InSitu CD204 Gene-SilencedDendriticCell, not Unmodiﬁed … · tration of WT DCs to the irradiated RM1 tumors failed to provide any additional beneﬁts (Fig. 2B, left). In contrast,

Therapeutic Discovery

In Situ Vaccination withCD204Gene-Silenced Dendritic Cell,not Unmodified Dendritic Cell, Enhances Radiation Therapyof Prostate Cancer

ChunqingGuo1,3,4,HuanfaYi1,3,4, Xiaofei Yu1,3,4, DamingZuo1,3,4, JieQian1,3,4,GaryYang5,BarbaraA. Foster6,John R. Subjeck7, Xiaolei Sun2,4, Ross B. Mikkelsen2,4, Paul B. Fisher1,3,7, and Xiang-Yang Wang1,3,4

AbstractGiven the complexity of prostate cancer progression and metastasis, multimodalities that target different

aspects of tumor biology, for example, radiotherapy in conjunction with immunotherapy, may provide the

best opportunities for promoting clinical benefits in patients with high-risk localized prostate cancer. Here,

we show that intratumoral administration of unmodified dendritic cells (DC) failed to synergize with

fractionated radiotherapy. However, ionizing radiation combined with in situ vaccination with DCs, in

which the immunosuppressive scavenger receptor A (SRA/CD204) has been downregulated by lentivirus-

mediated gene silencing, profoundly suppressed the growth of two mouse prostate cancers (e.g., RM1 and

TRAMP-C2) and prolonged the lifespan of tumor-bearing animals. Treatment of subcutaneous tumors with

this novel combinatorial radioimmunotherapeutic regimen resulted in a significant reduction in distant

experimental metastases. SRA/CD204-silenced DCs were highly efficient in generating antigen or tumor-

specific T cells with increased effector functions (e.g., cytokine production and tumoricidal activity). SRA/

CD204 silencing-enhanced tumor cell death was associated with elevated IFN-g levels in tumor tissue and

increased tumor-infiltrating CD8þ cells. IFN-g neutralization or depletion of CD8þ cells abrogated the

SRA/CD204 downregulation-promoted antitumor efficacy, indicating a critical role of IFN-g–producingCD8þ T cells. Therefore, blocking SRA/CD204 activity significantly enhances the therapeutic potency of

local radiotherapy combined with in situ DC vaccination by promoting a robust systemic antitumor

immunity. Further studies are warranted to test this novel combinatorial approach for translating into

improved clinical outcomes in patients with prostate cancer.Mol Cancer Ther; 11(11); 2331–41.�2012 AACR.

IntroductionProstate cancer is the most prevalent solid tumor and

the second leading cause of cancer death among men intheUnited States (1). Although themajority of patients arecurrently diagnosed with clinically localized disease,many patients develop biochemical failure after localdefinite therapy, for example, radiotherapy, perhaps dueto occult metastatic disease. Androgen deprivation ther-apy (ADT) in addition to radiotherapy improves overall

survival of patients at high risk for recurrence (2). Thisapproach, however, is associated with significant sideeffects. Given that patients with prostate cancer oftenpresent with low tumor burden, one potential treatmentis the incorporation of immunotherapy into a standardradiotherapy protocol (3). With recent approval of anantigen-presenting cell vaccine sipuleucel-T (Provenge)by the U.S. Food and Drug Administration, immunother-apy now represents an established treatmentmodality formetastatic prostate cancer (4, 5).

Several lines of evidence support an approach ofcombined radiotherapy and immunotherapy to achievelocal tumor control while at the same time generatingsystemic immune responses (6). Local irradiation not onlydebulks tumor but also generates an inflammatorymicro-environment. Tumor antigens released by dying tumorcells are captured and processed by specialized antigen-presenting cells [e.g., dendritic cells (DC)] in the contextof the costimulatory or "danger" signals, resulting ineffective T-cell activation (7). Radiotherapy also renderstumor cells more susceptible to recognition and attackby tumor-specific CTLs (8–10). Gulley and colleagues re-cently reported clinical trials in which radiotherapy com-bined with a prostate-specific antigen-targeted vaccine

Authors' Affiliations: Departments of 1Human & Molecular Genetics and2Radiation Oncology, 3VCU Institute of Molecular Medicine, 4VCUMasseyCancer Center, Virginia Commonwealth University, Richmond, Virginia;5Department of RadiationMedicine, LomaLindaUniversityMedicalCenter,Loma Linda, California; and Departments of 6Pharmacology and Thera-peutics and 7Cellular Stress Biology, Roswell Park Cancer Institute,Buffalo, New York

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Xiang-Yang Wang, Department of Human &Molecular Genetics, Virginia Commonwealth University School of Medi-cine, PO Box 980033, Richmond, VA 23298. Phone: 804-628-2679; Fax:804-628-1194; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0164

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on August 14, 2021. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 15, 2012; DOI: 10.1158/1535-7163.MCT-12-0164

http://mct.aacrjournals.org/

displayed a favorable toxicity profile and generatedsignificant T-cell responses in patients with prostatecancer (11, 12). Indeed, several phase II/III clinical trialsof radiotherapy in combination with therapeutic cancervaccines, for example, ProstAtakTM (NCT01436968),PSA/TRICOM (NCT00450619), L-BLP25 (Stimuvax,NCT01496131), for treatment of high-risk prostate cancerare ongoing.

