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Mol Cancer Ther. 2006;5:2013-2022
© 2006 American Association for Cancer Research

Research Articles: Therapeutics

Enhanced killing of pancreatic cancer cells by expression of fusogenic membrane glycoproteins in combination with chemotherapy

Dennis Hoffmann and Oliver Wildner

Department of Molecular and Medical Virology, Institute of Microbiology and Hygiene, Ruhr-University Bochum, Bochum, Germany

Requests for reprints: Oliver Wildner, Department of Molecular and Medical Virology, Institute of Microbiology and Hygiene, Ruhr-University Bochum, Room 6/40, Building MA, D-44801 Bochum, Germany. Phone: 49-234-32-27834; Fax: 49-234-32-14352. E-mail: Oliver.Wildner{at}ruhr-uni-bochum.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pancreatic cancer has a poor prognosis with an annual mortality rate close to the annual incidence rate. We evaluated whether the expression of measles virus fusogenic membrane glycoproteins (FMG) H and F will enhance chemotherapy. Using Chou-Talalay analysis, we showed in vitro in pancreatic cancer cells that the expression of FMG often synergistically enhances clinically relevant chemotherapy. Furthermore, cell fusion in combination with chemotherapy resulted in strongly enhanced Annexin V binding, an early marker for apoptosis, when compared with single treatment. We showed in an i.p. and s.c. pancreatic xenograft model that the administration of a replication-defective adenoviral vector Ad.H/F encoding tumor-restricted FMG in combination with gemcitabine significantly enhanced treatment outcome when compared with treatment with each compound individually. To improve tumor transduction efficiency, the Ad.H/F vector was also transcomplemented with an oncolytic replication-restricted adenovirus (Ad.COXbulletMK), resulting in significantly improved treatment efficacy. We assessed treatment efficacy by survival analysis or measuring growth, respectively. In the i.p. model, on day 120, three of eight animals treated with this novel triple therapy consisting of Ad.H/F, gemcitabine, and Ad.COXbulletMK were alive and tumor free. Treatment with Ad.H/F and Ad.COXbulletMK resulted in one long-term survivor. In all other treatment groups, there were no long-term survivors. The significantly improved therapeutic outcome of animals receiving the triple therapy was attributed to multiple factors, including most likely improved FMG expression throughout the tumor and enhanced sensitivity of the tumor cells to gemcitabine by adenoviral gene products but also FMG expression. Qualitatively similar results were obtained in a s.c. pancreatic xenograft model. [Mol Cancer Ther 2006;5(8):2013–22]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Annually, ~32,000 patients in the United States and ~50,000 in Europe will develop and also die from pancreatic cancer (1, 2). Pancreatic cancer is extremely aggressive locally and frequently metastasizes to the liver and peritoneum. Complete surgical resection currently offers the only potential for long-term survival. For patients with advanced or metastatic cancer of the pancreas, the most active chemotherapeutic agents are gemcitabine and 5-fluorouracil (5-FU), which have a response rate of 15% to 20% and 10% to 20%, respectively (3, 4). The thymidylate synthetase inhibition of 5-FU is enhanced by leucovorin (LV; ref. 5). Currently, gemcitabine is the new standard of care in pancreatic cancer. It has shown activity in pancreatic cancer refractory to 5-FU (6) and offers a slightly better survival than 5-FU (4). In addition, irinotecan (CPT-11; ref. 7) and combination chemotherapy regimens with infusional 5-FU/LV and oxaliplatin, known as FOLFOX (8), have been evaluated in clinical trials. However, minimal benefit in survival has been shown for these agents as responses are usually only partial remission and of short duration.

In this study, we evaluated in vitro by using Chou-Talalay analysis (9) and in two pancreatic xenograft models whether the expression of measles virus fusogenic membrane glycoproteins (FMG) H and F, encoded by a replication-defective adenoviral vector Ad.H/F, will enhance chemotherapy.

