CD73-Deficient Mice Have Increased Antitumor Immunity and ...MC38-ova, EG7, AT-3, and B16F10 tumor...

Microenvironment and Immunology

CD73-Deficient Mice Have Increased Antitumor Immunityand Are Resistant to Experimental Metastasis

John Stagg1,2, Upulie Divisekera1, Helene Duret1, Tim Sparwasser3, Michele W.L. Teng1,4,Phillip K. Darcy1,4, and Mark J. Smyth1,4

AbstractCD73 is a cell-surface enzyme that suppresses immune responses by producing extracellular adenosine. In this

study, we employed CD73 gene-targeted mice to investigate the role of host-derived CD73 on antitumorimmunity and tumor cell metastasis. We found that CD73 ablation significantly suppressed the growth ofovalbumin-expressing MC38 colon cancer, EG7 lymphoma, AT-3 mammary tumors, and B16F10 melanoma. Theprotective effect of CD73 deficiency on primary tumors was dependent on CD8þ T cells and associated with anincreased frequency of antigen-specific CD8þ T cells in peripheral blood and tumors and increased antigen-specific IFN-g production. Replicate studies in bone marrow chimeras established that both hematopoietic andnonhematopoietic expression of CD73 was important to promote tumor immune escape. Using adoptivereconstitution of T regulatory cell (Treg)–depleted DEREG (depletion of regulatory T cells) mice, wedemonstrated that part of the protumorigenic effect of Tregs was dependent on their expression of CD73.CD73-deficient mice were also protected against pulmonary metastasis of B16F10 melanoma cells afterintravenous injection. Unexpectedly, we found that the prometastatic effect of host-derived CD73 wasdependent on CD73 expression on nonhematopoietic cells. CD73 expression on nonhematopoietic cells, mostlikely endothelial cells, was critical for promoting lung metastasis in a manner independent from immuno-suppressive effects. Notably, in vivo blockade of CD73 with a selective inhibitor or anti-CD73 monoclonalantibody significantly reduced tumor growth and metastasis of CD73-negative tumors. Taken together, ourfindings indicate that CD73 may be targeted at multiple levels to induce anticancer effects including at the levelof tumor cells, Tregs, and nonhematopoietic cells. Cancer Res; 71(8); 2892–900. �2011 AACR.

Introduction

The release of extracellular ATP by phagocytes and injuredor stressed cells acts as a proinflammatory coactivator of theinflammasome via P2x7 receptor signaling (1, 2). In contrast,the phosphohydrolysis of extracellular ATP into adenosine,

essentially through the enzymatic activity of CD39 (NTPDase I)and CD73 (ecto-50-nucleotidase), acts as an immunosuppres-sive pathway through the activation of adenosine receptors (3–6). In the context of cancer, landmark studies by Sitkovsky andcolleagues have demonstrated that the accumulation of extra-cellular adenosine in tumors suppresses antitumor immuneresponses, essentially via the activation of A2A adenosinereceptors (7, 8). Extracellular adenosine levels are generallyconstant in most tissue but can rapidly increase in response tohypoxia and chronic inflammation (9). The process of extra-cellular adenosine accumulation in solid tumors has beenrecently reviewed (3). The accumulation of extracellular ade-nosine in tumors generates an immunosuppressive microen-vironment that effectively enhances tumor immune escape.Activation of A2A adenosine receptors on T cells has beenshown to inhibit T-cell–mediated cytotoxicity, cytokine pro-duction (10) and T-cell proliferation (11, 12) and to promoteT-cell anergy (13).

The importance of A2A adenosine receptors in regulatingantitumor immunity was first revealed by Ohta and colleagueswho demonstrated that mice genetically deficient in A2Aadenosine receptors spontaneously rejected immunogenictumors in a mechanism involving increased CD8þ T-cellresponses (8). We have recently demonstrated that one ofthe mechanisms contributing to the immunosuppressiveaccumulation of extracellular adenosine in tumors is the

