A novel CDC25B promoter based oncolytic adenovirus inhibited growth of orthotopic human pancreatic tumors in different preclinical models

Cancer Therapy: Preclinical

A Novel CDC25B Promoter–Based OncolyticAdenovirus Inhibited Growth of OrthotopicHuman Pancreatic Tumors in Different PreclinicalModelsHelga L.Weber1,2, Manuel Gidekel2,3, Santiago Werbajh1, Edgardo Salvatierra1,Cecilia Rotondaro1, Leonardo Sganga1, Gabriela Acosta Haab4, David T. Curiel5,Eduardo G. Cafferata1, and Osvaldo L. Podhajcer1

Abstract

Purpose:We decided to construct a novel oncolytic adenoviruswhose replication was driven by the CDC25B promoter for its usein preclinical models of pancreatic cancer.

Experimental Design:We placed the essential E1A gene undercontrol of the CDC25B promoter. Based on preliminary data, wepseudotyped the adenovirus with a chimeric fiber of serotypes5/3. We investigated the in vitro lytic effect and the in vivotherapeutic efficacy in combination with gemcitabine on humanpancreatic tumor xenografts orthotopically growing in nudemiceand in tumors growing in Syrian hamsters. We also assessedbiochemical markers of hepatic toxicity and CA19.9 levels.

Results: AV25CDC exhibited a strong in vitro lytic effect onpancreatic cancer cells. In vivo administration of AV25CDC com-bined with gemcitabine in mice harboring subcutaneously grow-ing SW1990 pancreatic tumors almost abrogated tumor growth.

Nude mice harboring 15-day-old orthotopic tumors, treatedintratumorally or systemically with AV25CDC combined withgemcitabine, exhibited 70% to 80% reduction in tumor sizecompared with control mice that lasted for at least 60 days.Chemovirotherapy treatment induced a return to normal levelsof biochemical parameters of hepatic toxicity; these mice exhib-ited more than 90% reduction in CA19.9 serum levels comparedwith control. Chemovirotherapy efficacy was confirmed in miceharboring Mia PaCa-2 tumors and in Syrian hamster harboringHaP-T1 tumors.Weobserved that viral treatment disrupted tumorarchitecture and induced an increase inMMP-9 activity thatmightfacilitate gemcitabine penetrability.

Conclusion: These data demonstrate that AV25CDC is aneffective oncolytic agent candidate for pancreatic cancer chemovir-otherapy combination. Clin Cancer Res; 21(7); 1665–74. �2015 AACR.

IntroductionWorldwide, more than 200,000 people die annually of pan-

creatic cancer, making it the fourth leading cause of cancer-relateddeath in the United States (1, 2). Despite the increased under-standing of the molecular biology of the disease, the use ofthe purine analog gemcitabine remains the standard of care(3), with a median survival time that did not exceed 6.5 months(4). In fact, gemcitabine and erlotinib remained as the only twodrugs approved for use in the advanced disease with modestbenefit.

One rising area as a potential new therapeutic approach incancer are oncolytic viruses (OV). These OVs are engineered ornatural viruses with selective toxicity for malignant cells. AmongOVs, conditionally replicative adenoviruses (CRAd) were devel-oped based on the fact that the transcriptional activity of the E1Agene, that is essential for adenoviral replication is driven bytumor-specific promoters (5). Several CRAds have been evaluatedin preclinical trials for their potential therapeutic effect on pan-creatic cancer such as the cyclooxygenase 2 promoter-based CRAd(6), and especially the hTERT promoter-based CRAd (7). Severalpreclinical studies have demonstrated improved efficacy whenhTERT- or COX-2-promoter–based oncolytic adenovirus (OAV),were combined with gemcitabine (6, 8); the replication-selectivedl922-947 adenovirus, defective in pRb binding, improved micesurvival when combined with 5-FU and gemcitabine (9). At theclinical level it was of note that intratumorally injected ONYX-15OV combined with gemcitabine was well-tolerated in a phase I/IIclinical trial of patients with pancreatic cancer (10). The HSV-1OV, HF10, reached the clinic and was well tolerated in phase Itrials (11) while a clinical trial with the HSV-1-based OV, BioVexGMCSF, is running (12). Thus, despite the severity of the diseaseand the certain success of OVs in preclinical trials, only very few ofthem reached the clinics.

CDC25B phosphatase has been found to be overexpressed inmore than 70% of human pancreatic cancer samples, oftenassociated with high-grade tumors and poor prognosis (13);

1Laboratory of Molecular and Cellular Therapy, Fundaci�on InstitutoLeloir, IIBBA-CONICET, Argentina. 2Universidad de La Frontera,Temuco, Chile. 3Universidad Aut�onoma de Chile, Santiago, Chile.4Laboratory of Pathology, Hospital de Oncología Marie Curie, BuenosAires, Argentina. 5Division of Cancer Biology,Washington UniversitySchool of Medicine, St. Louis, Missouri.

