STING Ligand c-di-GMP Improves Cancer Vaccination against ... · Research Article STING Ligand...

Research Article

STING Ligand c-di-GMP Improves Cancer Vaccinationagainst Metastatic Breast Cancer

Dinesh Chandra1, Wilber Quispe-Tintaya1, Arthee Jahangir1, Denise Asafu-Adjei1, Ilyssa Ramos1,Herman O. Sintim3,4, Jie Zhou3,4, Yoshihiro Hayakawa5, David K.R. Karaolis2, and Claudia Gravekamp1

AbstractCancer vaccination may be our best and most benign option for preventing or treating metastatic cancer.

However, breakthroughs are hampered by immune suppression in the tumormicroenvironment. In this study, weanalyzed whether cyclic diguanylate (c-di-GMP), a ligand for stimulator of interferon genes (STING), couldovercome immune suppression and improve vaccination against metastatic breast cancer. Mice with metastaticbreast cancer (4T1 model) were therapeutically immunized with an attenuated Listeria monocytogenes (LM)–based vaccine, expressing tumor-associated antigen Mage-b (LM-Mb), followed by multiple low doses of c-di-GMP (0.2 mmol/L). This treatment resulted in a striking and near elimination of all metastases. Experimentsrevealed that c-di-GMP targets myeloid-derived suppressor cells (MDSC) and tumor cells. Low doses of c-di-GMPsignificantly increased the production of IL12 byMDSCs, in correlationwith improvedT-cell responses toMage-b,whereas a high dose of c-di-GMP (range, 0.3–3 mmol/L) activated caspase-3 in the 4T1 tumor cells and killed thetumor cells directly. On the basis of these results, we tested one administration of high-dose c-di-GMP (3mmol/L)followed by repeated administrations of low-dose c-di-GMP (0.2 mmol/L) in the 4T1 model, and found equalefficacy compared with the combination of LM-Mb and c-di-GMP. This finding correlated with a mechanism ofimproved CD8 T-cell responses to tumor-associated antigens (TAA) Mage-b and Survivin, most likely throughcross-presentation of these TAAs from c-di-GMP–killed 4T1 tumor cells, and through c-di-GMP–activated TAA-specific T cells. Our results demonstrate that activation of STING-dependent pathways by c-di-GMP is highlyattractive for cancer immunotherapy. Cancer Immunol Res; 2(9); 901–10. �2014 AACR.

IntroductionCancer vaccination has shown great promise for preventing

or treating metastatic cancer in mice and humans (1–3).However, immune suppression in the tumor microenviron-ment (TME) remains a potential limitation to immunotherapy.Myeloid-derived suppressor cells (MDSC) are one of the mostimportant players in mediating TME-associated immune sup-pression because they strongly inhibit T-cell and natural killer(NK)–cell responses (4–6), with tumor-associated macro-phages, Tregs, and M2 macrophages also playing a role (4–8).Adjuvants reducing immune suppression are of great value

for cancer vaccination. c-di-GMP (30,50-cyclic diguanylic acid),also known as cyclic diguanylate, could be such an adjuvant.c-di-GMP is a bacterial intracellular signaling molecule thatwas initially identified by the Benziman laboratory in the

bacterium Acetobacter xylinum (renamed Gluconacetobacterxylinus; ref. 9). Various in vitro and in vivo animalmodel studiesusing chemically synthesized c-di-GMP demonstrated that c-di-GMP has potent immunomodulatory effects on cellularcomponents of both innate and adaptive immunity in infec-tions with such bacteria as Klebsiella pneumoniae (10–12).Recently, stimulator of interferon genes (STING) has beenidentified as the sensor for c-di-GMP (13). STING is a trans-membrane protein expressed in macrophages and dendriticcells (DCs; refs. 14–16). STING is mainly expressed in thethymus, heart, spleen, placenta, lung, and peripheral leuko-cytes, but is poorly expressed in the brain, skeletal musclecolon, small intestine, liver, and kidneys (14). Because of thestrong immunomodulatory effects of c-di-GMP, we evaluatedwhether STING-dependent c-di-GMP could improve cancervaccination through bypassing immune suppression andstimulating T-cell responses in mice with metastatic breastcancer.

As a vaccine, we used a highly attenuated Listeria mono-cytogenes (LM) bacterium expressing tumor-associated anti-gen (TAA) Mage-b (Mb), which was developed in an earlierstudy (3). This attenuated LM is different from wild-type LM(17, 18) in that the attenuated LM does not multiply in normaltissues and is naturally cleared by the immune system within 3to 5 days (5, 19, 20). Mage-b is highly expressed in metastasesand primary breast tumors of the 4T1 model (3), and ishomologous with human melanoma-associated antigen

1Department of Microbiology and Immunology, Albert Einstein Collegeof Medicine, Bronx, New York; 2Karagen Pharmaceuticals, Frederick;3Program inOncology, University ofMaryland,Marlene andStewartGreen-ebaumCancerCenter, Baltimore; 4Department ofChemistry andBiochem-istry, University of Maryland, College Park, Maryland; and 5Department ofApplied Chemistry, Aichi Institute of Technology, Toyota, Aichi, Japan

D. Chandra and W. Quispe-Tintaya contributed equally to this article.

Corresponding Author: Claudia Gravekamp, Albert Einstein College ofMedicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-430-4048; Fax: 718-430-8711; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-13-0123

�2014 American Association for Cancer Research.