Authors' Affiliations: 1Cancer Immunology Program, TrescowthickLaboratories, Peter MacCallum Cancer Centre, East Melbourne, Victoria,Australia; 2Centre de Recherche du Centre Hospitalier de l’Universit�e deMontr�eal, Facult�e de Pharmacie et Institut du Cancer de Montr�eal, Mon-treal, Quebec, Canada; 3Institute of Infection Immunology, TWINCORE,Centre for Experimental and Clinical Infection Research; a joint venturebetween the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover, Germany; and 4Department ofPathology, University of Melbourne, Melbourne, Victoria, Australia

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

Corresponding Author: John Stagg, Centre de Recherche du CentreHospitalier de l’Universit�e de Montr�eal, Facult�e de Pharmacie et Institut duCancer deMontr�eal, Montreal, Quebec, Canada. Phone: 514-890-8000, ext:25170; Fax: 613-9656-1411; E-mail: [email protected]; orMark J. Smyth, Cancer Immunology Program, Sir Donald and Lady Tres-cowthick Laboratories, Peter MacCallum Cancer Centre, Locked Bag 1,A'Beckett Street, 8006, East Melbourne, Victoria, Australia. Phone: 613-9656-3728; Fax: 613-9656-1411; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-4246

�2011 American Association for Cancer Research.

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expression of CD73 by tumor cells (14). CD73 is a glycosylpho-sphatidyl-inositol (GPI)-linked cell-surface enzyme constitu-tively expressed on endothelial cells, Foxp3þ T regulatory cells(Treg) and subsets of leukocytes, and is considered as the rate-limiting enzyme in the production of extracellular adenosine(15, 16). CD73 expression is induced by HIF-1a (hypoxia-inducible factor) activation, exposure to type I IFNs as wellas activation of Wnt signaling (17, 18). CD73 is foundexpressed in several types of cancer (3) and has been asso-ciated with increased glioma cell proliferation (19). Intrigu-ingly, CD73 expression is negatively regulated by estrogenreceptor (ER) signaling (20). As a result, breast cancer cellsdeficient in ER signaling express high levels of CD73. We haverecently demonstrated that CD73 expression on ER-negativemammary carcinoma cells significantly suppresses adaptiveantitumor immunity and promotes tumor cell metastasis.Furthermore, we demonstrated that tumor-derived CD73could be targeted for therapy with an anti-CD73 monoclonalantibody (mAb; ref. 14). Another independent group has sincecorroborated our findings in a mouse model of ovarian cancer,confirming that CD73 expression on breast and ovarian tumorcells promotes tumor immune escape (21).While the immunosuppressive effects of CD73 expression

on tumor cells have recently been studied, the effects of host-derived CD73 on antitumor immunity and tumor cell metas-tasis have not been investigated. In the non-hematopoieticcompartment, CD73 is mainly expressed on endothelial cellsand high endothelial venules (HEV), where it regulates leu-kocyte extravasation in peripheral organs (22) and inhibitsinflammation-induced lymphocyte migration into draininglymph nodes, respectively (22). In hematopoietic cells, CD73is most highly expressed on Foxp3þCD4þ Tregs and contri-butes to their immunosuppressive functions (11, 23). CD73can also be found on effector T and B cells at lower expressionlevels.We here report that CD73-deficient mice are significantly

protected against the development of subcutaneous tumorsand experimental lung metastases. We demonstrated thatCD73 expression on nonhematopoietic and hematopoieticcells significantly restricts CD8þ T-cell–mediated antitumorimmunity. Most importantly, we provide evidence that CD73expression on Tregs is important for Treg-mediated tumorgrowth. Unexpectedly, we observed that CD73-deficient micewere significantly protected against the development ofB16F10 lung metastases in a mechanism that was indepen-dent of CD73 expression on hematopoietic cells and indepen-dent of its immunosuppressive effects. We here propose thattargeted inhibition of host-derived CD73 can enhance anti-tumor immune responses by inhibiting Treg-mediated tumorgrowth and prevent the development of distant metastases.