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

E.G. Cafferata and O.L. Podhajcer contributed equally to this article.

Corresponding Author: Osvaldo L. Podhajcer, Fundaci�on Instituto Leloir,Avenida Patricias Argentinas 435, Buenos Aires C1405BWE, Argentina. Phone:54-11-4-863-4011; Fax: 54-11-4-865-2246; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-2316

�2015 American Association for Cancer Research.

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interestingly, most pancreatic and gastric cancer overexpressesonly this phosphatase isoform (13). Further studies demonstratedthatCDC25B levelswere augmented2.2 andmore than four timesin metastatic pancreatic cancer compared with primary tumorsand normal pancreatic tissue, respectively (14). Also, both malig-nant cells and cancer-associated fibroblasts exhibited stronglypositive staining for CDC25B (14). We constructed a novel CRAdnamed AV25CDC, in which the adenoviral E1A gene was placedunder the control of a 0.45-kb fragment of theCDC25B promoter.The combination of AV25CDC and gemcitabine exhibited thelargest therapeutic effect on orthotopically implanted humanxenografts tumors in nude mice and on tumors in Syrian ham-sters; AV25CDC therapeutic effect was accompanied by a strongdecrease in biochemical markers of hepatic toxicity and in thetumor biomarker CA19.9. Further evidence demonstrated thatAV25CDC treatment induced the disruption of tumor architec-ture that might have helped gemcitabine to penetrate deeper intothe tumor mass.

Materials and MethodsCell lines

Pancreatic cancer cells (BxPC-3, MIA PaCa-2, Panc-1, andSW1990), colorectal cancer cells (HT 29 and LoVo), gastric cancercells (MKN-45), andnormal cells (CCD1140sk,HFL1,WI38)werepurchased from the American Tissue Culture Collection (ATCC)between 2006 and 2008. All the cells were authenticated by ATCC.Upon arrival, cells were thawed, expandedonce (P1) to obtain 106

cells, and stored in liquid nitrogen in five vials. When necessary,each one of the vials at P1 was thawed and expanded to obtain 10vials (P2). Only P2 cells were used for the in vitro and in vivoexperiments. The HaP-T1 hamster cell line was kindly provided byDr. RubenHernandez Alcoceba (University of Navarra, Pamplona,Spain), andHaCaT cells were kindly provided by Fernando Larcher(Universidad Carlos III, Madrid, Spain). All the cell lines weregrown in the recommended medium supplemented with 10%fetal bovine serum (Natocor), 2 mmol/L glutamine, 100 U/mLof penicillin, and 100 mg/mL of streptomycin and maintained in a

37�C atmosphere containing 5% CO2. All the cells were routinelytested formycoplasma contamination by PCR, using the followingprimers: forward, 50-ACACCATGGGAGYTGGTAAT-30 and reverse,50-CTTCWTCGACTTYCAGACCCAAGGCAT-30. Our slightly mod-ified protocol (Tang and colleagues; ref. 15) is useful to detectcontamination with the following strains:M. arginine, M. orale, M.hyorhinis, M. fermentans, M. hominis, M. salivarium,M. argininin, andM. laidlawii.

Assessment of CDC25B, CONEXIN 26, and E1A mRNAexpression

Total RNA was extracted from each cell line to assess forCDC25B, CONEXIN 26, and E1A levels using quantitative real-time PCR. Detailed information is described in SupplementaryMaterials and Methods.

Assessment of viral replicationDNA was extracted from cells to assess for viral E4 levels by

quantitative real-time PCR as readout of viral particles. Total E4copies per sample were normalized with the amount of DNApresent in each sample and reported as E4 copies/ng of DNA (16).Detailed information is described in Supplementary Materialsand Methods.

Luciferase assayLuciferase activity following cells transduction by the nonre-

plicative viruses was measured using a Genios luminometer(TECAN) and normalized by protein concentration in the celllysate (Bio-Rad; ref. 16). Detailed information is described inSupplementary Materials and Methods.

In vitro assays combining AV25CDC with gemcitabineCells seeded in 96-well plates (2 � 103 cells per well) were

infected with AV25CDC for 24 hours followed by the addition offresh medium containing gemcitabine (20-deoxy-20,20-difluoro-cytidine monohydrochloride, Sandoz S.A). Five days later, cellviability was established with MTS as described (16). All assayswere carried out in six different replicates.

Cell viability assaysCellswereplated onto96-well plates at a density of 2�103 cells

per well and infected with the viruses at different multiplicity ofinfection (MOI). Six days after the amount of viable cells wasdetermined by theMTS assay (CellTiter 96 AqueousOne SolutionCell Proliferation assay (Promega). The plates were incubated for1 hour after which the absorbance of each well was read at awavelength of 490 nm. All assays were performed in quadrupli-cate, and each assay was repeated at least twice (17).

