Host miR155 Promotes Tumor Growth through a Myeloid ... · Host miR155 Promotes Tumor Growth through a Myeloid-Derived Suppressor Cell–Dependent Mechanism Siqi Chen1, Long Wang2,

Microenvironment and Immunology

Host miR155 Promotes Tumor Growth through aMyeloid-Derived Suppressor Cell–DependentMechanismSiqi Chen1, Long Wang2, Jie Fan1, Cong Ye1, Donye Dominguez1, Yi Zhang3,Tyler J. Curiel2, Deyu Fang4, Timothy M. Kuzel1, and Bin Zhang1

Abstract

miR155 is a regulator of immune cell development and func-tion that is generally thought to be immunostimulatory. How-ever, we report here that genetic ablation of miR155 renders miceresistant to chemical carcinogenesis and the growth of severaltransplanted tumors, suggesting that miR155 functions in immu-nosuppression and tumor promotion. Host miR155 deficiencypromoted overall antitumor immunity despite the finding ofdefective responses of miR155-deficient dendritic cells and anti-tumor T cells. Further analysis of immune cell compartmentsrevealed that miR155 regulated the accumulation of functionalmyeloid-derived suppressive cells (MDSC) in the tumor micro-

environment. Specifically, miR155 mediated MDSC suppressoractivity through at least two mechanisms, including SOCS1repression and a reduced ability to license the generation ofCD4þFoxp3þ regulatory T cells. Importantly, we demonstratedthatmiR155 expressionwas required forMDSC to facilitate tumorgrowth. Thus, our results revealed a contextual function formiR155 in antitumor immunity, with a role in MDSC supportthat appears to dominate in tumor-bearing hosts. Overall, thebalance of these cellular effects appears to be a root determinantof whethermiR155 promotes or inhibits tumor growth. Cancer Res;75(3); 519–31. �2014 AACR.

MiRNAs are evolutionarily conserved small noncoding RNAsthat posttranscriptionally modulate the expression of multipletarget genes and are hence implicated in a wide series of cellularanddevelopmental processes (1, 2).miR155 is processed from theB-cell integration cluster (BIC), a noncoding transcript primarilyupregulated in both activated B and T cells (3) and inmonocytes/macrophages upon inflammation (4, 5). Recent gene-targetingstudies of miR155 demonstrate a broad role for miR155 in theregulation of both immune cell development and function (6, 7).Indeed, miR155�/� mice have global immune defects due todefective B- and T-cell immunity and reduced dendritic cell (DC)function. Particularly, miR155-deficient DCs fail to present anti-gens efficiently (6) and produce cytokines (8), whereasmiR155 inCD4þ T cells regulates differentiation into the Th1, Th2, and Th17pathways (6, 9, 10). Furthermore, miR155 is required for CD8þ

T-cell responses to acute viral and bacterial challenges (11–14).In addition to these immunostimulatory effects, miR155 canalso exert some immunosuppressive effects, such as promoting

the development (15), or homeostasis and fitness (16) of regu-latory T cells (Treg), and expansion of functionalmyeloid-derivedsuppressive cells (MDSC; ref. 17). Thus, miR155 could modulateprotective immune responses and inflammation through distinctmechanisms.

miR155 dysregulation is closely related to cancer (4). miR155transgenic mice develop B-cell malignancy (18), and elevatedmiR155 expression was reported in several types of human B-celllymphomas (19). A correlation between increased miR155 anddevelopment of tumors such as leukemias, glioblastoma, andbreast, lung, or gastric cancers has been established recently (20,21). Therefore, targetingmiR155has beenproposedas a promisingapproach to treat both hematopoietic and solid cancers (22–24).However, the potent immunostimulatory effects of miR155 havealso been observed in the context of tumor. Notably, the roles ofmiR155 in effector CD8þ T cells (13, 25), tumor-infiltrating DCs(26, 27), and tumor-associated macrophages (28, 29) can bemodulated to potentiate cancer immunotherapies. Thus, whencancer is treated in a immunocompetent host by inhibitingmiR155, outcomes are difficult to predict. Importantly, underlyingmechanisms of host miR155 in modulating tumor growth are stillpoorly understood. We show here that host miR155 deficiencyhampers the accumulaiton of functional MDSCs and inducibleTreg cells in the tumor microenvironment, thereby promotingantitumor T-cell immunity and retarding tumor growth.

Materials and MethodsMice, cell lines, and reagents

C57BL/6 miR155�/�, CD45.1, and CD90.1 mice were pur-chased from the Jackson Laboratory, OT-I Rag1�/� and OT-IIRag1�/� mice from Taconic, and C57BL/6 miR155þ/þ mice fromNCI-Frederick. Dr. Hans Schreiber (University of Chicago)

1Robert H. Lurie Comprehensive Cancer Center, Department of Med-icine-Division of Hematology/Oncology, Northwestern UniversityFeinberg School of Medicine, Chicago, Illinois. 2Cancer Therapy andResearch Center, Department of Medicine, University of Texas HealthScience Center, San Antonio, Texas. 3Biotherapy Center, The FirstAffiliated Hospital of Zhengzhou University, Zhengzhou, Henan,China. 4Department of Pathology, Northwestern University FeinbergSchool of Medicine, Chicago, Illinois.

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

Corresponding Author: Bin Zhang, Northwestern University, Chicago, IL 60611.Phone: 312-503-2447; Fax: 312-503-0189; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-2331

�2014 American Association for Cancer Research.

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provided the MC38, EG7, B16F10, B16-SIY cell lines, anti-Gr1antibodies (RB6-8C5), and2C transgenicmice.Murine Lewis LungCarcinoma (LLC1) cells were purchased from ATCC (CRL-1642).LLC1 cells were infected with MIGR1-ovalbumin (OVA)-IRES-eGFP (30), and OVA-expressing cells (LLC1-OVA) were sortedtwice based onGFP expression.OVAproductionwas confirmedbyELISA (data not shown). All the cell lines were routinely tested formycoplasma infections by culture andDNA stain, andmaintainedin complete medium composed of RPMI 1640 with 5% FBS. Allanimal experiments were approved by institutional animal usecommittees of theUniversity of TexasHealth Science Center at SanAntonio and Northwestern University. The OVA-derived peptideOVA-I (SIINFEKL) was synthesized by GenScript. Dichlorofluor-escin diacetate (DCFDA), azoxymethane (AOM), and 5-fluoroura-cil (5-FU) were purchased from Sigma-Aldrich. Dextran sulfatesodium salt (DSS) was purchased from Affymetrix, Inc. All themAbs for flow cytometry were purchased from eBioscience andBioLegend. The Annexin V apoptosis detection Kit was fromBioLegend. The Kb/OVA tetramers were provided by the NationalInstitutes of Health Tetramer Core Facility. Depleting mAb cloneGK1.5 (anti-CD4), clone 53.6.7 (anti-CD8a), and clone PK136(anti-NK1.1) were purchased from Bio X Cell. Nw-hydroxy-nor-Arginine (Nor-NOHA) and arginase I activity kit were purchasedfrom Cayman Chemical Company.

