Preclinical Evaluation of Sequential Combination of ... · Oncolytic virus are engineered to take advantage of genetic mutations of speciﬁc tumor suppressor genes or oncogenes in

Large Molecule Therapeutics

Preclinical Evaluation of Sequential Combinationof Oncolytic Adenovirus Delta-24-RGD andPhosphatidylserine-Targeting Antibody inPancreatic Ductal AdenocarcinomaBingbing Dai1, David Roife1, Ya'an Kang1, Joy Gumin2, Mayrim V. Rios Perez1,Xinqun Li1, Michael Pratt1, Rolf A. Brekken3, Juan Fueyo-Margareto4,Frederick F. Lang2, and Jason B. Fleming1

Abstract

Delta-24-RGD (DNX-2401) is a conditional replication-com-petent oncolytic virus engineered to preferentially replicate in andlyse tumor cells with abnormality of p16/RB/E2F pathway. In aphase I clinical trial, Delta-24-RGD has shown favorable safetyprofile and promising clinical efficacy in brain tumor, whichprompted us to evaluate its anticancer activity in pancreatic ductaladenocarcinoma (PDAC), which also has high frequency ofhomozygous deletion and promoter methylation of CDKN2Aencoding the p16 protein. Our results demonstrate that Delta-24-RGD can induce dramatic cytotoxicity in a subset of PDAC celllines with high cyclin D1 expression. Induction of autophagy andapoptosis by Delta-24-RGD in sensitive PDAC cells was con-firmed with LC3B-GFP autophagy reporter and acridine orangestaining aswell asWestern blotting analysis of LC3B-II expression.

Notably, we found that Delta-24-RGD induced phosphatidylser-ine exposure in infected cells independent of cells' sensitivity toDelta-24-RGD, which renders a rationale for combination ofDelta-24-RGD viral therapy and phosphatidylserine targetingantibody for PDAC. In a mouse PDAC model derived from aliver metastatic pancreatic cancer cell line, Delta-24-RGD signif-icantly inhibited tumor growth compared with control (P <0.001), and combination of phosphatidylserine targeting anti-body 1N11 further enhanced its anticancer activity (P < 0.01)possibly through inducing synergistic anticancer immuneresponses. Given that these 2 agents are currently in clinicalevaluation, our study warrants further clinical evaluation ofthis novel combination strategy in pancreatic cancer therapy.Mol Cancer Ther; 16(4); 662–70. �2016 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC) remains one of

the leading causes of cancer-related death (1). Althoughadvances have been made, the overall 5-year survival rate ofpatients with PDAC remains less than 7% mainly due to latediagnosis and limited number of available systemic agents.New effective therapeutic agents for this cancer are still urgentlyneeded.

Oncolytic virus are engineered to take advantage of geneticmutations of specific tumor suppressor genes or oncogenes in

cancer cells to allow preferential replication within and lysis ofcancer cells while sparing normal cells (2, 3). Multiple types ofoncolytic viruses including Ad5/3 adenovirus, herpes simplexviruses (HSV), vaccinia virus, Newcastle disease virus, andmeasles virus have been evaluated clinically (3). The firstFDA-approved HSV1-based oncolytic virus T-VEC carrying agranulocyte-macrophage colony-stimulating factor (GM-CSF)gene significantly improved durable response rate of patientswith unresectable melanoma compared with GM-CSF treat-ment alone (16.3% vs. 2.1%, P < 0.0001; ref. 4). The approvalof T-VEC for late-stage melanoma brings the hope in oncolyticvirus–mediated immunotherapy for cancer treatment. Delta-24-RGD is an adenovirus-based oncolytic virus with a deletionof 24 basepairs in the E1A region and a modification in virusfiber with a RGD-4C motif to enhance its infection of cancercells independent of the expression of coxsackievirus andadenovirus receptor (CAR; refs. 5, 6). Adenovirus E1A genecodes a 19-kDa protein that binds to RB protein, thus releasingE2F factor from RB/E2F complex for cell-cycle progression. Thedeletion of 24 basepairs in E1A region suppresses virus repli-cation in normal cells but not in cancer cells with defect of p16/RB/E2F pathway. Delta-24-RGD has shown promising antican-cer effect by stimulating anticancer immune response in braintumor patients (7) and is currently in phase II clinical trials withcombination of chemotherapy for brain tumor. Because p16/RB/E2F pathway is also frequently altered in pancreatic cancer

1Department of Surgical Oncology, The University of Texas MDAnderson CancerCenter, Houston, Texas. 2Department of Neurosurgery, The University of TexasMD Anderson Cancer Center, Houston, Texas. 3Hamon Center for TherapeuticOncology Research, UT Southwestern Medical Center, Dallas, Texas. 4Depart-ment of Neuro-Oncology, The University of Texas MD Anderson Cancer Center,Houston, Texas.