"In vivo" studiesAll the in vivo studieswere approved by the Institutional Animal

Care and Use Committee of Instituto Leloir (Protocol #30OP)that has an approved Animal Welfare Assurance as a foreigninstitution with the Office of Laboratory Animal Welfare, NIHnumber A5168-01. Whole-body images of each mouse wereobtained by the Bioluminescence Assay using an in vivo biolumi-nescent system (IVIS50; Xenogen) and the Living Image 2.20.1Software (18, 19).

"In vivo" studies on subcutaneous tumorsFive- to six-week-old female and male athymic N:NIH (S)-nu

mice (obtained from the animal facility of the Faculty of

Translational Relevance

Pancreatic cancer exhibits a high mortality rate, and cur-rently no effective therapy is available. We have developed anoncolytic adenovirus named AV25CDC in which E1A expres-sion is driven by the human CDC25B gene promoter.AV25CDC was used solely or combined with gemcitabine totreat nudemice harboring orthotopically established SW1990human pancreatic cancer xenografts. Mice treated intratumo-rally or systemically with AV25CDC þ gemcitabine exhibitedup to 80% reduction in tumor size compared with controlmice, and showed a return to normal levels of biochemicalparameters of hepatic toxicity; these mice also exhibited morethan 90% reduction in CA19.9 serum levels compared withcontrol mice. Further studies confirmed these findings inadditional preclinical models, such as Mia PaCa-2 xenograftsin nude mice and HaP-T1 tumors in Syrian hamsters. Thesefindings provide a proof of concept for the combined use ofAV25CDC and gemcitabine as a powerful therapeutic modal-ity in pancreatic cancer.

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Veterinary, University of La Plata, Argentina) were subcutane-ously (s.c.) injected in one flank with 5 � 106 cells of SW1990.When the average tumor volume reached 100 mm3, micereceived 1 � 109 viral particles per mouse of AV25CDC or PBSadministered intratumorally on days 1, 4, and 7 after tumorcells' injection. For the combination of AV25CDC with gemci-tabine, mice were injected with AV25CDC followed by intra-peritoneal (i.p.) administration of gemcitabine (15 mg/kg for 5days) starting 1 day after the last AV25CDC injection. In vivoexperiments were performed following approval of the Insti-tutional Animal Care and Use Committee (IACUC); all animalsunder study received food and water ad libitum.

Orthotopic xenograft modelMice were anesthetized with i.p. injection of 80 mg/kg

ketamine (Aveco Co., Inc.) and 10 mg/kg xylazine (RugbyLaboratories, Inc.). A small (1 cm) lateral subcostal laparotomywas performed. A total of 1 � 105 SW1990 cells, suspended in50 mL PBS þ Matrigel 20% v/v (Matrigel Basement MembraneMatrix, BD Biosciences), were injected beneath the capsule ofthe pancreas, and the abdominal wall and skin were closed. Forintratumor treatment, mice received 1 � 109 viral particles permouse of AV25CDC. For systemic treatment the mice receivedPBS (50 mL) or 1010 viral particles/mouse in PBS (50 mL) in tailvein. For the combination with gemcitabine, mice were injectedi.p. with gemcitabine (15 mg/kg for 5 days) starting 1 day afterthe last AV25CDC injection. The last day of the experiment (40or 60 days after first virus administration) animals were bledand serum was used to determine serum markers in a special-ized laboratory. Mice were euthanized following institutionalguidelines, tumors were removed, weighed, and fixed in 10%buffered formalin for immunohistochemical studies; part ofthe tumor was kept for E4 quantification. None of the animalsshowed any signs of toxicity or weight loss throughout theexperiment.

Five-week-old male Syrian golden hamsters (weight, 70–80 g)were obtained from and housed at the animal facility of theNational Commission of Atomic Energy, Argentina. The animalswere anesthetizedwith i.p. injection of 80mg/kg ketamine (AvecoCo., Inc.) and 10 mg/kg xylazine (Rugby Laboratories, Inc.). Asmall (1 cm) lateral subcostal laparotomywas performed.HaP-T1cells (5 � 105), suspended in 100 mL PBS þ Matrigel 20% v/v(Matrigel Basement Membrane Matrix, BD Biosciences), wereinjected beneath the capsule of the pancreas, and the abdominalwall and skin were closed. Animals were anesthetized and thenadministered a single intrajugular injection of 1 � 1010 viralparticles per hamster of AV25CDC. For the combination ofAV25CDC with gemcitabine, hamsters were injected withAV25CDC followed by i.p. administration of gemcitabine(15 mg/kg for every day for 3 days) starting 1 day after theAV25CDC injection. At the end of the experiments (40 days afteradenoviral injection) hamsters were euthanized following insti-tutional guidelines. Tumors were removed, weighed, cut, andfixed in 10%buffered formalin for immunohistochemical studiesand the other half frozen for E4 quantification. Animals showedno signs of toxicity or weight loss throughout the experiment.