Analysis of cells by flow cytometryAll samples were initially incubated with 2.4G2 to block

antibody binding to Fc receptors. Single-cell suspensions werestained with 1 mg of relevant mAbs and then washed twice withcold PBS. Reactive oxygen species (ROS) detection by DCFDAstaining was conducted as described by Marigo and colleagues(31). The Annexin V staining, Kb/OVA tetramer staining, Foxp3staining, and intracellular IFNg staining were performed as pre-viously described (32). Samples were conducted on a MACS-Quant Analyzer (Miltenyi Biotec), and data were analyzed withFlowJo software.

In vivo killing assayAnalysis of tumor antigen-specific effector CTL activity in vivo

was performed as previously described (32). Briefly, OVA-I (SIIN-FEKL) peptide-pulsed eFluor 450high and SIY-peptide-pulsedeFluor 450low splenocytes were mixed at a ratio of 1:1, and atotal of 2 � 107 cells were injected i.p. into recipient animals.Draining lymph nodes (DLN) and spleen were then harvested 24hours after adoptive transfer, and eFluor 450 fluorescence inten-sity was analyzed by flow cytometry.

MDSC suppressive assaySplenic MDSCs from tumor-bearing wild-type (WT) or

miR155�/� mice were selected using CD11b MicroBeads (Milte-nyi Biotec), and tumor-infiltrating CD115þCD11bþGr1þ orCD115�CD11bþGr1þ MDSCs were sorted by a BD FACSAriacell sorter from LLC1-bearing mice. MDSCs were added at differ-ent ratios to OT-I or 2C splenocytes stimulated with 0.5 mg/mLOVA-I or SIY peptides for 3 days, and 3[H] thymidine uptakewas measured. For experiments that examined the effect of argi-nase inhibitors, nor-NOHA (NW-hydroxyl-nor-l-arginine, 0.5mmol/L) were added at the beginning of the culture. To evaluateMDSC tolerogenic activity on in vivo T-cell function, naive OT-1CD90.1 cells (2 � 106 per mouse) were transferred to CD90.2congeneic recipients, which were s.c. immunized, 2 days later,

with 10mgOVA-I peptides in incomplete Freund's adjuvant (IFA).MDSCs (2 � 106) from MC38 tumor–bearing WT or miR155�/�

mice, either pulsed or not with OVA-I peptides, were transferredon the same day of the immunization. DLNs were collected 10days after immunization and stimulated with 0.5 mg/mLOVA-I invitro for 3 days to measure T-cell proliferation by 3[H] thymidineuptake and IFNg-secreting CD8þ T cells by flow cytometry.

Arginase activityArginase activity was measured in cell lysates using the com-

mercially available QuantiChrom Arginase Assay Kit (BioAssaySystems) according to the manufacturer's instructions.

Bone marrow–derived MDSC generationTibias and femurs from C57BL/6 mice were removed using

sterile techniques, and bone marrow (BM) cells were flushed. Toobtain BM-derived MDSCs, cells were cultured with GM-CSF (40ng/mL, Biolegend) and IL6 (40 ng/mL, Biolegend) for 4 days. BM-derived MDSCs were selected using CD11b or Gr1 MicroBeads(Miltenyi Biotec).

RNA extraction and real-time PCRTotal RNA was extracted using Trizol reagent (Invitrogen)

according to the manufacturer's instructions. miR155 expressionwas detected by a TaqMan MicroRNA Assay kit (Applied Biosys-tems). The cDNA synthesis was performed using SuperScriptOne-Step RT-PCR (Invitrogen).Quantitative real-time PCRwas used toquantify a series of MDSC-associated genes by SYBR Green (Bio-Rad), and relative abundance of each mRNA was normalized toGAPDH mRNA.

Transfection of BM-derived MDSCsThe transfection of primary BM cells was performed according

to the instructions of the manufacturer (AMAXA). BM cells weretreated with GM-CSF (40 ng/mL, Biolegend) for 24 hours, fol-lowed by the transfection with 1 mmol/L pre-miR155/BIC (P-MDSC; Ambion), 2 mmol/L miR155 inhibitor miRNA (I-MDSC;Dharmacon), or control oligonucleotides (C-MDSC; Dharma-con) by AMAXA. For knockdown of SOCS1, specific and respec-tive control siRNAs used for transfection were from Santa CruzBiotechnology. To recover, cells were cultured for additional 72hours in the presence of GM-CSF (40 ng/mL; Biolegend) and IL6(40 ng/mL; Biolegend) after transfection. After selection withCD11b or Gr1 MicroBeads (Miltenyi Biotec), these GM-CSF andIL6-conditioned BM-derived MDSCs were tested for suppressiveassay.

Treg inductionSplenic WT or miR155�/� CD4þCD62Lþ na€�ve T cells were

selected with a CD4þCD62Lþ T-cell isolation kit (Miltenyi Bio-tec), and injected i.v. at 5 � 106 per mouse into CD45.1 micefollowed by a s.c. injection of 106 LLC1-OVA cells. The conversionof transferred T cells to Foxp3þ cells (CD45.2þ) in DLN andspleen from LLC1-OVA tumor–bearing mice were detected byflow cytometer 9 days after tumor cell injection. For MDSC-mediated Treg induction, splenic WT and miR155�/� Gr1þ

CD11bþMDSCs fromLLC1 tumor–bearingmicewere coculturedwith OT-II splenocytes at a 1:4 ratio for 5 days in the absence orpresence of 2 ng/mL TGFb, and induced CD25þFoxp3þ cellsamong total CD4þ T cells were subsequently determined by flowcytometry.

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AOM and DSS treatmentFor the colitis-associated colon cancer model, mice were given

10 mg/kg AOM via i.p. injection. One week later, 2.5% DSS wasgiven in the drinking water for 7 days followed by 14 days ofnormal water for a total of three cycles. Colons were harvested,flushed of feces, cut longitudinally, and fixed in 10% bufferedformalin overnight. The colons were scored with the aid of amagnifier for the number of colonic neoplasms to determine theincidence (number of animals with at least one tumor) andmultiplicity (number of tumors per animal) of neoplasms. Tumorarea was also evaluated based on length and width.