Materials and Methods

Cell lines, mice, and antibodiesAll mice were bred and maintained at the Peter MacCallum

Cancer Centre. EG7, MC38-ova, AT-3, and B16F10 mousetumor cell lines have been previously described (24–27) andwere authenticated by flow cytometry before use. C57BL/6

CD73-deficient mice were kindly provided by Dr. Linda H.Thompson (Oklahoma Medical Research Foundation, Okla-homa City, OK). C57BL/6 DEREG (DEpletion of REGulatoryT cells) transgenic mice were kindly provided by Dr. TimSparwasser (Institute of Infection Immunology, Hannover,Germany). CD73 expression was assessed using phycoery-thrin (PE)-conjugated anti-mouse CD73 mAb (clone TY/23;BD Bioscience). PE-conjugated anti-mouse CD45.1 (A20),fluorescein isothiocyanate (FITC)-conjugated anti-mouseGr1 (RB6-8C5), Pacific Blue–conjugated anti-mouse CD4(L3T4), PE-conjugated anti-mouse CD25, and allophycocya-nin (APC)-conjugated anti-mouse CD8 (53-6.7) were pur-chased from BD Bioscience; FITC-conjugated anti-mouseCD11b (M1/70), FITC-conjugated anti-mouse Foxp3,and APC-conjugated anti-mouse CD45.2 (104) were pur-chased from eBioscience. Purified anti-CD73 mAb (cloneTY/23; provided by Dr. Linda H. Thompson, OklahomaMedical Research Foundation), anti-CD4 mAb (GK1.5),anti-CD8 mAb (53-6.7 and 53-5.8), and control Ig (cloneMAC4) were produced in house. PE-conjugated MHC (majorhistocompatibility complex) class I/SIINFEKL tetramerswere purchased from Dr Andrew Brooks (University ofMelbourne).

In vivo treatmentsEG7, MC38-ova, AT-3, and B16F10 tumor cells were injected

subcutaneously into syngeneic C57BL/6 wild-type, CD73-defi-cient, or DEREG mice at the indicated doses. The followingmAbs were injected intraperitoneally at 100 mg per dose at theindicated time points: anti-asialoGM1 (Wako Chemicals),anti-CD8, anti-CD4, anti-CD73, and control Ig. B16F10 cellswere injected intravenously at the indicated dose and lungmetastases were counted under dissectingmicroscope 2weekslater after fixation in Bouin's solution. Where indicated, micewere treated with a,b-methyleneadenosine 50-diphosphate(APCP; Sigma) at 20 mg/kg i.v., anti-CD73 mAb (200 mgi.v.), or control Ig (200 mg i.v.). For bone marrow (BM)reconstitution, wild-type and CD73-deficient mice were irra-diated (10 Gy from a 137Cs source) and injected i.v. with 2� 106

BM cells derived from congenic wild-type or CD73-deficientmice. BM chimera mice were housed in microisolators for8 weeks before analysis and experimentation.

Treg reconstitution and suppression assayCD4þ CD25þ T cells were isolated from the spleens of wild-

type and CD73-deficient mice using a mouse Treg-isolation kitand AutoMACS (Miltenyi) according to the manufacturer'sinstructions. Cells were at least 90% pure as determined byflow cytometry. DEREG mice were injected with 1 mg ofdiphtheria toxin (DT) intraperitoneally 3 days prior to intra-venous injection of 2 � 106 purified Tregs, and then with500 ng of DT on days 0 and 7. For suppression assays,accessory CD4� cells, T effector cells (CD4þ CD25þ), andTregs (CD4þ CD25þ) were isolated from spleen cells on anAutoMACS (Miltenyi). Accessory cells were treated with mito-mycin C (Sigma) and T effector cells were stained with 5 mmol/L carboxyfluorescein succinimidyl ester (CFSE; Invitrogen). Atotal of 5 � 104 T effector cells were cocultured with 5 � 104

CD73-Deficient Mice Are Resistant to Cancer

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accessory cells and unlabeled Treg at different ratios andstimulated with 1 mg/mL anti-CD3 (clone 145-2C11). Respon-der cell proliferation was assessed by flow cytometry after 96hours.

Tumor-infiltrating lymphocytesEG7 tumors were excised, minced with scissors, and incu-

bated 1 hour at 37�C for in PBS containing collagenase type 4(Worthington Biochemical) and DNase I (Roche). Tumor cellsuspensions were passed through a 70-mm cell strainer,washed twice in PBS, and resuspended in PBS 2% serumfor flow cytometric analysis. Anti-CD16/32 mAb (clone2.4G2) was used to block Fc receptors. Flow cytometry wasperformed on a LSR II (BD Bioscience) and analyzed using thesoftware program FCS Express.

