Research Article

Exosomes Shuttle TREX1-SensitiveIFN-Stimulatory dsDNA from IrradiatedCancer Cells to DCsJulie M. Diamond1,2, Claire Vanpouille-Box2, Sheila Spada2, Nils-Petter Rudqvist2,Jessica R. Chapman3, Beatrix M. Ueberheide3,4, Karsten A. Pilones2, Yasmeen Sarfraz2,Silvia C. Formenti2, and Sandra Demaria2,5

Abstract

Radiotherapy (RT) used at immunogenic doses leads toaccumulation of cytosolic double-stranded DNA (dsDNA) incancer cells, which activates type I IFN (IFN-I) via the cGAS/STING pathway. Cancer cell–derived IFN-I is required torecruit BATF3-dependent dendritic cells (DC) topoorly immu-nogenic tumors and trigger antitumor T-cell responses incombination with immune checkpoint blockade. We havepreviously demonstrated that the exonuclease TREX1 regulatesradiation immunogenicity by degrading cytosolic dsDNA.Tumor-derived DNA can also activate cGAS/STING-mediatedproduction of IFN-I by DCs infiltrating immunogenic tumors.However, how DNA from cancer cells is transferred to thecytoplasm of DCs remains unclear. Here, we showed thattumor-derived exosomes (TEX) produced by irradiatedmouse

breast cancer cells (RT-TEX) transfer dsDNA to DCs andstimulate DC upregulation of costimulatory molecules andSTING-dependent activation of IFN-I. In vivo, RT-TEX elicitedtumor-specific CD8þ T-cell responses andprotectedmice fromtumor development significantly better than TEX fromuntreated cancer cells in a prophylactic vaccination experi-ment. We demonstrated that the IFN-stimulatory dsDNAcargo of RT-TEX is regulated by TREX1 expression in the parentcells. Overall, these results identify RT-TEX as a mechanismwhereby IFN-stimulatory dsDNA is transferred from irradiatedcancer cells to DCs. We have previously shown that theexpression of TREX1 is dependent on the RT dose size. Thus,these data have important implications for the use of RT withimmunotherapy. Cancer Immunol Res; 1–11. �2018 AACR.

IntroductionIt is well recognized that harnessing the power of the immune

system with immune checkpoint blocking antibodies (ICB) leadsto the successful elimination of cancer in a subset of patientsacross different malignancies. However, for the majority ofpatients, additional interventions are required to elicit sufficientantitumor T cells and achieve responses to ICBs (1). Among them,tumor-targeted radiotherapy (RT) has shown synergy with ICB inpreclinical studies and promising results in patients and iscurrently under investigation in several trials (2, 3). Priming ofantitumor T cells by RT is due, at least in part, to its ability toinduce an immunogenic cell death (ICD) of the cancer cells (4, 5).

ICD is a type of regulated cell death associated with the exposureand release of danger signals that activate DCs to uptake andcross-present tumor antigens to T cells (6). One of the criticalsignals for the spontaneous activation of antitumor T cells againstimmunogenic tumors, as well as RT-induced T-cell priming, isdsDNA (7, 8). Cytosolic tumor-derived dsDNA is sensed by cyclicGMP-AMP synthase (cGAS) in DCs to generate cGAMP requiredfor the activation of the adaptor STING (stimulator of interferongenes), resulting in the production of IFNb and induction ofseveral interferon-stimulated genes (7). DC phagocytosis of thecancer cells with the blockade of the CD47-signal regulatoryprotein alpha (SIRPa) axis was shown to result in the releaseof DNA into the DC cytosol (9). However, the mechanismswhereby dsDNA derived from the cancer cells reaches the cytosolof DCs in the absence of CD47-SIRPa blockade remain incom-pletely understood.

Exosomes are membrane microvesicles (30–100 nm) secretedfrom all cell types, which provide a sophisticated means of localand distal intercellular communication (10). Tumor-derived exo-somes (TEX) have been shown to shuttle a variety of bioactivemolecules between cells, including proteins, miRNAs, andmRNAs (11–15). Importantly, TEX also carry dsDNA, which ispresent in cancer cell–derived exosomes in variable but largerquantities than in exosomes derived from normal cells (16).Overall, the molecular profiles of exosomes reflect their cell oforigin. For instance, TEX carry tumor antigens that can be trans-ferred to DCs and elicit tumor-specific immune responses (14).However, for the most part, TEX have been shown to promoteimmunosuppressive and protumorigenic effects, a dichotomythat may depend on the molecular context of the parent and

1Department of Pathology, New York University School of Medicine, New York,New York. 2Department of Radiation Oncology, Weill Cornell Medicine, NewYork, New York. 3Proteomics Laboratory, New York University School ofMedicine, New York, New York. 4Department of Biochemistry and MolecularPharmacology, New York University School of Medicine, New York, New York.5Department of Pathology and Laboratory Medicine, Weill Cornell Medicine,New York, New York.

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

Current address for J.R. Chapman: Department of Pathology, Memorial SloanKettering Cancer Center, New York, New York.

Corresponding Author: Sandra Demaria, Weill Cornell Medicine, 1300 YorkAvenue, Box 169, New York, NY 10065. Phone: 646-962-2092; Fax: 212-746-0000; E-mail: szd3005@med.cornell.edu

doi: 10.1158/2326-6066.CIR-17-0581

�2018 American Association for Cancer Research.

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recipient cells (13, 17, 18). Notably, it has been shown thattreatment-induced molecular changes in cancer cells can lead tothe production of TEX with similarly altered cargo that can betransferred to recipient cells (19–21). Thus, we hypothesized thatkey molecular changes that occur in cancer cells treated withradiationmay be detected in the TEX that they produce and resultin enhanced TEX immunogenicity.

