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CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

A Virus-Infected, Reprogrammed Somatic Cell–DerivedTumor Cell (VIReST) Vaccination Regime Can PreventInitiation and Progression of Pancreatic Cancer A C

Shuangshuang Lu1, Zhe Zhang1, Pan Du1, Louisa S. Chard2, Wenli Yan1, Margueritte El Khouri2,Zhizhong Wang1, Zhongxian Zhang1, Yongchao Chu1, Dongling Gao1, Qinxian Zhang3, Lirong Zhang3,Ai Nagano2, Jun Wang2, Claude Chelala2, Jing Liu4,5, Jiekai Chen4,5, Pentao Liu6, Yunshu Dong7,Shengdian Wang7, Xiaozhu Li7, Jianzeng Dong8, Nick R. Lemoine1,2, Duanqing Pei4,5, and Yaohe Wang1,2

ABSTRACT◥

Purpose: Pancreatic cancer remains one of the most lethalcancers, and late detection renders most tumors refractory toconventional therapies. Development of cancer prophylaxis maybe the most realistic option for improving mortality associated withthis disease. Here, we develop a novel individualized prophylacticand therapeutic vaccination regimen using induced pluripotentstem cells (iPSC), gene editing, and tumor-targeted replicatingoncolytic viruses.

Experimental Design: We created a Virus-Infected, Repro-grammed Somatic cell-derived Tumor cell (VIReST) regime. iPSCsfrom healthy cells were induced to pancreatic tumor cells usingin situ gene editing via stable provision of KRasG12D and p53R172H

tumor driver mutations. These cells were preinfected with oncolyticAdenovirus (AdV) as prime or Vaccinia virus (VV) as boost,to improve vaccine immunogenicity, prior to delivery of vaccinesin a sequential regime to young KPC transgenic mice, genetically

programmed to develop pancreatic cancer, to prevent and delaydisease development.

Results: Tumor cells preinfected with oncolytic AdV as primeor VV as boost were the best regime to induce tumor-specificimmunity. iPSC-derived tumor cells were highly related inantigen repertoire to pancreatic cancer cells of KPC transgenicmice, suggesting that an individual's stem cells can provide anantigenically matched whole tumor cell vaccine. The VIReSTvaccination primed tumor-specific T-cell responses, resulting indelayed disease emergence and progression and significantlyprolonged survival of KPC transgenic mice. Importantly, thisregime was well-tolerated and nontoxic.

Conclusions: These results provide both proof of concept and arobust technology platform for the development of personalizedprophylactic cancer vaccines to prevent pancreatic malignanciesin at-risk individuals.

IntroductionThe immune system has long been recognized as critical for

maintaining control over nascent tumor cells. Despite this, tumors

have an inherent ability to overcome immune constraints and developinto malignant disease (1). Advances in our understanding of theevolutionary mechanisms by which tumors escape control haveresulted in groundbreaking new therapeutics, including checkpointblockade and CAR-T therapies that are redefining survival rates formany patients with cancer. However, there has been little success inapplying this understanding to the generation of effective therapeuticsfor some of the most treatment-resistant tumors such as pancreaticductal adenocarcinoma (PDAC), which continues to have extremelyhigh mortality rates (2). Our improved understanding of cancerprogression in recent years suggests a long period of clinical latencyassociated with solid tumors (3); thus, a more meaningful approach tocontrol may be to develop prophylactic strategies that can be imple-mented in this window of opportunity to provoke the immune systemintomaintaining strict control of the tumor, preventing progression toincurable disease. Theoretically, prophylactic vaccination should bemore effective than therapeutic control as nascent tumors lack thecomplex immune tolerance mechanisms of established tumors; how-ever, although preventative strategies have proven effective for cancersin which the etiologic agents are known (4, 5), for most nonviralcancers, it remains ineffective, largely due to the lack of appropriatetumor antigens and an effective approach to induce robust antitumorimmunity against the antigens. Vaccination strategies relying onpresentation of known tumor-associated antigens (TAA) have shownlittle success due to inappropriate choices of TAAs and the inability ofthese regimes to overcome tumor-induced immune suppression.Autologous whole cancer cell vaccines are more successful in theirtargeting of tumor cells as these vaccines can present a large number ofrelevant antigens in the appropriate HLA context, but requirement for

1National Center for International Research in Cell and Gene Therapy, Sino-British Research Centre for Molecular Oncology, Academy of Medical Sciences,ZhengzhouUniversity, Zhengzhou, China. 2Centre forMolecularOncology, BartsCancer Institute, Queen Mary University of London, London, United Kingdom.3School of Basic Medical Sciences, Academy of Medical Sciences, ZhengzhouUniversity, Zhengzhou, China. 4CAS Key Laboratory of Regenerative Biology,South China Institute for StemCell Biology and Regenerative Medicine, Guangz-hou Institutes of Biomedicine and Health, Chinese Academy of Sciences,Guangzhou, Guangzhou, China. 5Guangzhou Regenerative Medicine and HealthGuangdong Laboratory, Guangzhou, China. 6School of Biomedical Sciences, LiKa Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.7CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, ChineseAcademyof Sciences, Beijing, China. 8Department of Cardiology, BeijingAnzhenHospital, Capital Medical University, Beijing, China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corrected online March 23, 2021.

Corresponding Authors: Yaohe Wang, Queen Mary University of London, JohnVane ScienceCentre, Charterhouse Square, LondonEC1M6BQ,UnitedKingdom.Phone: 4420-7882-3596; Fax: 4420-7882-3840; E-mail:[email protected]; and Duanqing Pei, [email protected]

Clin Cancer Res 2020;26:465–76

doi: 10.1158/1078-0432.CCR-19-1395

�2019 American Association for Cancer Research.

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sufficient viable biopsy material limits application to few therapeuticand no prophylactic settings. An allogeneic whole-cell vaccineapproach offers more scope for preventative use, but does not accountfor the extensive heterogeneity of tumors and may confer selectivepressures that promote tumor divergence (6). These limitations dem-onstrate a necessity for far more personalized approaches that accountfor genetic heterogeneity between individuals and are suitably immu-nogenic to promote robust antitumor immunity for long-lastingprevention of tumor escape.

To address the challenge of ensuring appropriate immunogenicityof prophylactic vaccination, we investigated the use of replicatingoncolytic viruses (OV). OV are a class of naturally occurring orgenetically modified viruses that selectively replicate in and kill tumorcells, and it is widely recognized that tumor-specific T-cell activationplays a crucial role in OV-mediated therapeutic efficacy. Infected cellvaccines, whereby cancer cells are preinfected with replicating tumortropic virus prior to delivery as a vaccine, have been shown to provokehigh levels of antitumor immunity that was not achieved when cellswere deliveredwithout infection (7), suggesting that replicating virusescan provide relevant danger signals required for vaccine immunoge-nicity. We have recently demonstrated that therapeutic prime–boostdelivery of Adenovirus (AdV) andVaccinia virus (VV), sequentially, isan effective regime for elimination of PDAC in vivo (8). The under-lying mechanisms responsible for the efficacy of specific sequentialadministration remain unclear, but we show here that both viruses cantrigger complementary aspects of immunologic cell death that likelypromote the strong immunogenicity of the regime. Here, we show thatinfection of autologous tumor cells with replicating AdV or VV, anddelivery of these cells prophylactically in a prime–boost manner usingAdV-infected cells as a prime and VV-infected cells as a boost, caninduce significant antitumor immune responses and delay diseaseprogression in a transgenic mousemodel of PDAC, demonstrating theintrinsic value of OV as vaccine adjuvants. However, as discussed,requirement for biopsy material limits the scope of autologous vac-cination regimes to therapeutic and not prophylactic use.