Statistical analysisStatistical analyses are detailed in SupplementaryMaterials and

Methods.

ResultsAV25CDC exhibited an in vitro cytocidal activity on pancreaticcancer cells

Because the sequence of the human version of the CDC25Bpromoter was not available, we designed oligonucleotides pri-mers based on the Cdc25B murine promoter and cloned twodifferent fragments of the human genomic sequence of 0.25 kband 0.45 kb extensions (Supplementary Fig. S1A); the largestregion of homology between the murine and the human versionextends from�110 toþ4 of the murine promoter that includes aTATA box, a NFY site and two SP1 sites (Supplementary Fig. S1A),which were described as functional in the murine promoter (20).

In parallel, we established the infective capacity of severaladenoviral vectors carrying different engineered chimeric fibers.Using luciferase expression as a reporter gene, we demonstratedthat the strongest activity was associated with the adenoviruscarrying the chimeric fiber of serotypes 5/3 (Fig. 1A). Therefore,we constructed nonreplicative adenoviral vectors containing thechimeric 5/3 fiber expressing luciferase downstream of the 0.2 kb(AV25CDCs Luc 5/3) or the 0.45 kb (AV25CDCLuc 5/3)CDC25Bpromoter variants. We observed that the strongest luciferaseactivitywas associatedwith the virus carrying the 0.45 kbCDC25Bpromoter variant (Fig. 1B). The transcriptional activity of the 0.45kb promoter correlated with CDC25BmRNA levels, as the largestactivity was observed in Panc-1 pancreatic cancer cells that exhib-ited the highestCDC25BmRNA levels (Supplementary Table S1).

Thus, we constructed a CRAd named AV25CDC in which E1Awas cloned downstream of the 0.45 kb CDC25B promoter. E1Aexpression was confirmed byWestern blot analysis after infectionof SW1990 pancreatic cancer cells with AV25CDC (Supplemen-tary Fig. S1B). AV25CDC was highly lytic on Panc-1, MIA Paca2,and BxPC3 cells even at lowMOIs of 0.1–1, whereas SW1990 cellswere more resistant to the lytic activity as AV25CDC was effectiveonly at a starting MOI of 10 (Fig. 1C). Using E4 copy number as areadout, we observed up to 5-fold increase in AV25CDC viralparticles 72 hours after infection confirming that the virus canindeed replicate in SW1990 cells (Fig. 1C, inset). Wild-typeadenovirus 5/3 (Ad5/3WT) was more effective than AV25CDCin BxPC3 and SW1990, whereas both viruses exhibited a similaractivity in Panc-1 and MIA PaCa-2 cells (Fig. 1C). AV25CDC alsoexhibited a strong cytopathic effect on HT29 and LoVo coloncancer cells and MKN45 gastric cancer cells (SupplementaryFig. S1C). On the other hand, AV25CDC showed a largely atten-uated lytic effect on nonmalignant cells that express very lowlevels of CDC25B, compared with Ad5/3WT (Fig. 1D).

The combination of AV25CDC and gemcitabine exhibited thelargest therapeutic effect on subcutaneously establishedhumanpancreatic tumor xenografts

Treatment with the nucleoside analog gemcitabine (GEM) isthe mainstay chemotherapeutics for human pancreatic cancer(21). In initial in vitro studies, we observed that MIA Paca-2and SW1990 cells were largely resistant to GEM with an IC50 of1 mmol/L (Fig. 2A) that seemed to correlate with the lackof connexin-26 expression that was shown to facilitate cell tocell passage of GEM through gap junctions (Fig. 2A, inset).Further studies demonstrated that the combination ofAV25CDC þ GEM was more effective than each single agentalone, on the in vitro growth inhibition of SW1990 cells(Fig. 2B). Based on this evidence, we assessed initially whether

A Novel Oncolytic Adenovirus for Pancreatic Cancer

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a combined chemovirotherapy could be more effective thansingle agent treatment of s.c. established SW1990 tumors. Toavoid any effect of GEM on the cell cycle that would hamper

AV25CDC replication, the virus was administered 24 hours inadvance of GEM in all the combination treatments. The com-bination of AV25CDC þ GEM reduced tumor growth up to

Figure 1.In vitro infectivity and lytic activity of AV25CDC. A, levels of infectivity of nonreplicative adenoviral vectors with modified capsid on pancreatic cancer cells. Resultsare expressed in relative units of Renilla luciferase (RLU) normalized per milligram of total protein. B, luciferase activity of nonreplicative adenovirus whoseactivity is driven by the 0.25 kb CDC25B promoter (AV25CDCs Luc 5/3) and the 0.45 kb CDC25B promoters (AV25CDC Luc 5/3) in pancreatic cancer cell lines.Cell extracts were assayed 48 hours after cells' transduction. Results are expressed in relative units of Firefly luciferase (RLU) normalized to Renilla. C and D,in vitro lytic effect of AV25CDC on malignant and nonmalignant cells. Cells were cultured for 6 days in the presence of different MOIs of AV25CDC; cell viability wasdetermined by the MTS assay in three independent experiments. Data are expressed in all cases as the mean�SD. Inset, E4 gene copies of Ad5/3WT and AV25CDCfollowing replication in SW1990 cells. E4 gene copies were determined at 5 and 72 hours as a measurement of viral replication (�� , P < 0.001).

