Inhibition of Fatty Acid Oxidation Modulates ......Research Article Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and

Research Article

Inhibition of Fatty Acid Oxidation ModulatesImmunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances CancerTherapiesFokhrul Hossain1,2, Amir A. Al-Khami1, Dorota Wyczechowska1, Claudia Hernandez1,Liqin Zheng1, Krzystoff Reiss1,3, Luis Del Valle1,4, Jimena Trillo-Tinoco1, Tomasz Maj5,Weiping Zou5, Paulo C. Rodriguez1,6, and Augusto C. Ochoa1,7

Abstract

Myeloid-derived suppressor cells (MDSC) promote tumorgrowth by inhibiting T-cell immunity and promoting malignantcell proliferation and migration. The therapeutic potential ofblocking MDSC in tumors has been limited by their heteroge-neity, plasticity, and resistance to various chemotherapy agents.Recent studies have highlighted the role of energy metabolicpathways in the differentiation and function of immune cells;however, the metabolic characteristics regulating MDSC remainunclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosup-pressive function, and test whether its inhibition blocks MDSCand enhances antitumor therapies. Using several murine tumormodels, we found that tumor-infiltrating MDSC (T-MDSC)increased fatty acid uptake and activated fatty acid oxidation(FAO). This was accompanied by an increased mitochondrial

mass, upregulation of key FAO enzymes, and increased oxygenconsumption rate. Pharmacologic inhibition of FAO blockedimmune inhibitory pathways and functions in T-MDSCand decreased their production of inhibitory cytokines. FAOinhibition alone significantly delayed tumor growth in aT-cell–dependent manner and enhanced the antitumor effectof adoptive T-cell therapy. Furthermore, FAO inhibition com-bined with low-dose chemotherapy completely inhibitedT-MDSC immunosuppressive effects and induced a significantantitumor effect. Interestingly, a similar increase in fatty aciduptake and expression of FAO-related enzymes was found inhuman MDSC in peripheral blood and tumors. These resultssupport the possibility of testing FAO inhibition as a novelapproach to block MDSC and enhance various cancer therapies.Cancer Immunol Res; 3(11); 1236–47. �2015 AACR.

IntroductionStromal cells in the tumor microenvironment promote tumor

growth and metastatic spread, limit the antitumor responseto immunotherapy, and protect tumors from the effect of che-motherapy and radiotherapy (1–3). Prominent in the tumor

microenvironment are tumor-infiltrating myeloid-derived sup-pressor cells (T-MDSC) that, in addition to blocking T-cell func-tion and protecting tumors from the effect of chemotherapy andradiotherapy, support the expansion of regulatory T cells (Treg;refs. 4, 5), further enhancing this highly immunosuppressivemicroenvironment. T-MDSC use several mechanisms to blockT-cell function, including the depletion of L-arginine by arginase I,the induction of T-cell apoptosis by nitric oxide (NO), and thesynthesis of peroxynitrite (PNT; refs. 6–8). The plasticity ofMDSCand the redundancy of thesemechanisms have been shownby thefact that blocking one specific immunosuppressive mechanisminduces the upregulation of the remaining pathways and onlyresults in a partial recovery of T-cell function. Thus, therapiesaimed at inhibitingMDSChavebeen limited tomyelosuppressivechemotherapeutic agents (gemcitabine and 5-fluoruracil) andmulti-tyrosine kinase inhibitors (sunitinib; refs. 9, 10). Therefore,better approaches to inhibit MDSC and enhance cancer therapies,in particular cancer immunotherapy, are needed.

The last decade has seen major progress in understanding theenergy metabolic pathways used by different immune cell sub-populations (11–14). Effector T cells are highly glycolytic, where-as Tregs and memory T cells use fatty acid oxidation (FAO;refs. 15–17). Similarly, M1 macrophages and granulocytes pref-erentially use glycolysis (18), whereas M2 macrophages rely onFAO (11, 19, 20). In the present study, we aimed to characterizethe energy metabolic pathway(s) used by T-MDSC, establish its

1Stanley S. Scott Cancer Center, Louisiana State University HealthSciences Center, New Orleans, Louisiana. 2Department of Biochem-istry and Molecular Biology, Louisiana State University HealthSciences Center, New Orleans, Louisiana. 3Department of InternalMedicine, Louisiana State University Health Sciences Center, NewOrleans, Louisiana. 4Department of Pathology, Louisiana State Uni-versity Health Sciences Center, New Orleans, Louisiana. 5Departmentof Surgery, University of Michigan, Ann Arbor, Michigan. 6Departmentof Microbiology, Immunology and Parasitology, Louisiana State Uni-versity Health Sciences Center, New Orleans, Louisiana. 7Departmentof Pediatrics, Louisiana State University Health Sciences Center, NewOrleans, Louisiana.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

F. Hossain and A.A. Al-Khami contributed equally to this article.

Corresponding Author: Augusto C. Ochoa, Louisiana State University HealthSciences Center, 1700 Tulane Avenue, Room910, NewOrleans, LA 70112. Phone:504-210-2828; Fax: 504-210-2970; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-15-0036

�2015 American Association for Cancer Research.

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impact on the immunosuppressivemechanisms, and test whetherinhibition of this pathway would block MDSC and enhanceantitumor therapies. The results showed that, upon infiltratingthe tumor, MDSC increased the incorporation of fatty acids andactivated FAO. This was accompanied by an increased mitochon-drial biogenesis, upregulation of key FAO enzymes, and increasedoxygen consumption rate (OCR). Inhibition of FAO blockedthe tolerogenic function and immunosuppressive mechanismsof T-MDSC and resulted in a T-cell–dependent inhibition oftumor growth. More importantly, FAO inhibition enhanced theantitumor effect of low-dose chemotherapy and adoptive cellulartherapy (ACT). Therefore, targeting FAO represents a novelapproach to globally inhibiting the function of T-MDSC andenhancing the antitumor effect of various cancer therapies.

