

40. Monga SP. b-Catenin signaling and roles in liver homeostasis, injury, andtumorigenesis. Gastroenterology 2015;148:1294–310.

41. Yang J, Mowry LE, Nejak-Bowen KN, Okabe H, Diegel CR, Lang RA, et al.b-catenin signaling in murine liver zonation and regeneration: a Wnt-Wntsituation! Hepatology 2014;60:964–76.

42. Wang Y, He L, Du Y, Zhu P, Huang G, Luo J, et al. The longnoncoding RNA lncTCF7 promotes self-renewal of human liver cancerstem cells through activation of Wnt signaling. Cell Stem Cell 2015;16:413–25.

43. Bengochea A, de Souza MM, Lefrancois L, Le Roux E, Galy O, Chemin I,et al. Common dysregulation of Wnt/Frizzled receptor elements in humanhepatocellular carcinoma. Br J Cancer 2008;99:143–50.

44. Feng Y, Ren J, Gui Y, Wei W, Shu B, Lu Q, et al. Wnt/b-catenin-promotedmacrophage alternative activation contributes to kidney fibrosis. J Am SocNephrol 2018;29:182–93.

45. Akcora B €O, Storm G, Bansal R. Inhibition of canonical WNT signalingpathway by b-catenin/CBP inhibitor ICG-001 ameliorates liver fibrosis invivo through suppression of stromal CXCL12. BiochimBiophys Acta 2018;1864:804–18.

46. Kwon C, Cheng P, King IN, Andersen P, Shenje L, Nigam V, et al. Notchpost-translationally regulates beta-catenin protein in stem and progenitorcells. Nat Cell Biol 2011;13:1244–51.

47. Paschos KA, Majeed AW, Bird NC. Natural history of hepatic metastasesfrom colorectal cancer–pathobiological pathways with clinical signifi-cance. World J Gastroenterol 2014;20:3719–37.

48. Wen SW, Ager EI, Christophi C. Bimodal role of Kupffer cellsduring colorectal cancer liver metastasis. Cancer Biol Ther 2013;14:606–13.

49. Carethers JM, Jung BH. Genetics and genetic biomarkers in sporadiccolorectal cancer. Gastroenterology 2015;149:1177–90.

50. Qian BZ, Li J, Zhang H, Kitamura T, Zhang J, Campion LR, et al. CCL2recruits inflammatory monocytes to facilitate breast-tumour metastasis.Nature 2011;475:222–5.

51. Kitamura T, Doughty-Shenton D, Cassetta L, Fragkogianni S, Brownlie D,Kato Y, et al. Monocytes differentiate to immune suppressive precursors ofmetastasis-associated macrophages in mouse models of metastatic breastcancer. Front Immunol 2017;8:2004.


Ye et al.




2019;79:4160-4172. Published OnlineFirst July 2, 2019.Cancer Res Yu-Chen Ye, Jun-Long Zhao, Yi-Tong Lu, et al. Hepatocellular CarcinomaAlternative, CCR2-Independent Tumor-Associated Macrophages in NOTCH Signaling via WNT Regulates the Proliferation of

Updated version

10.1158/0008-5472.CAN-18-1691doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2019/07/02/0008-5472.CAN-18-1691.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/79/16/4160.full#ref-list-1

This article cites 51 articles, 13 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/79/16/4160To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-18-1691

http://cancerres.aacrjournals.org/content/suppl/2019/07/02/0008-5472.CAN-18-1691.DC1

http://cancerres.aacrjournals.org/content/79/16/4160.full#ref-list-1

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/79/16/4160


notch signaling via wnt regulates the proliferation of … · carcinoma. introduction macrophages...

Documents