The intratumoral injection of replication-defective viral vectors results in low tumor transduction efficiency because the dispersion of replication-deficient vectors is largely confined close to the initial site of injection (10). Several factors are responsible for this phenomenon, including the interconnection of tumor cells by tight junctions; high density of cellular receptors and binding of viral particles by these receptors effectively removes them from the intracellular space, decreasing the driving gradient for particle diffusion (11).

Previously, it has been shown by several groups that replication-competent vectors, or replication-defective vectors rendered replication-competent by transcomplementation of missing genes pivotal for vector replication, have the potential to overcome poor tumor transduction efficiency (12–16). Thus, we transcomplemented the FMG-encoding vector with the oncolytic replication-restricted adenovirus vector Ad.COXbulletMK (17). In cells transduced with both the replication-defective vector encoding FMG and the replication-competent oncolytic vector, the replication-defective vector will be transcomplemented to allow vector amplification and intratumoral spread, resulting in improved tumor transduction (12–16, 18, 19). In addition, oncolytic adenoviruses are also potent chemosensitizers (20).

Intratumoral expression of fusogenic glycoproteins lead to syncytia formation of infected cells with adjacent cells, thereby increasing the dispersion of viruses throughout the tumor, lateral spread of the transgene, virus release, and immunogenicity of tumor cells (21–25). For cancer gene therapy, glycoproteins from HIV-1 (23), gibbon ape leukemia virus (22, 24), and measles virus (25) have been evaluated. In measles virus, the hemagglutinin (H) protein mediates attachment to its receptor on the target cell (CD46 or CD150; refs. 26, 27) and thus triggers conformational changes in the virus fusion (F) glycoprotein (28). This leads to a biologically active fusogenic form of the F protein that interacts with the host cell membrane, causing virus-cell or cell-cell fusion (29). Both H and F proteins are necessary for fusion to occur.

To improve safety and therapeutic index of replication-competent adenoviral vectors, it has been attempted to restrict vector replication to tumor cells. One approach is based on the replacement of viral promoters by tumor/tissue–specific promoters, which are critical for replication. In this transcriptional targeting strategy, initially only the adenovirus E1A gene has been placed under the transcriptional control of a tumor-specific promoter (30–33). Recent studies showed enhanced tumor-restricted adenoviral replication and lysis by placing the viral E1A and E4 genes under the transcriptional control of heterologous promoters (17, 34–36).

To our knowledge, this is the first report analyzing the interaction of viral fusogenic glycoproteins and chemotherapy for the treatment of cancer. To enhance in vivo transduction efficiency, we also transcomplemented the replication-defective FMG-encoding vector with the oncolytic replication-restricted adenovirus vector Ad.COXbulletMK. This novel triple therapy had the highest antineoplastic efficacy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Cell Culture
The permanent human cell lines cfPac1, Panc1, 293, and HeLa were obtained from American Type Culture Collection (Manassas, VA).

We established short-term cultures of pancreatic cancer cells, designated PPC#1, PPC#2, and PPC#3, from patients who underwent surgery for treatment of histologically confirmed pancreatic cancer. The polyclonal cell lines cfPac1-Luc3 and Panc1-Luc3, which constitutively express firefly luciferase, were generated by transduction with an murine leukemia virus–based retrovirus encoding luciferase (GC Luc3 EN; ref. 37) and subsequent neomycin selection.

Viruses
The replication-defective adenoviral vector Ad.Luc encoding firefly luciferase (pGL3basic, Promega, Madison, WI) driven by the hCMV-IE promoter was generated with the AdEasy system (38). In the tumor-restricted, replication-competent adenovirus Ad.COXbulletMK, the adenoviral E1 and E4 gene products, which are essential for viral replication, are transcriptionally targeted by the cyclooxygenase-2 (–327/+59; ref. 39) and midkine (MK –1,009/+25; ref. 40) promoter, respectively, as described previously (17).