Materials and MethodsHuman peripheral blood cells

Samples were obtained from consented patients and donorsunder approved Institutional Review Board protocols. Peripheralbloodmononuclear cellswere separated onFicoll-Paque Plus (GEHealthcare Life Sciences). MDSC (CD14neg CD33þ HLA-DRneg

CD66bþ) were sorted using a BD FACSAria (BD Biosciences).Polymorphonuclear granulocytes (PMN) were isolated by sus-pension over 3% dextran, as described (21).

Mouse strains, cell lines, and therapeutic modelsC57BL/6 mice (8–10-week-old females) were purchased

from Harlan laboratories, and OT-1 T cell antigen receptor(TCR) transgenic mice were from The Jackson Laboratory. Lewislung carcinoma (3LL) and MCA-38 colon adenocarcinoma(American Type Culture Collection) were cultured in RPMI1640 (Lonza-Biowhittaker) supplemented with 10% FBS(Hyclone), 25 mmol/L HEPES, 4 mmol/L L-glutamine, and100 U/mL penicillin, streptomycin (all from Life Technolo-gies). 3LL cells expressing ovalbumin (3LL-OVA) were gener-ated, as previously described (22). 3LL and MCA-38 cells wereperiodically tested and validated to be Mycoplasma free.

In vivo tumormodelswere generated by injectingC57BL/6mices.c. with 1� 106 3LL cells or 2.5� 105 MCA-38 cells, followed bydaily i.p. injections of 50mg/kg of the FAO inhibitors etomoxir orranolazine (Sigma-Aldrich). For depletion of T-cell subsets, micewere injected i.p. with 500 mg/mouse anti-CD4 (GK1.5) or anti-CD8 (2.43) (BioXCell) 1 day before and 2 days after tumorinjection, followed by injection of 250 mg/mouse every 5 daysthroughout the experiment. To test the synergy between FAOinhibition and low-dose chemotherapy, 3LL or MCA-38 tumor–bearing mice were treated daily for 6 days with etomoxir, startingon day 1 after tumor injection, followed by a single i.p. dose ofcyclophosphamide (CTX; Sigma-Aldrich) at 200 mg/kg on day 7.To test the effect on established tumors, tumor cells were allowedto grow for 5 days, followed by etomoxir injections on days 6through 10 (5 days) and a single injection of CTX (200mg/kg) onday 11. To determine the effect of FAO inhibition on ACT, micewere injected s.c. with 1 � 106 3LL-OVA cells and treated withetomoxir daily throughout the experiment. OT-1 T cells (2.5 �106) were adoptively transferred on day 14, and mice were vacci-nated with 100 mg/mouse SIINFEKL peptide (American PeptideCompany) on day 15 after tumor injection. Ten days after thetransfer, spleenswere recovered and challengedwith SIINFEKL for24hours, afterwhich theyweremonitored for IFNg production by

Elispot (R & D systems). Activated OT-1 T cells were generated bystimulating splenocytes from OT-1 TCR transgenic mice in com-plete media with SIINFEKL peptide (1 mg/mL) and IL2 (100U/mL) for 3 days. Tumor volume was measured using calipersand calculated using the formula: [(small diameter)2 � (largediameter) � 0.5]. Experiments using animals were approved bythe LSU-Institutional animal care and usage committee.

Isolation of T cells and MDSCCD3þ T cells were isolated from spleens of C57BL/6mice using

aT-cell–negative isolationkit (Life Technologies). Purity exceeded95%. For T-MDSC, tumors were digested with DNAse and Lib-erase (Roche USA) at 37�C for 1 hour, and T-MDSC were isolatedfrom tumor single-cell suspensions, as described (23). Similarly,splenic MDSC were isolated from spleens of tumor-bearing mice,and normal myeloid cells (nMC) were isolated from spleens ofcontrol mice. The purity of different MDSC preparations rangedfrom90% to 99%. Ly6Cþ and Ly6GþMDSC subsets were isolatedby flow cytometric cell sorting. For the generation of bonemarrow–derived MDSC (BM-MDSC), bone marrow cells wereharvested from femurs and tibias of control mice and culturedwith G-CSF (100 ng/mL), GM-CSF (20 ng/mL), and IL13 (80 ng/mL), as previously described (24). Cytokines were purchasedfrom R&D Systems. When indicated, etomoxir (100 mmol/L) wasadded on day 2 of culture.

MDSC suppression of T cellsCD3þ T cells were labeled with 1 mmol/L carboxyfluorescein

diacetate succinimidyl ester (CFSE; Molecular Probes; Life Tech-nologies) andwere coculturedwithBM-MDSCor T-MDSCat a 4:1T-cell:MDSC ratio, in the presence of plate-bound anti-CD3 (145-2C11) and anti-CD28 (37.51; 1 mg/mL each; BD Biosciences).T-cell proliferationwasmeasured after 72 hours byCFSE dilution.When indicated, IFNg production was assessed by ELISA(Biolegend).

Flow cytometryAnti-human antibodies used to characterize cell subpopula-

tions were: anti-CD33 (VM53), anti-HLA-DR (G46-6), anti-CD66b (80H3; Beckman Coulter), and anti-CD14 (61D3;eBioscience). Mouse antibodies specific for CD11b (M1/70), Gr1(RB6-8C5), Ly6C (AL-21), Ly6G (1A8), CD8 (53-6.7), andCD45.1 (A20) were obtained from BD Biosciences. A Live/Deadstain kit was fromMolecular Probes (Life Technologies). AGalliosflow cytometer (Beckman Coulter) was used for flow cytometryacquisition. Samples were analyzed with FlowJo software(TreeStar).