Induction of immunity against self-antigens is oftenrestricted by multiple intrinsic inhibitory checkpoints ormolecules (13, 14). Knowledge of these inhibitory regu-lators has led to great progress in development of immu-notherapeutic strategies for cancer treatment (15). Wehave identified an immunosuppressive pathway in DCsinvolving the scavenger receptor SRA/CD204, which candampen immune responses against several cancers,including prostate cancer (16, 17). SRA/CD204 attenuatesthe immunostimulatory capability ofDCs and subsequentactivation of CTLs in the context of vaccine therapy andtumor immunity (18, 19). Recently,we showed that silenc-ing SRA/CD204 in primary DCs markedly enhanced thepotency of DC vaccines in mounting an effective antitu-mor T-cell response (20).

Considering the fact that SRA/CD204 absence signifi-cantly increases the immunogenicity of ionizing radia-tion–treated prostate cancer cells (17), we hypothesizethat local radiotherapy-induced cancer cell damage incombination with in situ vaccination with SRA/CD204-downregulated DCs could achieve improved systemicantitumor efficacy. In the present study, we provide thefirst experimental evidence that intratumoral administra-tion ofSRA/CD204-silencedDCsprofoundly enhances thecontrol of radiotherapy-treated local prostate cancer aswell as its metastases, which is mainly mediated by IFN-g–producingCD8þCTLs. Our data support the concept ofinhibiting SRA/CD204as anovel strategy tooptimizeDC-targeted immunotherapy that may be rationally com-bined with radiotherapy for treatment of human prostatecancer.

Materials and MethodsMice and cell lines

Wild-type (WT) C57BL/6 mice were obtained fromNational Cancer Institute (Bethesda, MD). SRA/CD204knockout mice (SRA�/�), OT-I mice bearing TCR specificfor OVA257–264 (SIINFEKL)were purchased fromThe Jack-son Laboratory. All experiments andprocedures involvingmice were approved by the Institutional Animal Care andUse Committee of Virginia Commonwealth University(Richmond,VA).Androgen-refractorymouseprostatecan-cer cell lines, RM1 (21) kindly provided by Dr. TC Thomp-son (Baylor College of Medicine, Houston, TX) andTRAMP-C2 (22), were used. RM1 cells expressing OVA(17) or luciferase were generated in our laboratory. All celllines were maintained in Dulbecco’s Modified Eagles’Media supplemented with 10% FBS (Hyclone), 2 mmol/LL-glutamine, 100 U/mL penicillin, and 100 mg/mL strep-

tomycin. These cells have been screened for mycoplasmaand other pathogens before in vivo use. No authenticationof the cell lines was conducted by the authors.

Short hairpin RNA–mediated gene silencing in DCsLentiviral vector encoding mouse SRA/CD204 short

hairpin RNA (shRNA)were packagedusing Phoenix cellsco-transfected with pLKO.1 constructs and pMD.G andpCMVDR8.91 (20). DCs were generated by culturingmouse bone marrow (BM) cells in the presence of gran-ulocytemacrophage colony-stimulating factor (GM-CSF).Lentiviral infection of primary DCs was carried out aspreviously described (20, 23). Briefly, bone marrow cellswere cultured in media containing recombinant mouseGM-CSF (20 ng/mL, Peprotech). Day 3 BM-DCs wereinfected with lentiviruses in the presence of 8 mg/mLpolybrene for 3 hours. Cells were then resuspended andplated in 12-well plates at 1 � 106 cells/mL. Day 8 DCswere collected and used for studies.

Tumor treatment and immunization protocolsMice were injected with 1 � 105 RM1 tumor cells or

2 � 106 TRAMP-C2 tumor cells subcutaneously in theright hind leg. When tumors reached 4 to 5 mm indiameter, mice were anesthetized using ketamine(80 mg/kg)/xylazine (10 mg/kg) and locally irradiatedwith a Picker 60Co source at 1 Gy/min. Mice received asingle dose of 10 Gy on each of 3 consecutive days. Forin situ DC immunization, mice were injected with DCs(2 � 106) into the tumors 24 and 72 hours after the lastradiation treatment. In some experiments, CD4þ, CD8þ

T cells, or natural killer (NK) cells were depleted byintraperitoneal (i.p.) injection of GK1.5, 2.43 or PK136monoclonal antibodies (mAb), respectively, as previ-ously described (16).