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90% (Fig. 2C) compared with control mice. Surprisingly, GEMalone had no therapeutic effect at all, whereas the virus alonehad an intermediate effect (Fig. 2C). We confirmed thatAV25CDC was able to replicate inside the tumor mass as weobserved a 2-fold increase in E4 copy number at day 7 after asingle intratumor administration of AV25CDC (Fig. 2C, inset).We next assessed the fractional tumor volume (FTV) to estab-lish whether the combined chemovirotherapy effect was syn-ergistic (22). Indeed, an FTV value of 5.7 indicated that thechemovirotherapy combination exerted a synergistic therapeu-tic effect (Supplementary Table S2). Because GEM alone had noeffect at all, it was likely that viral administration 24 hours inadvance facilitated GEM penetration inside the tumor mass.

Combination of AV25CDC and gemcitabine was highlyefficient on orthotopically xenografted human tumors

To further define the therapeutic efficacy of AV25CDC, weadministered 5 � 105 Matrigel-embedded SW1990 cells expres-sing luciferase directly into the mice pancreas. Fifteen days later,mice were split in four groups that received either PBS; GEM aloneadministered i.p. three times; a single intratumoral (i.t.) injectionof 1� 109 viral particles of AV25CDC; or AV25CDC followed 24

hours later by GEM (AV25CDC þ GEM). Mice were sacrificed 25days later and an autopsywas performed that included removal ofthe entire tumor area. Microscopic analysis of control tumorsshowed an infiltratingmucinous adenocarcinoma of the pancreasand areas of remnant pancreatic acinar cells (data not shown).Macroscopic analyses evidenced a clear reduction in tumor sizeespecially between mice treated with AV25CDC þ GEM com-pared with control and GEM-treated mice (Fig. 2D, left). Thecombination of AV25CDC þ GEM induced the largest reductionof 75% in tumor weight, compared with control mice, althoughno statistically significant differences in tumor size were observedbetween the AV25CDC-treated group compared with the combi-nation of the OAV þ GEM (Fig. 2D, right); even not when micewere injected i.t. twice with the OAV (Supplementary Fig. S1D).The presence of adenoviral E4 gene copies inside the tumor massat the end of the experiment confirmed that AV25CDC replicatedin vivo (Supplementary Fig. S1D, inset).

We also assessed biochemical parameters associatedwith organtoxicity. Control mice exhibited increased serum levels of amy-lase,GOT-AST andGPT-ALT indicating that both the pancreas andthe liver were compromised by tumor growth (Table 1). GEMtreatment induced a reversion to normal levels of GPT-ALT but

Figure 2.In vitro and in vivo effects of AV25CDCand GEM. A, gemcitabine cytotoxicity.Cells treated for 72 hours withincreasing concentrations ofgemcitabine.Weassessed the viabilityusing the MTS assay. Inset, connexin26 mRNA expression. B, in vitroefficacy of the combination ofAV25CDC with GEM. SW1990 cellswere treated either with AV25CDCalone or combined with gemcitabine.Six days later, cell viability wasassessed with the MTS assay. C, in vivostudies on s.c. established tumors.Nude mice harboring s.c. SW1990tumors were treated as described inMaterials and Methods. Animals weresacrificed when tumors reachedapproximately 2,500 mm3. Data areexpressed as mean � SD (� , P < 0.05;�� , P < 0.01 at day 80). Inset,assessment of E4 gene copies presentat day 7 in s.c. SW1990 tumorsfollowing one intratumoradministration of AV25CDC(�� , P < 0.01). D, left, representativemacroscopic images of tumorsobtained at autopsy frommice treatedeither with PBS, GEM, AV25CDC, orAV25CDCþ GEM. Arrow, tumor mass.Right, tumor weight assessed at theend of the experiment after autopsy.(� , P < 0.01; �� , P < 0.001.)






not of GOT-AST and amylase, indicating that the pancreas and theliver remained highly compromised (Table 1). However, treat-ment with AV25CDCor AV25CDCþGEM induced a reversion tonormal levels in all the biochemical parameters tested (Table 1).Interestingly, control mice exhibited huge serum levels of thetumor biomarker CA 19.9 released by SW1990 cells. At the end ofthe experiment, the combined treatment with AV25CDC þ GEMinduced almost 95% reduction in CA 19.9 levels compared withthe control, confirming that the largest therapeutic benefit wasobserved following the combined chemovirotherapy treatment(Table 1).