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

Corresponding Author: Jason B. Fleming, Department of Surgical Oncology,Unit 0107, The University of Texas M.D. Anderson Cancer Center, 1515 HolcombeBlvd., Houston, TX 77030. Phone: 713-745-0890; Fax: 713-745-1921; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-16-0526

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

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due to the deletion, mutation, or promoter methylation ofCDKN2A gene which encodes the p16 protein, we hypothesizethat Delta-24-RGD could be an active agent for pancreaticcancer therapy, especially for the tumors with abnormalp16/RB/E2F pathway.

Phosphatidylserine, a membrane phospholipid, is localized inthe inner leaflet of a plasmamembrane in normal nontumorigeniccells but is presented on the surface of apoptotic cells and cancercells within the tumormicroenvironment (8, 9). Although a signalfor cell engulfment, phosphatidylserine is known to dampen theimmune response. Phosphatidylserine exposure on the outermembrane also occurs during viral cellular infection and replica-tion. Monoclonal antibodies have been raised to target phospha-tidylserine and investigated as anti-viral therapy (10). Recent datafrom an animal model of melanoma demonstrated that combin-ing phosphatidylserine-targeting antibodies improved the effec-tiveness of immune checkpoint inhibitors, suggesting that anti-bodies to phosphatidylserine can reverse its immune dampeningsignals (11). Phosphatidylserine-targeting antibodies, bavituxi-mab, have also been raised to target phosphatidylserine-expressingtumor cells and investigated in phase I clinical trials of severalsolid tumors systems including metastatic breast and lung cancers(12, 13). Together, these studies suggest that anti-phosphatidyl-serine antibodies could augment the anticancer effects of oncolyticvirus therapy.

In this study, we evaluated the anticancer activity of Delta-24-RGD in multiple pancreatic cancer cell lines and primary pan-creatic cells established from patient-derived xenograft tumors(PDX) and explored potential predictive biomarkers for sensitiv-ity.We found thatDelta-24-RGD induced dramatic cytotoxicity ina subset of pancreatic cancer cell lines with high expression ofcyclin D1 and induced phosphatidylserine exposure in infectedcells. In addition, combination with a phosphatidylserine-target-ing antibody further enhanced the anticancer effects of Delta-24-RGD in vivo, possibly through stimulating immune responses.Our study demonstrates that Delta-24-RGD could be a promisingagent, alone or in combination, for pancreatic cancer therapy.

Materials and MethodsReagents, cell lines, and animals

Cell culture medium RPMI-1640 was purchased fromHyClone. FBS and MTT cell viability reagents were purchasedfrom Life Science Technologies. Penicillin/streptomycin/neomy-cin (PSN) Antibiotic Mixture 100� was purchased from Sigma.Antibodies for PARP, cleaved PARP, caspase-9, cleaved caspase-9,caspase-7, cleaved caspase-7, Ki-67, and b-actin were purchasedfrom Cell Signaling Technology. E1A antibody and phosphati-dylserine antibody [4b6] for in vitro immunofluorescent stainingwere purchased from Abcam. CD68 and NKp46 antibodies werefrom Biolegend. Phosphatidylserine antibody 1N11, also knownas PGN635, a fully human anti-phosphatidylserine antibody wasprepared by Dr. Rolf A. Brekken's laboratory (University of TexasSouthwesternMedical Center, Dallas, TX) and was used for in vivostudy (14). Conventional pancreatic cancer cell lines were fromATCC, and primary pancreatic cancer cells were established in ourlaboratory as described previously (15). All primary pancreaticcancer cells were authenticated with unique fingerprinting. NOD/SCID and nude mice (female, 6 weeks) were purchased from theNational Cancer Institute (NIH, Bethesda, MD) and JacksonLaboratories.

Adenovirus infectionDelta-24-RGD and Ad-GFP-RGD virus were prepared as

described previously (16). For cell infection with Ad-GFP-RGDor Delta-24-RGD, 1 � 104 cells were seeded in 6-well plates, andafter 24 hours, cells were infected with adenovirus at differentmultiplicity of infections (MOI).

Cytotoxicity crystal violet stainingCells (1 � 104) were seeded in 6-well plates and infected with

Delta-24-RGD or Ad-GFP-RGD control virus at different MOIs,and then 10 days postinfection, cells were fixed with ice-coldmethanol for 10minutes. Cells werewashedwith PBS and stainedwith 0.5% crystal violet solution under room temperature for 10minutes. Theplateswere rinsedwithwater until no color cameoff.The plates were dried at room temperature for overnight.