Here, we demonstrated that TEX released from TSA mousebreast carcinoma cells irradiated with 8 Gy � 3 (RT-TEX), anoptimally immunogenic dose and fractionation for this tumor(22, 23), have an altered molecular composition comparedwith TEX from untreated cells (UT-TEX). We have previouslyshown that 8 Gy � 3 RT induces the accumulation of dsDNA inthe cytosol of TSA cells that activates the IFN-I pathwayvia cGAS/STING (23). Consistent with our hypothesis, onlyRT-TEX carried dsDNA capable of inducing IFN-I productionby DCs in a STING-dependent fashion. IFN-stimulatorydsDNA carried by RT-TEX was regulated by the expression ofthe three-prime repair exonuclease 1 (TREX1) in the parentcells, and RT-TEX elicited significantly better protective antitu-mor immunity than UT-TEX when used to vaccinate mice.Overall, these results provide a mechanism whereby dsDNAderived from irradiated cancer cells reaches the cytoplasmof DCs and highlight the importance of TREX1 as a regulatorof RT immunogenicity (24).

Materials and MethodsMice

Six-week-old BALB/c and C57BL/6 female mice and B6(Cg)-Tmem173tm1.2Camb/J (STING-deficient) mice were purchasedfrom The Jackson Laboratory. All mice were maintained underpathogen-free conditions in the animal facility at Weill CornellMedicine (New York, NY). All experiments were approved bythe Institutional Animal Care and Use Committee of WeillCornell Medicine.

Tumor cell cultureTSA is a BALB/c mouse–derived mammary carcinoma (provid-

ed by P.L. Lollini, University of Bologna, Bologna, Italy; ref. 25).A20 is a BALB/cmouse–derived B-cell leukemia/lymphoma (pro-videdbyG. Inghirami,Weill CornellMedicine,NewYork; ref. 26).B16.Flt3L cells, genetically engineered to stably secrete Flt3-ligand(27), were a gift from Boris Reizis (NYU School of Medicine, NewYork). TSA cell derivatives containing a doxycycline-inducibleTrex1 gene (TSAKI Trex1), TSA cells expressing a doxycycline-induc-ible shRNA nonsilencing construct (TSAshNS), and shRNA con-structs targeting Cgas (TSAshcGAS) and Sting (TSAshSTING) werepreviously described (23). Cells were authenticated by morphol-ogy, phenotype, and growth and routinely screened forMycoplas-ma (LookOut Mycoplasma PCR Detection kit, Sigma-Aldrich).Cells were cultured in DMEM (Invitrogen Corporation) supple-mented with L-glutamine (2 mmol/L), penicillin (100 U/mL),streptomycin (100 mg/mL), 2-mercapthoethanol (2.5� 105 mol/L), and 10% FBS (Life Technologies). For B16.Flt3L culture,nonessential amino acids (Invitrogen Corporation) andsodium pyruvate (1 mmol/L; Invitrogen Corporation) wereadded. Cells were cultured for the minimum time requiredto achieve sufficient expansion, approximately 1 week or 3 to 5passages for the preparation of exosome stocks in variousexperiments.

Exosome isolation and purificationTSA cells were cultured in complete medium as described

above. To induce the expression of Trex1 or to induce the knock-down of Cgas and Sting, 7 � 105 TSAKI Trex1, TSAshNS, TSAshcGAS,and TSAshSTING cells were plated in T75 flask in media containingdoxycycline (4mg/mL) 4 days prior to RT, as previously described(23). Cells either received Sham RT or 8 Gy on 3 consecutive daysusing the Small Animal Radiation Research Platform (SARRPXstrahl Ltd). Following the last radiation exposure, culture medi-um was replaced with DMEM-containing antibiotic solutions asoutlined above and10%exosome-depleted FBS (Exo-FBS, SystemBiosciences, Inc.). Supernatants from sham- or RT-treated cellswere collected 48 hours later, and exosomes were isolated bysequential centrifugation as previously described (28). Briefly, cellsupernatant was sequentially centrifuged at 2000 � g for 20minutes at 4�C and 10,000 � g for 30 minutes at 4�C to removecells and cellular debris. The supernatant was then centrifuged at100,000 � g for 70 minutes at 4�C, and the pellet containingexosomes was collected, washed in cold PBS (Invitrogen), andcentrifuged again at 100,000 � g for 70 minutes at 4�C. Thewashed exosomes were further purified by a sucrose step gradientas described (29). Total exosomal protein was measured by theBradford Protein Assay (BioRad). The yield was 0.4 to 0.6 mg TEX/106 TSA cells. To confirm the morphological appearance ofpurified TEX by transmission electron microscopy (TEM), 5 mLof TEX in PBS were placed onto glow-discharged (Bench TopTurbo, Denton Vacuum) home-made carbon-coated 400 meshCu/Rh grid (Ted Pella Inc.). After staining with 1% uranyl acetatein distilled water (Polysciences, Inc.), the grids were examinedunder Philips CM-12 electron microscope and photographedwith a Gatan (4k � 2.7k) digital camera.

Mass spectrometryTEX protein samples were prepared in biological triplicate for

each group (UT-TEX and RT-TEX), with 400 mg of exosomalprotein per sample. Briefly, sucrose-purified TEX were lysed inSDS detergent containing buffer and prepared for mass spectro-metric analysis using Filter Assisted Sample Preparation proce-dure (30). The tryptic peptides were eluted, desalted, and aliquotsof the peptide mixtures were injected onto an Acclaim PepMap100 (2 cm � 75 mm ID) trap column in line with an EASY Spraycolumn (50 cm � 75 mm ID; PepMap RSLC C18, 2 mm) with theautosampler of an EASY nLC 1000 interfaced to a Q ExactiveOrbitrap mass spectrometer (Thermo Fisher Scientific). Peptideswere gradient eluted using the following gradient (solvent A: 2%acetonitrile in 0.5% acetic acid and solvent B: 90% acetonitrile in0.5% acetic acid): 5% to 40% in 60 minutes, 40% to 100% in 10minutes, and followed by 100% for 20 minutes. High-resolutionfullMS spectra were acquiredwith a resolution of 70,000, an AGCtarget of 1e6, with amaximum ion time of 120ms, and scan rangeof 400 to 1500 m/z. Following each full MS scan, 20 high-resolution HCD MS/MS spectra were acquired. The MS/MS spec-tra were collected at a resolution of 17,500, an AGC target of 5e4,maximum ion time of 120 ms, one microscan, 2.0 m/z isolationwindow, fixed first mass of 150 m/z, dynamic exclusion of 30seconds and a normalized collision energy of 27.