Experimental lung metastasesC57BL/6 mice bearing s.c. RM1 tumors were estab-

lished with lung metastases by intravenously injecting5 � 104 RM1-luciferase cells in PBS (without Ca2þ

or Mg2þ). Only tumors in the flank were treated.Mice were injected i.p. with D-luciferin (150 mg/kg,Caliper Life Sciences) and examined using a XenogenIVIS Imaging System. Lung metastatic nodules werealso enumerated. In the case of TRAMP-C2 tumor,1 � 106 cells were injected i.v. to generate pulmonarymetastases.

In vitro T-cell stimulationDCs were pulsed with tumor cell lysates (1:1 ratio) for

5 hours. After washing, serially diluted DCs were incu-bated with 5 � 104 OT-I cells in a round-bottom, 96-wellmicrotiter plate. Cells were cultured for 60 hours andpulsed with 3H-thymidine (0.5 mCi/well) during the last16 hours of culture period. T-cell proliferation wasassessed on the basis of 3H-thymidine incorporation.Levels of interleukin (IL)-2 in the supernatants weredetermined using ELISA.

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Enzyme-linked immunosorbent spot assay andintracellular IFN-g stainingSplenocytes or lymph node cells from treated mice

were analyzed for the frequency of antigen-specificIFN-g–secreting CD8þ T cells using enzyme-linkedimmunosorbent spot (ELISPOT) assays as previouslydescribed (19). For intracellular cytokine staining, cellswere stimulated with OVA257–264 peptides (AnaSpecInc.) for 3 days, followed by treatment with brefeldinA (BD GolgiPlug; BD Biosciences). Cells were thenstained with antibodies for CD8 and IFN-g and sub-jected to fluorescence-activated cell-sorting (FACS)analysis (19).

Cytotoxicity assaysAn in vivo CTL assay to determine antigen-specific

cytolytic activity of CD8þ T cells was conducted as pre-viously described (18). Briefly, naive splenocytes thatwere labeled with 12.5 mmol/L carboxyfluorescein succi-nimidyl ester (CFSE; i.e., CFSEhigh cells) using CellTrace5-(and 6-) CFSE Cell Proliferation Kit (Molecular Probes)and pulsed with OVA257–264 served as targets. Controlcells were labeled with 0.5 mmol/L CFSE (i.e., CFSElow

cells) and pulsed with irrelevant peptides. A mixture ofthese cell populations (total 5 � 106 cells) at 1:1 ratio wasinjected i.v. into treated mice, followed by FACS analysisof splenocytes 16 hours later.

Statistical analysisData are presented as mean� SD. Differences between

groups within experiments were analyzed using 2-tailedunpaired Student t test. Animal survival data wereanalyzed using log-rank test. P < 0.05 was consideredstatistically significant.

ResultsSRA/CD204 dampens the therapeutic effectivenessof conventional radiotherapyWepreviously showed thatmouse prostate tumor RM1

cells treated with ionizing radiation displayed increasedimmunogenicity in SRA�/� mice (17). Therefore, wesought to determine whether absence of SRA/CD204 intumor-bearing mice could amplify the antitumor efficacyof local radiotherapy. As previous reported, RM1 tumorsgrew aggressively at a similar rate in wild-type (WT) andSRA�/�mice before treatment (17). However, RM1 tumorgrowth was profoundly inhibited by radiotherapy inSRA�/�mice comparedwithWTmice (Fig. 1), suggestingthat SRA/CD204 ablation sensitizes RM1 tumors toradiotherapy.

In situ vaccination using SRA/CD204-deficient DCsenhances the effectiveness of radiotherapy bypromoting CD8þ T-cell activationSRA/CD204 is expressed in antigen-presenting cells,

and enhanced antitumor immunity in SRA�/� mice hasbeen shown to result from increased functions of SRA�/�

DCs exposed to tumor cell lysates (17) or stress proteins(19). We tested whether improved tumor control could beachieved by intratumoral delivery of SRA�/� DCs fol-lowing radiotherapy (Fig. 2A). Our initial studies showedthat fractionated radiotherapy (i.e., 3 consecutive 10 Gy)delayed tumor growth (Fig. 2B, left). Direct injections ofWTor SRA�/�DCswithout radiotherapyhadno effect ontumor growth (data not shown). Surprisingly, adminis-tration of WT DCs to the irradiated RM1 tumors failed toprovide any additional benefits (Fig. 2B, left). In contrast,in situ vaccination with SRA�/� DCs after radiotherapyresulted in increased tumor suppression compared withradiotherapy alone or radiotherapy plus WT DCs (Fig. 2,middle). The enhanced tumor control inmice treatedwithradiotherapy plus SRA�/� DCs was also associated withsignificantly improved animal survival (Fig. 2B, right).