CancerImmunology

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(MAGE; ref. 21). MAGE is expressed in 90% of all breast cancers(22). LM is an intracellular pathogen that delivers the vaccineantigen directly into antigen-presenting cells (APC) such asmacrophages with high efficiency (23). The vaccine antigenproduced by LM is processed and presented as short peptidesvia the MHC class I and II pathways generating both CD4 andCD8T-cell responses (24). Killing of tumor cells occurs throughCD8 T cells. Although semiprophylactic immunizations withLM-Mb (one before and two after tumor development) werehighly effective againstmetastatic breast cancer, this effectwasless abundant with a more clinically relevant immunizationprotocol of three exclusive therapeutic vaccinations (aftertumor development; ref. 20) due to the strong immune sup-pression in the TME. Therefore, reducing immune suppressionand improving T-cell responses to TAAs in the TME was themost important goal in this study, and c-di-GMP seemed anextremely suitable candidate.

Here, we demonstrate that c-di-GMP exhibits variousmechanisms to combat metastatic breast cancer. Low dosesof c-di-GMP provided strong adjuvant effects in LM-Mb vac-cinations by reducing theMDSC population (highly expressingSTING), by converting a subpopulation of immune-suppres-sing MDSCs into an immune-stimulating phenotype pro-ducing IL12, and by improving CD8 T-cell responses to TAAMage-b delivered through LM. High doses of c-di-GMP acti-vated caspase-3 and killed tumor cells directly. This uniquecombination of therapeutic low doses of c-di-GMP and LM-Mbresulted in an almost complete elimination of the metastases.Moreover, one high-dose c-di-GMP treatment followed bymultiple low doses of c-di-GMP in a therapeutic setting wasequally effective compared with LM-Mb þ c-di-GMP, andshowed improved CD8 T-cell responses to Mage-b and Survi-vin, most likely through cross-presentation of TAAs of c-di-GMP–killed tumor cells and through activation of the T cells bymultiple low doses of c-di-GMP. These dramatic results with c-di-GMP are highly promising for human clinical application.

Materials and MethodsMice

Normal female BALB/cmice (3months) were obtained fromCharles River and maintained in the animal husbandry facilityof Albert Einstein College of Medicine according to the guide-lines of the Association for Assessment and Accreditation ofLaboratory Animal Care and the Albert Einstein Institute forAnimal Studies. All mice were kept under biosafety level 2conditions, as required for LM.

Cells and cell cultureThe 4T1 cell line, derived from a spontaneous mammary

carcinoma in a BALB/c mouse (25), was cultured in DMEMsupplemented with 10% FBS, 1 mmol/L mixed nonessentialamino acids, 2mmol/L L-glutamine, insulin (0.5 HSP units/mL),penicillin (100 units/mL), and streptomycin (100 mg/mL). Thiscell line was developed by Dr. Fred Miller, Karmanos CancerInstitute (Detroit, MI), and an early passage (passage 25) waskindly provided in June 2004. The 4T1 cell line is extremelymetastatic in BALB/cmice (syngeneic background strain). Thisextrememetastatic character has been verified in BALB/c mice

within the past 2 months. The cell line has a characteristicgrowth pattern, i.e., it forms mammosphere-like balls in cellculture. This observation has been verified within the past 2months by inverted lightmicroscopy. The 4T1 cell line expressesTAAMage-b and has been verified within the past 6 months byRT-PCR. The 4T1 cell line is Mycoplasma free.

The HEK293T cell line was purchased from the ATCC (CRL-11268) in 2009. This is a human epithelial kidney cell line. Thiscell line is often used as a negative control for STING expres-sion in Western blots. In this study, HEK293T was negativefor STING expression by Western blotting. The HEK293T cellline is Mycoplasma free.

Plasmids, LM, and c-di-GMPThe LM-Mb strainwas developed in an earlier study (3). This

was constructed in the prfA-negative XFL-7 strain (referred toas LM in this study), which lacks the positive regulatory factorA that is a centralmediator of virulence (26). The vaccine strainwas transformed with LM plasmid pGG-34, which encodesprfA and amino acids 311–660 of murine Mage-b fused to anoncytolytic form of Listeriolysin O (LLO; refs. 3, 27). Com-plementation of prfA expression by the plasmid does not fullyrestore virulence, but enforces retention of the plasmid duringinfection (26, 27).

c-di-GMP (provided by D. Karaolis, Karagen Pharmaceuti-cals, Frederick,MD), used in these studies, was synthesized andprepared as previously described (28). Stock solutions of c-di-GMPwere generated at 3mmol/L in saline (equals 150 nmol in50 mL). For in vivo experiments, 0.2 mmol/L per mouse wasused, and for in vitro experiments, a range of 9.3 mmol/L to 75mmol/L was used as indicated in the text.

Immunization and tumor challengeVarious immunization protocols have been tested in a met-

astatic breast tumor model 4T1, as described previously (3).Semiprophylactic protocol A. Mice were immunized i.p.

with c-di-GMP(3mmol/L equals 150 nmol in 50mL) ondays 0, 7,and 14, andwith LM-Mb (107 CFU) i.p. on days 1, 8, and 15, while4T1 tumor cells (105) were injected into the mammary fat padon day 3.