Cell viability MTT assayCells were seeded at the density of 1� 103 cells per well in a 96-

well plate, and 24 hours later, the cells were infected with Dleta-24-RGD at MOIs of 0.1, 0.3, 1, 3, and 10. Cell viability wasmeasured with MTT assay as described previously (15). The dosethat causes 50% of cells death (IC50) of infected cells comparedwith noninfected control was calculated with GraphPad Prismsoftware (6.0).

Immunofluorescent staining and FACS analysisCells were harvested with trypsinization and washed with PBS

containing 10% FBS. About 2 � 106 cells in 100 mL FACS bufferwere added in polystyrene round-bottom tube and incubatedwith another 100mL Fcblockingbuffer on ice for 20minutes. Cellswere centrifuged at 1,500 rpm for 5 minutes at 4�C and resus-pended with 100 mL FACS buffer. Primary antibodies were addedin the buffer with a concentration of 1 mg/mL. Cells were incu-bated for 60 minutes at 4�C. After incubation, cells were washedwith FACS buffer 3 times by centrifugation at 1,500 rpm andresuspended in 200 mL cold FACS buffer. Diluted fluorochrome-labeled secondary antibody in FACS buffer was added to the cellsand incubated them for 30 minutes at 4�C. Cells were washed 3times with FACS buffer and resuspended in 200 mL FACS buffer.FACS analysis of fluorescence intensity was performed withFACSCalibur (BD), and the results were analyzed with FlowJo10 software (FlowJo).

Autophagy assayCells (1�105) seeded in thewells of chamber slidewere treated

with PBS or Delta-24-RGD virus at 1 MOI for 3 to 5 days, and 10mL LC3B-GFP virus (BacMam 2.0, Thermo Fisher) was added intothe well and incubated for 24 hours. Cells were checked underfluorescence microscope and LC3-GFP–positive autophagosomewere counted. For acridine orange staining and flow cytometricanalysis, cells (1� 106) in 6-well plates were infected with Delta-24-RGD virus at MOIs of 0.1, 1, and 10 for 5 days. Cells wereharvested, washed with PBS, and incubated with acridine orange(1 mg/mL) for 15 minutes. Cells were washed with PBS twice andresuspended in 400 mL PBS for FACS analysis.

Analysis of virus replicationCells (1 � 105) were seeded in 6-well plates and infected with

Delta-24-RGD at 1 MOI. Cells were harvested and counted, andcell lysates were collected at days 1, 3, and 7 postinfection. VirusDNA was isolated and purified with Adeno-X qPCR Titration Kit

Combination of Delta-24-RGD and 1N11 in PDAC

www.aacrjournals.org Mol Cancer Ther; 16(4) April 2017 663




fromClontech Company. The copy numbers of adenovirus in thecells were quantified with the kit following manufacturer'sinstruction and normalized by cell number.

Western blottingCells after treatment or infection were washed with cold PBS,

and tissue lysates were extracted with RIPA buffer and quantifiedfor protein concentration with bicinchoninic acid (BCA)method.Thirty to 50micrograms of protein was used forWestern blotting.Detailedmethods can be found in our previous publication (17).

Immunohistochemical and immunofluorescent staining ofparaffin-embedded tumor tissues

Tumor tissues harvested from mice were fixed for overnight.Paraffin-embedded tumor tissues were cut into 5-mm sections.Immunohistochemical or immunofluorescent staining was per-formedusing themethods described previously (18). Imageswerecaptured with an Olympus DP72 camera and CellSens softwareon an Olympus BX51 microscope.

Animal experiment in vivoAnimal experiment protocol (00001089-RN00) was reviewed

and approved by The University of Texas MD Anderson CancerCenter (Houston, TX) institutional review board and in accor-dance with the Guidelines for the Care and Use of LaboratoryAnimals published by the NIH. MDA-PATC53 cells were har-vested, and 2 � 106 cells in 100 mL PBS were mixed with equalvolume of Matrigel (Invitrogen) and implanted into nude mice.When tumors reach the size of about 100 mm3, mice wererandomly divided into 4 groups with 5 mice in each group. Micewere treatedwithPBSorDelta-24-RGDadenovirus 3 timesweeklyby intratumoral injection of 1 � 108 plaque-forming units (pfu)virus. Treatment with 1N11 antibody was started after the third

virus treatment by intraperitoneal injection of 100 mL antibodywith the dose of 1 mg/kg. A total of 3 doses of 1N11 wereadministrated. The tumor volumes were calculated using theformula: length � width2 � 0.52. When tumors reached the sizeof 12 mm in diameter, all mice were sacrificed, and tumors wereharvested for histochemical analysis.