To determine whether the increased tumor control bycombined radiotherapy and SRA�/� DC vaccine corre-lated with enhanced T-cell activation, RM1 tumorsexpressing a non-"self" model antigen OVA (RM1-OVA)were used to facilitate monitoring of antigen-specific Tcells. The frequency of IFN-g–producing CD8þ cells reac-tive with OVA257–264 epitope increased in mice treatedwith radiotherapy plus WT DCs. However, SRA�/� DCswere much more efficient than WT DCs in generatingOVA-specific CD8þ T cells when combined with radio-therapy (Fig. 2C).

Tumor immune evasion often involves a complex arrayof immunosuppressive mechanisms including inhibitionof T-cell–stimulating activity of DCs in the tumorenvironment. We compared the capability of WT andSRA�/� DCs to stimulate OVA-specific CD8þ T cells inthe presence of tumor-mediated immunosuppression.When cocultured with naive OT-I cells in RM1 tumor–conditionedmedia, OVA257–264-pulsed SRA�/�DCswereconsistently more effective than WT DCs in stimulating

Figure 1. SRA/CD204 ablation improves the effectiveness of radiotherapyagainst established prostate tumors. RM1 tumor–bearing WT andSRA�/� mice (n ¼ 5) were treated with or without radiotherapy (RT).��, P < 0.01. The results shown are representative of at least 3independent experiments.

Improving Radioimmunotherapy by Targeting CD204

www.aacrjournals.org Mol Cancer Ther; 11(11) November 2012 2333




OT-I cell proliferation andproduction of cytokine IL-2 andIFN-g (Fig. 2D). Interestingly, exposure of WT DCs withtumor-conditioned media caused upregulation of SRA/CD204 protein levels (data not shown).

SRA/CD204 silencing in DCs enhances cross-presentation of antigen from dying tumor cells

BM-DCs were genetically modified by infection withlentiviral vectors encoding shRNA specific for SRA/

CD204 or scrambled shRNA as previously described(20). SRA/CD204-silenced DCs (DC-SRA shRNA) pro-ducedmore pro-inflammatory cytokine IL-6 (Fig. 3A) andchemokine IP-10 (Fig. 3B) than scrambled shRNA-treatedDCs (i.e., DC-scram) upon exposure to dying RM1 tumorcells. The mRNA levels of the inflammatory genes (il6,ip10 and ifnb) similarly increased in DC-SRA shRNA(Supplementary Fig. S1A–S1C). Elevation of IL-6 expres-sion was further confirmed by intracellular cytokine

Figure 2. SRA/CD204 absence in DCs enhances the antitumor efficacy of radiotherapy (RT) combined with DC vaccination. A, scheme for the combinedradiotherapy and in situ DC vaccination. B, RM1 tumor–bearing mice (n ¼ 5) were treated with radiotherapy alone, radiotherapy plus WT DCs,or left untreated. WT DC vaccine failed to enhance tumor inhibition following radiotherapy (left). In contrast, administration of SRA�/� DC vaccinecombined with radiotherapy led to improved tumor control (middle) and survival rate (right). ��, P < 0.01, radiotherapy þ WT DC versus radiotherapy þSRA�/� DCs. C, enhanced T-cell activation by radiotherapy and SRA�/� DC vaccination. Splenocytes (top) and lymph node cells (bottom) from treatedRM1-OVA tumor–bearing mice were stimulated with OVA257–264 peptides. The frequency of IFN-g–producing CD8þ T cells was measured usingELISPOT (�, P < 0.01). D, resistance of SRA�/� DCs to tumor-mediated immunosuppression. OVA257–264-loaded DCs were cocultured with OT-I cells inthe presence of RM1 tumor–conditioned media (TCM) at indicated concentrations. T-cell proliferation was assessed using 3H-thymidine incorporationassays (top). Index ratios of T-cell proliferation driven by DCs (SRA�/� DCs/WT DCs) are also presented (bottom). In addition, the levels of IL-2and IFN-g in the supernatants were determined using ELISA (�, P < 0.01).

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staining (Supplementary Fig. S1D). These results are con-sistent with our previous observations that lack of SRA/CD204 sensitizes DCs to inflammatory signals associatedwith cell injury (17). In addition, DC-SRA shRNA pulsedwith RM1-OVA tumor lyastes were more efficient thanmock cells in stimulating OVA-specific OT-I cell activa-tion (Fig. 3C and D).