Peptide identification, protein grouping, and protein quanti-tation was performed using the label-free quantitation (LFQ)algorithm in the MaxQuant software suite version 1.5.0.12searched against a Uniprot Mus musculus database (31, 32). Forthe first search, the peptidemass tolerance was set to 20 ppm, and

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for the main search, peptide mass tolerance was set to 4.5 ppm.Trypsin-specific cleavage was selected with two missed cleavages.Both peptide spectral match and protein FDR were set to 1% foridentification. Carbamidomethylation of cysteine was added as astatic modification. Oxidation of methionine and acetylation ofN-termini were allowed as variable modifications. Protein quan-titation was performed using unique and razor peptides. The dataset was filtered to include proteins identified with two or moreunique and/or razor peptides and proteins that were detected inall three replicates of at least one treatment. A two-sidedStudent t test was performed, correcting for multiple testingby controlling for FDR at 5% using a permutation method.Proteins with a q value of <0.05 were considered significant. Atotal of 684 proteins met the filter requirements, and of those,158 were defined as significant by a q value of <0.05. Prote-omics data have been uploaded to the MassIVE (accessionnumber MSV000081341) and ProteomeXchange (accessionnumber PXD007069) repositories.

Pathway analysisQIAGEN's Ingenuity PathwayAnalysis software (IPA; Ingenuity

Systems) was used to determine upstream regulators and impacton canonical pathways (Fisher exact test, P values <0.05). Apathway or upstream regulator was determined activated orinhibited for |z-scores| � 2 (positive z-score: activation; negativez-score: inhibition).

TEX vaccination and tumor challengeBALB/c mice were vaccinated s.c. near the base of the tail

with TEX (20 mg/mouse, n ¼ 6/group). Mice received boostvaccinations 7 and 14 days later. PBS was used for control mice.Seven days after the last immunization, mice were challenged s.c. with 1� 105 TSA tumor cells. Tumor growth was monitored 2to 3 times weekly for 4 weeks. Perpendicular tumor diameterswere measured with a Vernier caliper, and tumor volumeswere calculated as length � width2 � 0.52. Mice were sacrificed20 days after tumor inoculation for analysis of the immuneinfiltrate.

Analysis of tumor-infiltrating CD8þ T cellsTumors were enzymatically digested using Miltenyi's Mouse

Tumor Dissociation Kit on a gentleMACS Octo Dissociator(Miltenyi Biotec). Following dissociation, red blood cell lysiswas performed, and the cell suspension was filtered through a40-mm strainer to remove large debris. Cells were blocked withCD16/32 antibody to Fc gamma III and II receptors (ThermoFisher Scientific, Clone 93) and were then stained for flowcytometry analysis using Fixable Viability Dye eFluor 506(Thermo Fisher Scientific), APC-Cy7–conjugated anti-CD45(Affymetrix, eBioscience; Clone I3/2.3), VioBlue-conjugatedanti-CD8a (Miltenyi Biotec; Clone 53–6.7), and R-PE–labeledPro5 MHC class I pentamers linked to the MuLV env gp70423-431 (AH1 peptide, SPSYVYHQF; ProImmune Ltd.). Con-trol pentamers were loaded with irrelevant MCMV IE1 168-176peptide (YPHFMPTNL). After staining, cells were fixed with 4%paraformaldehyde, and samples analyzed using a MACSQuantAnalyzer 10 (Miltenyi Biotec), and FlowJo software (version8.8.7). Cell density was calculated by dividing the totalnumber of cells staining positive for a given marker for thetumor volume.

Analysis of tumor-reactive CD8þ T cells from spleenT cells were isolated from the spleen of TSA tumor-bearingmice

using a Pan T-Cell Isolation Kit II (Miltenyi Biotec), following themanufacturer's protocol. A total of 1 � 106 T cells were thenculturedwithMouse T-Activator CD3/CD28Dynabeads (ThermoFisher Scientific) in 1 mL T-cell medium supplemented withhuman rIL2 (10 U/mL; provided by the Biological ResourcesBranch, Developmental Therapeutics Program, Division ofCancer Treatment and Diagnosis, National Cancer Institute) for4 days in a 24-well tissue culture plate. Beads were removed bymagnetic separation, and activated T cells were then cultured with1� 104 irradiated TSA or A20 lymphoma cells at effector to targetratio 20:1 for 4 hours. GolgiPlug containing Brefeldin A (1 mL per1mL culture media, BD Biosciences) was added for 8 hours toinhibit intracellular protein transport. T cells were then washedand stained with Fixable Viability Dye eFluor 506 (Thermo FisherScientific). Cells were then stained for surface markers withAPCcy7-conjugated anti-CD45 (Affymetrix, eBioscience; CloneI3/2.3) and Vio-Blue-conjugated anti-CD8a (Miltenyi Biotec,Clone 53-6.7). Intracellular staining was then performed for IFNgusing an Intracellular Fixation and Permeabilization Buffer Set(Thermo Fisher Scientific) according to the manufacturer's pro-tocol followed by staining with FITC-conjugated anti-IFNg(Thermo Fisher Scientific; Clone XMG1.2). Cells were analyzedas described above.

TEX labeling and uptake kinetics by DC in vivoPurified TEXwerefluorescently labeledwith PKH67membrane

dye (Sigma-Aldrich), following the manufacturer's protocol.Labeled exosomes were washed in 30 mL of exosome-free mediaand collected by ultracentrifugation at 100,000� g for 70minutesat 4�C. Absence of dye contamination was verified using a PBScontrol. Labeled TEX (10 mg) was injected s.c. near the base of thetail. Thedraining inguinal lymphnodewasharvested 24, 48, or 72hours after injection for flow cytometric analysis (n ¼ 3 mice/group at each time point). Cell suspensions were stained withBV421-conjugated anti-CD11c (BioLegend; Clone N418), andanalyzed as described above.