Tumor challenge and treatmentsB16F10, B16-SIY, LLC1, LLC1-OVA, MC38, or EG7 cells (1 �

106) in suspension were injected s.c. For MDSC depletion, 3 daysafter tumor cell injection, mice were injected i.p. by 5-FU (50mg/kg)oranti-Gr1antibodies (RB6-8C5,200mg)onceevery4days.Depletion of CD4þ T cells, CD8þ T cells, or natural killer (NK) cellswas achieved by twice a week i.p. injection of depleting mAb cloneGK1.5 (anti-CD4, 200 mg), clone 53.6.7 (anti-CD8a, 200 mg) orclone PK136 (anti-NK1.1, 200 mg) starting one day before tumorchallenge. Flow cytometry confirmed depletion efficiency of targetcells for 3 days following injections. For adoptive transfer ofMDSCs, splenic Gr1þCD11bþ MDSCs from tumor-bearing WT ormiR155�/�micewere injected i.v. at 5� 106 permouse into LLC1-bearing mice at days 7 and 14. For adoptive transfer of Tregs (33),splenic CD4þCD25þ Treg cells were selected with a CD4þCD25þ

regulatory T-cell isolation kit (Miltenyi Biotec) from WT ormiR155�/� mice, and i.v. injected at 2 � 106 per mouse intoLLC1-bearing mice on days 7, 14, and 20. The size of tumor wasdetermined at 2- to 3-day intervals. Tumor volumesweremeasuredalong 3 orthogonal axes (a, b, and c) and calculated as abc/2.

Statistical analysisMean values were compared using an unpaired Student

two-tailed t test. Probability values >0.05 were considerednonsignificant.

ResultsHost miR155 promotes tumor growth

The immunoregulatory role of miR155 has been well docu-mented in numerous experimental settings. However, the specificcontributions of endogenous miR155 to antitumor immunityand tumorigenesis are poorly understood. We compared chem-ically-induced tumor and transplanted tumor growth in miR155-deficient (miR155�/�) versus syngeneic, WT (miR155þ/þ) mice.Mice were given AOM and DSS, as previously described, topromote colorectal carcinogenesis (34). Upon AOM and DSSchallenge, miR155�/� mice exhibited less acute body weight losscomparable with WT mice (data not shown). AOM and DSSproduced colonic tumors in all 8 WT mice, but in 3 of 8miR155�/�mice. Themultiplicity of colonic neoplasms (numberand size of tumors) was also significantly decreased inmiR155�/�

mice. However, there was no sinificant difference in colon lengthbetween untreated WT andmiR155�/�mice (Fig. 1A). Moreover,WT and miR155�/� mice given AOM alone or DSS alone had nomacroscopic colonic tumors (data not shown). We next studiedthe role of host miR155 on transplantable tumor growth inmiR155�/� mice. MC38 colon cancer cells (Fig. 1B) and LLC1Lewis lung carcinoma cells (Fig. 1C) were s.c. inoculated into WT

ormiR155�/�mice. Tumors injected intomiR155�/�mice exhib-ited delayed growth compared with those in control mice (Fig. 1Band C). In addition, miR155 deficiency was also effective ininhibiting the growth of immunogenic LLC1-OVA (Fig. 1D).Similarly, the growth of lyphoma EG7 (expressing OVA antigen)tumors was inhibited in miR155�/�mice (Fig. 1E). However, thesizes of B16-SIY melanoma (expressing SIY antigen) were com-parable between the WT and miR155�/� mice at multiple timepoints (Supplementary Fig. S1A), suggesting that the role of hostmiR155 in tumor growth is tumor-dependent.

Host miR155 deficiency enhances antigen-specific antitumorT-cell immunity

Given the importance of miR155 in immune regulation, wenext examined the phenotype and cytokine profile of tumor-infiltrating immune cells in tumor-bearing mice. At 19 days aftertumor inoculation, we found no significant alterations in thepercentages of B cells (CD19þ), NK cells (NK1.1þ), or myeloidDCs (CD11bþCD11cþ) in local infiltrates of EG7 (Fig. 2A) orLLC1-OVA (Supplementary Fig. S2A) tumors in miR155�/� ver-sus WT mice. Interestingly, remarkably fewer tumor-infiltratingCD8þ, CD4þ lymphocytes were found in miR155�/� versus WTmice (Fig. 2A and Supplementary Fig. S2A). Although percentagesof tumor-infiltrating IFNgþCD8þ T cells were comparablebetween groups, tetramer staining showed a greater number ofOVA-reactive (tumor-specific) CD8þ T cells in EG7-bearing (Fig.2B and C) or LLC1-OVA–bearing (Supplementary Fig. S2B andS2C) miR155�/� mice than WT mice. We next examined thecytolytic function of tumor antigen-specific T cells. Target celllysis in vivo was remarkably improved in DLN of EG7 tumor–bearing miR155�/� mice compared with tumor-bearing WTmice(Fig. 2D). To assess the roles of CD4þ, CD8þ, and NK cells in thetumor-inhibiting effects observed in miR155�/� mice, mice wereinoculated with EG7 cells, and subsequently received depletinganti-CD4, anti-CD8a, or anti-NK1.1 antibodies against CD4þ orCD8þ T cells, or NK cells, respectively. Notably, the tumor-inhibiting advantage of host miR155 deficiency was primarilydependent on CD8þ cells, but independent of CD4þ cells or NKcells (Fig. 2E). Thesedata suggest that loss ofmiR155expression inmice results in the enhanced antitumor T-cell immunity thatcontributes to the inhibition of immunogenic tumor growth.

Previous studies have demonstrated the involvement ofmiR155 in the DCs (26, 27) and T cells (13, 25) in controllingtumor growth. As expected, we found that tumor-associatedmiR155�/� DCs expressed less MHC-II (Supplementary Fig.S3A), and induced less antigen-specific CD8þ T-cell prolifera-tion compared with WT DCs (Supplementary Fig. S3B). Sim-ilarly, tumor-infiltrated miR155�/� CD8þ T cells sorted fromLLC1-OVA tumors displayed reduced response to DCs pulsedwith OVA-I peptides in vitro (Supplementary Fig. S3C). In thisimmune cell–specific context, it is of interest that we observedintrinsic defects in miR155�/� tumor–associated DCs andantitumor T cells.

miR155 is required for MDSC accumulation in tumor-bearingmice

Although above data suggest a cell-intrinsic role of miR155in tumor-associated DCs and antitumor T cells, host miR155deficiency promoted overall antitumor T-cell immunity andinhibited tumor growth. In search of a cellular mechanism forthe miR155-mediated tumor-promoting effect, we investigated