Therapeutic protocol B (high-dose LM-Mb and c-di-GMP). Mice were injected with 4T1 tumor cells (105) in themammary fat pad on day 0, immunized i.p. with c-di-GMP (3mmol/L equals 150 nmol in 50 mL) on days 3, 10, and 17, andwith LM-Mb (107 CFU; i.p.) on days 4, 11, and 18.

Therapeutic protocol C (low-dose LM-Mb and c-di-GMP). 4T1 tumor cells (0.5 � 105) were injected into themammary fat pad on day 0, and c-di-GMP (0.2 mmol/L equals0.01 nmol in 50 mL) was administered every day i.p., starting onday 3, whereas LM-Mb (104 CFU) was administered i.p. on days4, 7, 10, 13, and 16.

Therapeutic protocol D (high- vs. low-dose c-di-GMP).4T1 tumor cells (0.5� 105) were injected into the mammary fatpad on day 0, and one high dose of c-di-GMP (3 mmol/L equals150 nmol in 50 mL) was administered on day 4, followed by i.p.low-dose c-di-GMP (0.01 nmol) every day for 16 days. A detailedschematic view of all immunization protocols is shown inSupplementary Fig. S1A–S1D. All mice were euthanized on day

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19 andanalyzed for thenumber ofmetastases and tumor growth.Primary tumors extend to the chest cavity lining andmetastasespredominantly to the mesenteric lymph nodes, and less fre-quently to the diaphragm, portal liver, spleen, and kidneys (20).

Isolation of MDSCsMonocytic and granulocytic MDSCs (mMDSC and gMDSC,

respectively) were isolated from spleen cell suspensionsaccording the manufacturer's instructions (Myeloid-DerivedSuppressor Cell Isolation Kit; Miltenyi Biotec) as describedpreviously (5). For separation of the magnetically labeled cells,the Automacs Proseparator (Miltenyi Biotech) was used. Asdetermined by flow cytometry, the purity of the isolatedgMDSC was �90% and of mMDSC �85%.

ELISPOT and cytokine ELISASpleen cells were isolated from vaccinated and control mice

with 4T1 tumors for analysis byELISPOTasdescribedpreviously(3). To detect LM-induced T-cell responses, 2� 105 spleen cellsfrom vaccinated or controlmicewere infected with 2� 105 CFUof LM for 1 hour, and subsequently treated with gentamicin(50 mg/mL) until the end of restimulation (72 hours). To detectTAA-specific T-cell responses, 4� 105 spleen cells of vaccinatedor control mice were transfected with pcDNA3.1-Mage-b pluspCMV-GM-CSF using lipofectamin 2000, and cultured for72 hours as described previously (3). Also, Survivin66–74 peptide(GWEPDDNPI) was used for restimulation of the spleencells from c-di-GMP–treated and saline control mice in thepresence or absence of MHC class I antibodies (EBioscience).Briefly, 4 � 105 spleen cells were mixed with or without anti-MHC class I antibodies (1 mg/mL) and then restimulated withSurvivin66–74 peptide (100 mg/mL) for 72 hours. After 24 hours,IL2 (25 U/mL; BD Pharmingen) was added to both spleencultures, to enrich for TAA-specific T cells. At the end of the72 hours, the frequency of IFNg-producing cells was determinedby ELISPOT according to standard protocols (BD Pharmingen)using an ELISPOT reader (CTL Immunospot S4 analyzer; Cel-lular Technology, Ltd). To determine the frequency of IFNg-producingCD8 T cells, spleen cells were depleted for CD8 T cellsusing magnetic bead depletion techniques according to themanufacturer's instructions (Miltenyi Biotech). All antibodieswere purchased from BD Pharmingen.The in vitro production of IL12p40 by gMDSC and mMDSC

was measured by ELISA as described previously (5). For thispurpose, 500,000 gMDSCs or mMDSCs were incubated withdifferent concentrations of c-di-GMP.After 72hours, the levels ofIL12 in the culture supernatant were determined by ELISAaccording to the manufacturer's instructions (BD Pharmingen).

Depletion of CD8 T cells in vivoCD8 T cells were depleted in 4T1 tumor–bearing mice with

400 mg of anti-CD8 antibodies (H35; ref. 29; kindly provided byG. Lauvau,DepartmentofMicrobiology and Immunology, AlbertEinstein College of Medicine, Bronx, NY) during c-di-GMPtreatment (five injections 3days apart). Allmicewere euthanized2 days after the last anti-CD8 treatment, and analyzed for tumorweight and number of metastases. After depletion, the percent-age of CD8 T cells in the spleen was less than 1%. As control,isotype-matched rat antibodies against HRPN were used.