Isolation of primary DCsTo improve the yield of DCs, C57BL/6 female mice were

injected s.c. with 7 � 105 B16.Flt3L, a cell line genetically engi-neered to stably secrete fms-related tyrosine kinase 3 ligand (Flt3L;refs. 27, 33). Spleenswere harvested 14days later andprocessed asdescribed (34). Briefly, to prepare single cell suspensions, spleenswereminced in RPMI, and the obtainedmixture was filtered usinga Falcon 40-mm strainer (Corning). Following red blood cell lysisby ACK buffer, the cell suspension was filtered again. Afterwashing in PBS, cells were blocked with CD16/32 antibody toFc gamma III and II receptors (ThermoFisher Scientific;Clone93),followed by incubation with CD11c microbeads ultrapure(Milteny Biotec) and loaded onto a MACS column (MiltenyBiotec) for the positive selection, as outlined in the kit protocol.

DC culture with TEXPrimary CD11cþ DCs were cultured with purified TEX (30 mg

TEX per 1 � 106 DCs) or with TLR3 agonist poly:ICLC (0.025mg/mL; Sigma-Aldrich) for 48 hours in medium supplementedwith exosome-depleted FBS. Cells were collected for analysis byflow cytometry and quantitative PCR with reverse transcription(qRT-PCR), as described below. Cell culture supernatants were

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collected formeasurement of secreted IFNb usingHigh SensitivityMouse IFNb ELISA Kit (PBL Assay Science).

For phenotypic analysis, 2� 105 DCs were washed and stainedwith Fixable Viability Dye (eFluor 506, Thermo Fisher Scientific),FITC–anti-CD11c (Thermo Fisher Scientific; Clone N418), PE–anti-CD40 (Thermo Fisher Scientific; Clone 1C10), PE-Cy7–anti-CD80 (Thermo Fisher Scientific; Clone 16-10A1), and PacificBlue–anti-CD86 (Thermo Fisher Scientific; Clone GL1), acquiredby a MACSQuant Analyzer (Miltenyi Biotec), and analyzed withFlowJo software (version 8.8.7).

Quantitative PCR with reverse transcription (qRT-PCR)Total RNA from DCs was isolated using RNeasy Micro Kit

(Qiagen). Real-time PCR was performed using the AppliedBiosystems 7500 real-time PCR cycler (Thermo Fisher Scientific).One microgram of RNA was used for cDNA synthesis, performedwith SuperScript IV VILO Master Mix (Thermo Fisher Scientific),followed by real-time RT-PCR with the DyNAmo Flash SYBRGreen qPCR Kit (Thermo Fisher Scientific), according tothe manufacturer's protocol. Samples were normalized to house-keeping genes, and expression on untreated cells was assigneda relative value of 1.0. The PrimePCR SYBR Green assayprimers (BioRad) used in this study were UniqueAssay ID:qMmuCED0050444 for mouse Ifnb1, qMmuCID0023356 formouse Mx1, qMmuCED0046382 for mouse Ifnar1, andqMmuCED0027497 for mouse Gapdh. Data were analyzed withthe 7500 Dx Instrument's Sequence Detection software (ThermoFisher Scientific). To calculate the relative gene expression, the 2(�DDCt) method was used. Experiments were performed inbiological triplicate, and statistical significancewas assessed usingunpaired, two-tailed t test.

Quantification of exosomal dsDNAdsDNA was quantified from 5 mg of TEX, with or without

DNase I pretreatment (0.15 units/mL, Sigma-Aldrich) using theSpectraMax Quant AccuBlue Pico dsDNA Assay Kit (MolecularDevices) as described (23). The samples were prepared accordingto themanufacturer's protocol and analyzed using the FlexStation3 multimode microplate reader.

Statistical analysisData are presented as mean� SEM. For comparisons with only

two groups, P values were calculated using unpaired Student two-

tailed t tests. The two-way ANOVA test was used for tumor growthcurve analyses. The SEMare indicated by errors bars for each groupof data. Differences were considered significant at P values below0.05. All data were analyzed using GraphPad Prism software(GraphPad version 7).

ResultsExosomes secreted by irradiated cancer cells show an alteredproteomic profile

TEX can carry multiple tumor antigens and deliver them torecipient DCs for presentation to T cells (14). However, TEX havelimited immunogenicity, which can be increased bymodificationof the parent cells, for example, by heat stress (35), suggesting thatincreased adjuvanticity of cancer cells can be transferred to theexosomes they secrete.

We have shown that RT can increase tumor immunogenicityand induce the generation of tumor-specific CD8þ T cells thatmediate the regression of the irradiated tumors, as well as syn-chronous nonirradiated tumors in synergy with ICB (22). Becausethe proimmunogenic effects of RT are dependent on the dose andfractionation (23), for these experiments, we used an optimal RTregimen of 8 Gy� 3 to irradiate themouse breast cancer cells TSAand test whether this treatment altered the molecular composi-tion of the secreted TEX. TEX were isolated from the supernatantsof untreated and RT-treated TSA cells using a two-step procedureto achieve a high purity and theirmorphology verified by electronmicroscopy (Fig. 1A). Next, the proteomic profile of RT-TEX wascompared with UT-TEX using label-free quantitation mass spec-trometry (LFQ-MS). This analysis revealed significant differencesbetween the two groups, with RT-TEX containing 114 proteinsnot detected in UT-TEX (Supplementary Table S1 and Supple-mentary Excel Sheet 1; entire data set available at Proteome-Xchange ID: PXD007069). The shared proteins (27.6%) werefound at significantly different levels in the two groups (q <0.05). To understand the biological context of the differencesbetween UT-TEX and RT-TEX, the IPA tool was used to compareall the proteins identified in each TEX group. Whereas 153pathways were shared between the two groups, 10 pathwayswere unique to RT-TEX and 15 pathways were unique toUT-TEX (Fig. 1B). Interferon signaling was one of the 10 path-ways unique to RT-TEX (Table 1), suggesting that RT-TEX cargoreflects the cellular environment of irradiated TSA cells, which

Figure 1.