The advent of stem cell technology, especially induced pluripotentstem cells (iPSC), offers novel alternatives for the development ofwhole-cell–based preventative vaccination protocols, but therapeuticvaccination using iPSCs has historically failed to elicit protective

advantages (9). Although a recent report of the use of undifferentiatediPSCs to induce antitumor immune responses in murine cancermodels validates the continued exploration of iPSC-based vaccinationregimes (10), the associated antitumor efficacy has remained disap-pointing. We hypothesize that the tumor antigen profile presented byvaccination with undifferentiated iPSCs is insufficient to confer robustprotective antitumor immunity.

Here, we sought to utilize iPSC technology to generate whole tumorcell vaccines in which the unique characteristics of an individual'stumor were preserved. The development of lineage-specific cells fromsomatic cells can now be achieved consistently in vitro (11), andtransformation of iPS-differentiated pancreatic cells into tumor cellsby lentiviral transduction of KRasG12D and p53R172H, driver mutationscommon to a spectrum of cancers, has been reported (12), althoughthis regimen demonstrated a low transduction efficiency and randominsertion of the driver mutation genes. We have developed an alter-native technological platform for derivation of pancreatic cancer cellsfrom healthy somatic cells by in situ gene editing of iPSCs using stableknock-in of inactive KRasG12D and p53R172H prior to lineage differ-entiation and transformation. The derived murine tumor cells dis-played high antigenic similarities to PDAC cell lines derived from therelated KPC or KP transgenic mouse model of pancreatic cancer,demonstrating that this process models neoantigen accrual, based onthe genetic and epigenetic profile of autologous stem cells, during theearliest stages of tumorigenesis. These cells can supply a large numberof relevant neoantigens and TAAs for induction of specific antitumorimmunity. As such we can generate an effective alternative to autol-ogous whole-cell vaccines that is antigenically compatible with eachpatient, but suitable for provision in a preventative setting that can, viaimmune surveillance mechanisms, detect initiation of malignanciesand prevent their development.

By combining OV with the iPSC technology platform, we createda Virus-Infected Reprogrammed Somatic cell-derived Tumor cell(VIReST) vaccination regime. Prophylactic prime vaccination withAdV-infected reprogrammed iPSC tumor cells followed by boostervaccination with the VV-infected cells was extremely effective forprevention of PDAC development and progression in a robust trans-genic model that faithfully recapitulates the disease progression andthe associated complex immune-suppressive environment. This plat-form provides a realistic prospect for cancer prevention in at-riskindividuals, which may be the most constructive approach to affectsurvival statistics for this disease.

Materials and MethodsCells and viruses

Tail-tip fibroblasts from LSL-KRasG12D/þ; Trp53R172H/þ; Pdx-1-Cre(KPC) or wild-type (WT) littermate mice and murine embryonicfibroblasts (MEF) from E13.5 WT mice were cultured using DMEMsupplemented with 10% FBS. Embryogenic stem cells or iPSCswere cultured using either mES medium (DMEM supplementedwith 15% FBS; GEMINI), leukemia inhibitory factor (GEMINI;1,000 U/mL), b-mercaptoethanol (0.45 mmol/L), 1� nonessentialamino acids, 1� Glutamax, 1� sodium pyruvate, or KSR medium(mES medium with 15% Knockout Serum Replacement replacing15% FBS). MEF feeder cells were inactivated by mitomycin C(MMC) treatment (Melonepharma; 20 mg/mL for 2.5 hours).

The murine PDAC cell line DT6606 was established from LSL-KRasG12D/þ; Pdx-1-Cre mice that had developed PDAC. The PDACcell lines TB11381, TB32043, and TB32047 were cultured from LSL-KRasG12D/þ; Trp53R172H/þ; Pdx-Cre mice that had developed PDAC

Translational Relevance

Less than 5% of patients diagnosed with pancreatic ductaladenocarcinoma (PDAC) survive for longer than 5 years; however,the lengthy time to progression associated with PDAC suggests awindow of opportunity for application of preventative vaccinationstrategies, prior to development of immune tolerance mechanismsinduced by tumors. Current cancer vaccines that could beemployed preventatively are limited to allogenic strategies, whichdo not account for inter- and intrapatient heterogeneity and mayactively promote tumor growth. Induced pluripotent stem cellsfrom identified “at-risk” individuals can be used to create autol-ogous tumor cells for preventative vaccination. The derived cellsare highly antigenically compatible, and by preinfecting inducedtumor cells prior to vaccine delivery using oncolytic viruses, theefficacy of antitumor immune induction is increased. Personalizedprophylactic vaccination is the most effective way to reduce cancerincidence and may be possible using the platform described in thenear future.

Lu et al.

Clin Cancer Res; 26(2) January 15, 2020 CLINICAL CANCER RESEARCH466




(13). These were kindly provided by David Tuveson (Cancer ResearchUK Cambridge Institute, now at Cold Spring Harbor Laboratory) andmaintained in DMEM supplemented with 5% FBS. CV1 (Africanmonkey kidney) cells and JH293 cells were obtained from the ATCCand maintained in DMEM containing 5% FBS. WT-KP, KPC, andpancreatic progenitor like cells (PPLC) were maintained in DMEMcontaining 10%FBS.All cell lineswere routinely tested for the presenceof Mycoplasma.

The thymidine kinase (TK)–deleted Lister strain VV (VVL15) wasdescribed previously (14). VVL15 was further modified by XhoI/EcoRI-mediated removal of the LacZ open-reading frame from theVV TK shuttle vector pSC65 (GenBank: HC193923.1), which wasreplaced with red fluorescent protein (RFP) derived by NheI/AflIIdigestion of pCMV-dsRED-Express (Clontech). The virus was pro-duced as described previously (15). Ad-Cre nonreplicative virus waspurchased fromVector Biolabs and propagated in our laboratory. WTAdenovirus serotype 5 (Ad5) was described previously (8).

Generation of iPSCsInducing tail-tip fibroblasts to pluripotency was carried out accord-

ing to the protocol of Dr. Duanqing Pei's laboratory (16). Retroviralvectors (pMX) containing the murine cDNAs of Oct4, Sox2, and Klf4were purchased from Addgene. These plasmids were transfected intoPlatE cells (provided by Dr. Jiekai Chen, Guangzhou Institute ofBiomedicine and Health, Chinese Academy of Science, cultured usingDMEM supplemented with 10% FBS) using calcium phosphate trans-fection for packaging before WT male mouse tail-tip fibroblasts andKPC male mouse tail-tip fibroblasts were infected with the retro-viruses. After 48 hours, cells were cultured in iCD1 media as detailedpreviously (16), and this day was indicated as d0. At d10, high-qualityiPSC clones were picked under a microscope according to theirmorphology and passaged.

Introduction of KRAS and p53 mutations in WT iPSCspBlueScript-PGK-LSL-K-RasG12D and pBlueScript-PGK-LSL-

p53R172H plasmids (kind gifts from Dr. David Tuveson, ColdSpring Harbor Laboratory) containing LoxP-flanked transcriptionstop signals were used to modify WT iPSCs. We modified thepuromycin-resistant gene of pBlueScript-PGK-LSL-P53R172H to neo-mycin resistance. For cell modification, the KRas plasmid was NotI-linearized and electroporated into iPSCs. The cells were selected usingpuromycin (2 mg/mL) and genotyped by PCR using primers flankingthe LoxP-Stop-LoxP (LSL) cassette and the genome of right homol-ogous arm. KRas_forward (F): 50-CCATGGCTTGAGTAAGTCTGC-30; KRas_reverse (R): 50-TGACTGCTCTCTTTC-30 (PCR band 4,100bp); the mutant site was amplified and sequenced using the followingprimers: amplification primer: F: 50-GCCTGCTGAAAATGACT-GAGTAT-30; R: 50-CTCTATCGTAGGGTCATAC-30 (PCR band180 bp). Sequencing primer: 50-CTCTATCGTAGGGTCATAC-30.The PCR fragment was sequenced in GENWIZ, Inc. The p53 plasmidwas linearized using NotI and electroporated into the KRasG12D/þ

iPSCs with TALENS targeting the p53 homologous site. TALENStarget sequences were 50-GGAAGGCCAGCCCTGGTTG-30 and 50-GTAAGAAAATGTTGGCTGGG-30. The cells were selected by G418(400 mg/mL) and genotyped by PCR using primers flanking thegenome of the left homologous arm and the LSL cassette. P53_F:50-CACGCTTCTCCGAAGACTGG-30; P53_R: 50-TATGCTATA-CGA AGTTATGTCG-30 (PCR band 1,900 bp). The mutant site wasamplified and sequenced using the following primers: amplificationprimer: F: 50-TCTCTTCCAGTACTCTCCTCC-30; R: 50-AATTACA-GACCTCGGGTGGCT-30 (PCR band 500 bp); sequencing primer:

50-AAGCTATTCTGCCAGCTGGCG-30. The PCR fragment wassequenced at GENWIZ, lnc.