Radiotherapy combined with SRA/CD204-silencedDC vaccine results in enhanced therapeutic efficacyagainst local tumors and distant metastasesUsing a mouse prostate cancer RM1 model, we evalu-

ated the antitumor response augmented by radiotherapyand in situ vaccination with SRA/CD204-silenced DCs.DC-SRA shRNA, but not DC-scram, promoted growthinhibition of RM1 tumors and prolonged the survival ofradiotherapy-treated mice (Fig. 4A). The similar resultswere also obtained in a different mouse model of prostatecancer (Fig. 4B), TRAMP-C2, which was derived fromspontaneousprostate cancers arising inTRAMPmice (22).Although local radiotherapy of the primary tumor can

prevent development of subsequent systemic metastases,tumor radiation fails to control pre-existing systemicdisease. We used an experimental lung metastasis modelto assess the ability of the combination therapy to controldistant metastases. RM1-luciferase tumor cells wereinjected i.v. into WT mice 1 day before the implantation

of s.c. RM1 tumors. Treatment of s.c. tumors with radio-therapy alone or radiotherapy plus DC-scram vaccinepartially inhibited lung metastases. However, radiother-apy plus DC-SRA shRNA was far more effective thanother treatment modalities in controlling metastases (Fig.4C and D, left). The result was also validated using themetastasis model of TRAMP-C2 tumor (Fig. 4D, right).Mice receiving radiotherapyplusDC-SRA shRNAdidnotdisplay any signs of pathology or autoimmune toxicity inthe prostate (Supplementary Fig. S2A) and other organs(data not shown).

Radiotherapy combined with DC-SRA shRNAvaccination enhances activation of antigen-specificCTLs

The enhanced antitumor effect observed after the com-binatorial treatment using DC-SRA shRNA prompted usto examine systemic immune responses. Upon stimula-tion with OVA257–264 peptide, splenocytes from RM1-OVA tumor–bearing mice treated with radiotherapyplus DC-SRA shRNA exhibited increased proliferation(Fig. 5A, top) and IL-2 production (Fig. 5A, bottom)comparedwith those frommice treatedwith radiotherapyplus DC-scram. Mice receiving radiotherapy plus DC-SRA shRNA also showed increased frequency of OVA-specific, IFN-g–expressing CD8þ T cells, as shown byintracellular IFN-g staining assays (Fig. 5B) and ELISPOT

Figure 3. SRA/CD204 silencing inDCs promotes cross-presentationof RM1 tumor cell–associatedantigens. A and B, SRA/CD204downregulation renders DCs moreresponsive to stimulation with dyingtumor cells. DCs were infected withlentiviruses encoding scrambledshRNA or SRA shRNA and co-cultured with RM1 cell lysatesprepared after ionizing radiation.Protein levels of IL-6 (A) and IP-10 (B)in the media were determined usingELISA. The efficiency ofSRA/CD204silencing inDCswas assessed usingimmunoblotting (A, inset). C and D,increased CD8þ T-cell activation bySRA/CD204-silenced DCs. DCswere pulsed with RM1-OVA celllysates and cocultured with naiveOT-I cells. T cell proliferation wasassessed using 3H-thymidineincorporation assays (C). The IL-2concentration in the supernatantwas determined using ELISA (D).�, P < 0.01. The results shownrepresent 3 experiments.






assays (data not shown). Considering that OVA is a non-"self" antigen and there is no pre-existing tolerance inmice, we assessed T-cell recognition of 6 transmembraneepithelial antigen of the prostate (STEAP), an antigenhighly expressed in advanced human prostate cancersand mouse prostate tumors (24, 25). Integration of DC-SRA shRNA vaccine into the radiotherapy protocol effec-tively primed CD8þ T cells recognizing mSTEAP326–335

(Fig. 5C, left) or RM1 tumor cells (Fig. 5C, right). T-cellfunctions assays showed that radiotherapy-treated RM1-OVA tumor–bearingmice receiving DC-SRA shRNAvac-cine developed a stronger cytolytic response againstOVA257–264-pulsed targets in vivo (Fig. 5D) and RM1-OVAtumor cells in vitro (Supplementary Fig. S2B) comparedwith other treatment modalities.

SRA/CD204 silencing enhanced antitumor efficacy isdependent on IFN-g–producing CD8þ T cells

Terminal deoxynucleotidyl transferase–mediateddUTP nick end labeling (TUNEL) assays showed thatradiotherapy plus DC-SRA shRNA induced more RM1tumor cell death than radiotherapy alone or radiotherapyplus DC-scram. In parallel, radiotherapy plus DC-SRAshRNA also resulted in a significant increase in tumor-infiltratingCD8þ cells (Fig. 6A). Levels ofCD4þ cells in the

tumor tissues were comparable among treatment groups(Supplementary Fig. S3). Interestingly, increased tumorinfiltration by CD11cþ cells, presumably DCs, was alsoseen in mice receiving SRA/CD204-silenced DCs (Supple-mentary Fig. S3). These tumor-resident CD11cþ cellswere unlikely to be ex vivo genetically engineered DCsbecause the majority of DCs injected post-radiotherapymigrated out of the tumor site within 24 hours (data notshown).