Isolation and proteomic profiling ofTEX. A, Schema of the TEX isolationmethod. TSA mouse breast cancercells were left untreated (UT) orirradiated with 8 Gy � 3 (RT) andsupernatants were collected after 48hours (h) for TEX purification asindicated. Purified TEX were analyzedby transmission electron microscopy(EM) and analyzed by LFQ-MS. B,Venn diagram illustrating the numberof shared and unique pathwaysrepresented in each TEX group.White:RT-TEX; dark gray: UT-TEX; light gray,overlap.

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display cancer cell–intrinsic IFN-I pathway activation (23).Finally, among the 153 shared pathways, 21 were predictedto be activated in RT-TEX compared with UT-TEX, whereas only3 pathways were predicted to be inhibited (SupplementaryTable S2).

STING-dependent activation of dendritic cells by exosomesfrom irradiated cancer cells

IFN-I is essential for activation of DCs and priming of antitu-mor T cells (36, 37). Because immunogenic RT induces IFN-Iproduction by cancer cells (23), and the proteomics analysisindicated that IFN-I pathway was selectively upregulated in RT-TEX, we next tested the effects of RT-TEX onDC activation. To thisend, primary CD11cþDCs were isolated from the spleens ofmiceand cultured ex vivowith TEX for 48hrs. DCs cultured with RT-TEXorwith the powerful TLR3 agonist poly:ICLC showed significantlyincreased cell surface expression of costimulatory moleculesCD40, CD80, and CD86 (Fig. 2A and B). RT-TEX and poly:ICLCalso induced expression of Ifnb1, Mx1, and Ifnar1 genes andsecretion of IFNb by DCs (Fig. 2C and D). In contrast, UT-TEXwere unable to induce DC activation and IFNb production (Fig.2A–D). Thus, RT-TEX exhibited adjuvant properties that were notseen in UT-TEX.

The STING pathway has been implicated in induction of IFN-Iproduction by DCs exposed to irradiated cancer cells (8). Todetermine whether RT-TEX activate DCs through this pathway,TEX were cocultured with primary DCs isolated from STING-deficient (Sting–/–) mice. Sting–/– DCs were able to upregulateIfnb1, Mx1, and Ifnar1, and produce IFNb when stimulated withpoly:ICLC. However, they failed to respond to RT-TEX, indicatingthat STING is an essential mediator of the adjuvant activity ofRT-TEX (Fig. 3).

TREX1-sensitive dsDNA carried by exosomes causes IFN-Iactivation in recipient DCs

Cancer cell–derived DNA has been shown to be responsiblefor the STING-mediated activation of IFN-I in DCs (7, 8).DsDNA can be present inside exosomes as well as associatedwith their outer membrane (16). To determine the amount andlocation of dsDNA carried by TEX, dsDNA was measured beforeand after treatment with DNase I. This revealed that a largeportion of dsDNA in RT-TEX is external, surface-associatedDNA, but RT-TEX also contain significantly more internaldsDNA compared with UT-TEX (Supplementary Fig. S1A). To

determine if the external DNA was responsible for the induc-tion of IFN-I in recipient DCs, RT-TEX were treated with DNaseI before culture with DCs. No significant decreases in the abilityof DNase-treated RT-TEX to induce IFN-I pathway activationor IFNb secretion by DCs were seen compared with untreatedRT-TEX (Supplementary Fig. S1B and S1C), demonstrating thatexternal DNA did not contribute to the IFN-stimulatory effectsof RT-TEX.

We have shown that 8 Gy � 3 RT induces the accumulation ofdsDNA in the cytosol of the irradiated TSA cells, where it activatescGAS leading to downstream STING-mediated IFN-I induction.The IFN-stimulatory cytosolic dsDNA is degraded by TREX1,which is induced by single RT doses above 12 Gy (23). Thus, totest if the IFN-stimulatory dsDNA cargo of RT-TEX originatesfrom the cytosolic fraction that is sensitive to TREX1, we usedTSA cells transduced with a doxycycline-inducible Trex1 cDNA(TSAKI Trex1). Doxycycline treatment of these cells, which we havedemonstrated abrogates the cytoplasmic accumulation of dsDNAinduced byRT (23), decreasedmarkedly the internal dsDNA cargoof RT-TEX (Fig. 4A) and abrogated RT-TEX's ability to induce IFNbsecretion by DCs and Ifnb1, Mx1, Ifnar1 gene expression (Fig. 4Band C).

Because TREX1 removes the substrate for cGAS activation, wenext asked if the abrogated IFN-stimulatory activity of RT-TEXproduced by doxycycline-treated TSAKI Trex1 cells was entirelydue to loss of dsDNA in the TEX cargo, or could it be explainedby loss of cGAMP or other factors activated downstream ofSTING. To this end, RT-TEX were isolated from TSA cells withshRNA-mediated downregulation of cGAS or STING and testedfor the ability to activate DCs. RT-TEX derived from cGAS- orSTING-deficient TSA cells were equally effective as RT-TEX fromcontrol TSA cells expressing a nonsilencing construct at induc-ing the expression of Ifnar1 in recipient DCs and only slightlyless effective at inducing Ifnb1 (Supplementary Fig. S2). Over-all, these data demonstrate that dsDNA is responsible for theincreased adjuvanticity of RT-TEX and is regulated by TREX1 inthe parent cells.