The replication-defective adenoviral vector Ad.H/F encodes the measles virus FMG under the transcriptional control of the tumor-specific human telomerase reverse transcriptase gene promoter (–548/+5; ref. 41). For this, the CMV-IE promoter of the measles virus H and F bicistronic expression plasmid pGC-H/IRES/F (kindly provided by Roberto Cattaneo, Mayo Clinic College of Medicine, Rochester, MN; ref. 42) was replaced with the human telomerase reverse transcriptase promoter from the plasmid pBTdel-548 (kindly provided by Izumi Horikawa, National Cancer Institute, NIH, Bethesda, MD; ref. 41) and inserted into the multiple cloning site of pAdTrack (38), creating pAdTrack human telomerase reverse transcriptase H/F. Infectious viral vector was generated with the AdEasy-1 system, as described previously (38).

The reference strain VR-5 (American Type Culture Collection) was used as wild-type adenovirus type 5. The vectors Ad.Luc and Ad.H/F were amplified in 293 cells; the Ad.COXbulletMK vector and Ad5 in HeLa cells were purified as described previously (43). The vector Ad.H/F was generated and produced in the presence of the synthetic fusion inhibitory peptide Z-D-Phe-Phe-Gly-OH (10 µmol/L; Bachem AG, Bubendorf, Switzerland; ref. 44).

The bioactivity of the Ad.Luc vector was determined on 293 cells by bioluminescence and expressed as gene transfer units (GTU)/mL (45). The titer (GTU/mL) of the Ad.H/F vector, which encodes green fluorescent protein by a second transcription unit, was determined on cfPac1, Panc1, PPC#1, PPC#2, PPC#3, and 293 cells in the presence of the synthetic fusion inhibitory peptide by flow cytometry (46). The bioactivity of the Ad.COXbulletMK was determined by TCID50 (45) using cfPac1 and 293 cells.

Viral Transduction Efficiency
Cells were seeded at a concentration of 1 x 103 per well in 100 µL into 96-well flat-bottomed culture plates. Twelve hours later, cell monolayers were transduced with a defined amount (3 x 102 GTU) of Ad.Luc, resulting in 30% of the maximum obtainable luciferase activity in 293 cells. Twenty-four hours posttransduction, cell monolayers were washed with PBS and luciferase assay was carried out as described above.

Analysis of Combined Drug Effects
Cells were treated with serial dilutions of each drug individually or with a fixed ratio of both drugs simultaneously at doses that corresponded to Formula, 1, and 1 Formula times the respective ED50. When the two drugs were administered at a fixed ratio, the dose of the combination required to produce fractional survival (f) could be separated into the components (D)1 and (D)2 of drugs 1 and 2, respectively. For each level of cytotoxicity (f = 0.95, 0.90, ..., 0.05), a variable called the combination index (CI) was calculated using the software CalcuSyn (47) according to the Chou and Talalay equation (9, 48):

Formula
CI < 1 indicates synergy, whereas a CI = 1 shows an additive effect, and CI > 1 indicates antagonism (9, 48). In this equation, the dose reduction index (DRI) defines the extent of drug dose reduction possible in a combination for a given degree of effect compared with the dose of each drug alone (48, 49). When the concentration-effect relationship follows the principle of mass action, the median-effect plot should be linear. Linear correlation coefficients (r) were generated for each curve to determine the applicability of the data to this method of analysis. In all synergy experiments, r was >0.9.

Thirty-six hours after treatment, cytotoxicity of the cfPac1-Luc3 and Panc1-Luc3 cell lines were determined by bioluminescence. For cells not genetically engineered to express luciferase, cytotoxicity was analyzed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega), as recommended by the manufacturer.