Extracellular flux analysisOCR and extracellular acidification rate (ECAR)weremeasured

using XF-24 and XFe-24 Extracellular Flux Analyzers, respectively(Seahorse Bioscience) following the manufacturer's instructions.OCR was measured in XF media containing 11 mmol/L glucoseand 1 mmol/L sodium pyruvate under basal conditions andin response to 1 mmol/L oligomycin, 1 mmol/L carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), and 0.1 mmol/Lrotenone plus 0.1 mmol/L antimycin A. ECAR was measured inXF media containing 2 mmol/L L-glutamine under basal condi-tions and in response to 10 mmol/L glucose, 1 mmol/L oligomy-cin, and 100 mmol/L 2-Deoxy-D-glucose (2DG).

Inhibition of Fatty Acid Oxidation Blocks MDSC Function

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Glucose and fatty acid uptake assaysGlucose uptake was measured using a flow cytometry–based

assay, in which single-cell suspensions were incubated with100 mmol/L fluorescent 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2NBDG) for 2 hours, followed bystaining with different cell surfacemarkers (16). Fatty acid uptakein MDSC was determined by a fluorometric fatty acid uptake kit(Abcam). Cells were serum-deprived for 1 hour at 37�C andincubated with fatty acidmixture for 30minutes; the fluorescencesignal was measured in a microplate reader.

Measurement of mitochondrial mass and production ofsuperoxide, reactive oxygen species, nitrite, PNT, and ATP

Staining with Mitotracker green was used to determine mito-chondrial mass, Mitosox red to determine mitochondrial super-oxide, and 20,70-Dichlorofluorescein Diacetate (DCFDA) to deter-mine cellular reactive oxygen species (ROS;Molecular Probes; LifeTechnologies). Nitrite production was evaluated using standardGriess reagent (Molecular Probes; Life Technologies). PNT pro-duction was assessed by quantifying nitrotyrosine residues fromcell lysates by ELISA (Millipore). The levels of ATPwere quantifiedusing a bioluminescence assay kit (Molecular Probes; LifeTechnologies).

Real-time PCRTotal RNA was isolated using the RNAeasy Mini Kit (Qiagen).

cDNA was generated using the iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed for carnitine palmitoyltrans-ferase 1 (CPT1), Acyl CoA dehydrogenase (ACADM), peroxisomeproliferator-activated receptor gamma coactivator 1-beta(PGC1b), and3-hydroxyacyl-CoAdehydrogenase (HADHA)withTaqman primers from Applied Biosystems. Gene expression wascalculated relative to 18s rRNA using 2–DDCT method.

Western blotWestern blot analysis was performed using standard protocols.

Proteins were electrophoresed in 8% TrisGlycine gels, transferredto polyvinylidene difluoride membranes, and immunoblottedwith antibodies against arginase I (19; BD Biosciences) andb-actin (AC-74; Sigma).

Bio-Plex cytokine assays and CPT1 enzyme activityCytokines and chemokines were assessed in cell lysates using a

Bio-Plex immunoassay (Bio-Rad Laboratories). CPT1 activity wasmeasured in cell lysates based on the release of CoA-SH frompalmitoyl CoA using thiol reagent 5,5-dithio-bis 2-nitrobenzoicacid (DTNB; Sigma; ref. 25).

Cancer stem cell sphere assay and biomarkersMouse cancer cells (3LL and MC38) were cultured for 7 days

in ultra-low attachment polystyrene 6-well plates (Corning;#3471). The cells from nearly confluent regular cell culturewere washed twice in PBS and resuspended in X-Vivo 20serum-free medium (Lonza; #04-448Q). The cells were seededin 3 mL of X-Vivo 20 medium (2.0 � 103 MC38 cells or 1 �103 3LL cells) with or without etomoxir (ET) (100 mmol/L).After 7 days, spheres were counted with phase contrast micro-scope, with exclusion of spheres below 50 mm (which can becell aggregates).

RT-PCRSpheres were collected in 5 mL tubes, centrifuged, and resus-

pended in 4 mL of 0.25% Trypsin, 2.21 mmol/L EDTA in HanksBalanced Salt Solution (Cellgro; #25-053-Cl). After 30-minuteincubation at 37�C, cells were centrifuged and resuspended inTrizol reagent (Life Technologies; #15596-026). Samples wereprocessed according to the manufacturer's instructions. Reversetranscriptionwas performedwith ClonedAMVFirst-Strand cDNASynthesis Kit (Life Technologies; #12328-040). qPCR was per-formed with Fast SYBR Green Master Mix (Life Technologies;#4385612), using the following primers: Mouse pouf51 (OCT3/4), forward primer 50-GGAGGGATGGCATACTGTGG-30 and re-verse primer 50-ACCTTTCCAAAGAGAACGCC-30; Mouse nanog,forward primer 50-TCGAATTCTGGGAACGCCTC-30 and reverseprimer 50-CAGGTCTTAACCTGCTTATAGCTCA-30; Mouse Sox2,forward primer 50-GGAGGAGAGCGCCTGTTTTT-30 and reverseprimer 50-CTGGCGGAGAATAGT-TGGGG-30; mouse GAPDH,forward primer 50-ATGACATCAAGAAGGTGGTG-30 and reverseprimer 50-CATACCAGGAAATGAGCTTG-30.