Statistical analysisThe significance of differences between different treatment

groups was analyzed by two-way ANOVA or t test (two tailed),and P < 0.05 was considered as significant. Correlation of cyclinD1 expression with sensitivity of cell lines to Delta-24-RGD viruswas analyzed with Pearson correlation method. All statisticalanalyses were done with GraphPad Prism 6.0 (GraphPad Soft-ware Inc.).

ResultsOncolytic virus Delta-24-RGD induced dramatic anticanceractivity in pancreatic cancer cells

To test the cytotoxicity of Delta-24-RGD in pancreatic cancercells, 4 cell lines, BxPC3, PANC1,MiaPaCa2, andMDA-PATC53, aprimary cell line established in our laboratory, were infected withAd-GFP-RGD control and Delta-24-RGD at different MOIs fol-lowed by crystal violet staining. Infection of cells with Delta-24-RGD virus induced dramatic cytotoxicity effects in PANC1, Mia-PaCa2, and MDA-PATC53 cells but not in BxPC3 cells (Fig. 1A).We then used the cell viability assay to test cytotoxicity of Delta-24-RGD in 6 classic and 6 primary pancreatic cancer cell linesderived from PDAC PDXmodels (Fig. 1B and C). Six of 12 testedcell lines were sensitive to Delta-24-RGD. Notably, PANC1,MiaPaCa2, and AsPC1 have similar sensitivity as human gliomacell line (U251),whichwasused as apositive control as it has beenpreviously shown to be sensitive to Delta-24-RGD (5). The IC50

Figure 1.

Oncolytic virus Delta-24-RGD(D-24-RGD) induced cytotoxicity inpancreatic cancer cells. A, Cytotoxiccrystal violet staining. Cells (1 � 104)were seed in 6-well plate and infectedwith D-24-RGD or Ad-GFP-RGDcontrol at different MOIs, and then 10days postinfection, cells were fixedwith 4% formalin and stained withcrystal violet.B andC,MTT cell viabilityassays in multiple conventional andprimary pancreatic cancer cell lines.Cells were seed at 1 � 103 in 96-wellplates and infected with D-24-RGDwith indicated MOIs, and then 10 daysafter infections, cells viability wasanalyzed with MTT assay.

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values of Delta-24-RGD in each of the cell lines were calculated(Supplementary Table S1). We used the IC50 less than 10MOIs asa sensitivity cutoff. On the basis of this cutoff, of the 12 linestested, MDA-PATC53, MiaPaCa2, PANC1, MDA-PATC108,AsPC1, and MDA-PATC118 are sensitive to Delta-24-RGD. Theseresults suggest that Delta-24-RGD induced dramatic cytotoxicityin a subset of pancreatic cancer cells.

CyclinD1 expression is associatedwith sensitivity of PDAC cellsto Delta-24-RGD

To determine the molecular markers that correlate with sensi-tivity of pancreatic cancer cells to Delta-24-RGD (SupplementaryTable S1), we analyzed the protein expressions of the majorcomponents of p16/RB/cyclin D1/CDK4 pathway using Westernblotting. The expression of p16 was low in all of the tested cells,except MDA-PATC43 (Fig. 2A), which is consistent with high-frequency deletion and promoter methylation of p16 gene inpancreatic cancer. This result indicates that lowornoexpressionofp16 is not a predictor of sensitivity to Delata-24-RGD. Theexpression of RB gene was low in all the sensitive cells, whereas2 of 5 resistant cells also have low RB expression, suggesting thatlowRB expression alone is not a predictor of sensitivity either (Fig.2A). Overall, the expression of cyclin D1 and CDK4 was higher insensitive cells than in resistant cells (Fig. 2B). Using Pearsoncorrelation analysis, we found that cyclinD1 expression is reverse-ly correlated with the IC50 of Dela-24-RGD (R ¼ �0.8506; Fig.2C). To examine for differences in virus replication betweensensitive and resistant cells, virus copy number was analyzed witha PCR-based assay. As was shown in Fig. 2D, virus copy numberincreased dramatically over time in 2 sensitive cell lines, Mia-PaCa2 and MDA-PATC53, but not in the resistant BxPC3 cells,suggesting that replication of Delta-24-RGD in infected cells isassociated with its cytotoxicity effects.