Host miR155 Promotes Tumor Growth

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the well-defined immunosuppressive immune cell subsets intumor, including MDSCs and Tregs. We observed that intratu-moralGr1þCD11bþMDSCswere consistently decreased in LLC1-OVA–bearingmiR155�/�mice in comparison withWTmice (Fig.3A and B). Further analysis revealed significant reductions in thepercentage of CD11bþGr1þ cells from miR155�/� mice com-pared with WT controls in spleen and peripheral blood (Fig. 3C).We also tested other tumor models, including EL4, B16F10, andLLC1, and found that miR155�/� mice have much fewer splenicMDSCs than WT mice (Supplementary Fig. S4A). These resultsconfirmed thatmiR155 is required forMDSCaccumulationundertumor-bearing conditions because no significant differences werenoted in the percentages of splenic CD11bþGr1þ cells betweentumor-free miR155�/� and WT mice (data not shown). MDSCconsists of ly6G�ly6Chigh (monocytic) and ly6Gþly6Clow (gran-ulocytic) subpopulations (31, 35). Of note, the preferentialreduction of the splenic (Supplementary Fig. S4B and S4C) andtumor-infiltrating (Fig. 3D) monocytic ly6G�ly6Chigh subset wasobserved in LLC1-OVA–bearing miR155�/�mice compared withWT mice. These results suggest miR155 is required for tumor-associated MDSC accumulation particularly with a monocyticphenotype. To dissect the role of miR155 further in regulatingMDSC accumulation, we stainedwith Ki67 (Fig. 3E) and AnnexinV (Fig. 3F) to test the proliferative ability and apoptotic status of

MDSCs within the tumor microenvironment, respectively. Nosignificant differences in both granulocytic andmonocytic MDSCsubsets were found between WT mice and miR155�/� mice.

Given the critical function of miR155 in promoting myeloidlineage commitment in hematopoietic stem cells and myeloidprogenitors (36), we asked whetherMDSC differentiation requiresmiR155. To evaluate differentiation of myeloid cells in the pres-ence of tumor-derived factors, BM cells frommiR155�/�mice andtheirWT littermateswere culturedwithGM-CSF for 5days in tumorcell–conditioned medium (TCM). As expected, tumor-derivedfactors significantly reduced the differentiation of DCs andmacro-phages and increased the generation of Gr1þCD11bþ MDSCs inWT populations (Fig. 3G), consistent with previous observation(37). In contrast, TCM failed to inhibit the differentiation ofmyeloid progenitor cells appreciably from miR155�/� mice (Fig.3G), suggesting an important role of miR155 in MDSC differen-tiation in the tumor microenvironment.

miR155 is required for MDSC suppressive function duringtumor growth

To determine whether miR155 is required for MDSC suppres-sive function, we purified CD11bþ cells fromMC38-bearingmiceand cocultured with OT-I splenocytes. Notably, the miR155�/�

MDSC appreciably lost their capacity to suppress proliferation of

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Figure 1.Tumor growth is inhibited in miR155�/� mice. A, chemically-induced colon tumor incidence in WT and miR155�/� mice. Mice were given no treatment, orDSS and AOM as described in Materials and Methods. Length of colon, size, and numbers of colon polyps were recorded (n¼ 5). B–E, mice (n¼ 3–5 per group) wereinoculated s.c. with 106 MC38 (B), LLC1 (C), LLC1-OVA (D), or EG7 (E) cells. Data (mean � SEM) are representative of at least five independent experiments.� , P < 0.05; �� , P < 0.01.

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antigen-specific T cell in vitro, whereas WT MDSC remainedstrongly suppressive (Fig. 4A). To corroborate these findingsfurther, splenic MDSCs were isolated from EG7 (Fig. 4B) orB16-SIY (Supplementary Fig. S1B) tumor–bearing WT ormiR155�/� mice. As expected, miR155�/� MDSCs were unableto inhibit antigen-specific T-cell proliferation in vitro comparedwith WT MDSCs. Emerging data show that the degree of immu-nosuppression varies among populations of MDSCs isolatedfrom different organs, and intratumoral MDSCs are the mostimmunosuppressive (38). In the LLC1 tumor model, CD115 actsas a function marker for MDSCs (39). Thus, we sorted CD115þ

CD11bþ cells and CD115�CD11bþ cells from LLC1 tumor tis-sues, and compared their suppressive activity between WT andmiR155�/� mice (Fig. 4C). Consistent with previous results,intratumoralWTCD115þCD11bþ cells but not CD115�CD11bþ

cells were inhibitory. In contrast, miR155�/� CD115þCD11bþ

cells were unable to suppress T-cell proliferation (Fig. 4B). Toevaluate MDSC tolerogenic activity on antigen-specific CD8þ Tcells in vivo, MDSCs from tumor-bearing WT or miR155�/� mice,either pulsed or not with OVA-I peptides, were transferred on thesame day of the immunization. DLNs were collected 10 days afterimmunization and stimulated in vitro to measure T-cell prolifer-ation (Fig. 4D) and enumerate CD8þ T cells producing IFNg (Fig.4E). Both the number of transferred CD90.1þ cells and number ofIFNg-secreting CD8þ T cells in DLNs were significantly reduced inmice that receivedMDSCs derived fromWT tumor–bearingmice,but not miR155�/� tumor–bearing mice.

miR155 is upregulated and functions in cytokine-inducedMDSCs

It is generally accepted that MDSCs are elicited by tumor-derived factors (e.g., GM-CSF, IL6) from precursors present in

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Figure 2.Host miR155 deficiency enhances antigen-specific antitumor T-cell immunity. A, percentage and absolute number of CD4þCD3þ, CD8þCD3þ, Gr1þCD11bþ,CD3�CD19þ, and CD49bþNK1.1þ cells in tumor infiltrates of WT or miR155�/� mice collected 21 days after inoculation with EG7 tumor cells (n ¼ 5). B, CD8þIFNgþ

T-cell frequency in spleen, DLN, and tumor from EG7-bearing WT or miR155�/� mice 21 days after tumor inoculation (n ¼ 3–6). C, representative flowcytometric analysis of tumor antigen-specific CD8þ T cells from EG7-bearing WT or miR155�/� mice. Frequency of tetramerþ cells, specific for the OVA epitopeSIINFEKL in CD8þ infiltrates from mice in B, was summarized. D, representative flow cytometric analyses of in vivo antigen-specific killing capacity ofantitumor T cells from EL4- or EG7-bearing WT and miR155�/� mice. Equal numbers of eFluor 450high SIINFEKL peptide-pulsed and eFluor 450low SIY-peptide-pulsed WT splenocytes were adoptively transferred into tumor-bearing mice. Numbers denote percentage of SIINFEKL peptide-pulsed target cell killingin DLN. The percentage of killing for EG7-bearingmice in DLNwas calculated as described inMaterials andMethods (n¼ 3). E, inmiR155�/�mice (n¼ 5), depletion ofCD4þTcells, CD8þT cells, or NK cellswas achievedby twiceweekly i.p. injection of control Ig, anti-CD4, anti-CD8, or anti-NK1.1 depletingAbs, respectively, beginning1 day before tumor challenge. Data are representative of two independent experiments. � , P < 0.05; �� , P < 0.01.