Analysis of Apoptosis
Fourteen hours after treatment, Annexin V binding and caspase-3/7 activity of Panc1 or cfPac1 cells was analyzed with the Annexin V-FITC Apoptosis Detection kit I (BD, Mansfield, MA) or Caspase-Glo 3/7 assay (Promega), respectively, according to the instructions from the manufacturer.

Bioluminescence Imaging
To assess the cancer treatment response, nude mice were anesthetized (ketamine/xylazine 95 and 12 mg/kg i.p., respectively), and K+ D-luciferin (150 mg/kg body weight; BD) was injected i.p. 10 minutes before imaging using a photon counting system (Hamamatsu Photonics, Herrsching am Ammersee, Germany) and HPD-LIS 2.6 software (Hamamatsu Photonics). An integration time of 10 minutes was used for luminescent image acquisition.

Animal Studies
Six- to eight-week-old female athymic nude (BALB/c nu/nu) mice were obtained from Janvier (Le Genest-St-Isle, France). For the i.p. pancreatic cancer model, 1 x 106 viable cfPac1-Luc3 cells were injected in 1 mL PBS into the peritoneal cavity. Six days later, animals were assigned randomly to groups of eight animals. One animal of each group underwent bioluminescence imaging to confirm peritoneal carcinomatosis at this time point. One day later, therapy was initiated. Animals treated with Ad.H/F vector or Ad.COXbulletMK, alone or in combination with gemcitabine, received per treatment cycle 2 x 108 TCID50 or GTU determined on cfPac1, respectively. Animals treated with Ad.H/F in combination with Ad.COXbulletMK received per treatment cycle 1 x 108 TCID50 plus 1 x 108 GTU (determined on cfPac1) alone or in combination with gemcitabine. All viral vectors were administered i.p. on day 0 and day 2 in 3 mL 4% (w/v) polyethylene glycol (Sigma-Aldrich, St. Louis, MO) in PBS. For the s.c. xenograft model, mice received s.c. 1 x 107 cFPac1-Luc3 cells in 100 µL into the hind flanks. When the tumors reached a volume of ~200 mm3, animals were randomly assigned to treatment groups (n = 6). Viral vectors were given intratumoral at the same dose as in the i.p. model on 3 consecutive days in 100 µL PBS. At least weekly, minimum and maximum perpendicular tumor axes were measured using vernier calipers, and tumor volume was calculated using the formula l x w2 x 0.5. The skin thickness of 0.4 mm was subtracted from the measurements.

In both tumor models, gemcitabine (125 mg/kg/d) was given i.p. daily for 5 consecutive days starting on day 4 in 3 mL 0.9% NaCl solution.

Statistical Methods and Median-Effect Analysis
The statistical software package SPSS 13 (SPSS, Inc., Chicago, IL) was used for data analysis with indicated tests. Analysis of combined effects of multiple drugs, described by Chou and Talalay (9), was done with the software CalcuSyn 2.0 (Biosoft, Cambridge, United Kingdom).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Efficient In vitro Transduction of Pancreatic Cancer Cells with Ad5-based Vectors
In this study, we analyzed whether the efficacy of chemotherapy can be enhanced by expression of measles virus FMGs (H and F). Because our strategy relies on the transduction of pancreatic cancer cells by an adenoviral vector to express measles virus FMGs, we first analyzed the transduction efficiency of permanent pancreatic cancer cell lines and of short-term cultures of pancreatic cancer cells with a replication-defective adenoviral vector encoding firefly luciferase (Ad.Luc). Cell monolayers were transduced with a defined amount (3 x 102 GTU) of Ad.Luc, resulting in 30% of the maximum obtainable luciferase activity in 293 cells 24 hours posttransduction. The ED30 was chosen to minimize double transductions and saturation effects. As shown in Fig. 1 , transduction of the established pancreatic cancer cell lines (cfPac1 and Panc1) and the short-term pancreatic cancer cell cultures (PPC#1, PPC#2, and PPC#3) with Ad.Luc resulted in 4.59 x 106 ± 1.6 x 105, 5.78 x 106 ± 2.3 x 105, 2.67 x 106 ± 2.2 x 105, 3.60 x 106 ± 3.8 x 105, and 2.17 x 106 ± 2.8 x 105 RLU/103 cells, respectively. By Student's t test, transduction efficiency of short-term pancreatic cancer cultures with the Ad5-based vector was significantly lower than that of permanent pancreatic cancer cells lines (P < 0.05).