To further confirm AV25CDC efficacy mice harboring ortho-topic SW1990 tumors were split in six different groups thatreceived PBS, PBS þ GEM, OAV (i.t.), OAV (i.t.) þ GEM, OAV(i.v.), and OAV (i.v.) þ GEM. Mice were followed for additional60 days when control mice were considered as not survivors.Confirming theprevious studies, the combinationof AV25CDCþGEM exhibited the largest therapeutic effect regardless of whetherthe OAV was administered i.t. or systemically, although GEMcombined with i.t. administered OAV was slightly superior(Fig. 3A). Interestingly, the chemovirotherapy treatment wasstatistically significantly more efficacious than the OAV or GEMtreatments as single agents (Fig. 3A). Moreover, the chemovir-otherapy combination also showed the complete reversion tonormal levels of all the serummarkers of organ toxicity (data notshown). Mice follow up with bioluminescence imaging demon-strated that the chemovirotherapy treatment that included i.t.administered OAV combined with GEM seemed to completelyarrest tumor growth (Fig. 3B and C). Similar studies performedwith mice harboring orthotopic Mia PaCa-2 tumors confirmedthat the combination of AV25CDC þ GEM was more efficaciousthan each single agent alone (Fig. 4A); the combination of GEMwith i.t. administered OAV was slightly superior to the combina-tion of GEM with systemically injected OAV although not statis-tically significant difference was observed (Fig. 4A). On the otherthe combination of the OAV administered i.t. combined withGEM exhibited statistically significant higher efficacy than theOAV alone (Fig. 4A).

Further confirmation of the therapeutic efficacy of the OAVwasattempted with HaP-T1 pancreatic cancer cells on Syrian ham-sters. First, we confirmed that HaP-T1 cells indeed expressed highlevels ofCDC25B (Supplementary Fig. S2A insert). AV25CDCwasable to lyse HaP-T1 cells in vitro at MOIs similar to that of Ad5WTand Ad5/3WT (Supplementary Fig. S2A, left); moreover,AV25CDC lytic effect was enhanced in the presence of GEM(Supplementary Fig. S2A right). For the in vivo studies Syrianhamsters harboring 15-day-old orthotopic Hap-T1 tumors weresplit in four groups that received either AV25CDC Luc 5/3; GEMalone; a single systemic administration of 1 � 1010 viral particlesof AV25CDC; or AV25CDC þ GEM. The nonreplicative adeno-viruswasused as a control as it hadno effect on the growthofHap-T1 tumors (data not shown). Hamsters were followed for 40 days

when control animals were considered not survivors. The resultsconfirmed that the combined chemovirotherapy exhibited thelargest statistically significant therapeutic effect compared withthe control or the OAV or GEM treatments as single agents(Fig. 4B). E4 gene copies assessment in the tumormass confirmedthat AV25CDC indeed replicated in vivo (Fig. 4C). In comparison,we observed only neglectable levels of viral particles in lung,kidney, spleen, and liver that are permissive for adenoviral rep-lication (Fig. 4C).Moreover, nohistologic abnormalitywas foundin the normal organs of all treated Syrian hamsters (Supplemen-tary Fig. S2B). In addition, AV25CDC was unable to replicate inexplants obtained from four different normal Syrian hamsterorgans compared with Ad5/3WT, which replicated in all of them(Supplementary Fig. S2C).

AV25CDC administration disrupted tumor architectureThe whole in vitro and in vivo data clearly demonstrated a

cooperative effect between AV25CDC and GEM suggesting thatthe prior administration of the OAVmight facilitate GEM activity.On the other hand, wewere unable to see any stimulatory effect ofGEM on the transcriptional activity of the CDC25B promoter(Supplementary Fig. S3A) nor on the cell-surface expression of theadenoviral serotype 3 receptors Desmoglein-2 and CD46 (Sup-plementary Fig. S3B).

Similar to human tumors, pancreatic tumor xenografts growings.c. or orthotopically in nude mice contained tumor cells nestssurroundedbyadense extracellularmatrix ofpacked collagenfiberswith intermingledfibroblasts. Thesefibers weremore prominent ins.c. tumors compared with orthotopic tumors (compare Fig. 5Aaand 5Ab). Histologic analysis of the tumor mass 24 hours afteradministration of AV25CDC showed a severe disruption of thetumor architecture with large necrotic areas devoid of collagenfibers both in the s.c. (Fig. 5AC) as in the orthotopic tumorstreated i.t. or systemically (Fig. 5AD and 5Ae, respectively).Deep analysis evidenced tumor architecture disruption in largeareas accounting for 10% to 40% of the entire tumor mass (datanot shown). Interestingly, systemic or i.t. administration of theAd5/3WT virus also disrupted tumor architecture (Supplemen-tary Fig. S3C). Immunohistochemical analysis at 24hours showednodifference in the amount of infiltrating neutrophils andmacro-phages (the infiltrate was devoid of NK cells), that located mostlyat the tumor periphery (data not shown). The disruption of tumorarchitecture was increasingly evident at 7 days after OAV admin-istration (Fig. 5Bb and 5Bd) compared with control tumors(Fig. 5Ba and 5Bc). Interestingly, tumor architecture disruptionwas associated with highly increased levels of activated MMP-9(Fig. 5C) inside the E1A-expressing tumor mass (Fig. 5D).