Human biopsies and immunohistochemistryA total of 23 archival biopsy samples from patients with colon

adenocarcinoma, clear cell kidney carcinoma, and breast ductalcarcinoma were obtained from the Louisiana Cancer ResearchCenter (LCRC) tissue biorepository. All samples were de-identi-fied. Immunohistochemistry was performed using avidin–bio-tin–peroxidase (Vector Laboratories). Briefly, our protocolincludes deparaffinization in xylene, re-hydration through des-cending grades of alcohol up to water, nonenzymatic antigenretrieval in Citrate buffer, pH 6.0 for 30 minutes at 95�C, andendogenous peroxidase quenchingwithH2O2 inmethanol for 20minutes. After PBS wash, samples were blocked with 5% normalgoat serum in 0.1%PBS/BSA. Primary antibodies includedmousemonoclonal antibodies againstHADHA(1:500dilution; Abcam),CD66b (1:100 dilution; LS-B7134; Lifespan Biosciences), CPT1(1:200 dilution; 8F6AE9; Abcam), and HLA-DR (1:100 dilution;L243; Santa Cruz; Biotechnology). After PBS washing, sectionswere incubated with a biotinylated anti-rabbit secondary IgG for20 minutes, incubated with avidin–biotin–peroxidase (ABC)complexes, and developed with diaminobenzidine (Sigma). Allsections were counterstained with hematoxylin, dehydrated inalcohol, cleared in xylene, and mounted with Permount (Fisher).

Multi-labeling and confocal microscopyFor immunofluorescence, double labeling of paraffin-embed-

ded sections, deparaffinization, antigen retrieval, and blockingwere performed as described above (endogenous peroxidase wasomitted). Sections were then incubated with a first primary anti-body overnight at room temperature and rinsed in PBS. A fluores-cein-conjugated secondary antibody was incubated for 1 hour inthe dark. After washing with PBS, a second primary antibody wasincubated overnight in the dark, followed by rinsing with PBS andincubation with a second rhodamine-tagged secondary antibodyfor 1 hour. Sections were washed in PBS, mounted in aqueousmounting media with DAPI (Vector Laboratories), and visualizedin a confocal microscope (Olympus FV1000).

Statistical analysisData were analyzed by either Student t test or one-way ANOVA

followed by Tukey posttest using Graph-Pad Prism analysis

Hossain et al.

Cancer Immunol Res; 3(11) November 2015 Cancer Immunology Research1238




software. Results were expressed as mean� SEM, and P values arepresented in the figures as �,P <0.05; ��,P < 0.01 or ��,P < 0.001).

ResultsTumor microenvironment induces fatty acid oxidation inT-MDSC

The harsh tumor microenvironment created in part by theincreased metabolic rate of tumor cells can result in a significantmetabolic stress on other cells including T-MDSC. We aimed todetermine the metabolic characteristics of T-MDSC and establishwhether they could regulate the immunosuppressive functions ofthese cells. To identify the metabolic characteristics of T-MDSC, asingle-cell suspension of s.c. 3LL tumor was simultaneouslylabeled with the fluorescent glucose analogue 2NBDG, andcell-surface markers for MDSC, defined as CD11bþ Gr1þ

CD11c� F480�. Control cells included nMCs from spleens of

na€�ve mice and MDSC from spleens of tumor-bearing mice(splenic MDSC; Fig. 1A; Supplementary Fig. S1A). The nonmye-loid fraction containing mostly tumor cells had a significantlyincreased 2NBDG uptake (Fig. 1A, last panel; SupplementaryFig. S1A), whereas that in nMCs, splenic MDSC, and T-MDSCwas all lower. Instead T-MDSC (but not nMCs or splenic MDSC)had a high fatty acid uptake, which was similar in the Ly6Cþ

monocytic MDSC (M-MDSC) and the Ly6Gþ granulocytic MDSC(G-MDSC) subpopulations (Fig. 1B). T-MDSC had an increasedmetabolic activity overall as shown by the increased OCR andECAR, reflecting an increased FAO and glycolysis (Fig. 1C). How-ever, the ratio of OCR/ECAR clearly demonstrated a preferentialincrease in OCR, which confirmed the metabolic reprogramingtoward fatty acid oxidation (Fig 1D). In addition, the increasedOCR in T-MDSC was accompanied by an increase in mitochon-drial biogenesis as shown by higher staining with Mitotrackerand enhanced production of superoxide (Mitosox) and ROS

Figure 1.T-MDSC increase fatty acid uptake, activateFAO, and increasemitochondrial biogenesis and function. A, 2NBDG incorporationwas tested inCD11bþGr1þ splenocytesfrom control normal mice (nMC) and from 3LL-bearing mice (splenic MDSC), and 3LL tumor single-cell suspensions (T-MDSC). B, fatty acid uptakewas measured in CD11bþ, Ly6Cþ, and Ly6Gþ populations sorted from control spleens, spleens from 3LL-bearing mice, and 3LL tumor single-cell suspensions.C, OCR and ECAR were measured under basal conditions and after addition of the indicated drugs. D, OCR/ECAR ratios. E, mitochondrial biogenesis andfunction were evaluated by assessing mitochondrial mass (Mitotracker), mitochondrial superoxide (Mitosox), and cellular ROS (DCFDA). F, quantitative RT-PCRanalysis of CPT1, ACADM, PGC1b, and HADHA expression. G–I, immunosuppressive mechanisms were determined by (G) Western blot for arginaseI, (H) NO production as measured by Griess reagent, and (I) PNT production as measured by nitrotyrosine ELISA. J, immunosuppressive function as shown bythe ability of MDSC to inhibit the proliferation of T cells stimulated with anti-CD3/CD28. Data, mean � SEM and representative of at least three independentexperiments. �� , P < 0.001.






(DCFDA; Fig. 1E; Supplementary Fig. S1B–S1D). Moreover, T-MDSC displayed a significantly elevated expression of genesassociated with FAO, including CPT1, ACADM, PGC1b, andHADHA (Fig. 1F).

Themetabolic reprograming was paralleled by an upregulationof arginase I, elevated production of NO and PNT (Fig. 1G–I;Supplementary Fig. S1E), and increased ability to inhibit T-cellproliferation (Fig. 1J). In addition, T-MDSC produced higherlevels of G-CSF, GM-CSF, IL1b, IL6, and IL10, cytokines knownto promote and sustain MDSC development; however, they alsoproduced higher levels of IL12 (Supplementary Fig. S1F).