Differentiation of iPSCs into PPLCiPSCs were cultured in mES medium on an MMC-treated MEF

feeder layer. For differentiation, iPSCs were cultured for two passageswithout feeder cells, dissociated into single cells using 0.25% trypsinbefore culture in Medium 1 (Med1; 25% Nutrient mixture F12, 75%IscovemodifiedDulbecco's media, 1�N-2 supplement, 1�Glutamax,0.05% BSA, and 0.45 mmol/L b-mercaptoethanol) at 5� 105 cells/mLin low adhesion plates to form embryoid bodies (EB). Forty-eighthours later, EBs were cultured using 0.1% gelatin-coated plates inMed1 supplemented with 100 ng/mL Activin A and 3 mmol/LChir99021 for 24 hours. Medium was replaced with Med1 supple-mented with 100 ng/mL Activin A. Cells were cultured for 48 hours toform definitive endoderm (DE). DE were cultured in Med2 (DMEM,1� B27 supplement, and 1� Glutamax) with 2 mmol/L retinoic acid(Sigma); 1 mmol/L A83-01 (Stemgent); 0.5 mmol/L Cyclopamine(Tocris); 50 ng/mL Noggin (R&D Systems); and 280 mmol/L VitaminC (Vc; Sigma) for 48 hours. Mediumwas replaced withMed2 contain-ing 1 mmol/L A83-01; 0.5 mmol/L cyclopamine; 50 ng/mLNoggin; and280 mmol/L Vc (Sigma) for 48 hours to induce differentiation intoPPLC (17, 18).

Transformation of PPLCs into tumor cellsThe modified WT-KP PPLCs were infected with nonreplicating

Ad5-Cre (50 pfu/cell) to remove the LSL cassette at day 9 andcultured in DMEM containing 10% FBS for 10 passages to facilitatetheir transformation to pancreatic tumor cells. The KPC PPLCswere directly cultured in DMEM containing 10% FBS for 10passages to allow natural transformation to tumor cells. p53and KRas were genotyped at passage 3 by PCR using primersto detect a WT band of 290 bp (P53) and 285 bp (KRas) or themutant band of 330 bp (P53) and 325 bp (KRas). P53g_F-50-AGCCTGCCTAGCTTCCTCAGG-30; P53g_R-50-CTTGGAGAC-ATAGCCACACTG-30 . KRasg_F-50- GGGTAGGTGTTGGGATA-GCTG-30; KRasg_R-50-TCCGAATTCAGTGACTACAGATGTA-CAGAG-30.

Additional methods can be found in the Supplementary Methods.

ResultsTumor cells preinfected with two different OVs and deliveredsequentially can engender superior tumor-specific immunity

It has been demonstrated that OV infection of whole tumor cellvaccines significantly enhances their immunogenicity (7), and we andothers have shown that the administration of two antigenically distinctviruses sequentially provides superior therapeutic control over tumorgrowth compared with use of one virus alone (8, 19). To determinewhether OV infection of induced tumor cells provides a beneficialadjuvant in a prophylactic setting, we investigated the underlyingmechanisms for the improved efficacy associated with in situ admin-istration of AdV and VV.

As defined byKroemer and colleagues, features of immunogenic celldeath (ICD), known to be key for effective induction of antitumorimmunity, comprise HMGB1 release, calreticulin (CRT) exposure,and ATP release (20). Multiple studies have confirmed that OVs arepotent inducers of ICD (21); hence, we investigated the induction ofeach of these features upon infection with AdV and VV. Encourag-ingly, we found that both viruses were able to induce ATP release48 hours after infection of pancreatic tumor cells (Fig. 1A), although

A Novel Prophylactic Vaccination for Pancreatic Cancer

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Syngeneic tumor cells preinfected with AdV and VV are effective at priming antitumor immune responses that can protect mice against tumor challenge whendelivered sequentially.A,DT6606 cellswere infectedwith Ad5 at MOI 500 particles per cell (ppc; Ad500) or 100 ppc (Ad100); VVL15 at 1 pfu/cell (VV1) or 0.1 pfu/cell(VV0.1); treatedwith 2 mmol/L ofmitoxantrone (MTX) or left uninfected (Mock). Supernatants were collected 48 hours after treatment and assessed for ATP release.Mean luminescence� SD is shown. Resultswere analyzed using two-wayANOVAwith post hocBonferroni testing (n¼ 4).B,DT6606 cellswere infectedwith Ad5 atMOI 500 and 1,000 ppc; VVL15 at MOI 1 and 10 pfu/cell or treated with 2 mmol/L of mitoxantrone (MTX) or left untreated (Mock). At 48 hours after infection, cellsupernatants were tested for content in HMGB1 by Western blot (n ¼ 3). C, DT6606 cells were infected with Ad5 at MOI 500 ppc; VVL15 at MOI 1 pfu/cell or leftuninfected (Mock). Cellswere harvested at the indicated timepoints and assessed for CRTexposure byflowcytometry. Experimentswereperformed in triplicate, andthe mean � SEM is shown. D and E, Three-month-old KPC mice were vaccinated subcutaneously with a homologous prime–boost regime using DT6606 cellspreinfectedwithAd5, VVL15 (VV), orMMCor heterologous regimewithAd5preinfected cells as aprime followedbyVVL15 preinfected cells as aboost (Ad5/VV). As acontrol, na€�ve KPC mice of the same age were injected with PBS subcutaneously on the days of infection. (Continued on the following page.)

Lu et al.





only AdV was able to elevate these levels significantly compared withmock treatment. Only VVL15-RFPwas able to induceHMGB1 releaseand CRT exposure on pancreatic tumor DT6606 cells (Fig. 1B andC).Thus, it appears that both viruses can trigger certain features of ICD,which may complement each other to improve immunogenicity whenviruses are used sequentially as opposed to individually.