Flow cytometry analysisImmune cells from spleen, blood, lymph nodes, or tumors

of treated and control mice were isolated as describedpreviously (30). To identify MDSCs, anti–CD11b-Alexa488/PerCP-cy5.5 and anti–Gr1-PerCP-cy5.5 (clone RB6-8C5) anti-bodies were used. The CD11bþGr1high population representsthe gMDSC population and the CD11bþGr1low the mMDSCpopulation. To analyze various subsets of immune cells of4T1 tumor–bearing mice involved in antitumor responses inspleens, tumors, and lymph nodes, anti–CD3-Alexa488, anti–CD4-PerCP-cy5.5, anti–CD8-APC, anti–CD49d-PE (NK cells),anti–CD19-FITC (B cells), and anti–CD11c-FITC antibodieswere used. All cell populations in the tumor were analyzedwithin the CD45-positive population using anti–CD45-FITC/APC antibody. For the maturation of MDSCs and DCs in thespleens of tumor-bearing mice, we used anti–CD80-APC,anti–CD86-PE, and anti-MHC class II-PE antibodies. Todetect the production of intracellular cytokines, the cyto-fix/cytoperm Kit from BD Pharmingen was used accordingto the manufacturer's instructions, and antibodies to IFNgand IL12 were used. Appropriate isotype controls were usedfor each sample. Depending on the sample size, 10,000 to50,000 cells were acquired by scanning using a FluorescenceActivated Cell Sorter (flow cytometry; Beckton and Dick-inson; Excalibur), and analyzed using FlowJo 7.6 software.Cell debris and dead cells were excluded from the analysisbased on scatter signals and use of Fixable Blue or GreenLive/Dead Cell Stain Kit (Invitrogen). All antibodies werepurchased from BD Pharmingen or eBiosciences.

MTT test and cell death4T1 cells or HEK293T cells (2,000 cells in 0.1 mL) were

cultured with different doses of c-di-GMP as indicated in thetext for 24 hours, then cell viability was analyzed by the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT)method at a wave length of 570 nm. Cell death in c-di-GMP–treated cultureswas calculated by subtracting percent live cellsfrom nontreated cells (100%).

Caspase-3 analysis4T1 tumor cells were treated with different concentrations

of c-di-GMP as indicated in the text, fixed in 10% neutralbuffered formalin, and permeabilizedwith 0.1%TritonX100 for30 minutes. Endogenous peroxidase activity was quenchedusing 3% hydrogen peroxide for 10minutes, and blocked by 5%normal donkey serum and 2% BSA for 1 hour. The cells werethen stained by IHC methods, using a primary antibody toactive caspase-3 (rabbit anti-mouse IgG; Cell Signaling Tech-nology) 1:50 dilution for 1 hour at room temperature, followedby a secondary antibody conjugated with horseradish perox-idase (HRP) for 1 hour at room temperature (Invitrogen), andthen followed with diaminobenzidine as the final chromogen.All slides were briefly counterstained with hematoxylin.

STING analysisExpression of STING was analyzed by Western blotting.

Tissues were homogenized with 1 mL of PBS containing0.1% Triton-X100, 2 mmol/L EDTA, and protease inhibitorsby using Mini Bead Beater for 2 to 5 � 30 seconds. Cultures of

c-di-GMP Improves Vaccination against Breast Cancer

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4T1 tumor cells andMDSCs were centrifuged and resuspendedin 400 mL of ice-cold lysis buffer (1 mol/L HEPES, 0.5 mol/LEDTA, 0.1 mol/L EGTA, 2 mol/L KCl 0.1 mol/L DTT, cocktail ofprotease inhibitors 5 mg/mL, and 10% NP-40). The lysates (20mg) were resuspended in 10% SDS-polyacrylamide gels, andproteins were electro-transferred to PVDF membranes. Mem-branes were probed with rabbit antibodies against STING (CellSignaling Technology). Antibody to b-actin was used to ensureequal loading. Membranes were incubated with HRP-conju-gated anti-rabbit goat IgG secondary antibodies (Cell SignalingTechnology), and detection was obtained using a chemilumi-nescence detection kit (Thermo Scientific).

Statistical analysisTo statistically analyze the effects of LM-Mb and/or c-di-

GMP on the growth of metastases and tumors and on immuneresponses in the mouse models, the unpaired t test and theMann–Whitney test were used. �, P < 0.05; ��, P < 0.01; ��, P <0.001; and ��, P < 0.0001. Values of P <0.05 were consideredstatistically significant.

ResultsHigh doses of c-di-GMP and LM-Mb completelyeliminated metastatic breast cancer without T-cellcontribution in a semiprophylactic setting

Immune suppression in theTME is amajor problem in cancervaccination (5, 7), and adjuvants that can reduce or convertimmune suppression are greatly needed. c-di-GMP has shownpromising immune stimulatory effects on innate and adaptiveimmune responses in bacterial infections (10). To analyzewhether c-di-GMP could also induce immune stimulatoryeffects on the immune system in cancer vaccination, tumor-bearing mice were immunized with LM-Mb and c-di-GMP insemiprophylactic setting (protocol A). This combination proto-col completely eliminated the metastatic breast cancer (Fig. 1AandB), a result that couldnot be obtainedwith c-di-GMPor LM-Mb alone. Most interestingly, this striking result was not due toimprovedCD8T-cell responses toMage-b andLM. In contrast, itseemed that c-di-GMP did not increase but decreased Mage-b–specific (Fig. 1C) or LM-specific (Fig. 1D)CD8T-cell responses inthe spleen of vaccinated and control mice. Because c-di-GMPalone was highly effective against metastases but did notimprove T-cell responses, we further investigated the mecha-nism of action of c-di-GMP. We also tested LM-Mb and c-di-GMP therapeutically and found that the number of metastaseswas reduced by 73% and tumor growth by 33% compared withthe saline-only group (Supplementary Fig. S2A and S2B).