IFN-g release assaysEG7 cells (106) were injected in the footpad of wild-type and

CD73-deficient mice and 5 days later, popliteal lymph nodecells were harvested, plated in 96 U-well plates (3 � 105 cellsper well), and restimulated with 1 mg/mL ova protein (Sigma).Cell supernatants were harvested 72 hours later and IFN-glevels measured by ELISA (eBioscience).

Results

CD73-deficient mice are resistant to primary tumorgrowth

To investigate the contribution of host-derived CD73 ontumorigenesis, we first compared the growth rate of varioussyngeneic tumors in wild-type and CD73-deficient mice.MC38-ova, EG7, AT-3, and B16F10 tumor cells were injectedsubcutaneously into wild-type and CD73-deficient mice andtumor size monitored over time. As shown in Figure 1, CD73-deficient mice were greatly resistant to the growth of sub-cutaneous MC38-ova and EG7 tumors. In comparison, CD73-deficient mice were more modestly resistant to subcutaneousAT-3 and B16F10 tumors (Fig. 1). Notably, tumor resistancedid not correlate with CD73 expression on tumor cells (Sup-plementary Fig. S1).

Because of the immunosuppressive function of CD73, wenext assessed whether tumor resistance of CD73-deficientmice was mediated by an increased antitumor immuneresponse. For this purpose, CD73-deficient mice were injectedwith depleting antibodies against CD8þ, CD4þ, or naturalkiller (NK) cells and subsequently injected with MC38-ova orEG7 tumor cells. As shown in Figure 2A, the resistance of

Figure 1. CD73-deficient mice areresistant to the development ofsubcutaneous tumors.A, ovalbumin-expressing MC38tumor cells (MC38-ova; 5 � 105

cells) were injectedsubcutaneously to wild-type andCD73-deficient B6 mice andtumor growth monitored over time(data represent means of 5 miceper group � SE; representative of3 experiments is shown). B, sameas A except mice were injectedwith 106 EG7 tumors cells (datarepresent means of 6 mice pergroup � SE; representative of 2experiments is shown). C, same asA except mice were injected with 5� 105 AT-3 tumor cells (datarepresent means of 6 mice pergroup � SE). D, same as A exceptmice were injected with 2 � 105

B16F10 tumor cells (datarepresent means of 5 mice pergroup � SE; representative of 2experiments is shown). *, P < 0.05by Mann–Whitney test.

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CD73-deficient mice towardMC38-ova tumors was dependenton CD8þ T cells, but independent of CD4þ T cells or NK cells.Similar results were obtained for EG7 tumors (SupplementaryFig. S2A). Notably, CD73-deficient mice were not significantlyresistant to parental MC38 tumors, which lack ova and arethus less immunogenic than MC38-ova tumors. The increasedimmunogenicity of MC38-ova tumors is evidenced by theirability to generate endogenous CD8þ T-cell–mediated control,which is not observed for parental MC38 tumors (Supplemen-tary Fig. S2B and C). Our data thus suggest that host-derivedCD73 is important in suppressing existing CD8þ T-cell–mediated antitumor responses.

We next compared the systemic frequency, tumor infiltra-tion, and function of antigen-specific CD8þ T cells in CD73-deficient and wild-type mice. Flow cytometric analysisrevealed that tumor-bearing CD73-deficient mice had anincrease proportion of tumor-specific CD8þ T cells in per-ipheral blood (Fig. 2B). Tumor-specific CD8þ T cells were alsofound more abundantly infiltrated in the tumors of CD73-deficient mice (Fig. 2C). In contrast, tumor infiltration byFoxp3þ CD4þ Tregs was similar in CD73-deficient and wild-type mice (Fig. 2D). Consistent with an increased CD8þ T-cell–mediated antitumor immune response, antigenic restimula-tion of tumor-draining lymph node cells isolated from