Delta-24-RGD induced autophagy in pancreatic cancer cellsPrevious studies have found that oncolytic virus induces

autophagy-mediated cancer cells death (19). To investigatewhether the cytotoxicity induced by Delta-24-RGD in pan-creatic cancer cells is also mediated by autophagy, LC3B-GFPexpression reporter was introduced into the cells after 5 daysof infection with Delta-24-RGD. Infection of Delta-24-RGDindeed dramatically induced autophagosome formation evi-denced by LC3B-GFP punctae in Delta-24-RGD–sensitive andnot in -resistant cells (Fig. 3A and B). Acridine orangestaining further confirmed that acidic vesicular organelles(AVO) were significantly increased in sensitive cells afterinfection with Delta-24-RGD (Fig. 3C). In addition, Delta-24-RGD induced dramatic expression of LC3B-II, an autop-hagy marker, in sensitive cells (Fig. 3D). The result alsoshowed that high MOI of Delta-24-RGD induced cleavedPARP, caspase-7, and caspase-9, indicating that caspase acti-vation was induced (Fig. 3D).

Delta-24-RGD adenovirus induced phosphatidylserineexposure in pancreatic cancer cells

Phosphatidylserine is an aminophospholipid distributed ininner leaflet of cell membrane. It is externalized to the outerleaflet when cells undergo apoptosis. Exposure of phosphati-dylserine is a signaling for phagocytosis of apoptotic cells.Phosphatidylserine exposure on a virus's envelop or vesicle is astrategy called apoptotic mimicry for virus entry into host cells(20–22). Previous studies have revealed that some virusescan induce exposure of phosphatidylserine on the membraneof infected cells, which has been explored as a strategy forantivirus therapy (10). Phosphatidylserine exposure on cancer-specific endothelial cells and cancer cells was also reported andplays a role in cancer-related immunosuppression (8, 23–25).

Figure 2.

Cyclin D1 expression is associated withthe sensitivity of pancreatic cancer cellsto Delta-24-RGD (D-24-RGD). A,Western blotting assay. The expressionof the major components in p16/RBpathways, including p16, RB, cyclin D1,and CDk4, was analyzed with Westernblotting. B, Relative expression level ofcyclin D1 in resistant and sensitivepancreatic cell lines was analyzed usingthe ratio of the density of cyclin D1 overb-actin. C, Correlation of cyclin D1expression with cells' sensitivity wasanalyzed with Pearson correlation. D,D-24-RGD virus replication. D-24-RGDvirus replications in the cells afterinfection were analyzed using PCR-based assay to check virus copy numberin BxPC3, MiaPaCa2, and MDA-PATC53cells as described in Materials andMethods. NS, not significant;� , P < 0.05; �� , P < 0.01; �� , P < 0.001.






Targeting phosphatidylserine with an anti-phosphatidylserineantibody has been under clinical investigation for cancertherapy (11, 26, 27). To check whether Delta-24-RGD canalso induce phosphatidylserine exposure in infected pancreaticcancer cells, immunofluorescent staining followed by flowcytometric analysis of Detla-24-RGD–infected cells was per-formed with a phosphatidylserine-specific antibody. As wasshown in Fig. 4A, infection with Delta-24-RGD virus inducedphosphatidylserine exposure in both resistant and sensitivepancreatic cancer cells. Further microscopic analysis also con-firmed that Delta-24-RGD induced phosphatidylserine exter-nalization to the outer leaflet of the cell membrane in 3 PDACcell lines (Fig. 4B). These results suggest that Delta-24-RGD caninduce phosphatidylserine exposure, which may not dependon the sensitivity of infected cells.

Combination of Delta-24-RGD and phosphatidylserine-targeting antibody induced enhanced antitumor activity in vivo

Previous studies have shown that phosphatidylserine exposurein tumor cells induces immunosuppression and targeting phos-phatidylserine promotes anticancer immune response (11, 26).We hypothesize that the combination of Delta-24-RGD oncolyticvirus with phosphatidylserine-targeting antibody may furtherenhance anticancer effects through both antibody-dependentcell-mediated cytotoxicity (ADCC) and enhanced anticancerimmune response. To confirm this hypothesis, mice bearingtumors derived from a sensitive cell line MDA-PATC53 were firsttreated with 1 � 108 pfu of Delta-24-RGD for 3 times by intra-tumoral injections. Then, mice were treated with intraperitonealinjections of phosphatidylserine-targeting antibody 1N11, a fullyhuman phosphatidylserine antibody. The detailed treatment

Figure 3.