Figure 1
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  		Figure 1. Transduction efficiency of pancreatic cancer cells. Cell monolayers were transduced with a defined amount (3 x 102 GTU) of a replication-defective adenoviral vector encoding firefly luciferase (Ad.Luc), resulting in 30% of the maximum obtainable luciferase activity in 293 cells 24 h after transduction. We compared the transduction efficiency of established pancreatic cancer cell lines (cfPac1 and Panc1) with that of short-term cultures of pancreatic cancer cells (PPC#1, PPC#2, and PPC#3). Columns, mean from three independent experiments; bars, SD.

 
Synergism between FMG Expression and Chemotherapy In vitro
Next, we determined in vitro whether the expression of measles virus H and F encoded by a replication-defective adenovirus vector will enhance chemotherapy. To minimize unwanted effects, the expression of H and F was transcriptionally driven by the human telomerase reverse transcriptase gene promoter, which is tumor specific, as previously reported (50, 51). The activity of the human telomerase reverse transcriptase promoter was similar in all tested pancreatic cancer cell lines (data not shown).

The Ad.H/F doses of transduction were adjusted based on the titer determined by GFU for each cell line individually. The ED50 concentrations (fraction of affected cells is 0.5) were determined for each drug (Table 1 ). For Ad.H/F, the 6:1 ratio of particles to GTU was used to calculate the molarity, which translates into 6.022 x 1023 viral particles = 1 mol (45). This provided the basis for the appropriate ratio of Ad.H/F and chemotherapeutic drugs. The ratio was kept constant when the drugs were studied in combination. The interaction of the drugs was analyzed with the software CalcuSyn (47). The dose-effect curves and combination index plots are shown in Figs. 2 and 3 . In the combination index plots, values >1 indicate antagonistic drug effects, whereas those <1 are considered synergistic. A combination index of 1 suggests additive effects of both drugs. The effects of drug combinations on the ED50 concentrations are presented in Table 2 . For example, in cfPac1-Luc3 cells treated with Ad.H/F and gemcitabine, the dose of Ad.H/F and gemcitabine needed to achieve the ED50 was reduced 44.32- and 119.77-fold, respectively, when compared with single-agent treatment. More striking results were obtained with Ad.H/F and FOLFOX in Panc1-Luc3 and cfPac1-Luc3. In both tested permanent pancreatic cancer cell lines, the expression of FMG resulted in enhanced cytotoxicity of the used chemotherapeutic drugs (Fig. 3A).


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  		Table 1. ED50 of single agents

 

Figure 2
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  		Figure 2. Dose-effect curves. Permanent pancreatic cancer cell lines cfPac1-Luc3 and Panc1-Luc3 (A) and short-term cultures of pancreatic cancer cells, PPC#1, PPC#2, and PPC#3 cells (B), were exposed in vitro for 36 h to irinotecan (Irino), gemcitabine (Gem), 5-FU/LV (6:1), and FOLFOX (LV/5-FU + Ox; 2.5:8:0.5), alone or in combination with a replication-defective adenoviral vector encoding measles virus H and F (Ad.H/F) at a constant molar ratio. Cytotoxicity was assessed by bioluminescence.

 

Figure 3
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  		Figure 3. Analysis of combined drug effects. To quantitate the interaction between the treatments, CI values versus cytotoxicity plots were generated for the permanent pancreatic cancer cell lines (A) and short-term cultures of pancreatic cancer cells (B) from the median-effect plots (25). For these curves, CI < 1 defines a synergistic interaction and CI > 1 defines antagonistic drug effects. The straight line at CI = 1 represents the additive effects of both drugs. The CI values for the ED50 are indicated.