Effect of FAO inhibition on MDSC functionWe then explored the effect of FAO inhibition on the induction

and function of MDSC using BM-MDSC from normal C57BL/6mice. Bone marrow precursors were activated with G-CSF, GM-CSF, and IL13 and incubated with or without etomoxir, a specificinhibitor of CPT1, which is the first and rate-limiting enzyme inthe FAO cycle. Incubation of BM-MDSC with etomoxir loweredCPT1 enzymatic activity (Supplementary Fig. S2A), but did notalter the proportion of G-MDSC or M-MDSC, and did not induceapoptosis or block proliferation of BM-MDSC (SupplementaryFig. S2B–S2D). Etomoxir, however, decreased the basal andmaximal OCR in BM-MDSC (Fig. 2A), diminished fatty aciduptake (Fig. 2B), and decreased ATP levels by approximately40% to 50% (Fig. 2C; Supplementary Fig. S2E). Furthermore,etomoxir-treated BM-MDSC had a significantly decreased abilityto block T-cell proliferation (Fig. 2D) and had a lower expression

and activity of arginase I (Fig. 2E and F). NO levels were notdetected in BM-MDSC generated in vitro, and PNT levels remainedunchanged (data not shown).

We next tested the effect of FAO inhibition in vivo on theaccumulation, metabolism, and function of T-MDSC. The initialexperiments tested the effect of daily i.p. injections of etomoxir(50mg/kg) intoC57BL/6micebearing s.c. 3LL tumors, starting onday 1 after tumor injection andup today 20, atwhich time tumorswere harvested. Mice did not show any overt toxicity at this dose.Etomoxir treatment decreased the enzymatic activity of CPT1 inT-MDSC in vivo (Supplementary Fig. S3A), but did not alter thepercentage of total T-MDSC or that of G-MDSC and M-MDSCsubsets infiltrating the 3LL tumors (Fig. 3A) or significantlyincrease their apoptosis. Similar data were found in MCA-38colon carcinoma (data not shown). However, treatment withetomoxir significantly inhibited fatty acid uptake (Fig. 3B) andATPproduction (Fig. 3C) anddecreasedOCRandECAR (Fig. 3D).This suggested that inhibition of FAO decreased the overallmetabolic activity of T-MDSC and that T-MDSC appeared to beunable to compensate by increasing glycolytic stress response.More importantly, FAO inhibition in vivo decreased the immu-nosuppressive function of T-MDSC, as demonstrated by theirinability to block T-cell proliferation and IFNg production(Fig. 3E). This correlated with a lower expression and productionof arginase I, ROS, NO, and PNT (Fig. 3F–I; Supplementary Fig.S3B). In addition, T-MDSC from etomoxir-treatedmice producedsignificantly lower levels of cytokines critical to the induction anddifferentiation of MDSC, such as G-CSF, GM-CSF, IL6, and IL10

Figure 2.FAO inhibition impairs the function of BM-MDSC. BM-MDSC were generated in vitro, as described in Materials and Methods, in the absence or presence ofetomoxir (100 mmol/L). A, OCR was measured under basal conditions and after the addition of the indicated mitochondrial regulators. Four days after culture,fatty acid uptake (B), ATP levels (C), immunosuppressive function of BM-MDSC (D), arginase I expression (E), and arginase I activity (F) were assessed.Data, mean � SEM and representative of at least three independent experiments. �� , P < 0.01; �� , P < 0.001.

Hossain et al.





(Fig. 3J). In contrast, the levels of IL1b and IL12 remainedunchanged. Treatment with etomoxir also diminished the accu-mulation of CD4þ FoxP3þ Tregs in the spleens of mice (Supple-mentary Fig. S3C); minimal numbers of Tregs were found infil-trating 3LL tumors. Cumulatively, these results suggest that inhi-biting FAO blocks the immunosuppressive mechanisms andfunction of T-MDSC. Genetic confirmation of the effect of CPT1inhibition could not be done at present because CPT1 knockoutmice are embryonic lethal and conditional CPT1 knockouts arenot yet available for testing.

Antitumor effect of FAO inhibitionFAO inhibitors are used to treat severe coronary disease. There-

fore, we tested the effect of FAO inhibitors, administered indifferent regimens in mice bearing s.c. 3LL lung carcinoma orMCA-38 colon carcinoma. Mice that started etomoxir treatmentalone 1 day after tumor implantation and up to day 20 had asignificant delay in tumor growth, compared with controls(Fig. 4A and B). Histologic studies showed that tumors fromcontrol mice had areas of necrosis, whereas tumors from eto-moxir-treated mice had minimal necrosis, but were infiltrated bymononuclear cells (Fig. 4C, top). Tunel assay showed that tumorsfrom etomoxir-treated mice had a higher number of cells under-going apoptosis (Fig. 4C, middle). Immunohistochemical stain-ing showed no changes in CD31þ endothelial cells or in thenumber of blood vessels, suggesting that etomoxir did not affectangiogenesis (Fig. 4C, bottom). We also tested the effect of asecond FAO inhibitor, ranolazine, that specifically blocks thetrifunctional enzymeHADHA, which catalyzes the last three steps

of the FAO cycle (26). Mice treated with ranolazine had a similarantitumor effect as did etomoxir (Fig. 4D). Clonogenic assaysshowed that FAO inhibition with etomoxir or ranolazine did notinhibit the in vitro growth of 3LL orMCA-38 cells (SupplementaryFig. S4A); etomoxir treatment did not change the number ofcancer stem cells (stem cell spheres) or the expression of cancerstem cells markers Sox2, Nanog, or Oct3/4 (Supplementary Figs.S3D and S4B).However, the depletion of CD4þ orCD8þ T cells inetomoxir-treated mice virtually abrogated the antitumor effect ofetomoxir (Fig. 4E; Supplementary Fig. S5A), suggesting that theantitumor effect was at least in part mediated by T cells. We alsotested the possibility that etomoxir would augment T-cell func-tion; however, in vitro cultures of T cells with etomoxir failed toshow changes in proliferation, cytokine production, or cytotoxicfunction (or cytotoxic proteins; Supplementary Fig. S5B–S5D).