ELISA of tumor tissues showed that the levels of IFN-g ,a cytokine critical for effective antitumor immunity, weresignificantly higher in mice receiving radiotherapy plusDC-SRA shRNA vaccination than other treatment groups(Fig. 6B, left). Removal of CD8þ cells by antibody deple-tion substantially decreased the intratumoral levels ofIFN-g , whereas depletion of CD4þ or NK1.1 cells onlyshowed a slight effect (Fig. 6B, middle). Intracellularstaining showed that tumor-infiltrating CD8þ cells frommice treated with radiotherapy and DC-SRA shRNAdisplayed markedly increased expression of IFN-g (Fig.6B, right) as well as granzyme B (Supplementary Fig. S4).Following radiation treatment, mice receiving DC-SRAshRNA showed a dramatic increase in the percentage ofIFN-gþCD8þ cells comparedwith IFN-gþCD4þ cells in thespleen (Supplementary Fig. S5). Furthermore, depletion

Figure 4. In situ vaccination with SRA/CD204-silenced DCs enhances the antitumor efficacy of radiotherapy. A, RM1 tumor–bearing mice (n ¼ 5) receivedradiotherapy alone, radiotherapy plus DC-scram or DC-SRA shRNA, or were left untreated. Tumor growth (left) and survival of RM1 tumor-bearingmice (right)were monitored. ��, P < 0.01, radiotherapy þ DC-SRA shRNA versus radiotherapy þ DC-scram. B, enhanced suppression of TRAMP-C2 prostate tumor bySRA/CD204-targeted combinatorial therapy. �, P < 0.01. C and D, treatment of local prostate tumors with radiotherapy (RT) plus DC-SRA shRNA reducesdistant metastases. Mice (n ¼ 5) were simultaneously established with s.c. "primary" RM1 tumors in the flank and lung metastases through i.v. injection ofRM1-luciferase cells. Tumors in the flank were subjected to treatment only. The metastases in the lungs were assessed using bioluminescence imaging (C).Lung tissues were collected fromRM1-tumor (left) or TRAMP-C2 tumor (right)-bearingmice following treatment of s.c. tumors, andmetastatic tumor nodulesin the lungs were counted (D). �, P < 0.05; ��, P < 0.01. Data are representative of 2 experiments with similar results. NS, not statistically significant.

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of CD8þ, not CD4þ or NK1.1 cells, abolished SRA/CD204silencing-enhanced antitumor efficacy (Fig. 6C). Finally,blocking IFN-g using neutralizing antibodies also abro-gated the therapeutic efficacy of the combinatorial ther-apy (Fig. 6D), suggesting that IFN-g–producing CD8þ

cells represent a key factor mediating SRA/CD204 silenc-ing-promoted antitumor immunity.

DiscussionGiven that radiotherapy is the primary treatment

option for high-risk localized prostate cancer and thebiochemical relapse rate of many of these patients ishigh, combining radiotherapy with immunotherapyrepresents an attractive strategy to improve clinical out-comes. In the present study, we provide the first exper-imental evidence that silencing SRA/CD204 in DCsmarkedly enhances antitumor effectiveness of radio-therapy combined with in situ DC vaccination. Theamplified systemic antitumor immunity by SRA/CD204downregulation in DCs not only confers an additionalinhibitory effect on radiotherapy-treated tumors but

also limits experimental metastatic outgrowth. Ourstudies establish the feasibility and efficacy of this novelcombinatorial radioimmunotherapy in the 2 preclinicalmodels of prostate cancer.

DCvaccine represents a promising immunotherapeuticapproach for cancer eradication (26). While DCs are oftenloaded ex vivo with tumor antigens or tumor lysates toinduce antitumor immune responses in conventional DCvaccination, in situ DC vaccination in a setting of localradiotherapy has also been reported to provide additionalantitumor benefits (27). The failure of WT DCs or DC-scram to synergize with local radiotherapy for controllingestablished RM1 tumor in our initial studywas unexpect-ed, which may be attributed to its poorly immunogenicnature or the immunosuppressionmediatedby this tumorline. However, absence or silencing of SRA/CD204 in DCswas able to effectively restore T-cell–mediated antitumorimmune responses when these modified DCs were intro-duced to the tumors treated with ionizing radiation. Thisfinding was also confirmed using a second mouse modelof prostate cancer (i.e., TRAMP-C2), which lends further

Figure 5. SRA/CD204 silencing enhances tumor-specificT-cell responses after radiotherapy (RT) combinedwith in situDCvaccination. A, increased activationof antigen-specific CD8þ T cells. RM1-OVA tumor-bearing mice were treated with radiotherapy alone or radiotherapy plus DC vaccines. Splenocytes werestimulated with OVA257–264 and analyzed for T-cell proliferation (top) and IL-2 production (bottom). B, the percentage of IFN-g–producing CD8þ T cells wasassessed using intracellular cytokine staining assays. C, enhanced immune cell recognition of prostate tumor antigen STEAP and RM1 tumor cells.Splenocyteswere stimulatedwithmSTEAP326–335 (left) or irradiatedRM1 cells (right). IL-2 or IFN-g productionwas assessed using ELISA. �,P < 0.01. Data arerepresentative of 2 experiments in which at least of 3 mice of each group were analyzed. D, increased cytolytic activity of effector T cells. One week after DCvaccination, RM1-OVA tumor–bearing mice (n¼ 3) were injected i.v. with OVA257–264-pulsed, CFSE

high splenocytes mixed with CFSElow splenocytes pulsedwith irrelevant peptides. Spleens were analyzed 16 hours later using flow cytometry. The percentage of killing of antigen-positive target is shown inparentheses. Representative histograms from 3 experiments with similar results are shown.






support to the previously proposed role of SRA/CD204 asan immunosuppressor capable of attenuating DC func-tions (16–20, 28).