DiscussionWe have developed a novel and potent replicative oncolytic

adenovirus that was able to inhibit the in vivo growth of locally

Table 1. Serum markers of organ toxicity and tumor growth

TreatmentsBiochemical parameters Naive PBS GEM AV25CDC AV25CDC þ GEM

CA 19.9 (UI/mL) 73.2 � 11.3 38,591 � 9,571 14,041 � 6,265 9,964 � 4,984 2,630 � 1,864Amylase (U/L) 769.0 � 124 1,242 � 133 1,151 � 386 809.0 � 170 680.0 � 5.3GOT (AST; UI/L) 212.5 � 47 305.0 � 137 280 � 64 180.3 � 37 184.0 � 55.0GPT (ALT; UI/L) 48.3 � 6.8 74.5 � 26 47,0 � 17 46.0 � 14 40.0 � 5.0

NOTE: Data are expressed as mean � SD.

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advanced orthotopically growing human pancreatic tumor xeno-grafts in nude mice and pancreatic tumors in Syrian hamsters.

It was of note that GEM had almost no effect on s.c. tumorsand only a partial one on orthotopic tumors, but its combina-

tion with the OAV exhibited the largest therapeutic effect that inmost cases was also statistically significant different comparedwith the effect exerted by each single agent. Different studieshave reported that the innate resistance of pancreatic cancer tosystemic therapies is related to the poor delivery of chemothera-pies due to a dense stromal matrix (23–25). Gene expressionanalysis of GEM resistance in pancreatic cancer demonstratedenrichment in stroma-related genes (26). Targeting the stromaalso enhanced macrophage-mediated anti-stroma depletionthat increased GEM efficacy (27). The present in vivo studiesdemonstrated a remarkable disruption in tumor architecturefollowing viral treatment that was coincidental with theincreased activation mainly of MMP9. The capacity to disruptthe tumor matrix was also observed upon administration of awild-type virus pseudotypedwith a chimeric fiber 5/3 suggesting

Figure 3.In vivo efficacy of AV25CDC (combined or not with GEM) on orthotopicSW1990 mcherry-Luc tumors. A, tumor weight of orthotopic tumors wasassessed 60 days after the initiation of the different treatments at autopsy.B, representative bioluminescent imaging. Mice harboring orthotopicSW1990 mcherry-Luc tumors were treated as depicted in the panels andfollowed for 60 days. C, tumor growth curves are plotted. Luminescenceintensities are expressed in photon counts/second/ROI. (� , P < 0.05;�� , P < 0.01; �� , P < 0.001.) Figure 4.

In vivo efficacy of AV25CDC (combined or not with GEM) on orthotopicallypancreatic tumors. A, tumor weight of MIA PaCa-2 orthotopic tumors wasassessed 60 days after the initiation of the different treatments at autopsy.(�� , P < 0.01; �� , P < 0.001.) B, similar to A but with HaP-T1 tumorsorthotopically growing in Syrian hamsters. (�� , P < 0.01; �� , P < 0.001.) C, E4gene copies in different hamster organs obtained at autopsy of animalstreated with AV25CDC. Data are expressed as mean � SD (n ¼ 4).






that this is an intrinsic characteristic of adenovirus activity. Thischange in tumor architecture would certainly be permissive forGEM penetration deep inside the tumor mass and provide anexplanation for the superior therapeutic efficacy of the chemo-virotherapy combination. Because no increase in the inflamma-tory infiltrate was observed at 24 hours following AV25CDCadministration, it is likely that the enhanced MMP9 activity andthe disruption of tumor architecture occurs as a direct effect ofthe OAV on malignant cells. The fact that AV25CDC was able tolyse in vitro WI38 and HFL1 fetal fibroblasts that resemblecancer-associated fibroblasts (CAF) indicates that AV25CDC canbe also active on CAF in a clinical setting. In addition, we cannotrule out that E1A can also sensitize pancreatic cancer cells toGEM (28).