Effects of FAO inhibition plus low-dose chemotherapy or ACTT-MDSC have been shown to decrease the antitumor effects of

chemotherapy and radiotherapy (27) and impair the therapeuticeffect of various forms of immunotherapy (1). Therefore, wetested whether FAO inhibition modulated the antitumor effectsof low-dose chemotherapy or ACT. In the initial model, etomoxirwas given only for 6 days after tumor implantation, followed by asingle dose of CTX (200 mg/kg) on day 7. Results showed anincreased antitumor effect on both 3LL and MCA-38 tumor–bearing mice treated with FAO inhibitors plus chemotherapy(Fig. 5A and B). This antitumor effect was also seen when treatingmice with "established" 3LL tumors (6–7 days after tumorimplantation), in which etomoxir was started on day 6 after

Figure 3.FAO inhibition in vivo decreases fatty acid uptake, ATP production, and immunosuppressive mechanisms in T-MDSC. C57BL/6 mice bearing s.c. 3LL tumors weretreated i.p. with etomoxir (50 mg/kg) or PBS for 20 days starting 1 day after tumor injection, and tumors were harvested on day 21. A, tumor single-cellsuspensions were stained for total T-MDSC (CD11bþ Gr1þ), G-MDSC (CD11bþ Ly6Gþ Ly6CInt), and M-MDSC (CD11bþ Ly6Glo Ly6Chi). Sorted CD11bþ GR1þ cellswere tested for fatty acid uptake (B), ATP levels (C), and OCR and ECAR (D). E, immunosuppressive function of T-MDSC was tested by their ability tosuppress T-cell proliferation (left) and IFNg production (right). Expression of arginase I (F) and production of ROS (G), NO (H), and PNT (I) were measuredin T-MDSC. J, cytokines were measured in T-MDSC lysates using a Bio-Plex immunoassay. Data, mean � SEM and representative of at least three independentexperiments. P values: � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






tumor inoculation and given for 5 days (until day 10), followedby a single dose of CTX (Fig. 5C). A similar effect was seen withranolazine (Fig. 5D).

Furthermore, the combination of etomoxir and ACT, usingOT-1 T cells to treat OVA-expressing 3LL tumors, resulted in asignificantly better antitumor effect (Fig. 5E). The increased effi-cacy of T-cell immunotherapy in etomoxir-treatedmice correlatedwith a higher number of adoptively transferred OT-1 T cells(CD45.1þ) infiltrating the tumors and increased number of cellsproducing IFNg (Fig. 5F and G).

Increased fatty acid uptake and expression ofCPT1 andHADHAin peripheral blood and T-MDSC from cancer patients

Peripheral blood MDSC from 23 patients with cancer (breast,renal cell carcinoma, bladder cancer, and colon cancer) wereisolated and tested for fatty acid uptake. PMNs from normaldonors and from the same patients were used as controls. Inaccordance with previous reports (21, 28), G-MDSC (CD14neg

CD33þ HLA-DRneg CD66bþ) were increased in the peripheralblood of these patients (Fig. 6A and B). Similar to murine T-MDSC, human G-MDSC had an increased fatty acid uptake (Fig.6C), and although M-MDSC also incorporated fatty acids, they

represented <0.9% of the total circulating MDSC (data notshown). We then examined the expression of CPT-1 and HADHAin biopsies from patients with colon adenocarcinoma, renal cellcarcinoma, and breast ductal carcinoma. Immunohistochemistryfor CPT-1 demonstrated its presence with a punctate cytoplasmicpattern (consistent with its expression in the mitochondria) intumor cells and amarkedly increased expression shown as a solidpattern in inflammatory cells (Fig. 6D, left). Double labeling withanti-CPT1 (fluorescein) and anti-CD66b (rhodamine) showedthat the CD66bþ T-MDSC expressed significantly higher levels ofCPT1, compared with tumor cells (Fig. 6D, right "merge" plots).Similarly, immunohistochemistry for HADHA showed theexpression of the enzyme in both tumor cells and inflammatorycells in the stroma (Fig. 6E, left). Confocal microscopy followingdouble labeling with anti-HADHA (fluorescein) and anti-CD66b(rhodamine) showed that all CD66bþMDSCwere alsoHADHAþ

(Fig. 6E, right). These results suggest that human T-MDSChave similar metabolic characteristics as murine T-MDSC. Theclinical and biologic significance of these results in patientswith cancer is yet to be determined. However, the results supportthe possibility of testing FAO inhibitors as adjuvants to variouscancer therapies.

Figure 4.FAO inhibition in vivo significantlydelays tumor growth. C57BL/6 micebearing s.c. 3LL or MCA-38 tumorswere treated with 50 mg/kg etomoxiri.p. daily for 20 days, and tumors weremeasured every 2 to 3 days. A and B,tumor growth in control andetomoxir-treated mice (data ¼ mean� SEM; n ¼ 5 mice/group from 3independent experiments;P<0.01). C,3LL tumors harvested on day 21 werestained with hematoxylin & eosin,examined for apoptosis by TUNELassay, and tested for CD31 expressionby immunohistochemistry (n¼ 3micefrom 2 independent experiments. D,C57BL/6 mice bearing s.c. 3LL tumorswere treated (i.p.) daily for 20 dayswith 50 mg/kg of ranolazine (data,mean� SEM; n¼ 5mice/group from3independent experiments;P < 0.01). E, 3LL tumor–bearing micewere treated with etomoxir plusdepleting antibodies for CD4 or CD8.Controls received IgG isotype (data,mean � SEM; n ¼ 5 mice/groupfrom two independent experiments).��, P < 0.01.