High doses of c-di-GMP activated caspase-3 in 4T1 tumorcells

Because high doses of c-di-GMP had an effect on themetastases and primary tumor in vivo, we determined whetherc-di-GMP could kill tumor cells directly. We found that 75mmol/L (equals 15 nmol in 200 mL) of c-di-GMP reduced thegrowth of 4T1 tumor cells in vitro by 70% (MTT test; Fig. 2A),and 750 mmol/L (equals 150 nmol in 200 mL) by 92% (Supple-mentary Fig. S3). To analyze the mechanism of tumor cellkilling in more detail, we determined caspase-3 expression at

various doses of c-di-GMP. We found that 37.5 and 75 mmol/L(equals 7.5 and 15 nmol in 200 mL) of c-di-GMP inducedcaspase-3 expression in 40% and 20% of the 4T1 tumor cells,respectively (Fig. 2B and C). Moreover, most of the remainingtumor cells died. Because c-di-GMP acts through interactionwith its sensor STING, we analyzed 4T1 tumor cells for STINGexpression by Western blotting. As shown here, STING isexpressed in 4T1 tumor cells and bone marrow cells (positivecontrol), and even at much higher levels in the 4T1 metastasesand primary tumor, whereas STING could not be detected inHEK293T cells (negative control; Fig. 2D). c-di-GMP had sig-nificantly less effect on the viability and cell death of STING-negative HEK293 cells than of STING-positive 4T1 at doses of37.5 and 75 mmol/L (Supplementary Fig. S4).

Low doses of c-di-GMP improved efficacy of LM-Mb andT-cell responses to Mage-b in a therapeutic setting

On the basis of the results described above, we hypothesizedthat if high doses could kill tumor cells directly, it may be toxicfor T cells as well. Therefore, we tested whether low doses of c-di-GMP were effective against the metastases and couldimprove T-cell responses. In addition, we combined this witha clinically more relevant therapeutic immunization protocol(protocol B). Various lower doses of c-di-GMP (range, 0.5–2mmol/L) were effective against metastases and the primarytumor (Supplementary Fig. S5A and S5B), but when combinedwith LM-Mb, c-di-GMP did not improve T-cell responses toMage-b. Therefore, we further decreased the dose of c-di-GMP(range, 0.2–200 mmol/L) and found that the administration of0.2 mmol/L of c-di-GMP every day for 2 weeks was the mosteffective. Also, LM seemed to be more effective therapeuticallyat a lower dose (104 CFU) once every 3 days than a high dose(107 CFU) once per week (Supplementary Fig. S5C and S5D).On the basis of these results, we administered 0.2 mmol/L ofc-di-GMP daily with 104 CFU of LM-Mb every 3 days in atherapeutic setting (Supplementary Fig. S1C) in all remainingstudies. As shown in Fig. 3A and B, this combinationwas highlyeffective against the metastases but less vigorous against theprimary tumor. The number of metastases in the group of LM-Mb þ c-di-GMP was significantly reduced compared with allother groups, whereas the tumor weight in the group with LM-Mb þ c-di-GMP was significantly reduced compared with thesaline-only group. Finally, we found that therapeutic treatmentof tumor-bearing mice with 0.2 mmol/L of c-di-GMP signifi-cantly improved T-cell responses to LM-Mb in vivo and in vitroafter restimulation with Mage-b (Fig. 3C and D).

c-di-GMP targets MDSCsMDSCs represent a major population in the TME that

strongly suppresses T-cell activation (4–6). Here, we testedwhether low doses of c-di-GMP had an effect on MDSCs. Wefound a significant reduction in the number ofMDSCs (35%) inblood compared with that in the saline group when combinedwith LM-Mb, but separately this effect was less abundant andnot significant (Fig. 4A). Because T-cell responses wereimproved by the administration of low doses of c-di-GMP, weanalyzed the production of IL12 by MDSCs. IL12 is known forstimulating na€�ve and mature T cells (31). Low concentrations

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of c-di-GMP (range, 0.05–10 mmol/L) increased the IL12 secre-tion in vitro by the granulocytic MDSCs (CD11bþGr1high) andevenmore bymonocyticMDSCs (CD11bþGr1low) isolated from4T1 tumor–bearing mice (Fig. 4B and C). Because c-di-GMPinteracts with STING, we also analyzed STING expression. Asshown in Fig. 4D, both mMDSCs and gMDSCs expressedSTING, but the expression was more abundant in mMDSCs.

One high dose followed by multiple low doses ofc-di-GMP is highly effective against metastases in atherapeutic settingOur results show that high doses of c-di-GMP killed tumor

cells directly and that low doses of c-di-GMP activated T-cell

responses in vivo. Here, we combined one high dose of c-di-GMP (3 mmol/L equals 150 nmol in 50 mL) with multipleinjections of low-dose c-di-GMP (0.01 nmol) every day for2 weeks, and found similar efficacy in reducing the numberof metastases, tumor weight (Fig. 5A and B), and tumor size(Supplementary Fig. S6) comparedwith the combination of lowdoses of LM-Mb and c-di-GMP (Fig. 3A and B). Compared withthe saline group, the c-di-GMP–treated mice had significantlyimproved CD8 T-cell responses to Mage-b in vivo (Fig. 5C)and in vitro (Fig. 5D). We then analyzed T-cell responsesto TAAs in more detail and restimulated spleen cells fromc-di-GMP–treated and control mice with immunodominantSurvivin66–74 peptide (GWEPDDNPI), matching the H2-D