Vaccination with RT-TEX induces protective antitumorimmunity

Next, we tested if the increased adjuvanticity of RT-TEX resultsin improved induction of antitumor immune responses in vivocompared with UT-TEX. First, the ability of TEX injected subcu-taneously to reach theDCs in the draining lymphnode (DLN)was

Table 1. Pathways unique to UT-TEX and RT-TEX

UT-TEX P RT-TEX P

CTLA4 signaling in cytotoxic T lymphocytes 0.0267 Folate polyglutamylation 0.0089Role of macrophages, fibroblasts, and endothelial cells in rheumatoid arthritis 0.0271 Methylglyoxal degradation III 0.0121Non–small cell lung cancer signaling 0.0321 Pentose phosphate pathway (nonoxidative branch) 0.0131Cytotoxic T lymphocyte-mediated apoptosis of target cells 0.0365 Interferon signaling 0.0242IL3 signaling 0.0423 Cardiac beta-adrenergic signaling 0.0243Oncostatin M signaling 0.0426 Folate transformations I 0.0297Aryl hydrocarbon receptor signaling 0.0431 Prostanoid biosynthesis 0.0297nNOS signaling in skeletal muscle cells 0.0454 Neuroprotective role of THOP1 in Alzheimer disease 0.0342Fatty acid biosynthesis initiation II 0.0454 Antioxidant action of vitamin C 0.0404Palmitate biosynthesis I (animals) 0.0454 CD28 signaling in T helper cells 0.0468Glycine biosynthesis I 0.0454S-methyl-50-thioadenosine degradation II 0.0454Nitric oxide signaling in the cardiovascular system 0.0462Ephrin A signaling 0.0490HIF1a signaling 0.0496

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evaluated. To this end, we used a well-established method tomonitor uptake of PKH67-labeled exosomes in vivo (38). At 24hours after injection, both UT-TEX and RT-TEX were taken up byDCs, but the percentage of DCs that acquired TEX, as indicatedby PKH67-positivity, was slightly higher in mice injected withRT-TEX than with UT-TEX (mean 14% versus 9.5%, P < 0.05;Fig. 5A and B). The percentage of positive DCs decreased similarlyin both groups at later time points.

Having confirmed that TEX injected subcutaneously couldreach the DCs in the DLNs where T-cell priming occurs, micewere vaccinated three times with UT-TEX or RT-TEX, followed bychallenge with live TSA cancer cells (Fig. 6A). All mice vaccinatedwith UT-TEX developed tumors, although tumor growth wasslightly but significantly slower compared with control nonvac-cinated mice (Fig. 6B and C). In contrast, 2 of 6 mice vaccinatedwith RT-TEX did not develop tumors, and the tumors in theremaining mice grew with a significantly larger delay compared

with both control and UT-TEX vaccinated mice (Fig. 6B and C).The tumors with delayed growth contained on average moreCD8þ T cells compared with untreated and UT-TEX–vaccinatedmice, and a larger number among them were specific for anendogenous antigen that is immunodominant in TSA tumor(ref. 25; Fig. 6D and E).

To assess for systemic tumor-specific T-cell responses, T cellsisolated from spleen were activated for 4 days in vitro andthen incubated with irradiated TSA cells or the unrelatedA20 lymphoma cells for 3 hours, followed by analysis of IFNgproduction by intracellular staining. A significant increase inTSA tumor-specific IFNgþ CD8þ T cells was evident in micevaccinated with RT-TEX compared with control and UT-TEXvaccinated mice, whereas no response was seen against A20cells (Fig. 6F and G). Overall, these results indicated that RT-TEX were a more powerful vaccine than UT-TEX and elicitedtumor-specific CD8þ T cells.

Figure 2.

RT-TEX induce activation and IFN-I production by primary DCs. 1� 106 CD11cþDCs isolated from spleenswere cultured for 48 hourswith TEX (30mg) or TLR3 agonistpoly:ICLC (0.025 mg/mL) and analyzed for (A and B) expression of costimulatory molecules and (C and D) IFN-I pathway activation. A, Histograms showingcostimulatory molecule expression on DCs cultured with PBS (CTRL: shaded gray), UT-TEX (gray line), RT-TEX (black line), and poly:ICLC (black broken line).B, Mean fluorescence intensity (MFI) � SEM of samples in each group (n ¼ 3/group). MFI was calculated after subtracting background. C, Gene expressionevaluated by qRT-PCR. D, IFNb measured by ELISA in the supernatant of DCs. Results are representative of two independent experiments. Using a Studenttwo-tailed t test; � , P < 0.05; �� , P < 0.005; ��� , P < 0.0005; ���� , P < 0.00005.

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DiscussionExosomes can carry a variety of tumor antigens and are under

investigation in the clinic as promising cell-free cancer vaccines(39). Exosomes secreted by cancer cells efficiently deliver tumorantigens to DCs (14), but their low/absent adjuvanticity andpotential for immunosuppressive and protumorigenic effectshave hampered their use as vaccines (13, 17, 18). Because radi-

ation can increase cancer cells' adjuvanticity, wehypothesized thatTEX produced by irradiated cancer cells would carry a cargodifferent fromTEXproducedbyuntreated cancer cells, anddisplayincreased immunogenicity. Proteomics analysis and vaccinationexperiments supported and validated both of these hypotheses.

Several proteins were found uniquely in RT-TEX, and otherswere present at different levels, indicating that different pathways

Figure 3.

IFN-I pathway activation by RT-TEX is STING-dependent. 1 � 106 CD11cþ DCs isolated from the spleens of STING-deficient mice were cultured for 48 hourswith TEX (30 mg) or TLR3 agonist poly:ICLC (0.025mg/mL) and analyzed for IFN-I pathway activation.A,Gene expression evaluated by qRT-PCR.B, IFNbmeasuredby ELISA in supernatant of DCs. Results are representative of two independent experiments. Using a Student two-tailed t test; � , P < 0.05; ���� , P < 0.00005.

Figure 4.

IFN-stimulatory dsDNA in RT-TEX isregulated by Trex1 expression inparent cells. A, Quantification ofinternal dsDNA carried by TEXsecreted by irradiated TSAKI Trex1 cellstreated or notwith doxycycline (DOX),as indicated, to induce Trex1. B and C,1 � 106 CD11cþ DCs were cultured for48 hours with TEX (30 mg) derivedfrom TSAKI Trex1 cells treated or notwith doxycycline (DOX; 4 mg/mL), asindicated, or with TLR3 agonist poly:ICLC (0.025 mg/mL) and analyzed forIFN-I pathway activation. B, IFNbmeasured by ELISA in the supernatantof DCs. C, Gene expression evaluatedby qRT-PCR. Using a Student two-tailed t test; �, P < 0.05; �� , P < 0.005;��� , P < 0.0005; ���� , P < 0.00005.