Next, we tested whether infecting autologous cells and deliveringthem using aVirus-Infected Cancer Cell Vaccination (VICCV) regimecould result in tumor-specific immunity. KPC transgenic mice wereimmunized with OV-infected DT6606 cells at weeks 0 and 4. Ex vivosplenocyte reactivation on exposure to growth-arrested DT6606(Fig. 1D) or unrelated CMT93 cells (Fig. 1E) demonstrated that onlya VICCV regime comprising delivery of AdV-infected tumor cells atweek 0 followed by VV-infected tumor cells delivered at week 4induced significant IFNg responses to syngeneic, but not unrelatedtumor cells. As proof of principle of the efficacy of this regime, wevaccinated immunocompetent C57/Bl6 mice using the VICCV regimedescribed above. Subsequent challenge 2 weeks post boost withDT6606 cells indicated that delivering Ad5-infected tumor cells asa prime and VV-infected tumor cells as a boost was the best regime toinduce tumor-specific immunity and prevent challenge tumor growth(Fig. 1F). We then investigated the use of syngeneic tumor cells in theVICCV regime to protect against disease progression in a morecomplex KPC transgenic mouse model. This demonstrated thatvaccination of KPC mice prior to disease emergence using AdV-infected TB11381 tumor cells, derived from the KPC mouse, followedby booster vaccination with VV-infected TB11381 tumor cells, wasable to significantly delay disease progression (Fig. 1G), demonstratingthe principle of using preinfected tumor cells, delivered in a prime–boost manner, as a prophylactic approach for anticancer vaccination.

iPSCs derived from healthy animals can be used to createantigenically relevant whole tumor cell vaccines

For development of a prophylactic vaccine strategy, it is necessary togenerate antigenically compatible, lineage-specific tumor cells usinghealthy somatic cells derived from an individual. We first confirmedthat iPSCs containing inactive tumor driver mutations (KRasG12D andp53R172H) remained pluripotent. Fibroblasts from KPC transgenicmice with endogenous, inactivated KRasG12D and p53R172H mutationswere induced following the strategy illustrated in Fig. 2A by infectionwith retroviruses expressing Oct4, Sox2, and Klf4 reprogrammingfactors (16, 22) to create KPC iPSCs (Supplementary Table S1). Theirpluripotency was confirmed using colony formation assays (Fig. 2B)and detection of iPSC markers by qPCR, which demonstrated reac-tivation of endogenousOct4, Sox2, andKlf4, with the concurrent loss ofexpression of exo-reprogramming factors (Fig. 2C).

To create a platform that can be applied prophylactically in additionto therapeutically, induction of tumor cells from healthy individualsvia iPSC reprogramming is required. We next isolated somatic cellsfrom WT mice (129J/Bl6 KPC littermates) that do not containendogenous genomic mutations. These cells were induced to plur-ipotency as described and demonstrated similar pheno- and genotypesin colony formation assays and qPCR, respectively (Fig. 2B and C).

Induced cells then underwent homologous recombination to intro-duce LSL-controlled common tumor driver mutations, creatingKRasG12D/p53R172H heterozygote subclones (WT-KP iPSCs; Supple-mentary Table S1; Supplementary Fig. S1A–S1C). Neither endogenousnor in situ insertion of the two silent mutations affected pluripotencyof the iPSCs as determined by expression of both Nanog and Oct4(Fig. 2D), generation of viable chimeric mice (SupplementaryFig. S1D), and teratoma formation after implantation into immuno-compromised mice (Fig. 2E). Next, WT iPSCs (derived from WTmice), KPC iPSCs (derived from KPC transgenic mice), or WT-KPiPSCs (derived fromWTmice iPSCs engineered to contain in situ LSL-controlled KRasG12D and p53R172H mutations) were effectively differ-entiated into pancreatic progenitor cells using the protocol indicated inSupplementary Fig. S2A and lineage differentiation confirmed usingimmunofluorescence analysis (Fig. 3A) or qPCR analysis (Supple-mentary Fig. S2B) of standard differentiation markers.

KRasG12D and p53R172H mutations were activated spontaneously indifferentiated KPC pancreatic progenitor cells due to pancreas-specificexpression of Pdx-1–activating Cre recombinase, resulting in trans-formed KPC PDAC cells (Supplementary Table S1). LSL-controlledKRasG12D/þ and p53R172H/þ mutations in WT-KP pancreatic progen-itor cells were activated by infection with nonreplicating Ad5 vectorexpressing Cre (AdCre) to create KP-AC (PDAC) cells (Supplemen-tary Table S1; Supplementary Fig. S2C). KPC and KP-AC cells weretumorigenic when inoculated into the flanks of nude (Fig. 3B) orimmunocompetent syngeneic mice (Fig. 3C), producing characteris-tically stroma-rich tumors with similar cell morphology to thoseresulting from implantation of DT6606 or TB11381 cells derived fromthe KC or KPC PDAC transgenic mouse models (Fig. 3D; Supple-mentary Table S1).

Most importantly, transcriptome analysis of reprogrammed tumorcell lines demonstrated highly similar gene expression profiles totransgenic mouse-derived tumor cell lines (Fig. 3E). Concordancebetween transformed and nontransformed iPSC cells (KP-AC or KPCvs. WT-iPSCs or WT-KP iPSCs) was low, and gene expressionconcordance never exceeded 30%, suggesting that untransformediPSCs were unsuitable as vaccination material as the antigen spectrumpresented would not confer a suitable level of protection. Interestingly,the concordance between the KP-AC or KPC tumor cells and theDT6606 tumor cell line, derived from KRAS driver mutation alone,was high. These results demonstrate that the generation of neoantigensupon tumorigenesis is not dependent on the driver mutation, but theepigenetics within each lineage. Furthermore, transformed lineage-specific cells provide the highest number of relevant neoantigens orTAAs and thus represent the most rational mechanism for creatingeffective whole cancer cell vaccination regimes.

OV-infected iPSC-derived tumor cells are immunogenic whenapplied prophylactically in vivo

Prophylaxis using autologous cells is possible using mouse modelsdue to the inbred nature of mouse colonies preventing genetic dis-crepancies that would restrict their use in a human prophylacticsetting. For translation to the clinical setting, cells that are matched

(Continued.) Fifteen days after the boost injection, splenocytes were cultured ex vivo with growth-arrested DT6606 cells (D) or growth-arrested CMT93 cells (E).Mean IFNg production� SEM is shown (n¼ 3). F, C57/Bl6 mice were subjected to immunization using DT6606 pancreatic cancer cells that were either treated withMMC only or preinfected with VV or AdV, followed by MMC treatment. Preinfected or MMC-treated cells were given twice, in different combinations, at a 4-weekinterval as the prime–boost immunization regime. Animalswere rechallenged 2weeks after the last injectionwith DT6606 tumor cells. The percentage of tumor-freeanimals at 3 weeks after challenge in each treatment group is shown (n ¼ 10/group). Statistical analysis was performed using one-way ANOVA test followedby a Tukey post-test. G, KPC mice were subjected to immunization using TB11381 pancreatic cancer cells that were preinfected with VV or AdV, followed byMMC treatment. Cells were given twice (AdV infected as a prime, VV infected as a boost) at a 4-week interval. Kaplan–Meier survival analysis followed by log-rank(Mantel–Cox) tests were used to assess significance (� , P < 0.05; �� , P < 0.01; �� , P < 0.001).






to each individual are required. Moreover, these cells must be obtainedprior to disease emergence to be of prophylactic use. Genotypicallymatched cells are currently only available via biopsy and thus limited totherapeutic use. In this regard, iPSC-derived tumor cells, which can bedeveloped from at-risk individuals, may be an effective alternativegiven the high levels of similarities between transcriptomes of derivedand endogenous tumor cells. To assess the potential of this approach, aVIReST immunization protocol was developed in which mice wereimmunized first with iPSC-derived tumor cells (KPC or KP-AC)

preinfected with AdV, followed at an interval of 4 weeks withiPSC-derived tumor cells preinfected with VV (Fig. 4A). Thesevirus-preinfected tumor cells were treated with MMC before immu-nization to prevent ongoing virus replication and tumor cell growth.Both AdV and VV were cytotoxic to (Supplementary Fig. S3A andS3B) and replicated efficiently in (Supplementary Fig. S3C–S3F)KPC and KP-AC tumor cell lines. Importantly, MMC treatment ofcells inhibited ongoing viral replication and prevented tumor cellproliferation (Supplementary Fig. S3G and S3H), but viral proteins