Figure 2. CD73-deficient micehave increased antitumor CD8þ

T cell responses. A, ovalbumin-expressing MC38 tumor cells(MC38-ova; 5 � 105 cells) wereinjected subcutaneously to CD73-deficient mice treated with controlIg or depleting antibodies to CD8þ

cells (clone 53-6.7), CD4þ cells(clone GK1.5), or NK cells (anti-asialo GM1). Depleting antibodieswere injected intraperitoneally1 day prior to tumor inoculationand on days 1, 7, and 14 aftertumor inoculation. Data representmeans of 5 mice per group � SE(representative of 2 experiments isshown). B, MC38-ova cells (5 �105) were injected subcutaneouslyto wild-type and CD73-deficientmice and peripheral bloodmononuclear cells (PBMC) wereanalyzed at day 9 for tetramer-reactive ova-specific CD8þ T cells(symbols represent individualmice, means � SE are shown).C, EG7 tumors were analyzed byflow cytometry for the presence ofova-specific CD8þ T cells(symbols represent individualmice, means � SE are shown).D, same as C, except that tumor-infiltrating Foxp3þ CD4þ cellswere analyzed (symbols representindividual mice, means � SE areshown). E, tumor-draining lymphnode cells from wild-type andCD73-deficient mice wererestimulated with ova for 72 hoursand IFN-g levels measured byELISA (data represent means of5 mice per group � SE). F, BMchimera mice were injectedsubcutaneously with 5 � 105

MC38-ova tumor cells and tumorgrowth monitored over time (datarepresent means of 8–11 mice pergroup � SE). *, P < 0.05 by Mann–Whitney test. WT, wild-type.






tumor-bearing CD73-deficient mice generated significantlygreater IFN-g production compared with restimulation ofwild-type tumor-draining lymph node cells (Fig. 2E).

CD73 is expressed on hematopoietic and nonhematopoieticcells. To further assess the contribution of CD73 expression onhematopoietic versus nonhematopoietic cells, we generatedCD73 BM chimera mice. BM reconstitution was confirmed byflow cytometric analysis 8 weeks after BM cell injection andbefore tumor cell inoculation (Supplementary Fig. S3). Asshown in Figure 2F, CD73 expression on either hematopoieticor nonhematopoietic cells significantly enhanced MC38-ovatumor growth, whereas CD73 expression in both hematopoie-tic and nonhematopoietic cells was most effective. Our datathus suggest that hematopoietic and nonhematopoieticexpression of CD73 promotes tumor immune escape.

CD73 expression on Tregs is critical for Treg-mediatedtumor growth

In hematopoietic cells, CD73 is most highly expressed onactivated Foxp3þ CD4þ CD25þ Tregs (23). We thus investi-gated the specific role of CD73 expression on Foxp3þ Tregsduring tumor growth. Foxp3þ Tregs develop normally inCD73-deficient mice, but their immunosuppressive functionis compromised (Fig. 3A). This is consistent with other studiesthat demonstrated that Tregs partly rely on CD73-mediatedproduction of extracellular adenosine for immunosuppression(11, 23, 28, 29). Nevertheless, the importance of CD73 in Treg-mediated tumor growth remains unknown. To assess the roleof CD73 expression by Tregs during tumor growth, we firstcompared the tumor-promoting effects of Tregs in wild-typeand CD73-deficient mice. As shown in Figure 3B, anti–folate

Figure 3. CD73 expression on Tregs is critical for Treg-mediated tumor growth. A, left, wild-type and CD73-deficient spleen cells were analyzed forFoxp3þCD4þCD25þ expression. Right, purified wild-type and CD73-deficient CD4þCD25þ T cells (Treg) were cocultured with CFSE-labeled wild-type CD4þ

CD25� T cells (Teff) and plate-bound anti-CD3 mAb. Teff cell division was measured by flow cytometry after 4 days (data represent means of triplicates� SE).B, wild-type and CD73-deficient mice were treated with anti-FR4 mAb or control Ig and injected subcutaneously with 106 MC38-ova tumor cells. Antibodies(10 mg i.v.) were injected intraperitoneally on days 2, 0, 4, 7, 11, and 14. Data represent means of 5 mice per group � SE. C, DEREG mice were treatedwith PBS or DT [days 3 (1 mg), 0 (500 ng), and 7 (500 ng)], injected subcutaneously with 106 MC38-ova tumor cells and treated with anti-CD73 mAb(100 mg i.p.) or control Ig (100 mg i.p.) on days 3, 7, 10, 14, 17, 21, and 24. Data represent means of 5 mice per group � SE. D, DEREG mice weretreated with PBS or DT (days 3, 0, and 7, as in C), injected subcutaneously with 106 MC38-ova tumor cells and intravenously with 2 � 106 wild-typeor CD73-deficient purified CD4þ CD25þ Tregs (tumor cells and Tregs injected at day 0). Data represent means of 5 mice per group � SE (representativeof 2 experiments is shown). *, P < 0.05 by Mann–Whitney test. WT, wild-type; cIg, control Ig.