hematopoietic organs such as the BM and possibly spleen (at leastin mice; refs. 40–42). GM-CSF alone (43) or the combination ofGM-CSF plus IL6 (44) has been used successfully to generateMDSCs in short-term culture in vitro from BM precursor cells.Interestingly, GM-CSF alone upregulated miR155 expressionduring the induction of BM-derived MDSCs. Moreover, a com-bination of GM-CSF and IL6 induced higher levels of miR155expression (Supplementary Fig. S5A). We next analyzed whethermiR155 affected cytokine-inducedMDSC function as observed intumor-bearing mice. As shown in Supplementary Fig. S5B,miR155�/� MDSCs failed to suppress antigen-specific T-cell pro-liferation in vitro compared with WT MDSCs. To examine furtherthe functional contribution of miR155 expression to the immu-noregulatory activity of MDSCs, we transfected BM cells with amiR155-specific inhibitor or a respective pre-miR155 (precursor)and analyzed the proliferative capacity of antigen-specific T cellsin the presence of cytokine-induced MDSCs (Supplementary Fig.S5C). To this end, pre-miR155, miR155 inhibitor, or control-transfected MDSCs were cocultured with responder T cells atdifferent ratios. As expected, control transfection in MDSCs did

not alter their suppressive capacity. In sharp contrast, treatmentwith miR155 inhibitors abrogated MDSC suppressive activity.Consistent with theses, overexpression of miR155 resulted instronger suppression of T-cell proliferation versus control-trans-fected MDSCs.

miR155 deficiency downregulates tumor-associated MDSCsuppressive pathways

To identify the factors by which miR155 regulated MDSCs, weanalyzed gene expressionprofiles inWTversusmiR155�/�MDSCsfrom LLC1-OVA–bearingmice.We used real-time PCR to evaluatemRNA levels of genes related to tumor angiogenesis, immuneresponses, and immune suppression (Fig. 4F). We found thatmmp9, vegf, inos, and arg1were down regulated, whereas socs1 andship1 were upregulated in miR155�/� MDSCs. On the basis ofprevious observations (45–47), VEGF and MMP-9 expressed byMDSCs contribute to the proangiogenic tumor microenviron-ment. Thus, our results raised the possibility that miR155expressed byMDSCs could promote tumor growth by stimulatingtumor angiogenesis. Because inducible nitric oxide synthase

59.5 10.6 37.4 3.8

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Figure 3.miR155 is required for MDSC accumulation in the tumor microenvironment. A, the percentage of tumor-infiltrating Gr1þCD11bþ MDSCs was determinedby flow cytometry from LLC1-OVA tumor–bearing mice. B, the absolute number of tumor-infiltrating MDSCs (n ¼ 3). C, the percentage of MDSCs in spleen(n ¼ 9) and peripheral blood (n ¼ 5) from LLC1-OVA tumor–bearing mice is summarized. D, percentage of CD11bþLy6GþLy6Clow (granulocytic) andCD11bþLy6G�Ly6Chigh (monocytic) MDSCs is indicated within plots and summarized (n ¼ 9). Flow cytometry analysis of expression of Ki-67 (E) andAnnexin V (F) on both granulocytic and monocytic tumor-infiltrating MDSCs (n ¼ 6). G, BM cells were cultured with GM-CSF and IL4 for 5 days incomplete culture medium or in the TCM. The cell phenotypes were examined by flow cytometry. Data are given as mean � SEM. � , P < 0.05; �� , P < 0.01;�� , P < 0.001. Data are representative of two independent experiments.

Chen et al.





(iNOS) and arginase-I in MDSCs are essential for their immuno-suppressive function, we asked whether downregulation of inos orarg1was implicated in the linkbetweenMDSC suppressive activityand miR155. We evaluated ROS levels within the population oftumor-infiltrating MDSCs. No significant differences in ROS pro-duction from MDSCs were found between miR155�/� and WTmice (Fig. 4G). However,miR155�/�MDSCs have a lower level ofarginase activity than WT counterparts (Fig. 4H). Moreover, inhi-bition of arginase-I with specific inhibitor nor-NOHA completelyabrogated suppressive activity of WT MDSCs, whereas the nor-NOHA treatment did not affect the miR155�/� MDSCs (Fig. 4I),suggesting that miR155 modulates arginase-dependent suppres-sive activity of MDSCs. Given the importance of CD115 and

CD124 (IL4Ra; ref. 48) in MDSCs, we also compared the expres-sion of CD115 and CD124 in both MDSC subsets from tumor-bearing mice. There were no significant difference betweenmiR155�/� andWTmice (Supplementary Fig. S4D). Taken togeth-er, our data indicate that miR155 is likely required for MDSC-mediated tumor angiogenesis and immunosuppression.

miR155 targets SOCS1 in MDSCsWe initially confirmed that splenic CD11bþ cells from tumor-

bearing mice had higher levels of miR155 expression than thosecounterparts from tumor-free mice (Fig. 5A), whereas no detect-able miR155 expression was found in tumor-bearing miR155�/�

mice (Fig. 5B), suggesting a link between miR155 upregulation

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Figure 4.miR155 is required for MDSC suppressive function during tumor growth. A, suppressive activity of MDSCs from MC38-bearing WT versus miR155�/� mice.Splenic Gr1þCD11bþMDSCs from either MC38-bearingWT ormiR155�/�micewere added at different ratios to OT-I splenocytes stimulatedwith OVA-I peptides for 3days, and 3[H] thymidine uptakewasmeasured. The suppressive activities of tumor-infiltratingMDSCs from EG7-bearing (B) or LLC1-bearing (C)WT andmiR155�/�

mice were assessed in a similar manner as described in A. D, to evaluate MDSC tolerogenic activity on in vivo T-cell function, naive OT-1 CD90.1 cells weretransferred to CD90.2 congeneic recipients, whichwere s.c. immunized, 2 days later, with OVA-I peptides in IFA. MDSCs fromMC38 tumor–bearingWT ormiR155�/�

mice, either pulsed or not with OVA-I peptides, were transferred on the same day of the immunization. DLNs were collected 10 days after immunization andstimulated with OVA-I in vitro to measure T-cell proliferation by 3[H] thymidine uptake. E, frequencies of CD8þCD90.1þ cells and IFNg-secreting CD8þ

T cells as determined by flow cytometry are summarized. F, real-time quantitative RT-PCR analysis of different gene expression in WT and miR155�/� MDSCs fromLLC1-OVA–bearing mice (n ¼ 5–14). G, ROS production was measured with DCFDA staining by flow cytometry and summarized within the granulocytic andmonocytic tumor-infiltrating MDSCs. H, arginase I activity of WT versus miR155�/� MDSCs. I, arginase I inhibitor nor-NOHA was able to blunt the suppressiveactivity of WT MDSC but not miR155�/� MDSCs. All samples had MDSC, and the ratio of T cell/MDSC was 2:1. � , P < 0.05; �� , P < 0.01. Data are representative oftwo independent experiments.