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Pluripotent stem cells can be induced from KPC orWT 129J/Bl6 mouse fibroblasts and geneticallymodified to contain silent KRas and p53 modifi-cations. LSL-KRasG12D/þ; LSL-Trp53R172H/þ; Pdx-1-Cre (KPC) or WT littermates (129J/Bl6) tail-tipfibroblasts were reprogrammed using retroviraltransduction of Oct4, Sox2, and Klf4 on day 0.A, Schematic showing the iPSC induction, mod-ification, differentiation, and transformation pro-cess. B, Representative light microscopy imagesfrom both cell lines are shown on days 0, 3, and10 after transduction and demonstrate develop-ment of iPSC clones in both cases (objectivemagnification 10�). C, qPCR detection of exo-reprogramming factor silencing and endo-reprogramming factor activation at iPSC passage3. Embryonic stem cells (ES) were used as apositive control and copy number relative toActin calculated. D, Immunostaining of Nanogand Oct4 in WT, WT-KP, and KPC iPSCs at pas-sage 3 after iPSC development. Nuclei werestained using DAPI (blue), and Nanog or Oct4is shown in green. Original objective magnifica-tion 10�. E, KPC, WT, and WT-KP iPSCs weresubcutaneously implanted into nude mice andallowed to grow. Resulting teratomas wereresected and analyzed using hematoxylin andeosin staining for the presence of characteristicgerm cell layers. Panels are from the sameteratoma section to demonstrate the inductionof multiple features of differentiation in eachteratoma.

Lu et al.





remained detectable in cell lysates 72 hours after infection (Supple-mentary Fig. S3I), demonstrating that this approach to vaccination isintrinsically safe with no compromise of vaccine immunogenicity.

Using the KPC model of PDAC, both KPC and KP-AC–basedVIReST applied prior to tumor lesion development could enhancesplenocyte IFNg production after ex vivo stimulation with differenttumor cell lines derived from this model (Fig. 4B). Elevated CD8þ

and CD4þ T-cell infiltrates were shown in the pancreas of VIReST-immunized animals after, but not during, VIReST treatment(Fig. 4C and D; Supplementary Fig. S4A). Analysis of the spleenand lymph node compartments indicated that there were increasedlevels of activated CD8þ and CD4þ T cells at both 1 and 3 weekspost-VIReST (Fig. 4E; Supplementary Fig. S4B), with a progressionfrom increased effector memory to central memory populations,that can rapidly expand following rechallenge, over time. Treatmentwas unable to alter the levels of regulatory T-cell (Treg) infiltrationinto the tumor (Supplementary Fig. S4C), and T-cell infiltrationinto the tumor was lost by 3 months after treatment (SupplementaryFig. S4D), suggesting avenues for improvement of this regime. Ofnote, virus-infected, nontransformed iPSCs did not induce signif-icant tumor-specific immunity against PDAC (SupplementaryFig. S4E and S4F).

VIReST delays tumor development and significantly prolongssurvival in transgenic mouse models of PDAC

We next investigated the effect of VIReST-based prophylaxis onmortality. Four-week-old KPC transgenic mice, who do not havetumor lesions at this age, were vaccinated as previously. Volumetricanalysis of pancreatic tumor burden revealed significantly delayedtumor development inVIReST-vaccinated animals (Fig. 5A andB). At6 weeks, tumor was detected in 50% of PBS-treatedmice versus 25% ofVIReST-treated animals. Progression in VIReST-treated animals wasdelayed compared with PBS treatment. This translated into a signif-icant survival advantage, with amedian survival time of untreatedmice(129 days) being increased to 195 days or 169 days (a 51% or 31%extension of lifespan) using KPC (Fig. 5C) or KP-AC (Fig. 5D)VIReST regimes, respectively. This demonstrates that the currentVIReST regimen can significantly postpone disease development andprogression in these complexmodels of cancer, a conclusion supportedby histologic analysis demonstrating delayed pancreatic intraepithelialneoplasia progression during VIReST treatment (SupplementaryFig. S5A–S5C). Of note, the use of iPSC-derived tumor cells comparedwith syngeneic tumor cells was more or equally effective at prolongingsurvival in this model (Supplementary Fig. S6A), although only iPSC-derived tumor cells have the potential for both therapeutic andprophylactic uses, although syngeneic therapy is limited to treatmentof established disease. This model was also used to demonstrate theimportance of virus infection of iPSC-derived tumor cells as statisticaltreatment efficacy, and induction of antitumor immunity was lostwhen virus infection was omitted from the regime (SupplementaryFig. S6B and S6C). The importance of early infiltration and activationof both CD8þ and CD4þ T-cell populations was confirmed in vivo bycomplete abrogation of the survival advantage afforded by VIReST inCD8þ-depleted (Fig. 5E) or CD4þ-depleted (Fig. 5F) PDAC trans-genic animals. Importantly, immunization did not result in evidentsigns of colitis or ileitis associated with autoimmune disorders, noweight loss was noted, there was no tumor growth detected at theimmunization site, and there was no difference in the amount ofcirculating anti-nuclear antibodies (ANA) detected (Fig. 5G). PDAC ischaracteristically unresponsive to immune checkpoint therapy; how-ever, given the influx of T cells into the tumor following VIReST

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iPSCs from healthy littermates can be used to derive antigenically compatiblelineage-specific tumor cells. A, iPSC markers (Oct4 and Nanog), DE markers(Foxa2 and Sox17), and PPLC markers (Sox9 and Pdx1) were analyzed usingimmunostaining of samples at days 0, 5, and 9. DAPI is shown in blue and theindicatedmarkers in green. Original objective magnification is 20�. B, Indicatedcells were subcutaneously inoculated into the flank of nude mice (KPC and KP-AC n ¼ 4; TB11381 n ¼ 3). Tumors were measured twice weekly, and the tumorvolumewas estimated. Mean tumor size� SEM is shown. C, Indicated cells weresubcutaneously inoculated into the flank of KPC littermates (129J/Bl6; n¼ 6/cellline). Tumor volume was estimated twice weekly. Mean tumor size � SEM isshown.D, Established tumorswere removed fromKPC littermates and analyzedusing hematoxylin and eosin staining. TB11381 and DT6606 tumors were ana-lyzed as controls. Objective magnification is 20� (top row) or 40� (bottomrow). E, Cell lines were subjected to RNA deep sequencing, and a transcriptomeexpression correlation matrix, based on 15,687 filtered genes, was generated.






therapy, and the reliance of this therapy on CD8þ and CD4þ T-cellsubsets, we investigated that addition of a-PD1 to the regime.Surprisingly, this addition had no impact on the efficacy of treat-ment, suggesting that eventual tumor escape from control occurs bymechanisms independent of PD1-PD-L1 checkpoint activation(Fig. 5H).

DiscussionLack of success in vaccination strategies to control tumor devel-

opment can be defined by two challenges. The first is derivation of largepools of relevant, immunogenic antigens that are not subject totolerance mechanisms. In the case of prophylactic strategies, immunepriming must occur prior to disease development to aid effective

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A VIReST regime using both KPC-and KP-AC–induced tumor cellsinduces potent antitumor immuneresponses. A, Schematic showingthe prime–boost vaccination proto-col. B–E, Four-week-old transgenicmice were immunized using a KPC orKP-AC VIReST regime. B, Threeweeks later, splenocytes were resti-mulated ex vivo with growth-arrested PDAC cells, or KRas ormesothelin (Meso)peptides and IFNgproduction measured using ELISA.Mean IFNg production � SEM isshown (n ¼ 3/group). Significancewas analyzed using one-wayANOVAwith Tukey post-test. C, CD8þ cellsand CD4þ T-cell infiltrate in the pan-creas were assessed using IHC at1 week, 2 weeks, and 3 weeks follow-ing the booster vaccination. D, CD8þ

cells from IHC sectionswere counted,and the average number per highpower field (HPF)� SEMwas plotted(n ¼ 3–4/group). CD4þ T-cell pres-ence in the whole tissue slice wasgraded as described in Materialsand Methods. Significance was ana-lyzed using one-way ANOVA withTukey post-test. E, T-cell subsets inspleens (Sp) and draining lymphnodes (LN) from KPC mice 3 weeksafter VIReST. Na€�ve T cell (CD44lo;CD62Lhi), TCM:Centralmemory T cell(CD44hi;CD62Lhi), and TEM: Effectormemory T cell (CD44hi;CD62Llo)were assessed. Significance wasanalyzed using one-way ANOVAwith Tukey post-test (n ¼ 3–4/group). � , P < 0.05; �� , P < 0.01;�� , P < 0.001; �� , P < 0.0001.