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receptor 4 (FR4)-mediated Treg depletion in wild-type mice(30) significantly abrogated MC38-ova tumor growth. In con-trast, Treg depletion in CD73-deficient mice had no effect.This suggested that Tregs in CD73-deficient mice failed topromote tumor growth. We hypothesized that this was due tothe fact that Tregs and host-derived CD73 had overlappingprotumorigenic effects; that is, CD73 expression on Tregs wasimportant for tumor growth. To directly assess this, we usedtransgenic DEREG mice, which express a DT receptor underFoxp3 promoter that allows for the complete depletion ofFoxp3þ Tregs after DT injection (30, 31). As shown in Figure3B and C, neutralization of CD73 induced comparable anti-tumor effects to Treg depletion with anti-FR4 mAb, and CD73neutralization was slightly less effective than total Treg abla-tion with DT. Taken together, these results were consistentwith the presence of overlapping protumorigenic effectsbetween Tregs and host-derived CD73.We next performed adoptive cell reconstitution of Treg-

depleted DEREG mice with purified CD4þ CD25þ Tregs iso-lated from wild-type or CD73-deficient mice. Flow cytometricanalysis confirmed adoptive cell reconstitution (Supplemen-tary Fig. S5). As shown in Figure 3D, only DEREG micereconstituted with wild-type Tregs, but not DEREG micereconstituted with CD73-deficient Tregs, could effectivelysupport MC38-ova tumor growth. Taken together, our datademonstrated that in the MC38-ova tumor model, part of theprotumorigenic effect of Tregs is dependent on CD73 expres-sion by Tregs.

CD73-deficient mice are resistant to B16F10 lungmetastasisWe next investigated the susceptibility of CD73-deficient

mice to develop experimental lung metastases followingintravenous injection of CD73-negative B16F10 melanomacells. As shown in Figure 4A, CD73-deficient mice were sig-nificantly resistant to the development of B16F10 lung metas-tases. Accordingly, CD73-deficient mice developed 3- to 4-foldless metastatic lung nodules after intravenous injection ofB16F10 cells. Unexpectedly, this resistance was maintainedin NK-cell–depleted or T cell–depleted CD73-deficient mice(Fig. 4A). This suggests that host-derived CD73 promotesB16F10 lung metastasis independently of its immunosuppres-sive effect on NK cells. To further assess the mechanism ofresistance of CD73-deficient mice against experimentalmetastasis, BM chimera mice were used. As shown in Figure4B, the lack of CD73 expression on nonhematopoietic cellswas largely responsible for the resistance of CD73-deficientmice against experimental B16F10 lung metastasis. Our datasuggest that CD73 expression on nonhematopoietic cells-–essentially endothelial cells—enhances the metastatic poten-tial of circulating tumor cells.

CD73-targeted therapy inhibits the growth andmetastasis of CD73-negative tumorsWe further assessed the therapeutic activity of CD73-

targeted therapy against CD73-negative tumors. Consistentwith the therapeutic effect observed against MC38-ovatumors (Fig. 3C), treatment of EG7 tumors with the CD73

inhibitor APCP also induced significant antitumor effect(Fig. 5A). We then assessed the therapeutic activity ofCD73 inhibition on the development of B16F10 lung metas-tases after intravenous tumor cell injection. As shown inFigure 5B, inhibition of CD73 with APCP or anti-CD73 mAbsignificantly suppressed the development of B16F10lung metastases in wild-type mice. Notably, only whenCD73 was blocked prior to B16F10 cell injection, and notafter B16F10 cell injection, was the development of lungmetastases suppressed. This further supports the conceptthat CD73 expression on endothelial cells promotes tumor