and MDSC induction upon tumor-bearing conditions. Interest-ingly, we detected enhanced levels of socs1 in miR155�/� MDSCscompared with WT MDSCs in the tumor-bearing mice but nottumor-free mice (Fig. 5C). To test the functional consequence ofelevated socs1 expression (as observed in the absence of miR155)onMDSC suppressive activity, we utilized siRNAs to knock downsocs1 expression in activated miR155�/� MDSCs. We found thatsocs1 knockdown by specific siRNAs completely restored suppres-sive activity of miR155�/� MDSCs compared with cells given ascrambled control (Fig. 5D). Thus, miR155 targets SOCS1 toregulate the suppressive function of MDSCs.

miR155 is required for MDSC-mediated Treg inductionBecause fewer tumor-infiltrating CD4þFoxp3þ Tregs were

found in tumor-bearing miR155�/� mice than in WT mice (Fig.6A and Supplementary Fig. S6), we tested a role for miR155 inTreg in immune suppression. As shown in Supplementary Fig.S7A, Tregs from either WT or miR155�/� mice potently sup-pressed proliferation of CD4þ T cells in vitro, consistent withprevious results (15, 16). Moreover, miR155�/� Tregs fromtumor-bearing mice had similar levels of CD39, CD73, CTLA-4, GITR, CD44, and CD62L expression compared with WT Treg(Supplementary Fig. S7B). To test further the tumor-promotingrole of Tregs, we performed adoptive transfer ofWTormiR155�/�

Tregs into LLC1-bearing mice. There was a subtle difference intumor growth between mice receiving WT and miR155�/� Tregs(Supplementary Fig. S7C). In addition, the role of miR155 intumor-mediated conversion of Tregs was also evaluated. Wefound similar conversion of CD4þ Foxp3þ cells in spleen andDLNs after tranfer of WT or miR155�/� CD62LþCD4þ naive Tcells into the tumor-bearing mice (Fig. 6B). Thus, these dataexclude a direct contribution of miR155 to Treg-mediated sup-

pressive function and tumor promotion, and tumor-mediatedconversion of Tregs.

MDSCs induce Treg expansion in tumor-bearingmice (39, 49).To determine whether miR155 mediates MDSC-mediated Treginduction, miR155�/� or WT MDSCs were cultured with OT-II Tcells plusOVA-II peptides. As expected,MDSCswere ineffective toinduce antigen-specific Treg in the absence of TGFb, but decreasedTreg cell induction was observed when comparing miR155�/�

with WT MDSCs in the presence of TGFb (Fig. 6C), indicating arole for miR155 in MDSC-mediated Treg induction.

miR155 expression by MDSC facilitates tumor growthAsmiR155was required forMDSCaccumulation and function,

we tested whether miR155 promoted tumor growth in anMDSC-dependent manner. We performed MDSC depletion in WT andmiR155�/�mice using either 5-FU (Fig. 7A) or depleting anti-Gr1antibodies (Fig. 7B) after LLC1 tumor challenge. Consistent withprior published data (50, 51), both 5-FU and anti-Gr1 efficientlydepleted CD11bþGr1þ populations, especially the Gr1hi popu-lation within tumor-bearing mice (Fig. 7A and B). Importantly,MDSC depletion greatly inhibited tumor growth in WT mice,indicating a tumor-promoting role forMDSCs. By contrast,MDSCdepletion minimally affected tumor growth in miR155�/� micecompared with WT mice at later time points (starting from day22; Fig. 7A and B).Moreover, adoptive transfer ofWTMDSCs intomiR155�/� mice resulted in faster tumor growth than transfer ofmiR155�/� MDSCs (Fig. 7C), further consistent with the directrole of miR155 on MDSCs in tumor growth. We did not expectmiR155�/� MDSCs to have migration defects. This notion issupported by the fact that miR155�/� MDSCs displayed equalability to traffic to the tumor site asWTMDSC (Fig. 7D), excludingthe possibility thatmiR155�/�MDSCmay not reach the tumor to

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Figure 5.miR155 targets socs1 within MDSCs. Aand B, miR155 expression wasmeasured by the real-timequantitative RT-PCR in splenic Gr1þ

CD11bþ cells fromWT na€�ve mice, LLC1tumor–bearing WT mice (A), andmiR155�/� tumor–bearingmice (n¼ 3;B). C, Socs1 gene expression wasmeasured by the real-timequantitative RT-PCR in splenic WT ormiR155�/� Gr1þCD11bþ cells fromna€�ve mice and LLC1 tumor–bearingmice (n¼ 5). D, to identify the functionof SOCS1 within Gr1þCD11bþ MDSC,miR155�/� MDSCs were transfectedwith siRNAs against SOCS1 or controloligos, and WT MDSCs were alsotransfected with control oligos byAMAXA. MDSCs 48 hours aftertransfection were added at differentratios to OT-I splenocytes stimulatedwith OVA-I peptides for 3 days, and3[H] thymidine uptakewas measured. Data are given asmean � SEM. Data are representativeof two independent experiments.� , P < 0.05; �� , P < 0.01.

Chen et al.





exert their effect. These results indicate that miR155 expression isrequired for MDSCs to facilitate tumor growth.

DiscussionmiR155 is required for development and function of both

innate and adaptive immune cells, and is thought to be largelyimmune stimulatory (52–54). However, to our surprise, chem-ically-induced tumor incidence and transplanted tumor growthwere decreased in miR155�/� mice. This was associated with anumber of immune phenotypic and functional alterations.

MDSCs and Treg cells are important immunosuppressivecells in the tumor microenvironment. Notably, there weresignificantly more MDSCs and Treg cells in tumor-bearing WTmice than in tumor-bearing miR155�/� mice. However, theprevalence of MDSCs and Tregs was similar in tumor-free WTversus miR155�/�mice. Our results indicate that miR155 couldregulate the development of MDSCs and Treg cells in thecontext of tumor, and in turn affect antitumor immuneresponses. In line with our observations, a recent study showed

that miR155 was upregulated in cytokine-induced MDSCs fromBM cultures and spleen MDSCs isolated from tumor-bearingmice, and promoted expansion of functional MDSCs (17).However, it remained unclear whether miR155 mediates inhib-tion of tumor growth in a MDSC-dependent manner despite itsdefined immune-stimulatory functions. In addition to regulat-ing immunosuppressive factors, we do not rule out the con-tribution of miR155 to tumor immunity through otherimmune components. Indeed, we observed the intrinsic defectsin miR155�/� tumor–associated DCs and antitumor T cells.Consistent with this concept, miR155 is required for activationof tumor-associated DCs (26, 27) and effector CD8þ T cell(13, 25) responsed to cancer. Moreover, ectopic miR155 expres-sion repolarized protumoral M2 macrophages toward an anti-tumor M1 phenotype (27), and increasing miR155 levels intumor-associated DCs by miRNAmimetics increased antitumorresponses (26). We do not exclude, however, that miR155insufficiency in other immune compartments may have similarprotumoral effects, as recently proposed for NK cells (55).