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Figure 1. Semiprophylactic immunizations with high doses of c-di-GMP and LM-Mb completely eliminated the metastatic breast cancer withoutimproving T-cell responses. BALB/cmice received one preventive immunization followed by two therapeutic immunizationswith high doses of c-di-GMP andLM-Mb according to immunization protocol A. All mice were euthanized 19 days after the first immunization and analyzed for the number of metastases(A) and tumor weight (B). In the same experiments, Mage-b–specific (C) and LM-specific (D) CD8 T-cell responses were analyzed in the spleen (pooled)by ELISPOT in vitro. The results shown here are the averages of three independent experiments (n ¼ 5 mice per group). Error bars, SEM. In A and B, allgroups were compared with LM-Mb þ c-di-GMP. In C and D, all groups not depleted for CD8 T cells were compared with LM-Mb þ c-di-GMP (�),whereas the CD8 T-cell–depleted groups were compared with the same groups without CD8 depletion (^). Mann–Whitney test. �, P < 0.05; ��, P < 0.01.Values of P < 0.05 were considered statistically significant.






haplotype of BALB/c mice, in the absence or presence of anti-MHC class I antibodies. Survivin is strongly expressed by 4T1tumor cells (32). We found a significant increase in the numberof IFNg-producing CD8 T cells to TAA Survivin compared withthat of the saline group, which was almost completely reducedwhen blocked with anti-MHC class I antibodies (Fig. 5E). Mostimportantly, we demonstrated a significant increase in thegrowth of tumor and metastases in mice that received c-di-GMP plus anti-CD8 antibodies compared with mice thatreceived c-di-GMP alone, but not compared with the salineand isotype control mice (Fig. 5F and G).

DiscussionIn the study presented here, we report the role of STING-

dependent pathways in cancer immunotherapy. For this pur-pose, we used LM-Mb as the vaccine and c-di-GMP as theSTING ligand in a metastatic breast cancer model, 4T1. In thepast, we have shown that LM-based vaccination is highlyeffective in stimulating innate and adaptive immune responsesin tumor-bearing mice when used in a semiprophylacticsetting (one immunization before and two after tumor devel-

opment; ref. 20). However, when used in a therapeutic setting(after tumor development), which is clinically more relevant,LM-based vaccination alonewas not able to overcome immunesuppression in the TME (19, 20), and clearly needs help tobypass this problem. Here, we demonstrate that STING ligandc-di-GMP overcomes immune suppression and stimulatesTAA-specific T cells in tumor-bearing mice.

To analyze the potential pathways of c-di-GMP in cancervaccination, we tested various immunization protocols ofLM-Mb and c-di-GMP in relation to efficacy and Mage-b–specific CD8 T-cell responses in 4T1 tumor–bearing mice.First, we tested LM-Mb and c-di-GMP in a semiprophylacticsetting. The results were spectacular. For the first time, weobtained a complete eradication of the metastatic breastcancer. This was not possible with either LM-Mb or c-di-GMP alone. Despite the complete elimination of the met-astatic breast cancer, we found that c-di-GMP did notstimulate but inhibited Mage-b–specific and LM-specificCD8 T-cell responses. Because c-di-GMP alone was alsohighly effective against metastases and even against primarytumors, we concluded that c-di-GMP itself had an effect onthe tumor cells. Indeed, we found that c-di-GMP strongly

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Figure 2. High doses of c-di-GMP killed 4T1 tumor cells directly and activated caspase-3. 4T1 tumor cells were incubated with various doses of c-di-GMP for24 hours (range, 9.3–75 mmol/L). The viability of the 4T1 tumor cells was assayed byMTT assay (A), or the number of 4T1 tumor cells with caspase-3 activationwas quantified by IHC (B). The result shown here is the average of three independent experiments. The number of caspase-3–positive cells was counted per100 cells. Error bars, SEM. An example of caspase-3 activation is shown by IHC (C). Also, the expression of STING was analyzed in the primary tumor,metastases, and the 4T1 cell line by Western blotting (D). Bone marrow (BM) cells and HEK293 were used as a positive and negative control, respectively.

Chandra et al.





reduced the viability of 4T1 tumor cells and induced acti-vation of caspase-3 in vitro at a dose of 75 mmol/L. Insupport of our results, Karaolis and colleagues found that c-di-GMP had an effect on growth factor–stimulated coloncancer cells in vitro (28). On the basis of these results, wehypothesized that if c-di-GMP is toxic for tumor cells, it mayalso be toxic for T cells and lower doses may stimulate theT cells. Therefore, we analyzed the effect of various lowdoses ofc-di-GMP on vaccine efficacy and on Mage-b–specific CD8T cells. We found that 0.2 mmol/L of c-di-GMP, administereddaily, significantly improvedCD8 T-cell responses toMage-b in

vivo and in vitro. Because c-di-GMP is secreted by various typesof bacteria such as A. xylinum, Pseudomonas aeruginosa, andVibrio cholerae (33), and because c-di-GMP serves as a dangersignal for the early detection of microbes (16), it may not besurprising that extremely low doses can be sensed by theimmune system.