Figure 4. CD73-deficient mice are resistant to experimental B16F10 lungmetastasis. A, wild-type and CD73-deficient mice were injectedintravenously with 2 � 105 B16F10 cells and 2 weeks later, B16F10 lungnodules were counted under a dissecting microscope (symbols representindividual mice; means� SE are shown; representative of 2 experiments isshown). In some groups, mice were depleted of NK cells or CD4þ/CD8þ Tcells with intraperitoneal injections at days 1 and 0 of 100 mg anti-asialoGM1 or anti-CD4 (GK1.5)/anti-CD8b (clone 53-5.8) mAbs, respectively(symbols represent individual mice; means� SE are shown). B, same as A,except that BM chimera mice were used and 105 B16F10 cells wereinjected intravenously (symbols represent individual mice; means� SE areshown). *, P < 0.05 by Mann–Whitney test. KO, knockout; ns, notsignificant.






cell metastasis. Taken together, our data suggest that inhi-bition of host-derived CD73 can effectively suppress tumorgrowth as well as the metastatic potential of circulatingtumor cells.

Discussion

We have previously shown that CD73 expression on breastcancer cells significantly inhibits adaptive tumor immunosur-veillance (14). The objective of this study was now to inves-tigate the role of host-derived CD73 on tumorigenesis. Wehere demonstrated that (i) CD73-deficient mice are resistantto the development of immunogenic tumors in a CD8þ T-cell–dependent manner; (ii) hematopoietic and nonhematopoieticexpression of CD73 each promote tumor immune escape in anon-redundant manner; (iii) CD73 expression on Foxp3þ

Tregs mediates part of the pro-tumorigenic effect of Tregs;(iv) nonhematopoietic expression of CD73, presumably onendothelial cells, enhances metastasis of circulating tumorcells to the lungs; and (v) targeted inhibition of CD73 sup-presses the subcutaneous growth and the metastasis of CD73-negative tumor cells.

An important aspect of this study was to use Treg recon-stitution of DEREGmice, which express the DT receptor underFoxp3 promoter, to directly demonstrate the importance ofCD73 expression on Tregs during tumorigenesis. At least inthe MC38-ova tumor model, extracellular adenosine produc-tion by Tregs appears to be an essential component of Treg-mediated immunosuppression. To our knowledge, this is thefirst time such reconstitution experiments have been used toreveal a key functional molecule on Tregs. Many mechanismsare involved in Treg-mediated immunosuppression. Our studydefines CD73 as an important Treg immunosuppressive factorfor promoting tumor growth. Future comparative studies withother important Treg molecules will now be of interest.

In lymphocytes, only Foxp3þ Tregs are endowed with thecomplete catalytic machinery able to produce extracellularadenosine. Accordingly, Foxp3þ Tregs constitutively expressCD39 (which hydrolyses ATP and ADP) and CD73 (whichhydrolyses AMP into adenosine). CD39/CD73 coexpression onTregs has been shown to be maintained by IL-2 (interleukin-2;ref. 32) and upregulated upon activation (23). CD39/CD73coexpression on Tregs converts proinflammatory extracellularATP into the potent immunosuppressor adenosine. Tregshave previously been shown to partly rely on the enzymaticactivity of CD39 for immunosuppression. Indeed, Deaglio andcolleagues (11) demonstrated that CD39-deficient Tregs haveimpaired immunosuppressive function in vitro and fail toblock skin allograft rejection in vivo. The data presented herefurther demonstrate the importance of CD73 on Tregs andsuggest that CD73 and CD39 have nonredundant immuno-suppressive effects.

The adenosine-mediated immunosuppressive function ofTregs has been shown to be essentially dependent on theactivation of A2A adenosine receptor of effector T cells. Whileextracellular adenosine activates A2A adenosine receptors oneffector T cells, Tregs can also accumulate cyclic AMP (cAMP)and transfer cAMP to effector T cells via gap junctions (33).Thus, extracellular adenosine production by Tregs couldtheoretically suppress effector T cells via 2 nonexclusivemechanisms: via activation of A2A adenosine receptors oneffector T cells and/or via activation of A2A adenosine recep-tors on Tregs followed by transfer of cAMP via gap junctions.