Delta-24-RGD (D-24-RGD) induced autophagy in pancreatic cancer cells. A, Autophagy assay with LC3B-GFP fusion protein. Cells were infected with D-24-GDR (1MOI) for 5 days followed by infection with LC3-GFP expression baculovirus for 24 hours, and then LC3B-GFP punctae (arrow indicated) were checked undermicroscope. B, Quantification of LC3B-GFP punctae. Punctae in 10 random views were counted under microscope. Results are mean � SD. �� , P < 0.01. C,Acridine orange staining. Cells were infected with D-24-RGD virus at different MOIs, and 5 days postinfection, cells were harvested and stained with acridine orangefollowed by flow cytometric analysis. Results are mean � SD. � , P < 0.5; �� , P < 0.01. D, Western blotting assay. Cells were infected with or Ad-GFP-RGD(Ad-GFP) control or D-24-RGD for 5 days, and cell lysates were harvested with RIPA buffer for Western blotting assay using the indicated antibodies.

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schedule is shown in Fig. 5A. The result showed that treatmentwith Delta-24-RGD alone significantly inhibited tumor growthcompared with nontreated control (P < 0.0001). Phosphatidyl-serine-targeting antibody 1N11 had a moderate antitumor effectthat was less effective than Delta-24-RGD alone (Fig. 5B and C).However, the combination of Delta-24-RGD virus and phospha-

tidylserine-targeting antibody was more effective than each agentalone (Delta-24-RGD plus 1N11 vs. 1N11 alone, P < 0.0001;Delta-24-RGDplus 1N11 vs.Delta-24-RGD, P< 0.01). Expressionof adenovirus antigen E1A was confirmed in Delta-24-RGD orcombination–treated tumors, indicating efficient virus infectionand caspase-related cell death induced by Delta-24-RGD in vivo

Figure 4.

Delta-24-RGD (D-24-RGD) inducedphosphatidylserine exposure inpancreatic cancer cells. A, Flowcytometric analysis ofphosphatidylserine. Cells wereinfected with D-24-RGD in 6-wellplates for 5 days, and cells wereharvested and stained withphosphatidylserine antibody andAlexa Fluor-488–conjugated secondantibody followed by flow cytometricanalysis. B, Microscopic analysis ofphosphatidylserine exposure. Cellswere grown and infected withD-24-RGD (1 MOI) in chamber slidefor 3 days. Cells were fixed with 4%formaldehyde and stained withphosphatidylserine antibody. The cellswere permeabilized with 0.1% TritonX-100, and the nuclei were stainedwith Hoechst 33342. Images weretaken under fluorescence microscope(40�, bar ¼ 50 mm).

Figure 5.

Evaluation of the combination ofDelta-24-RGD (D-24-RGD) and 1N11 invivo. A, Schedule of administration ofD-24-RGD and 1N11 antibody. Whentumor reach the size of about 100mm3, mice were divided into 4treatment groups and treated with D-24-RGD, 1N11, alone or in combinationas indicated schedule. B, Tumorgrowth curve. Tumor sizes weremeasured with caliper every 5 daysand calculated using the formula: d �l2 � 0.52. Two-way ANOVA was usedfor significance between 2 treatmentgroups. (Combo vs. 1N11, P < 0.0001;combo vs. D-24-RGD, P < 0.01; 1N11 vs.control,P<0.01; D-24-RGD vs. control,P < 0.0001). C, Tumors at the end ofthe experiments were harvested. D,Immunohistochemical staining wasperformed in tumor tissues withantibodies against adenovirus E1A andcleaved caspase-9 (ccs-9).






(Fig. 5C). Consistent with previous studies that phosphatidylser-ine-targeting antibody induces microphage activation (27), ourstudy shows enhanced staining of CD68, a marker of macro-phages, after treatment with 1N11 alone or after the combinationtreatment (Fig. 6). In addition, we also observed that infiltrationof activated NK cells was enhanced in the tumor tissue with singleor combined treatments (Fig. 6).