Local radiotherapy is accompanied by the release oftumor antigen and endogenous "danger" signals (29),such as stress proteins (30), oxidized low-density

Figure 6. SRA/CD204 silencing-enhanced RM1 tumor control depends on IFN-g production by CD8þ T cells. A, RM1 tumor cell death is associated withincreased tumor-infiltrating CD8þ cells. One week after DC vaccination, tumor cell death was examined by TUNEL assays. Nuclei were counterstainedwith 40,6-diamidino-2-phenylindole (DAPI). Cryosections of tumor tissues were stained with antibodies for CD8 (green). Scale bar indicates 50 mm.Quantitative analysis of infiltrating cells was conducted by counting positive cells in at least 5 randomly selected fields of view (right). �,P < 0.05. B, CD8þ cellsprimarily contribute to IFN-g production. IFN-g levels increased in RM1 tumor tissues after treatment with radiotherapy (RT) plus DC-SRA shRNA, asdetermined using ELISA (left). Depletion of CD8 cells before combined therapy reduced IFN-g levels in tumor tissues (middle). In addition, tumor-infiltratingCD8þ cells were analyzed for IFN-g expression with intracellular cytokine staining assays (right). C, CD8þ cells are required for SRA/CD204 silencing-enhanced tumor inhibition. RM1 tumor–bearingmice (n¼5)weredepletedof effector cell subsetsbefore the combinatorial therapy.D, IFN-g is critical forSRA/CD204 silencing-facilitatedantitumor activity.RM1 tumor–bearingmice (n¼5) receiving radiotherapy (RT) plusDC-SRAshRNAwere administratedwith IFN-gneutralizing antibodies or control IgG. NS, not statistically significant. �, P < 0.05; ��, P < 0.01. Data shown are representative of 2 experiments.

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lipoprotein (LDL; ref. 31), and high-mobility group box 1protein (HMGB1; ref. 32). It is likely that SRA/CD204limits the functional activation of DCs exposed to thesedamage-associatedmolecules or "danger" signals (33, 34).Out data support this hypothesis by showing that SRA/CD204 silencing renders DCs more responsive to stimu-lation with dying RM1 tumor cells, as evidenced byincreased expression of inflammatory mediators and T-cell–stimulating capability. These results also coincidewith our recent observation of significantly heightenedimmunogenicity of dying prostate tumor cells in SRA�/�

mice (17). In addition, lack of SRA/CD204 in DCs alsoenhances antigen cross-presentation, CTL activation, andantitumor responses induced by stress proteins as immu-nostimulators (16, 19).It was reported that dying tumor cells produced by

cancer therapies (e.g., radiotherapy or chemotherapy)augment a TLR4-dependent immune response (35, 36),which has been attributed to an essential role of TLR4signaling in processing and cross-presentation of tumorcell–associated antigens by DCs. Recently, our molecularstudies revealed that SRA/CD204 attenuated TLR4-engaged NF-kB signaling in DCs through blockade ofTRAF6 ubiquitination and oligomerization (37). The sim-ilar molecular mechanism may underlie SRA/CD204-mediated immune suppression in DCs upon stimulationwith "danger" molecules released by ionizing radiation–treated RM1 tumor cells. SRA/CD204 downregulationmay also enable DCs more resistant to tumor-mediatedimmune suppression, as shown in the current study and arecent report that SRA/CD204 contributes toDCdysfunc-tion in tumor-bearing mice and patients with cancer (28).In situDCvaccination described here can target the highlyindividualized tumor antigens as well as inflammatorysignals released from dying cancer cells following radio-therapy. Considering the immunomodulating effects ofradiotherapy, for example, sensitizing cancer cells to T-cell–mediated attack (8–10), andpromoting T-cell traffick-ing (38),weexpect that radiotherapyandconventionalDCvaccination with antigen-loaded, SRA/CD204-silencedDCs should also display enhanced synergistic antitumorefficacy.Combining radiotherapy and in situ vaccination with

SRA/CD204-silenced DCs is highly effective in stimu-lating a systemic, antigen-specific CTL-mediated anti-tumor immune response. As a result, a significantelevation in the number of tumor-infiltrating CD8þ

cells, the intratumoral IFN-g levels, and the frequencyof IFN-g–expressing CD8þ cells is achieved followingthe combinatorial therapy, which also positively corre-lates with increased tumor cell death. The supportingevidence also derives from the result showing thatantitumor efficacy of radiotherapy and DC-SRAshRNA vaccination is abrogated after IFN-g neutrali-zation or in the absence of CD8þ cells. Together withthe observation of diminished IFN-g level in the tumorbed upon CD8þ cell depletion, we conclude that CD8þ

T cells predominantly contribute to the improved

tumor control by SRA/CD204-silenced DC vaccine fol-lowing radiotherapy.