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DiscussionMDSC are chronic inflammatory cells that inhibit T-cell func-

tion and play an important role in promoting tumor growth andfacilitating the progression of chronic infections (4, 29). Thus,inhibiting MDSC has been pursued as a means of enhancing theeffect of cancer therapies, in particular immunotherapies. How-ever, MDSC have multiple immunosuppressive mechanisms,which they upregulate depending on the signals encountered inthe microenvironment of different tumors. MDSC in renal cellcarcinoma and colorectal carcinoma primarily produce arginase I,whereas MDSC in melanoma produce NO and PNT (21, 30, 31).

Targeted inhibition of any of these pathways has not resulted in asignificant therapeutic effect, limiting the anti-MDSC therapies tochemotherapeutic agents that suppress their production in thebone marrow. We therefore chose to study the metabolic char-acteristics ofMDSC and test whether inhibition of these pathwayscould have a therapeutic application.

In recent years, targeting the energy metabolism pathways oftumors and immune cells has gained interest because of itspotential to uncover novel prevention or therapeutic targets(32, 33). Thus, we aimed to characterize the energy metabolismpathway(s) of T-MDSC. These data presented here showed that

Figure 5.FAO inhibition is synergisticwith chemotherapy andACT. A andB, C57BL/6mice bearing s.c. 3LL (A) orMCA-38 (B) tumorswere treatedwith 50mg/kg etomoxir i.p.for 6 days (starting 1 day after tumor injection) plus a single injection of 200 mg/kg CTX on day 7. Tumor growth was followed for 21 days. C and D, micewith established tumors were treated with etomoxir (C) or ranolazine (D) i.p. for 5 days (days 6–10) followed by a single injection of CTX on day 11. E, forACT, mice with 3LL-OVA tumors were treated with etomoxir daily starting 1 day after tumor injection. The indicated groups received an i.v. injection of2.5 � 106 activated OT-1 cells, followed by vaccination with SIINFEKL (100 mg/mouse) on days 14 and 15, respectively. Tumor growth was monitored (datafor all combination therapies ¼ mean � SEM; n ¼ 5 mice/group from 2 independent experiments). F, tumors were isolated from mice receiving ACT and testedfor the numbers of tumor-infiltrating OT-1 cells (CD45.1þ). G, splenic T cells were stimulated ex vivo with SIINFEKL (1 mg/mL), and the frequency of IFNg-producingcells was tested by ELISPOT (data, mean � SEM; n ¼ 5 mice/group from one experiment). �� , P < 0.01.






Hossain et al.





highly immunosuppressive T-MDSC activate FAO. Inhibition ofFAO using agents approved for the treatment of coronary diseasefurther demonstrated the importance of this energy productionpathway on the immunosuppressive functions of MDSC. Themolecular mechanisms linking FAO with the regulatory mechan-isms in MDSC are currently unknown; however, the antitumoreffect in vivo supports the possibility of testing this therapeuticapproach in patients. The mechanisms by which FAO inhibitioncan cause anantitumor effect are several.Our data strongly suggestthat FAO inhibition blocks the immunosuppressive function ofT-MDSC and thus allow T cells to kill tumor cells. However, it isalso possible that FAO inhibition also decreases Treg function asproposed by Michalek and colleagues (34). In addition, recentreports have shown that some tumors, including pancreatic cancerstem cells and certain myeloid leukemias, rely on mitochondrialfunction for survival (35, 36). Although our data failed to dem-onstrate an inhibition of cancer stem cells in these two murinetumor models, it is still possible that FAO inhibition can affectmultiple cells that support the tumor microenvironment. Ourdata also suggest that, in addition to blocking MDSC function,FAO inhibition could promote antitumor responses because itdecreases the production of G-CSF, GM-CSF, and IL6, but doesnot alter the production of IL12. The net effect of these changes,therefore, appears to be a decrease in the overall immunosup-pressive microenvironment in favor of the development of anti-tumor responses. This is especially significant given the additiveeffect of etomoxir with CTX, which has been used to modulatethe immunosuppressive microenvironment. This combinationappears to fully inhibit the immunosuppressive function ofT-MDSC (Supplementary Fig. S6).

FAO is also important in other myeloid cell subsets. Elegantstudies from several laboratories have shown that M2 macro-phages primarily use FAO, whereas M1 macrophages useglycolysis (18–20). The pathophysiologic importance of thisobservation was recently demonstrated byHuang and colleagues,showing that inhibiting lipolysis by orlistat decreased the abilityof M2 macrophages to control parasitic infection (37). In addi-tion, Herber and colleagues recently showed that CD11cþ den-dritic cells infiltrating tumors incorporate oxidized lipids thatblock antigen processing in lysosomes and their assembly onMHC class II, therefore preventing effective antigen presentationand T-cell stimulation (12). This process was inhibited by 5-(tetradecycloxy)-2-furoic acid (TOFA), an inhibitor of fatty acidsynthesis. Another important myeloid subpopulation comprisesgranulocytes, which primarily use glycolysis as a source of ATP(38). Our data show that T-MDSC, but not splenic MDSC ornMCs, clearly upregulated FAO, suggesting that factors and/orsignals in the tumor microenvironment are responsible for thismetabolic reprogramming. In many cancer patients, G-MDSC aresignificantly increased in peripheral blood and tumors (28, 39),and as shown here, human G-MDSC have an increased fattyacid uptake and expression of FAO cycle enzymes CPT1 andHADHA.