DiscussionA recent clinical trial of Delta-24-RGD in patients with glio-

blastoma demonstrated favorable toxicity profile and remarkableclinical efficacy (7). This prompted us to evaluate its anticanceractivity in pancreatic cancer, as 85%of PDACshave an incompletep16/RB/E2F pathway due to promoter methylation and homo-zygous deletion of CDKN2A locus encoding p16 protein (28). Inour study, Delta-24-RGD induced dramatic cytotoxicity in morethan 50% of the tested cell lines. Specifically, 3 of 6 testedpancreatic cancer cell lines have sensitivity comparable to thatof the human glioma cell line U251 (Fig. 1A), which has beenshown, in previous study, to be a sensitive cell line to Delta-24-RGD in vitro and in vivo (19). One primary pancreatic cancer cellline,MDA-PATC53, whichwas established in our laboratory froma liver metastasis of pancreatic cancer (15), was particularlysensitive to Delta-24-RGD (Fig. 1A and C). Because most ofmetastatic pancreatic cancer cells are usually resistant to che-motherapies, this result suggests that Delta-24-RGD might be apromising agent for pancreatic cancer therapy. Importantly, atelomerase reverse transcriptase (TERT)-immortalized humanpancreatic duct epithelia cell line, HPNE-tert, was not sensitiveto Delta-24-RGD, suggesting that Delta-24-RGDmay not be toxicto normal pancreatic cells.

In this study, 2 conventional cell lines, BxPC3 and Hs776T,were not sensitive to Delta-24-RGD, and these 2 cell lines, based

on previous studies, have wild-type form of p16 gene (29).However, the genetic status of p16 seems to not be the principaldriver of cell sensitivity to Delta-24-RGD, as p16 was very low inalmost all tested cells lines (Fig. 2B). Likewise, RB expression wasnot found to be the determinant of sensitivity, although all thesensitive cells have low expression of RB (Fig. 2B). These resultssuggest that although Delta-24-RGD was originally engineered topreferably replicate in cancer cells with abnormality in p16/RBpathway, genetic or molecular changes of other factors might alsoaffect the sensitivity of cancer cells to Delta-24-RGD. We foundthat among the tested components of p16/RB pathway, theexpression level of cyclin D1 was most correlated with the sen-sitivity of PDAC cells toDelta-24-RGD. CyclinD1 can bind to andactivate CDK4/CDK6, which phosphorylates RB protein andpromotes the release of E2F and cell-cycle progression. Thispathway is negatively regulated by p16; however, in cells withdeletion or suppressed expression of p16, cyclin D1 becomes thekey driver for the activation of this pathway. This could explainwhy, in our study, high expression of cyclin D1 was closely linkedto Delta-24-RGD sensitivity. Considering that about 40% to 80%of pancreatic cancers have cyclin D1 overexpression or geneamplification (30, 31), we speculate that Delta-24-RGD couldbe active in a large fraction of pancreatic cancers.

Autophagy or autophagy-mediated apoptosis are a describedmechanism of oncolytic virus–induced cytotoxicity (19). In ourstudy, we usedmultiple approaches, including autophagy report-er LC3-GFP, acridine orange staining, and Western blotting toconfirm autophagy induction by Delta-24-RGD in pancreaticcancer cells (Fig. 3). Exposure of phosphatidylserine onenvelopedviruses or on the vesicular membrane of unenveloped viruses, astrategy called apoptosis mimicry, is a described method of virusinfection (32). In addition, some viruses induce phosphatidyl-serine exposure in infected cells, opening the possibility of usinganti-phosphatidylserine antibodies as an antiviral therapy (10).

Figure 6.

Combination of D-24-RGD and1N11 enhanced antitumorimmunoresponse. Immunofluorescentstaining was performed with tumortissues from treated or untreatedmice. Microphage marker CD68 andNK cell marker NKp46 were used tocheck microphage infiltrations intumor tissues. Images were takenunder fluorescence microscope(20�).

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However, the mechanism of virus-induced phosphatidylserineexposure in host cells has not been well studied. We found thatDelta-24-RGD induces phosphatidylserine exposure in both sen-sitive and resistance cells (Fig. 4). We speculate that oncolyticvirus–induced phosphatidylserine exposure could be caused byspecific viral genes, as it reports of E1B 19-kDa protein–inducedapoptotic mimicry in host cells to modulate innate immuneresponses (33). Alternatively, phosphatidylserine exposure couldbe related with metabolic changes induced by oncolytic virus(34). Further study is necessary to determine the mechanism ofadenovirus-induced phosphatidylserine exposure to develop fur-ther rationale treatment combinations with virus-mediated genetherapy.