Our findings are in contrast to the previous studyby Teitz-Tennenbaum and colleagues (39) in whichenhanced therapeutic efficacy of adoptively transferredTcells following radiotherapyandDCadministrationwasassociated with a selective increase in proliferation andfunction of CD4þ T cells. Several differences betweenthese 2 studies may contribute to this discrepancy.While our research is centered on SRA/CD204 silencing-enhanced endogenous tumor-reactive T-cell response,their study primarily focused on the activity of adoptivelytransferred T cells. Treatment regimens used in our study(i.e., radiotherapy plus genetically engineered DCs) arealso different from the one used by Teitz-Tennenbaumand colleagues (DC vaccination following radiotherapyand lymphodepletion plus adoptive T-cell transfer). Thedifferent observations could also derive from the use ofdifferent tumor models. It should be noted that the con-tribution of CD4þ T cells to tumor suppression was notdirectly examined in their study.

Radiotherapy is commonly combined with ADT fortreatment of patients with high-risk and locally advancedprostate cancer today (40). The addition of neoadjuvantand adjuvant ADT can significantly delay tumor progres-sion and improve disease-free and overall survival.Considering that immunotherapy (e.g., SRA/CD204-targeted DC vaccine) engages a complementary antitu-mor mechanism by targeting the host immune system, ittherefore has the potential to further promote the additiveor synergistic antitumor effects of multimodality therapy(e.g., radiotherapy plus ADT), leading to effective erad-ication of recurrence and metastases.

In summary, we have shown that selective downregu-lation of SRA/CD204 in DCs can overcome immunetolerance and enhance antitumor potency of radiotherapycombinedwith in situDC-based vaccination. Strategicallycombining radiotherapy with SRA/CD204-silenced DCscould alter or modify the tumor environment, shifting thebalance in favor of immune activation. Further studies arewarranted to determine whether multimodality therapycombining standard radiotherapy, SRA/CD204-targetedvaccines, and/or other novel approaches that effectivelybreak tumor-mediated immunoregulatory mechanisms(41) can be successfully translated into improved clinicaloutcomes.

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

Authors' ContributionsConception and design: G. Yang, P.B. Fisher, X.-Y. WangDevelopment ofmethodology:C.Guo,H. Yi, D.Zuo,G. Yang, X. Sun, R.B.MikkelsenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Guo, H. Yi, X. Yu, D. Zuo, J. Qian, G. Yang,X. Sun, R.B. MikkelsenAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): C. Guo, H. Yi, D. Zuo, B.A. Foster, P.B.Fisher, X.-Y. Wang






Writing, review, and/or revision of themanuscript:C. Guo, B.A. Foster, J.R. Subjeck, P.B. Fisher, X.-Y. WangStudy supervision: J.R. Subjeck, X.-Y. Wang

AcknowledgmentsThe authors thank Dr. Tim Thompson (Baylor College of Medicine) for

kindly providing us with the RM1 cell line.

Grant SupportThis work was supported in part by American Cancer Society Schol-

arship RSG-08-187-01-LIB (X.-Y. Wang), NIH CA129111 (X.-Y. Wang),

CA154708 (X.-Y. Wang), CA099326 (J.R. Subjeck), CA90881 (R.B. Mikkel-sen), CA097318 (P.B. Fisher), DOD Prostate Cancer Research ProgramW81XWH-10-PCRP-SIDA (P.B. Fisher and X.-Y. Wang), HarrisonEndowed Scholarship (X.-Y. Wang), and NCI Cancer Center supportgrants to Massey Cancer Center.

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 February 15, 2012; revised July 23, 2012; accepted August 8,2012; published OnlineFirst August 15, 2012.

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2012;11:2331-2341. Published OnlineFirst August 15, 2012.Mol Cancer Ther Chunqing Guo, Huanfa Yi, Xiaofei Yu, et al. Cancer

ProstateUnmodified Dendritic Cell, Enhances Radiation Therapy of Gene-Silenced Dendritic Cell, notCD204 Vaccination with In Situ

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InSitu CD204 Gene-SilencedDendriticCell, not Unmodiﬁed … · tration of WT DCs to the irradiated RM1 tumors failed to provide any additional beneﬁts (Fig. 2B, left). In contrast,