Preoperative CA 19.9 levels below certain threshold wereassociated with increased overall and recurrence-free survival(29). The evidence that the chemovirotherapy treatmentreduced CA 19.9 levels by 95% was a clear confirmation ofthe effective reduction in the tumor mass. Amylase levels havebeen historically associated in humans with pancreatic duct

obstruction and pancreatic cancer (30). The fact that the com-bined chemovirotherapy and at a lesser extent AV25CDC treat-ment but not GEM alone, reduced amylase levels, clearlyindicates that the treatment was able to reconstitute pancreaticfunction. GEM treatment reduced ALT but not AST levels.Previous studies in humans have shown that an increase inthe ratio of aspartate to alanine aminotransferase (AST/ALT) isan early marker of hepatic fibrosis (31). Thus, it is likely thatGEM treatment alone was unable to avoid tumor disseminationto the liver.

Previous studies have shown therapeutic efficacy of CRAdson human s.c. growing xenografts (32, 33). Only in few casesoncolytic activity was also assessed on s.c. tumors in combi-nation with GEM or 5-FU (34–36). Only a single study reportedthe use of a CRAd based on the Cox-2 promoter to systemicallytreat orthotopically injected human pancreatic cancer xeno-grafted but without combination with chemotherapy (37).Although the COX-2 promoter–based CRAd appeared veryefficient in vivo, this gene is highly expressed in inflammatorysituations raising the possibility of unwanted collateral effects

Figure 5.Microphotographs of s.c. and orthotopic tumors. A, 24 hour-old SW1990 tumors growing s.c. (a) and orthotopically (b) obtained from control mice treatedwith PBS.SW1990 tumors growing s.c. (c) or orthotopically (d and e), obtained from mice treated once with AV25CDC i.t. (c and d) or systemically (e). B, 7-day-oldtumors growing s.c. (a and b) or orthotopic (c and d) tumors obtained from control mice (a and c) or from mice treated i.t. with AV25CDC (b and d). Magnification,�10. Scale bar, 50 mm. C, MMP2 andMMP9 activation 24 hours after AV25CDC injection on tumormass. Parentheses, fold induction. D, E1A expression in tumormassafter adenoviral injection.

Weber et al.





on normal organs. It is of note that most pancreatic and gastriccancer tissues expressed only the CDC25B isoform (13) nar-rowing OAV specificity. Mostly important, all the studies per-formed in mice and Syrian hamsters demonstrated the lack oftoxicity of AV25CDC even when administered in combinationwith GEM. The chemovirotherapy combination was efficaciousregardless of whether the OAV was injected i.t or systemically,although i.t. injection, a usual procedure in pancreatic cancertreatment (10, 38), was more effective. The fact that mostpancreatic cancer are diagnosed at an advanced stage makesit unrealistic an intratumor administration unless a secondarysystemic immune response can be elicited. In this regard, fewoncolytic viral vectors were modified to express immunosti-mulatory genes such as GM-CSF (12). For systemic adminis-tration, it will be necessary to circumvent liver uptake andpreexisting immunity elicited by neutralizing antibodies, ade-novirus-specific T cells and NK response. One possibility relieson varying the administration routes (39); in addition, the useof carrier cells might act as shields to protect the virus from theimmune attack (40). AV25CDC seems to have the capacity toeliminate also colon cancer and gastric cancer cells with clearbiosafety parameters. Thus, it can be assumed that AV25CDCmight have a broader therapeutic use in different types ofcancers from gastrointestinal origin.

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

Authors' ContributionsConception and design: D.T. Curiel, E.G. Cafferata, O.L. PodhajcerDevelopment of methodology: C. Rotondaro, G. Acosta Haab, D.T. Curiel,O.L. PodhajcerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H.L. Weber, S. Werbajh, E. Salvatierra, L. Sganga,E.G. CafferataAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H.L. Weber, E. Salvatierra, E.G. Cafferata,O.L. PodhajcerWriting, review, and/or revision of themanuscript:H.L.Weber, E.G. Cafferata,O.L. PodhajcerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. GidekelStudy supervision: O.L. PodhajcerOther (awardee of grants): O.L. Podhajcer

Grant SupportThis work was supported by the PIA CTE-06, World Bank CONICYT Project,

Chile; the National Agency for Promotion of Science and Technology andAmigos de la Fundacion Leloir para la Investigacion en Cancer (AFULIC),Argentina; and the NIH Pancreatic Cancer SPORE grant 2P50CA101955, USA.H.L.Weber was the recipient of the Ph.D. fellowship 21070513 fromCONICYTand fellowship 75100015 from the Applied Cellular and Molecular BiologyPhD Program, Universidad de La Frontera.

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

Received September 17, 2014; revised December 16, 2014; acceptedDecember 18, 2014; published OnlineFirst January 8, 2015.

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Weber et al.




2015;21:1665-1674. Published OnlineFirst January 8, 2015.Clin Cancer Res Helga L. Weber, Manuel Gidekel, Santiago Werbajh, et al. Preclinical ModelsGrowth of Orthotopic Human Pancreatic Tumors in Different

Based Oncolytic Adenovirus Inhibited− PromoterCDC25BA Novel

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A novel CDC25B promoter based oncolytic adenovirus inhibited growth of orthotopic human pancreatic tumors in different preclinical models

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