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Figure 6.miR155 is required for MDSC-mediated Treg induction. A, representative dot plots of Foxp3 expression in LLC1-OVA tumor–infiltrating CD4þ cells. Thepercentage of Foxp3þ cells is indicated within plots and summarized (n ¼ 5). B, WT or miR155�/� CD4þCD62Lþ na€�ve T cells were transferred into CD45.1mice, followed by a s.c. injection of LLC1-OVA cells. The conversion of transferred T cells to Foxp3þ cells (CD45.2þ) in DLN and spleen from LLC1-OVAtumor–bearing mice was detected by flow cytometer 9 days after tumor cell injection. The levels of converted Foxp3 expression were determined bymean fluorescent intensity (MFI). Endogenous Foxp3þ cells (CD45.1þ) from host mice are shown as controls. C, WT and miR155�/� MDSCs from LLC1 tumor–bearing mice were cocultured with OT-II splenocytes at a 1:4 ratio for 5 days in the absence or presence of TGFb, and induced CD25þFoxp3þ cells amongtotal CD4þ T cells were subsequently determined by flow cytometry. Data are representative of two independent experiments. �� , P < 0.001.






Notably, our results on host miR155 deficiency and tumorgrowth differ froma recent study (25) using the EL4 tumormodel.Reasons for this discrepancy could include differences in thetumor cell lines that could alter the accumulation of distinctimmune cell subsets in the tumor microenvironment. We alsoused EG7 cells expressing the surrogate antigen OVA, rather thanparental (OVA-negative) EL4 cells in the prior report (25). Theseimmune differences could alter in vivo outcomes. Finally, giventhat miR155 regulation of one immune cell type can antagonizethe function of other cells, the balance of these effects maydetermine whether miR155 is beneficial or detrimental to tumorgrowth. In this regard, the prior study (25) focused on the intrinsicrole ofmiR155 in effector T cells, but didnot analyzeother distinctcellular subsets within the tumor such as MDSCs and Treg cellsthat promote tumor growth, as we showed in our study. Despitedefects in immunostimulatory activities observed in miR155�/�

effector T cells and DCs, they are still able to mount antitumor

responses. IncreasedmiR155 couldplay a critical role in balancinganti- and protumor immune components within the tumor. In agiven tumormodel system,miR155 could preferentially promoteMDSCs and Treg cells before potent antitumor T-cell immunity isestablished. Furthermore, the extent and regulation of tumor-induced immunosuppression including MDSC and Tregs couldvary in different tumor types and/or tumor stages. In support, weshowed that host miR155 dificiency inhibited the growth ofMC38 and LLC1 tumors rather than B16 tumors. Thus, it is likelythat miR155 plays dominant, MDSC-intrinsic roles in impairingantitumor T-cell immunity in these tumor models. Our datasuggest that the immune regulation of miR155 is highly con-text-dependent, and varies in thepresence of different cells, phasesof immune responses, and tumor model systems. Our studieshighlight the importance of evaluating the intrinsic contributionofmiR155 carefully inmajor immune cell subsets, wheremiR155could be either protective or deleterious to antitumor immunity.

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Figure 7.miR155 expression by MDSCs facilitates tumor growth. A,WT or miR155�/�mice were injected s.c. with 106 LLC1 tumor cells. Three days later, mice were injected i.p.by 5-FU or PBS (control; A) or anti-Gr1 antibodies (B) once every 4 days. MDSC depletion by 5-FU or anti-Gr1 depleting antibodies in vivo was determinedby flow cytometry and summarized (n ¼ 5). Tumor volume was measured and plotted at indicated times. NS, not significant. C, splenic Gr1þCD11bþ MDSCs fromtumor-bearing WT or miR155�/� mice were injected i.v. into LLC1-bearing mice at days 7 and 14. Mice receiving PBS without MDSCs were controls. D, MDSChoming to tumors. Equal numbers of splenicWTGr1þCD11bþMDSCs labeledwith CFSE andmiR155�/�MDSCs labeledwith eFluro450weremixed and transferred i.v.into LLC1 tumor–bearing mice. Either WTMDSCs labeled with CFSE alone or miR155�/�MDSCs labeled with eFluro450 alone were used as controls. Representativeflow cytometric analysis of CFSEþ cells versus eFluroþ cells in the tumor 24 hours after transfer. Frequencies of CFSEþ MDSCs (WT) and eFluroþ MDSCs(miR155�/�) among tumor tissues are summarized (n ¼ 5). � , P < 0.05; ��, P < 0.01; ��, P < 0.001. Data (mean � SEM) are representative of two independentexperiments.

Chen et al.





Although miR155 is required for Treg cell homeostasis in thepresence of limiting amounts of IL2, it is dispensable in non-competitive lymphopenic settings (16). Indeed, we showed noimpaired ability of miR155-deficient T cells to induce Foxp3 intumor-bearing hosts. Moreover, intact suppressive activity ofmiR155-deficient Tregs was observed, consistent with previousresults (15, 16). In addition to the direct inhibiton of T-cellproliferation, MDSCs can induce Treg expasion in tumor-bearingmice. Considering the importance of miR155 for functionalMDSC development, we tested whether miR155 is required forMDSC-mediated Treg induction. It appears that loss of miR155results in the reduced accumulation of MDSCs that not only caninhibit clonal expansion of activated effector T cells but alsoinduce tumor-specific Tregs to establish and maintain T-cellsuppression in tumor-bearingmice. Therefore, our results indicatethatmiR155 is likely involved in a close interaction ofMDSCs andTreg development during tumor progression.