The factors responsible for inducing FAO in the tumor micro-environment are still unknown. Tumor-derived extracts trigger anincreased synthesis of fatty acids in dendritic cells (12). In addi-tion, signaling through Stat6 and IL4, which promote MDSCdifferentiation, can also induce PGC1b and activate FAO (20).Our data using in vitro–derived BM-MDSC suggest that G-CSF orGM-CSFmay also induce FAO (data not shown). A previous studyshowed that GM-CSF and IL6, instead, upregulate AMPK andglycolysis in BM-MDSC (40). Recent reports suggest that lacticacid present in high concentrations in the tumor microenviron-ment can activate MDSC (29, 41); however, its role in inducingFAO is unknown. Thus, additional studies are needed to deter-mine the mechanisms causing the metabolic shift toward FAO inT-MDSC.

How FAO inhibition blocks MDSC immunosuppressive func-tions is not fully understood, especially given the fact that eto-moxir and ranolazine target different enzymes in this pathway.Etomoxir is a nonreversible inhibitor of CPT-1, the enzymeresponsible for the initial step of internalization of fatty acidsinto the mitochondria. Ranolazine is a piperazine derivative thatinhibits HADHA, a trifunctional enzyme that catalyzes the lastthree steps in FAO (26). Both drugs block fatty acid uptake, FAO,and ATP production. More importantly, however, the resultsdemonstrated a novel and potentially important adjuvant effectwhen combining FAO inhibition with chemotherapy and/orimmunotherapy. Phase I clinical trials with etomoxir for thetreatment of coronary disease showed toxicities characterizedby moderate increases in liver enzymes with chronic use (42).Ranolazine, however, is approved for the treatment of unstableangina. Other FAO inhibitors such as perhexiline (a CPT1inhibitor) and trimetazidine (an HADHA inhibitor) or lipaseinhibitors such as orlistat may, in the present context, inhibitMDSC. This could be an important development because thecurrent therapeutic approaches aimed at blocking MDSC relyprimarily on the use of chemotherapeutic agents, such asgemcitabine (43) and 5-fluorouracil (44), that suppress thebone marrow or the tyrosine kinase inhibitor sunitinib (45). Inaddition, certain chemotherapeutic agents increase rather thandecrease the accumulation of MDSC and promote the expres-sion of inhibitory pathways (46). Other approaches haveincluded the use of all-trans-retinoic acid (ATRA) in an attemptto differentiate MDSC into mature granulocytes (47). Thus,targeting the energy metabolic pathways in T-MDSC may pro-vide a broader range of effects by globally inhibiting severalimmunosuppressive mechanisms in T-MDSC, without causingbone marrow suppression, and allowing T cells to develop anantitumor function.

Results presented here support the possibility of testingapproved FAO inhibitors in the context of chemotherapy orimmunotherapy of cancer. It also highlights the importance ofstudying the metabolism of immune cells in diseases in whichMDSC function may be modulated by metabolic manipulation(11, 12), including other malignancies (4), trauma and/or sepsis

Figure 6.Peripheral blood MDSC from cancer patients have an increased fatty acid uptake, and T-MDSC express CPT-1 and HADHA. A and B, peripheral bloodMDSC (CD14neg CD33þ HLA-DRneg CD66bþ) from 23 patients with breast, renal cell carcinoma, bladder cancer, and colon cancer were measured by flowcytometry. C, MDSC were sorted and tested for fatty acid uptake. Control cells included PMNs from patients and normal controls. D, tumor samples from3 patients with colon carcinoma, renal cell carcinoma, and ductal carcinoma of the breast were tested for CPT1 by immunohistochemistry (left) andimmunofluorescence double labeling with CPT-1 (fluorescein) and CD66b (Rhodamine). E, tissues from D were tested for HADHA by immunohistochemistry(left) and immunofluorescence double labeling with HADHA (fluorescein) and CD66b (Rhodamine). � , P < 0.05.






(48), and chronic infections, such as HIV, leishmaniasis, andtuberculosis (49).

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

Authors' ContributionsConception and design: A.A. Al-Khami, P.C. Rodriguez, A.C. OchoaDevelopment of methodology: F. Hossain, A.A. Al-Khami, D. Wyczechowska,L. Zheng, K. Reiss, W. Zou, A.C. OchoaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Hossain, A.A. Al-Khami, D. Wyczechowska, C.Hernandez, L.D. Valle, J. Trillo-Tinoco, T. Maj, W. Zou, A.C. OchoaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Hossain, A.A. Al-Khami, D. Wyczechowska, C.Hernandez, L.D. Valle, J. Trillo-Tinoco, P.C. Rodriguez, A.C. OchoaWriting, review, and/or revision of themanuscript: F. Hossain, A.A. Al-Khami,K. Reiss, L.D. Valle, P.C. Rodriguez, A.C. Ochoa

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.A. Al-Khami, C. Hernandez, L. Zheng, P.C.Rodriguez, A.C. OchoaStudy supervision: A.A. Al-Khami, P.C. Rodriguez, A.C. Ochoa

Grant SupportThis study was funded in part by R01 AI112402, R01CA082689,

R01CA107974, and P20GM2013501 (to A.C. Ochoa) and partially supportedfrom LA CaTS Center (U54GM104940; to A.A. Al-Khami and A.C. Ochoa) andthe Al Copeland Foundation funds.

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

Received January 30, 2015; revised April 24, 2015; accepted May 20, 2015;published OnlineFirst May 29, 2015.

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2015;3:1236-1247. Published OnlineFirst May 29, 2015.Cancer Immunol Res Fokhrul Hossain, Amir A. Al-Khami, Dorota Wyczechowska, et al. Cancer TherapiesFunctions of Myeloid-Derived Suppressor Cells and Enhances Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive

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