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elimination of nascent tumor cells before their escape from immunecontrol via direct immunosuppression or HLA pathway loss. It is clearthat immune prevention at the earliest stages can overcome theimprinting of the immune system by previous, nonimmunogenicencounters that promote immune tolerance. Our vaccine, usingpatient-specific transformed iPSCs, potentiated using viruses to

enhance the immunogenicity, is designed to create immune responsesagainst the neoantigens that accrue upon initial development of cancersuch that the immune system can prevent development at the earlieststages. Fundamental to the success of the regime is the use of patient-matched iPSC technology, which provides the key to accessing uniqueneoantigens by modeling specific epigenetic changes that arise early in

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A VIReST regime using iPSC-derivedpancreatic tumor cells delays mor-tality in a transgenic model of can-cer. A–D, Four-week-old KPC trans-genic mice were immunized using aVIReST regime. A, After treatmentwith either PBS (n ¼ 4), theKPC VIReST regime (n ¼ 5), or theKP-AC VIReST regime (n ¼ 5), high-resolution ultrasound imagingwas employed to determine pancre-atic tumor volumes. The pancreasis outlined in white. Arrows indicatetumor deposits. K, kidney; S, spleen.B, Tumor volume � SEM was deter-mined by volumetric analysis,and significance was analyzed usinga two-way ANOVA with Bonferronipost hoc testing. KPC transgenicmice were treated with either theKPC (n ¼ 11; C) or KP-AC (n ¼ 18;D) VIReST regime, and long-termsurvival was monitored. Control ani-mals were treated with PBS (n ¼ 12).Kaplan–Meier survival analysis fol-lowed by log-rank (Mantel–Cox)tests was used to determine signif-icance. Four-week-old KPC micewere treated with the KPC VIReSTregime with or without CD8þ T-celldepletion (E) or CD4þ T-cell deple-tion (F), and long-term survivalwas monitored (n ¼ 11 per group).Kaplan–Meier survival analysis fol-lowed by log-rank (Mantel–Cox)tests was used to determine signif-icance. G, Sera of immunized miceand control mice were analyzedby ELISA for presence of ANAs.H, Four-week-old KPC transgenicmice were immunized using aVIReST regime and treated with200 mg/mouse a-PD1 1 weekafter prime and 1 week after boostinjections. Kaplan–Meier survival anal-ysis followed by log-rank (Mantel–Cox) tests was used to determinesignificance (� , P < 0.05; �� , P < 0.01;�� , P < 0.001; and �� , P < 0.0001).






disease genesis to drive stepwise progression of carcinogenesis viapatient-specific accrual of passenger mutations (23, 24) in a way thatallogenic vaccination regimes cannot. Using genotypically matchedcells ensures adaptive immunity is appropriately raised against neoan-tigens, and not irrelevant histocompatibility antigens and the epige-netic abnormalities specific to each individual play a seminal role in theearliest steps of cancer initiation (25), affecting both the initiation ofthe disease (26, 27) and the progression of the disease, for example thepatient-specific accrual of passenger nonsynonymous mutations dur-ing tumorigenesis (23). From our data, it is clear that driving tumor-igenesis with these two driver mutations results in an accuratemodeling of the neoantigen profile found in spontaneous PDAC fromtransgenic mice. Most importantly, we have demonstrated a high levelof similarity between transcriptomes of our KRas/P53-driven iPSC-based cells and DT6606 PDAC cells that were isolated from a 129J/C57Bl6 transgenic model driven solely by KRas. This provides furtherconfirmation that the epigenetics and not the driver per se account forthe specific pattern of neoantigens expressed. Clearly, the situation inhuman patients is far more complex than reflected in the mousemodel used in this study. Driving tumorigenesis by introducingfurther driver mutations is possible and the focus of our ongoingwork. Identification of genetic predisposition to PDAC can enableus to tailor iPSCs even more accurately, for example, BRCA2mutations, common in inherited familial atypical multiple molemelanoma (FAMMM) syndrome and LKB1/STK11mutations com-mon in inherited Peutz–Jeghers syndrome, both of which increasethe risk of PDAC development, provide options to tailor treatmentsto family history data that may increase the already high antigenicrelevance of the vaccine to the patient. However, the data presentedelegantly support this platform as suitable for development ofantigenically relevant vaccines for at-risk individuals.

The second challenge concerns antigen delivery, which mustpotentiate vaccine immunogenicity. OV is known to exert its mostpotent effects via activation of antitumor immune responses (7).The oncolytic process provides critical danger signals as a conse-quence of virus-induced ICD mechanisms, which initiate potentantitumor immune responses (28). Indeed, we found infection oftumor cells with both AdV and VV resulted in production of ICDsignals. Interestingly, the order of virus-infected cell administrationwas critical for treatment efficacy, and this phenomenon was alsoobserved during in vivo therapeutic use against establishedtumors (8). The underlying mechanism is not yet known; however,AdV is known to be extremely effective at TLR activation, resultingin early improvement of T-cell activation and Treg suppres-sion (29, 30). VV expresses a wide range of immune-modulatoryproteins that may either boost activated T-cell responses (31) oralternatively downregulate the induction of effective immuneresponses when administered before AdV.

Here, we demonstrate that sequential use of two distinct OV as partof a whole tumor cell vaccination regime is required to provideeffective prophylaxis against cancer progression in the absence ofdetectable autoimmunity.

Assessment of the efficacy of prophylaxis was carried out using atransgenic mouse model of PDAC that preserves the relationshipbetween developing tumors, the immune system, and surroundingtissues. Using these models, we showed that immunization using theinduced PDAC (KPC or KP-AC) cells alone was unable to preventdisease development, despite antigenic compatibility. However,when these cells were preinfected with OV and used in a VIReSTregime, a significant delay in mortality was achieved. This correlatedwith a delay in progression of PanINs to invasive disease and

demonstrated both a preventative and therapeutic effect of thevaccine in vivo.

Despite the potential of this regime, the protocol failed to affordcomplete protection from disease development in thesemodels. This islikely due to progressive failure of tumor-specific immunity over timeas we have observed that the increased infiltration of CD8þ T cellswithin tumors is lost 3months after the VV-infected tumor cells boost,and the inability to reduce the prolific Treg populations within thetumor (32). Interestingly, and perhaps surprisingly given the influx ofT cells prompted by VIReST and the reliance of the regime on theseadaptive immune cells, we found no improved impact when combin-ing the VIReST regime with a-PD1. This may suggest that eventualtumor escape from control occurs independently of PD1-PD-L1checkpoint activation, or may purely reflect the need for furtheroptimization of the delivery of checkpoint inhibitors such as a-PD1or other checkpoint pathways. Previous reports regarding clinicalefficacy of the allogeneic pancreatic tumor vaccine GVAX and Listeriavaccine against early-stage pancreatic intraepithelial neoplasms dem-onstrated that efficacy was far superior when inhibition of Tregs isinduced in parallel with vaccine delivery (32, 33). Therefore, combi-nation with immune checkpoint inhibition to mitigate immune sup-pression initiated early in tumorigenesis or coadministration withCD25 antagonistsmay further improve the efficacy ofVIReST (34, 35),although the timing of delivery ofmultiple agentswill need to be clearlydefined for maximal activity. A further consideration in this regard isthe myeloid-derived suppressor cell (MDSC) populations in prema-lignant or malignant lesions. Previous studies have reported thatMDSC levels in premalignancy correlates negatively with the devel-opment of antigen specific humoral and adaptive immune responsesafter therapeutic vaccination using a MUC1-directed vaccine (36).Mechanisms to reduce these populations specifically would benefitVIReST therapy. In addition, an awareness of MDSC levels in at-riskpopulations may direct treatment suitability. Of note, as genomesequencing provides more information about the genetic evolutionof cancers, potential immunogenic, and driver mutations, it will bepossible to modify iPSCs further, incorporating further initiating,tissue specific truncal mutations to increase the antigenic relevanceof the vaccine to patients or tailor treatments to family history (37). Anassessment of all these factors may vastly improve the development ofadequate levels of T-cell memory responses that confer long-termprotection to vulnerable populations.