Phosphatidylserine exposure induced by oncolytic viruses canhelp the clearance of infected cells by immune system. This offersan opportunity for combination therapy with phosphatidylser-ine-targeting antibodies. Phosphatidylserine-targeting antibo-dies, including bavituximab and 1N11, have been evaluated inmetastatic breast and lung cancers inphase I clinical trials showingpromising results (12, 13). In this study, we tested the combina-tion of Delta-24-RGD and 1N11, a fully human phosphatidyl-serine-targeting antibody similar to bavituximab, by using asequential combination strategy. Themajor consideration for thissequential combination strategy is that phosphatidylserine-tar-geting antibody may suppress virus production and spreading ifgiven concurrently. In a mouse pancreatic cancer model, sequen-tial combination of Delta-24-RGD and 1N11 was superior toeach single treatment alone (Fig. 5B). Mechanistically, combina-tion treatment induced macrophage-mediated anticancerimmune response, which was evidenced by increased staining ofmicrophage marker, CD68, in the tumors treated with singleand combination regimen. Stimulating of differentiation ofM2 tumor–associated microphage (TAM) to M1 TAM by phos-phatidylserine-targeting antibody has been reported previously(11, 27). In addition, modulating anticancer immune responseby Delta-24-RGD has been observed in patients with glio-blastoma and in immunocompetent mouse brain tumor models(7, 35–37). Notably, single treatment with Delta-24-RGD alsoinduced strong anticancer effects, whichmay involve direct tumorlyses, autophagy, as well as immune responses, as the nude miceused for the tumor model keep natural killer cells and macro-phages (38). Nevertheless, more pancreatic tumor animal mod-els, including immunocompetent mouse models, might beused to evaluate the anticancer effects induced by Delta-24-RGDas a single agent and in combination with other agents, such asphosphatidylserine-targeting antibodies.

In summary, in this study, we evaluated the anticancer activityof oncolytic virus Delta-24-RGD in pancreatic cancer cells in vitroand in preclinical animal models in vivo. We demonstrate thatDelta-24-RGD is active against pancreatic cancer cells, and cyclinD1 expression is associated with the vulnerability of pancreatic

cancer cells to oncolytic adenovirus treatment. In addition, wedemonstrate that Delta-24-RGD induced phosphatidylserineexposure, and combination of phosphatidylserine-targeting anti-body and Delta-24-RGD viral therapy induced enhanced antitu-mor activity. Further studies are needed for full understanding ofthemechanismof oncolytic virus–induced cytotoxicity, phospha-tidylserine exposure, and oncolytic virus–induced immuneresponse to develop rational combination therapies as well aspredictive biomarkers for patients' selection. Nevertheless, ourstudy demonstrates that Delta-24-RGD is a promising agent aloneor in combination, for the treatment of pancreatic cancer, adevastating disease with limited therapeutic regimens available.Given that Delta-24-RGD and phosphatidylserine-targeting anti-bodies are being evaluated in clinical trials and have favorabletoxicity profile, combination of these 2 agents could be quicklytranslated into clinical testing.

Disclosure of Potential Conflicts of InterestR.A. Brekken reports receiving a commercial research grant fromand serves as

a Medical and Scientific Advisor at Peregrine Pharmaceuticals. J. Fueyo-Margar-eto is shareholder of and is a consultant/advisory board member forDNAtrix. F.F. Lang reports receiving other commercial research supportfrom DNAtrix, Inc. and is a patent holder for Delta-24-RGD. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: B. Dai, J.B. FlemingDevelopment of methodology: B. Dai, D. Roife, Y. Kang, J. Gumin, X. Li,F.F. LangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Dai, D. Roife, M.V.R. Perez, M. PrattAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Dai, M.V.R. PerezWriting, review, and/or revision of the manuscript: B. Dai, R.A. Brekken,J. Fueyo-Margareto, F.F. Lang, J.B. FlemingAdministrative, technical, or material support (i.e., reporting or organiz-ing data, constructing databases): B. Dai, D. Roife, X. Li, R.A. Brekken,J. Fueyo-Margareto, F.F. Lang, J.B. FlemingStudy supervision: J.B. Fleming

Grant SupportThis work was funded by The Skip Viragh Family Foundation, the Various

Donors in Pancreatic Cancer Research Fund (to J.B. Fleming), the ResearchAnimal Support Facility—Houston under NIH/NCI award P30CA016672(to R.A. Depinho), the National Cancer InstituteP50 CA127001, the BroachFoundation for BrainCancer Research, theHoward and Susan Elias Foundation,and the Jason&PriscillaHiley Fund (to F.F. Lang). Thisworkwas also supportedin part by the NIH grant T32CA009599 (D. Roife and M.V. Rios Perez).

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

Received August 5, 2016; revised December 1, 2016; accepted December 15,2016; published OnlineFirst January 30, 2017.

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Dai et al.




2017;16:662-670. Published OnlineFirst January 30, 2017.Mol Cancer Ther Bingbing Dai, David Roife, Ya'an Kang, et al. Antibody in Pancreatic Ductal AdenocarcinomaAdenovirus Delta-24-RGD and Phosphatidylserine-Targeting Preclinical Evaluation of Sequential Combination of Oncolytic

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