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were represented and/or the represented pathways differed intheir predicted functional orientation (activated versus inhibited)between RT-TEX andUT-TEX.We found that RT-TEXwere capableof activating DCs, whereas UT-TEX were not. Thus, we furtherinvestigated the mechanisms underlying the increased adjuvan-ticity of RT-TEX. Depending on their nature, DAMPs are sensed bysurface, endosomal, or cytoplasmic receptors that are shared withpathogen-associated molecular patterns and converge into acti-vation of the IFN-I pathway in DCs (40). Various DAMPs aregenerated during ICD induced by cytotoxic treatments, includingRT (6). However, evidence indicates that in in vivo–irradiatedtumor dsDNA,which induces IFN-I via the cGAS/STINGpathway,is the critical signal for the priming of antitumor T cells (8, 23).Wehave shown that dsDNA accumulates in the cytosol of mouse andhuman cancer cells upon treatment with radiation and stimulatesIFN-I release by the cancer cells via the cGAS/STING pathway. Thereleased IFN-I signals to the cancer cells in an autocrine fashionand to host DCs, increasing their recruitment to the tumor andinducing their activation (23). Here, we demonstrated that irra-diated cancer cells could also signal by releasing TEX that carry thetumor dsDNA to the cytosol ofDCs, leading toDCs activation andpriming of antitumor T cells. Thus, RT-TEX provide a new mech-anism whereby RT increases tumor DNA-mediated IFN-I activa-tion in DCs.

Xu and colleagues (9) demonstrated that mitochondrial DNAfrom MC38 cells, which are phagocytosed by DCs when the "donot eat me" signal provided by CD47 is blocked, gains access tothe DC cytosol and stimulates cGAS/STING. Thus, it is likely thatdifferent pathways mediate the transfer of tumor-derived DNA toDCs. TEX might have a more important role in delivering tumor-derived DNA to DCs in the DLNs, whereas tumor cell phagocy-tosis by DCs may underlie priming of spontaneous T-cellresponses to immunogenic tumors that are infiltrated by sufficientDCs (7). The work of Xu and colleagues (9) also highlights theimportance of other mechanisms, such as CD47/SIRPa, in reg-ulating the access of the phagocytosed DNA to the cytosoliccompartment. It remains to be ascertained if access to the DC

cytosol of dsDNA contained inUT-TEX andRT-TEX is regulated bythe same or similar mechanisms. Further experimentation will berequired to determine the relative contribution of these differentmechanisms of tumor DNA delivery to DCs for the radiation-induced in situ vaccination.

Variable but conspicuous amounts of dsDNA have been foundin TEX produced by a number of cancer cells that were not treatedwith radiation (16). Similarly, UT-TEX contained some dsDNAbut did not stimulate DC activation and IFN-I release. TREX1upregulation in the parent cells reduced by approximately 50%but did not completely eliminate dsDNA carried by RT-TEX.However, the ability of RT-TEX to stimulate DCs was completelyabrogated. Thus, it is possible that the IFN-stimulatory dsDNAselectively present in RT-TEX and sensitive to the levels of TREX1in the parent cells has characteristics distinct from the dsDNApresent in TEX derived from untreated cancer cells. Thakur andcolleagues (16) performed an extensive characterization ofdsDNA present within TEX and found no bias for gene-encodingversus intergenic regions and no enrichment in specific regionscompared with genomic DNA. Interestingly, they also found thatdsDNA within TEX was methylated similar to genomic DNA.Thus,multiple factorsmay regulate the IFN-I stimulatory functionof dsDNA contained within RT-TEX, including abundance, struc-ture/epigenetic modifications, and efficiency of delivery to recip-ient cell cytosol. In relation to the latter, it is intriguing to considerif RT-TEX but not UT-TEX contain protein(s) or other factors(miRNA and mRNA) that regulate the access of the dsDNA to theDC cytosol, where it can stimulate the cGAS/STING pathway.Interestingly, exosomes derived from breast cancer cells treatedwith the topoisomerase I inhibitor topotecan were reported toproduce exosomes that carry DNA capable to activate DCs in aSTING-dependent way (41), suggesting that treatment-inducedDNA damage may be coupled with the activation of innateimmunity via a common pathway.

RT increases the expression of several antigens and expands theantigenic repertoire of cancer cells (42, 43). It is possible thatamong the more than hundred proteins selectively identified in

Figure 5.

Uptake of TEX by DCs in vivo. PKH67-labeled TEX (10 mg) were injected s.c.at the base of the tail, and the DLNswere collected 24, 48, and 72 hoursafter injection (n ¼ 3/group per timepoint). DLN cells were stained withanti-CD11c to identify DCs. A,Representative flow cytometry plotsgated on CD11cþ DCs. Boxes encasePKH67þ DCs. B, Mean percentage �SEM of TEXþ CD11cþ cells at differenttimes postinjection. Using a Studenttwo-tailed t test; � , P < 0.05.

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RT-TEX, proteins encoding antigenic peptides that could be deliv-ered to DCs are present. Thus, TEX could contribute to mediateantigenic spread, which has been reported to occur following RT(42, 44). Interferon signaling was one of the 10 pathways presentin RT-TEXbut notUT-TEX. Themain proteins identified inRT-TEXfrom this pathway were IFITM2, IFITM3, OAS1, and PSMB8.Although it cannot be excluded that each of these proteins couldperform functions in the recipient DCs that may contribute totheir activation and IFN-I production, the fact that RT-TEX pro-duced by TSAshcGAS and TSAshSTING cells were able to induce Ifnb1and Ifnar1 expression in DCs indicates that they are not required.However, because TEX are found in the peripheral blood, theproteomic signature of IFN-I pathway activation could be abiomarker of radiation-induced IFN-I activation in the tumorand possibly be used to predict which patients may respond tocombinations of radiation and ICB (23, 45).