One mechanism of the STING-dependent pathways is theproduction of cytokine IFNb in CD11cþ cells and macro-phages (14). Recently, it has been shown that IFNb isinvolved in the intratumoral accumulation of CD8aþ DCs,which is required for T-cell stimulation (34). DCs highly

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Figure 3. Therapeutic immunizations with low doses of c-di-GMP and LM-Mb almost completely eliminated all metastases and improved T-cell responses invitro and in vivo. BALB/c mice received five therapeutic immunizations with low doses of c-di-GMP every day and LM-Mb every 3 days, according toimmunization protocol C. Nineteen days after tumor challenge, mice were euthanized and analyzed for the number of metastases (A) and tumor weight (B).In the same experiments, CD8 T-cell responses to Mage-b were analyzed in the spleen (pooled) in vitro by ELISPOT (C), or in blood (pooled) in vivo withoutany restimulation by flow cytometry (D). The results shown here are the averages of three independent experiments (n ¼ 5 mice per group). Error bars,SEM. In A andB, all groups were comparedwith LM-Mbþ c-d-GMP. In C, all groups not depleted for CD8 T cells were comparedwith LM-Mbþ c-di-GMP (�),and the CD8 T-cell–depleted groups were compared with the same groups without CD8 depletion (^). In D, all groups were compared with LM-Mb þc-di-GMP (�). Mann–Whitney test. �, P < 0.05; ��, P < 0.01; ��, P < 0.001. Values of P < 0.05 were considered statistically significant.






express STING (14–16). We found that c-di-GMP increasedthe expression levels of maturation markers CD80/CD86 onDCs isolated from spleens of 4T1 tumor–bearing mice,which is important for presentation of TAAs and activationof TAA-specific T cells (Supplementary Fig. S7). In addition,we analyzed the effect of c-di-GMP on various subsets ofimmune cells in 4T1 tumor–bearing mice. The most prom-inent effect was the decrease in the CD4 T-cell populationand increase in the CD8 T-cell population in the lymphnodes due to c-di-GMP treatment in vivo compared with thatof the saline group (Supplementary Fig. S8 and Supplemen-tary Table S1). Because MDSCs are present in large numbersin patients with cancer and in mice with cancer, and becauseMDSCs play an important role in immune suppression, wealso analyzed the effect of c-di-GMP on MDSCs. First, wedemonstrated that STING is highly expressed on MDSCs.Moreover, we found that a low dose of STING ligand c-di-GMP induced the production of IL12 by MDSCs andimproved T-cell responses to TAA Mage-b when combinedwith LM-Mb vaccinations in tumor-bearing mice. BecauseIL12 is known to stimulate na€�ve and mature T cells (31), it ispossible that c-di-GMP in the 4T1 model has activated CD8T cells through the generation of IL12 by MDSCs. We alsofound that c-di-GMP–treated MDSCs could reduce immunesuppression and partly restore CD8 T-cell responses to CD3/CD28 stimulation compared with nontreated MDSCs (Sup-plementary Fig. S9). Interestingly, the combination of c-di-GMP and LM-Mb significantly reduced the MDSC popula-tion in blood, but less so with either c-di-GMP or LM-Mbalone. Reduction in the MDSC population may also contrib-ute to the reduction in immune suppression and conse-quently to reduced growth of tumor and metastases.Because MDSCs and immune suppression are present inalmost all cancers (6), c-di-GMP might be used as anadjuvant against other cancers and with other cancer vac-cines as well.

The exact mechanism of how the MDSC population inblood was decreased by LM-Mb and c-di-GMP immuniza-tions is not yet clear, but various mechanisms are possible.One option is that the combination of LM-Mb and c-di-GMPmay have destroyed the tumor cells in an early stage oftreatment and prevented tumor growth and thus the recruit-ment of MDSCs. Another option could be that because LM-Mb infects MDSCs (5) and c-di-GMP activates T cells, theLM-Mb–infected MDSCs were eliminated by the LM-acti-vated and Mage-b–activated T cells. Therefore, both reducedmigration of MDSCs toward smaller tumors in treatedcompared with nontreated mice, and elimination of LM-Mb–infected MDSCs by c-di-GMP–activated T cells maycontribute to the therapeutic efficacy, but more detailedanalysis is required.

On the basis of our results with high and low doses of c-di-GMP, we hypothesized that one administration of high-dosec-di-GMP could induce immunogenic tumor cell death,resulting in cross-presentation of TAAs from c-di-GMP–killedtumor cells by APCs to the immune system, and that repeatedlow doses of c-di-GMP could activate the TAA-specific T cells.We demonstrate that one high dose followed by multiple lowdoses of c-di-GMP, without LM-Mb vaccination, induced CD8T-cell responses to Mage-b in vivo and in vitro, and that thetreatment was equally effective against metastases comparedwith the combination of c-di-GMP and LM-Mb. We found asignificant increase in CD8 T-cell responses to Survivin inspleen cultures from mice that received one high dosefollowed by multiple low doses of c-di-GMP compared withthat from saline-treated control mice [4T1 tumor cells highlyexpress Survivin (32), and the cultures were restimulated withimmunodominant peptide Survivin66–74]. These CD8 T-cellresponses to Survivin were strongly reduced by blocking theinteraction of the Survivin peptide/MHC class I complex inthe restimulation assay with anti-MHC class I antibodies,indicating that c-di-GMP induced CD8 T-cell responses