For clinical translation, the determination of vaccine eligibility stillremains challenging. This technology has potential for therapeutic use,after tumor resection where adequate viable autologous material is notrecovered, as a mechanism to prevent tumor recurrence. Determina-tion of patients for primary prophylactic use is more complex;however, with the current lack of effective approaches for earlydiagnosis and screening, there has been a recent report of individualswith germline mutations or familial risk factors opting for radicalpancreatectomy to mitigate their risk (38, 39), and these would beobvious populations to benefit from the option of noninvasive pro-phylaxis. The European registry of hereditary pancreatitis and familialpancreatic cancer exists to stratify patients with pancreatic cancer forthe purpose of clinical intervention, and similar registries exist world-wide that stratify patients with pancreatic cancer by risk. Our expand-ing knowledge of the progression of genomic alterations and theinflammatory microenvironment that drive premalignancy are pro-viding unprecedented possibilities to identify at-risk individuals forearly intervention with cancer prevention strategies (40), and ambi-tious efforts are underway to detect early cancers via liquid biopsiesand analysis of circulating tumor DNA (41) in addition to a

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coordinated effort to determine effective serum or urine biomarkers todetect PDACearly in disease (42, 43), whichwill provide candidates forearly intervention strategies such as VIReST to prevent malignantprogression of nascent cancer.

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

Authors’ ContributionsConception and design: S. Lu, Y. WangDevelopment of methodology: S. Lu, P. Du, W. Yan, M. El Khouri, Y. WangAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): S. Lu, Z. Zhang, P. Du, M. El Khouri, Y. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Lu, Z. Zhang, P. Du, L.S. Chard, M. El Khouri,Z. Wang, A. Nagano, J. Wang, C. Chelala, J. Chen, Y. Dong, N.R. Lemoine, Y. WangWriting, review, and/or revision of themanuscript: S. Lu, L.S. Chard, J.Wang, J. Liu,Y. Dong, S. Wang, J. Dong, N.R. Lemoine, Y. Wang

Administrative, technical, or material support (i.e., reporting or organizing data,constructing databases): W. Yan, Z. Zhang, Y. Chu, D. Gao, Q. Zhang, J. Liu,S. Wang, X. Li, Y. WangStudy supervision: L. Zhang, J. Liu, P. Liu, D. Pei, Y. Wang

AcknowledgmentsThis project is supported by the National Key R&D program of China

(2016YFE0200800), the Nature Sciences Foundation of China (U1704282,81771776, and 31301007), and the core funding for development of the Celland Gene Therapy Program by Zhengzhou University. L.S. Chard is funded byThe MRC (MR/M015696/1). The authors are grateful to Professor David Tuvesonfor providing materials for this study.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 30, 2019; revised August 5, 2019; accepted October 24, 2019;published first November 25, 2019.

References1. Pandya PH,MurrayME, Pollok KE, Renbarger JL. The immune system in cancer

pathogenesis: potential therapeutic approaches. J Immunol Res 2016;2016:4273943.

2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin 2017;67:7–30.

3. de Bruin EC,McGranahanN,Mitter R, SalmM,WedgeDC, Yates L, et al. Spatialand temporal diversity in genomic instability processes defines lung cancerevolution. Science 2014;346:251–6.

4. Joura E, Bautista O, Luxembourg A. A 9-valent HPV vaccine in women. N Engl JMed 2015;372:2568–9.

5. Qu C, Chen T, Fan C, Zhan Q, Wang Y, Lu J, et al. Efficacy of neonatal HBVvaccination on liver cancer and other liver diseases over 30-year follow-up of theQidong hepatitis B intervention study: a cluster randomized controlled trial.PLoS Med 2014;11:e1001774.

6. Srivatsan S, Patel JM, Bozeman EN, Imasuen IE, He S, Daniels D, et al. Allogeneictumor cell vaccines: the promise and limitations in clinical trials. Hum VaccinImmunother 2014;10:52–63.

7. Lemay CG, Rintoul JL, Kus A, Paterson JM, Garcia V, Falls TJ, et al. Harnessingoncolytic virus-mediated antitumor immunity in an infected cell vaccine.Mol Ther 2012;20:1791–9.

8. Tysome JR, Li X, Wang S, Wang P, Gao D, Du P, et al. A novel therapeuticregimen to eradicate established solid tumors with an effective induction oftumor-specific immunity. Clin Cancer Res 2012;18:6679–89.

9. Li Y, Zeng H, Xu RH, Liu B, Li Z. Vaccination with human pluripotent stem cellsgenerates a broad spectrum of immunological and clinical responses againstcolon cancer. Stem Cells 2009;27:3103–11.

10. Kooreman NG, Kim Y, de Almeida PE, Termglinchan V, Diecke S, Shao N-Y,et al. Autologous iPSC-based vaccines elicit anti-tumor responses in vivo.Cell Stem Cell 2018;22:501–13.

11. Rostovskaya M, Bredenkamp N, Smith A. Towards consistent generation ofpancreatic lineage progenitors from human pluripotent stem cells. Philos TransR Soc Lond B Biol Sci 2015;370:20140365.

12. Huang L, Holtzinger A, Jagan I, BeGora M, Lohse I, Ngai N, et al.Ductal pancreatic cancer modeling and drug screening using humanpluripotent stem cell- and patient-derived tumor organoids. Nat Med2015;21:1364–71.

13. Hingorani SR, Wang L, Multani AS, Combs C, Deramaudt TB, HrubanRH, et al. Trp53R172H and KrasG12D cooperate to promote chromo-somal instability and widely metastatic pancreatic ductal adenocarcinomain mice. Cancer Cell 2005;7:469–83.

14. Denes B, Yu J, Fodor N, Takatsy Z, Fodor I, Langridge WH. Suppression ofhyperglycemia in NOD mice after inoculation with recombinant vacciniaviruses. Mol Biotechnol 2006;34:317–27.

15. Tysome JR, Briat A, Alusi G, Cao F, Gao D, Yu J, et al. Lister strain of vacciniavirus armed with endostatin-angiostatin fusion gene as a novel therapeutic agentfor human pancreatic cancer. Gene Ther 2009;16:1223–33.

16. Chen J, Liu J, Chen Y, Yang J, Chen J, Liu H, et al. Rational optimization ofreprogramming culture conditions for the generation of induced pluripotentstem cells with ultra-high efficiency and fast kinetics. Cell Res 2011;21:884–94.

17. Li K, Zhu S, Russ HA, Xu S, Xu T, Zhang Y, et al. Small molecules facilitate thereprogramming of mouse fibroblasts into pancreatic lineages. Cell Stem Cell2014;14:228–36.

18. Zhang Z, Chen X, Chang X, Ye X, Li Y, Cui H. Vaccination with embryonic stemcells generates effective antitumor immunity against ovarian cancer. Int J MolMed 2013;31:147–53.