Figure 5.CD73 inhibition suppresses the growth and metastasis of CD73-negative tumors. A, EG7 cells (106) were injected subcutaneously andmicewere treated with APCP (20 mg/kg i.v.) on day 5, 7, 9, and 12 (datarepresent means of 5 mice per group � SE). B, B16F10 cells (105) wereinjected intravenously and mice were treated with APCP (20 mg/kg i.v.),anti-CD73 mAb (200 mg i.v.), or control Ig (200 mg i.v.) 1 day prior and 2hours prior tumor inoculation. B16F10 lung nodules were counted under adissecting microscope 2 weeks later (symbols represent individual mice;means � SE are shown). C, same as B, except that 2 � 105 B16F10 cellswere injected at day 0 and anti-CD73mAb at days 1 and 0, or at days 1 and2 (symbols represent individual mice; means � SE are shown) *, P < 0.05by Mann–Whitney test. cIg, control Ig.

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A recent study by Sun and colleagues (34) investigated therole of host-derived CD39 on tumor growth and tumor cellmetastasis. Consistent with our study, Sun and colleagues (34)demonstrated that B16F10 metastasis was strongly inhibitedinmice with CD39-deficient nonhematopoietic cells. However,B16F10 metastasis was also inhibited in mice with CD39-deficient hematopoietic cells, in contrast to what we observedfor CD73. Sun and colleagues (34) demonstrated that Tregsinhibit NK-cell–mediated antitumor functions in a mechan-ism dependent on CD39 expression. In contrast, we showedthat host-derived CD73 promotes B16F10 metastasis indepen-dently of its effect on NK cells. Since both studies wereperformed with the same tumor model, this suggests thepresence of distinct nonredundant prometastatic effects forhost-derived CD39 and host-derived CD73. Further studiesshould clarify the mechanism by which nonhematopoieticexpression of CD73 promotes lung metastasis. Our hypothesisis that endothelial CD73 expression may promote transen-dothelial migration of tumor cells. While endothelial CD73 candecrease leukocyte attachment and migration into draininglymph nodes (22), it can also promote endothelial extravasa-tion in pathologic conditions. Accordingly, decreased T-cellextravasation was recently suggested as the cause of resis-tance of CD73-deficient mice to experimental autoimmuneencephalomyelitis (35). Through the production of extracel-lular adenosine and the activation of adenosine receptorson endothelial cells, Mills and colleagues (35) suggested thatendothelial CD73 can enhance expression of adhesion mole-cules and transendothelial migration.In conclusion, whereas the immunosuppressive effects of

tumor-derived CD73 have recently been studied, the effects ofhost-derived CD73 on antitumor immunity have not been

addressed. We here demonstrated an important role for CD73expression on Tregs for the suppression of adaptive antitumorimmune responses. Together with other studies, this suggeststhat the combined activity of CD39 and CD73 is a keycomponent of Treg-mediated suppression. We also demon-strated an important role for CD73 expression on nonhema-topoietic cells, presumably endothelial cells, on the metastaticpotential of circulating tumor cells. Finally, we demonstratedthat CD73 inhibition significantly inhibits the growth andmetastatic potential of CD73-negative tumor cells. We pro-pose that CD73 may be targeted at multiple levels to induceanticancer effects.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Nicole McLaughlin, Janelle Sharkey, and Michelle Stirlingfor technical support.

Grant Support

Funding support was provided from the National Health and MedicalResearch Council of Australia, the Susan Komen Breast Cancer Foundation,and the Victorian Breast Cancer Research Consortium. M.J. Smyth was sup-ported by an NH&MRC Australia Fellowship. M.W.L. Teng was supported by anNH&MRC Doherty Fellowship, and P.K. Darcy was supported by an NH&MRCCDA2 award.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 24, 2010; revised January 28, 2011; accepted February 1,2011; published OnlineFirst February 3, 2011.

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2011;71:2892-2900. Published OnlineFirst February 3, 2011.Cancer Res John Stagg, Upulie Divisekera, Helene Duret, et al. Are Resistant to Experimental MetastasisCD73-Deficient Mice Have Increased Antitumor Immunity and

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