In an effort to unravel the molecular basis for miR155'sfunction in the MDSCs, we found the miR155-targeted SOCS1to retain the suppressive activity of MDSCs. SOCS1 is defined asan important mechanism for the negative regulation of thecytokine–JAK–STAT pathway (56). Several studies have dem-onstrated that the expansion and suppressive function ofMDSC is mediated via the STATs (40–42). A recent studyreported that miR155 deficiency in Treg cells resulted inincreased SOCS1 expression accompanied by impaired activa-tion of STAT5 transcription factor in response to limitingamounts of IL2, and suggested that Foxp3-dependent regula-tion of miR155 maintains competitive fitness of Treg cells bytargeting SOCS1 (16). In line with these findings, our SOCS1shRNA experiments showed that defective suppressive activityby miR155�/� MDSCs could be complemented by knockdownof SOCS1 expression, which was elevated in these MDSCs. Ourdata indicate that SOCS1 could impair the suppressive functionof MDSCs when miR155 is absent, and at least partially explainwhy miR155 helps maintain MDSC activity. We also notedincreased SHIP-1 expression in miR155�/� MDSCs. Interest-ingly, SHIP-1 was recently reported as a target of miR155specifically in MDSC expansion (17), consistent with the pre-vious observation that myeloid-specific ablation of SHIPresulted in an increase in MDSC numbers (57). However, theprior study did not examine the importance of MDSC miR155status in tumor growth (17). To our knowledge, our data clearlyprovide the first evidence that cell-intrinsic MDSC miR155 isrequired for MDSCs to facilitate tumor growth, using bothadoptive transfer and MDSC depletion analyses. We showedinverse correlations between MDSC SHIP-1/SOCS1 andmiR155, suggesting both SHIP-1 and SOCS1 as target genesof miR155 during functional MDSC generation. As downregu-lation of either SHIP-1 or SOCS1 could increase STAT3 acti-vation (17, 58), which promotes functional MDSC expansion(37, 45), targeting both SHIP-1 and SOCS1 by miR155 wouldenhance STAT3 activity and MDSC accumulation. However, thebiology of miRNA signaling in MDSC development is likely tobe more complex. At this stage, we cannot exclude the involve-ment of additional targets other than SHIP-1 and SOCS1 oreven miRNAs other than miR155 in regulation of functionalMDSC induction.

miR155 expression is controlled by a wide range of inflam-matory factors, and transgenic overexpression of miR155results in cancer (18). Being oncogenic and pertinent to

inflammation, miR155 is considered as prototypical micro-RNA bridging inflammation and cancer development (4, 59).In support, we found that miR155 deficiency inhibited carci-nogenesis in the AOM and DSS-induced colorectal cancermodel. MiR155 deficiency could reduce colon inflammationthat is known to drive carcinogenesis in this model (60).Moreover, miR155 might promote tumor growth in an intrin-sic manner as this is an induced and not transplanted model.Nevertheless, miR155 positively regulates myeloid cell devel-opment by acting on BM progenitors during inflammatorystress. Particularly, our and other data (17) show that miR155is upregulated in MDSC either from tumor-bearing hosts orgenerated from BM progenitors by GM-CSF and IL6. Over-expression of miR155 enhanced, whereas depletion of miR155reduced the suppressive function of cytokine-induced MDSCs.Moreover, MDSC accumulation was impaired in tumor-bear-ing mice lacking miR155 and miR155-deficient MDSCs failedto inhibit T-cell functions. Thus, the induction of MDSC byproinflammatory mediators led to the novel hypothesis thatinflammation promotes the accumulation of functional MDSCby increased miR155 that downregulates immune surveillanceand antitumor immunity, thereby facilitating tumor growth.MDSCs also promote tumor progression through nonimmunemechanisms. Their release of MMP-9 and VEGF contributesto tumor angiogenesis. Given the decreased production ofMMP-9 and VEGF from miR155�/� MDSCs, further studieswill determine whether miR155 mediates MDSC-dependenttumor angiogenesis.

Extensive evidence indicates that miR155 functions as anoncomiR in many solid as well as hematologic tumors, and it isoften associated with poor prognosis (61, 62). Thus, it has beensuggested that therapeutic inhibition of miR155 could be aneffective strategy to treat cancer (22–24). However, in thesestudies, the contributions of immune regulation by miR155 totumor progressionwere unappreciable (23, 63, 64). Although ourdata expand the role of miR155 to MDSC-mediated tumorprotection, the cancer cell–intrinsic roles of miR155 in bothimmune and nonimmune conditions need further investigation.On the other hand, miR155 activation in effector T cells and DCsboosts antitumor immunity, demonstrating a potential beneficialrole for this miRNA during tumor progression. In this regard,besides its oncogenic activity, miR155 functions as a cell context–dependent "immunomiR" in orchestrating protumor or antitu-mor immune responses. Thus, our results suggest additionalinvestigations before considering miR155 manipulation for can-cer therapy. For example, cell-specific targeting of miR155 andconsideration of tumor effects on miR155-mediated outcomesmerit additional attention.

In summary, we investigated the role of host miR155 in AOMand DSS-induced colon carcinogenesis and multiple transplant-able tumor models. Our study identified a crucial cell-intrinsicrole ofmiR155and its target SOCS-1 inMDSCs anddemonstratedthat this miRNA is required by MDSCs to limit antitumor T-cellimmunity. Despite the evidence for an established role ofmiR155in effector T cells and DCs, this miRNA is closely linked to thedevelopment of MDSCs and Treg cells, triggers tumor immunesuppression, and thereby facilitates tumor growth. Our dataindicate that the biologic activities of miR155 are highly cellcontext–dependent, including tumor dependent. Further studieswill also be necessary to determine if host miR155 affects tumorangiogenesis and metastasis.






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

Authors' ContributionsConception and design: S. Chen, Y. Zhang, T.J. Curiel, D. Fang, B. ZhangDevelopment of methodology: S. Chen, L. Wang, J. Fan, D. Fang, T.M. Kuzel,B. ZhangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Chen, L. Wang, J. Fan, C. Ye, D. Dominguez,D. Fang, B. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Chen, T.J. Curiel, D. Fang, T.M. Kuzel, B. ZhangWriting, review, and/or revision of the manuscript: S. Chen, Y. Zhang,T.J. Curiel, D. Fang, T.M. Kuzel, B. ZhangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Wang, J. Fan, C. Ye, D. Dominguez, D. Fang,T.M. Kuzel, B. ZhangStudy supervision: D. Fang, B. Zhang

AcknowledgmentsThe authors thank the National Institutes of Health Tetramer Facility for

providing the Kb/OVA tetramers.

Grant SupportThis researchwas in part supported byNIH grant CA149669,OvarianCancer

Research Foundation Funds (LT/UTHSC/01.2011), the Northwestern Univer-sity RHLCCC Flow Cytometry Facility, Cancer Center Support Grants (NCICA060553 and CA054174), the Owens Foundation, and the Skinner Endow-ment the Holly Beach Public Library Association.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedAugust 13, 2014; revisedNovember 4, 2014; acceptedNovember 18,2014; published OnlineFirst December 10, 2014.

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2015;75:519-531. Published OnlineFirst December 10, 2014.Cancer Res Siqi Chen, Long Wang, Jie Fan, et al.

Dependent Mechanism−Suppressor Cell Host miR155 Promotes Tumor Growth through a Myeloid-Derived

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Host miR155 Promotes Tumor Growth through a Myeloid ... · Host miR155 Promotes Tumor Growth through a Myeloid-Derived Suppressor Cell–Dependent Mechanism Siqi Chen1, Long Wang2,

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