Finally, the finding that TREX1 expression in the parent celldetermined the ability of RT-TEX to activate the STINGpathway inrecipient DCs has important implications for the choice of radi-

ation doses and fractionation in the clinic, especially in trialstesting radiationwith immunotherapy. We have shown that thereis a threshold for single radiation doses above which, TREX1 isinduced and dsDNA is cleared from the cancer cell cytosol,abrogating cancer cell–intrinsic activation of IFN-I and subse-quent RT-induced abscopal responses (23). Data presented herestrongly suggest that this threshold, which ranges between 12 and18 Gy in different cancer cells tested, will also affect the inductionof the IFN-I pathway inDCs, bymodulating the dsDNA content ofRT-TEX. Interestingly, Deng and colleagues showed that a singledose of 20Gydidnot elicit IFN-I productionbyMC38 cancer cells,but it did elicit IFN-I production byDCs infiltratingMC38 tumorsvia the STING pathway (8). Consistent with this report, we foundthat 20 Gy RT-induced Trex1 expression, resulting in degradationof most dsDNA and precluding IFN-I activation in MC38 cells(23). Thus, it is possible that the residual dsDNA in MC38 cancercells was sufficient to activate DCs when delivered to DC cytosol,possibly by RT-TEX. Because no other radiation doses were testedin this study, it cannot be ruled out that radiation doses of 8 to 10

Figure 6.

Vaccination with RT-TEX induces protective antitumor immunity. Mice were vaccinated s.c. with 20 mg TEX and received boost vaccinations 7 and 14 dayslater (20 mg/boost), followed by challenge with a tumorigenic inoculum of 1� 105 TSA cells. A, Experimental schema depicting vaccination and challenge schedule.B, Tumor growth in each group postchallenge (n ¼ 6/group). C, Individual mouse tumor growth curves. Numbers indicate mice without tumor/total mice.D and E, Tumor-infiltrating T cells analyzed at day 20 postchallenge by flow cytometry. D,Density of CD8þ T cells in each treatment group (n¼ 3/group). E,Densityof tumor-specific CD8þ T cells as determined by Ld/AH1 pentamer staining. F and G, Spleen T cells harvested at day 20 postchallenge and tested for IFNgproduction by intracellular staining after in vitro activation and incubation with (F) 1 � 104 of either TSA cells or (G) the irrelevant target A20 lymphoma cells.All data are representative of two independent experiments. Using a Student two-tailed t test; � , P < 0.05; �� , P < 0.005; ���� , P < 0.00005.

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Gy, which induce optimal accumulation of dsDNA in the cytosolofMC38 cells (23), would have induced a stronger IFN-I responsein DCs. In support of this notion, the intratumoral delivery of aSTING agonist was required to achieve optimal T-cell–priming inmice treated with 20 Gy RT, suggesting a suboptimal DC activa-tion (8).

In conclusion, we showed that RT-TEX act as messengers of themolecular changes that took place in irradiated cancer cells.Together with tumor antigens, RT-TEX carried adjuvants thatactivated DCs and could prime protective antitumor T-cellresponses. These findings may lead to new vaccination strategiesand provide a rationale for testing, if circulating TEX in peripheralblood can be used as biomarkers of immunogenic changesinduced by RT in the tumor.

Disclosure of Potential Conflicts of InterestS.C. Formenti reports receiving a commercial research grant from Bristol-

Myers Squibb, Varian, Janssen, Regeneron, Eisai, and Merck and has receivedhonoraria from the speakers bureau of Bristol-Myers Squibb, Varian, Elekta,Janssen, Regeneron, GlaxoSmithKline, Eisai, Dynavax, AstraZeneca, andMerck.S. Demaria reports receiving a commercial research grant from Lytix Biopharmaand Nanobiotix and is a consultant/advisory board member for LytixBiopharma, AstraZeneca, AbbVie, and StemImmune. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: J.M. Diamond, S. DemariaDevelopment of methodology: J.M. Diamond, Y. Sarfraz, S. DemariaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.M. Diamond, C. Vanpouille-Box, S. Spada,J.R. Chapman, B.M. Ueberheide

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.M. Diamond, C. Vanpouille-Box, S. Spada,N.-P. Rudqvist, J.R. Chapman, B.M. Ueberheide, S.C. Formenti, S. DemariaWriting, review, and/or revision of the manuscript: J.M. Diamond,C. Vanpouille-Box, J.R. Chapman, S.C. Formenti, S. DemariaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.M. Diamond, K.A. Pilones, Y. SarfrazStudy supervision: S. Demaria

AcknowledgmentsThis work was supported by NIH R01CA201246 (to S. Demaria) and a grant

from The Chemotherapy Foundation (to S. Demaria). Additional fundingwas provided by the Breast Cancer Research Foundation award BCRF-16-054(to S.C. Formenti and S. Demaria) and S10 RR027619 (to S.C. Formenti).J.M. Diamond was supported, in part, by the Vittorio Defendi Fellowship inPathobiology from NYU School of Medicine, Department of Pathology. TheNYU Proteomics Laboratory is partially supported by the NYU School ofMedicine and the Laura and Isaac Perlmutter Cancer Center support grantP30CA016087 from the NCI.

The authors thank the laboratories of Dr. Robert J. Schneider at the NYUSchool of Medicine andDr. John P. Moore atWeill Cornell Medicine for the useof their ultracentrifuge machines. We thank Dr. Philippe Colin at Weill CornellMedicine for technical assistance with the ultracentrifuge. We also thankDr. Boris Reizis and his staff for providing the B16. Flt3-L cell line, and theNYU Langone School of Medicine DART Microscopy Lab, Alice Liang andKristen Dancel-Manning for their assistance with electron microscopy.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 12, 2017; revised April 1, 2018; accepted June 11, 2018;published first June 15, 2018.

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