19. Le Boeuf F, Diallo JS, McCart JA, Thorne S, Falls T, Stanford M, et al. Synergisticinteraction between oncolytic viruses augments tumor killing.Mol Ther 2010;18:888–95.

20. Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death in cancertherapy. Annu Rev Immunol 2013;31:51–72.

21. Guo ZS, Liu Z, Bartlett DL. Oncolytic immunotherapy: dying the rightway is a key to eliciting potent antitumor immunity. Front Oncol 2014;4:74.

22. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, et al.Generation of induced pluripotent stem cells without Myc from mouse andhuman fibroblasts. Nat Biotechnol 2008;26:101–6.

23. Fratta E, Sigalotti L, Covre A, Parisi G, Danielli R, Marie Nicolay HJ, et al.Epigenetic mechanisms in cancer formation and progression. In:MacalusoAGaM, editor. Cancer epigenetics: biomolecular therapeutics for human cancer.Hoboken, NJ: John Wiley & Sons, Inc.; 2011.

24. Langevin SM, Kratzke RA, Kelsey KT. Epigenetics of lung cancer. Transl Res2015;165:74–90.

25. Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of humancancer. Nat Rev Genet 2006;7:21–33.

26. Esteller M, Risques RA, Toyota M, Capella G, Moreno V, Peinado MA, et al.Promoter hypermethylation of the DNA repair gene O(6)-methylguanine-DNAmethyltransferase is associated with the presence of G:C to A:T transitionmutations in p53 in human colorectal tumorigenesis. Cancer Res 2001;61:4689–92.

27. Esteller M, Toyota M, Sanchez-Cespedes M, Capella G, Peinado MA,Watkins DN, et al. Inactivation of the DNA repair gene O6-methylgua-nine-DNA methyltransferase by promoter hypermethylation is associatedwith G to A mutations in K-ras in colorectal tumorigenesis. Cancer Res 2000;60:2368–71.

28. Jounai N, Kobiyama K, Takeshita F, Ishii KJ. Recognition of damage-associatedmolecular patterns related to nucleic acids during inflammation and vaccination.Front Cell Infect Microbiol 2012;2:168.

29. Yang Y, Huang CT, Huang X, Pardoll DM. Persistent Toll-like receptor signalsare required for reversal of regulatory T cell-mediated CD8 tolerance.Nat Immunol 2004;5:508–15.

30. Xagorari A, Chlichlia K. Toll-like receptors and viruses: induction of innateantiviral immune responses. Open Microbiol J 2008;2:49–59.






31. Schneider J, Gilbert SC, Blanchard TJ, Hanke T, Robson KJ, Hannan CM, et al.Enhanced immunogenicity for CD8þ T cell induction and complete protectiveefficacy of malaria DNA vaccination by boosting with modified vaccinia virusAnkara. Nat Med 1998;4:397–402.

32. Le DT, Wang-Gillam A, Picozzi V, Greten TF, Crocenzi T, Springett G, et al.Safety and survival with GVAX pancreas prime and Listeria Monocytogenes-expressing mesothelin (CRS-207) boost vaccines for metastatic pancreaticcancer. J Clin Oncol 2015;33:1325–33.

33. Keenan BP, Saenger Y, Kafrouni MI, Leubner A, Lauer P, Maitra A, et al. AListeria vaccine and depletion of T-regulatory cells activate immunity againstearly stage pancreatic intraepithelial neoplasms and prolong survival of mice.Gastroenterology 2014;146:1784–94.

34. Forni G.Vaccines for tumor prevention: a pipe dream? J Infect DevCtries 2015;9:600–8.

35. Chu NJ, Armstrong TD, Jaffee EM. Nonviral oncogenic antigens and theinflammatory signals driving early cancer development as targets for cancerimmunoprevention. Clin Cancer Res 2015;21:1549–57.

36. Ma P, Beatty PL, McKolanis J, Brand R, Schoen RE, Finn OJ. Circulatingmyeloid derived suppressor cells (MDSC) that accumulate in premalignancyshare phenotypic and functional characteristics with MDSC in cancer.Front Immunol 2019;10:1401.

37. LevineAJ, Jenkins NA,CopelandNG. The roles of initiating truncalmutations inhuman cancers: the order of mutations and tumor cell type matters. Cancer Cell2019;35:10–5.

38. Grant RC, Selander I, Connor AA, Selvarajah S, Borgida A, Briollais L, et al.Prevalence of germlinemutations in cancer predisposition genes in patients withpancreatic cancer. Gastroenterology 2015;148:556–64.

39. Iskandar ME, Wayne MG, Steele JG, Cooperman AM. Familial pancre-atic cancer: the case for prophylactic pancreatectomy in lieu of serialscreening and shared decision making. Case Rep Oncol Med 2014;2014:737183.

40. Kensler TW, Spira A, Garber JE, Szabo E, Lee JJ, Dong Z, et al. Transformingcancer prevention through precision medicine and immune-oncology.Cancer Prev Res 2016;9:2–10.

41. Aravanis AM, Lee M, Klausner RD. Next-generation sequencing of circulatingtumor DNA for early cancer detection. Cell 2017;168:571–4.

42. Song J, Sokoll LJ, Pasay JJ, RubinAL, LiH, BachDM, et al. Identification of serumbiomarker panels for the early detection of pancreatic cancer. Cancer EpidemiolBiomarkers Prev 2019;28:174–82.

43. Radon TP, Massat NJ, Jones R, Alrawashdeh W, Dumartin L, Ennis D, et al.Identification of a three-biomarker panel in urine for early detection of pan-creatic adenocarcinoma. Clin Cancer Res 2015;21:3512–21.


Lu et al.




CLINICAL CANCER RESEARCH | CORRECTION

Correction: A Virus-infected, ReprogrammedSomatic Cell–derived Tumor Cell (VIReST)Vaccination Regime can Prevent Initiationand Progression of Pancreatic CancerShuangshuang Lu, Zhe Zhang, Pan Du, Louisa S. Chard, Wenli Yan,Margueritte El Khouri, Zhizhong Wang, Zhongxian Zhang, Yongchao Chu,Dongling Gao, Qinxian Zhang, Lirong Zhang, Ai Nagano, Jun Wang,Claude Chelala, Jing Liu, Jiekai Chen, Pentao Liu, Yunshu Dong,Shengdian Wang, Xiaozhu Li, Jianzeng Dong, Nick R. Lemoine,Duanqing Pei, and Yaohe Wang

In the original version of this article (1), five immunofluorescence panels in Fig. 3A wereincorrect: DE-KPC-Oct4/DAPI, PPLC-KPC-Nanog/DAPI, PPLC-KPC-Sox17/DAPI,PPLC-WT-KP-Nanog/DAPI, and PPLC-WT-KP-Sox17/DAPI. In addition, the legendfor Fig. 2E did not provide clarification about the teratoma section used. These errors havebeen corrected in the latest onlineHTMLandPDF versions of the article. The authors regretthese errors.

Reference1. Lu S, Zhang Z, Du P, Chard LS, Yan W, El Khouri M, et al. A virus-infected, reprogrammed somatic cell–

derived tumor cell (VIReST) vaccination regime can prevent initiation and progression of pancreaticcancer. Clin Cancer Res 2020;26:465–76.

Published online May 3, 2021.Clin Cancer Res 2021;27:2663doi: 10.1158/1078-0432.CCR-21-0850�2021 American Association for Cancer Research.

AACRJournals.org | 2663



2020;26:465-476. Published OnlineFirst November 25, 2019.Clin Cancer Res Shuangshuang Lu, Zhe Zhang, Pan Du, et al. Progression of Pancreatic Cancer(VIReST) Vaccination Regime Can Prevent Initiation and

Derived Tumor Cell−A Virus-Infected, Reprogrammed Somatic Cell

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