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Figure 4. Therapeutic immunizations with low doses of c-di-GMP and LM-Mb reduced the percentage of MDSCs and c-di-GMP induced IL12 production byMDSCs. BALB/c mice were immunized with low doses of c-di-GMP and LM-Mb and challenged with 4T1 tumor cells according to immunization protocolC. Nineteen days after tumor challenge, mice were euthanized and the percentage of MDSCs (CD11bþGr1þ) was determined in blood in vivo by flowcytometry (A). An example of gating MDSC in blood is shown in Supplementary Fig. S10. In addition, gMDSCs (B) and mMDSCs (C) were isolated from 4T1tumor–bearing mice, incubated with various concentrations of c-di-GMP in vitro, and then analyzed for IL12 secretion in the culture supernatant byELISA (range, 0.05–10 mmol/L). Also, the expression of STING was analyzed in MDSCs by Western blotting (D). The results shown here are the averages ofthree independent experiments (n ¼ 5 mice per group). In A, all groups were compared with LM-Mb þ c-di-GMP. Error bars, SEM. Mann–Whitney test.��, P < 0.01. Values of P < 0.05 were considered statistically significant. Also, the expression of STING was analyzed in MDSCs by Western blotting.Bone marrow (BM) cells and HEK293 cells were used as positive and negative controls, respectively.

Chandra et al.





to TAA Survivin in vivo. The most convincing result demon-strating that c-di-GMP reduced growth of tumors and metas-tases through CD8 T-cell responses was shown by CD8 T-celldepletions in vivo. We found significantly larger tumors andmore metastases in 4T1 tumor–bearing mice that received c-di-GMP and anti-CD8 antibodies compared with mice thatreceived c-di-GMP or isotype control alone. In conclusion,these results strongly support our hypothesis that one admin-istration of a high dose of c-di-GMP could induce immuno-genic tumor cell death, resulting in cross-presentation ofTAAs of c-di-GMP–killed tumor cells by APCs to the immunesystem, and that repeated low doses of c-di-GMP couldactivate the TAA-specific T cells.In summary, we have demonstrated that the STING-acti-

vating ligand c-di-GMP improves vaccination or immunother-apy against metastatic breast cancer through multiple path-ways. We believe that results from this novel study provide arationale for the development of new directions in cancerimmunotherapy and in combination with agents capable of

inducing immunogenic tumor cell death such as chemother-apy, radiotherapy, and other therapies.

Disclosure of Potential Conflicts of InterestsD.K.R. Karaolis is the inventor of patents for use of cyclic dinucleotides to

modulate the immune system. No potential conflicts of interest were disclosedby the other authors.

Authors' ContributionsConception and design: D. Chandra, H.O. Sintim, C. GravekampDevelopment of methodology: D. Chandra, H.O. Sintim, C. GravekampAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Chandra, A. Jahangir, D. Asafu-Adjei, I. Ramos,H.O. Sintim, J. Zhou, C. GravekampAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Chandra, W. Quispe-Tintaya, H.O. Sintim,C. GravekampWriting, review, and/or revision of themanuscript:D. Chandra, W. Quispe-Tintaya, H.O. Sintim, C. GravekampAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): W. Quispe-Tintaya, D. Asafu-Adjei,H.O. Sintim, C. GravekampStudy supervision: H.O. Sintim, C. GravekampProvided compounds: Y. HayakawaOther (provided c-di-GMP): H.O. Sintim, J. Zhou

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Restimulation: Survivin66-74peptide Metastases: CD8 T-cell depletion in vivo Tumor: CD8 T-cell depletion in vivo

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Figure 5. One therapeutic high dose followed bymultiple low doses of c-di-GMP almost completely eliminated themetastases and improved T-cell responsesto TAAs. BALB/c mice received one high dose (3 mmol/L) of c-di-GMP followed by multiple low doses (0.2 mmol/L) of c-di-GMP every day according toimmunization protocol D. Nineteen days after tumor challenge, mice were euthanized and analyzed for the number of metastases (A) and tumor weight (B).In the same experiments, CD8 T-cell responses to Mage-b were analyzed in the spleen (pooled) in vitro by ELISPOT (C), or in blood (pooled) in vivowithout any restimulationby flowcytometry (D). The results shownhere are the averages of three independent experiments. In each experiment,n¼5mice pergroup. Unpaired t test. �, P < 0.05; ��, P < 0.01; ��, P < 0.001. Values of P < 0.05 were considered statistically significant. Spleen cells of c-di-GMP–treatedand saline control mice were restimulated with Survivin66–74 peptide (GWEPDDNPI) in the absence or presence of anti-MHC class I antibodies (E).Tumor-bearing mice were treated with c-di-GMP plus anti-CD8 antibodies (H35), and the number of metastases (F) and tumor weight (G) were comparedwith treatments with c-di-GMP alone, saline, or isotype controls. In this experiment, n ¼ 7 mice per group. Error bars in all graphs, SEM. Mann–Whitneytest. �, P < 0.05; ��, P < 0.01; ��, P < 0.001. Values of P < 0.05 were considered statistically significant. Error bars in all graphs, SEM.






Grant SupportThis work was supported by NIH grant (1RO1 AG023096-01), NCI grant (R21

AI090652-01), The Paul F Glenn Center for the Biology of Human Aging Research34118A, and National Science Foundation (NSF) 1307218 (to H.O. Sintim).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received August 13, 2013; revised April 28, 2014; accepted May 13, 2014;published OnlineFirst June 9, 2014.

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Chandra et al.




2014;2:901-910. Published OnlineFirst June 9, 2014.Cancer Immunol Res Dinesh Chandra, Wilber Quispe-Tintaya, Arthee Jahangir, et al. Metastatic Breast CancerSTING Ligand c-di-GMP Improves Cancer Vaccination against

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