 

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  		Table 2. The effects of drug combinations on the ED50 concentrations

 
Because permanent cell lines are often in continuous culture for several years and thus likely to have genetic aberrations, we verified our data in short-term cultures of primary pancreatic cancer cells (Figs. 2B and 3B). Two of the three primary cell cultures (PPC#2 and PPC#3) treated with Ad.H/F, in combination with gemcitabine or 5-FU/LV, showed synergy.

Increased Signs of Apoptosis after FMG Expression and Chemotherapy
For this, Panc1 cells were transduced with Ad.F/H at a multiplicity of infection of 5 (determined on Panc1 cells) and incubated with gemcitabine (25 nmol/L). Fourteen hours later, we analyzed early apoptotic events by Annexin V binding (Fig. 4A ) and caspase-3/7 activity (Fig. 4B) assays. As shown in Fig. 4A, incubation of the cells with Ad.H/F or gemcitabine resulted in 9.9 ± 0.5% and 6.5 ± 0.4% Annexin V–positive cells, respectively. When the cells were treated with Ad.H/F and gemcitabine, 58.3 ± 1.6% of the cells were Annexin V positive.


Figure 4
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  		Figure 4. Analysis of apoptosis. Panc1 cells were treated with gemcitabine alone or in combination with measles virus H and F encoding adenoviral vector Ad.H/F and/or the oncolytic Ad.COXbulletMK vector. Fourteen hours later, early apoptotic events were quantified using Annexin V binding (A) and caspase-3/7 activity assays (B). The results represent one of three experiments. Similar results were obtained with cfPac1 cells. The multiplicity of all viral vectors was 5, determined for each cell line individually.

 
As shown in Fig. 4B, incubation of the cells with Ad.H/F or gemcitabine resulted in ~1.2-fold increased caspase-3/7 activity when compared with untreated cells. Treatment of the cells with Ad.H/F and gemcitabine resulted in 2.01-fold increased caspase-3/7 activity.

Using plasmid DNA–encoding FMG, qualitatively similar results were obtained (data not shown). Transduction of the cells with Ad.Luc or Ad.H/F in the presence of the synthetic fusion inhibitory peptide (44) had no effect on the binding of Annexin V or caspase-3/7 activity (data not shown).

Fourteen hours postinfection in both apoptosis assays, the addition of Ad.COXbulletMK to the above-described treatment significantly changed neither Annexin V binding nor caspase-3/7 activity. Similar results were obtained using cfPac1 cells (data not shown).

FMG Expression and/or Transcomplementation Enhance In vivo Transduction
We evaluated, by bioluminescence imaging, whether the i.p. transduction efficiency of tumor cells can be enhanced by transcomplementation of the replication-defective Ad.Luc vector with the replication-restricted Ad.COXbulletMK (Fig. 5A ). On day 7, the luciferase activity Ad.Luc + Ad.COXbulletMK was 73.7-fold enhanced when compared with Ad.Luc alone. In this experiment, the i.p. administered dose of luciferase-encoding vector resulted in vitro in an ED10 of luciferase activity in the initial number of injected cfPac1 cells. The ratio of replication-competent to replication-defective was 1:1, as in the transcomplementation of Ad.H/F.


Figure 5
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  		Figure 5. Effect of transcomplementation or FMG expression on the i.p. photon emission after transduction with an Ad.Luc vector. Nude mice with pancreatic peritoneal carcinomatosis received i.p. only 1 x 106 GTU of the luciferase encoding vectors Ad.Luc resulting in vitro in an ED10 of luciferase activity in the initial number of injected cfPac1cells. A, bioluminescence tumor imaging 7 d after administration of the replication-defective luciferase encoding Ad.Luc vector alone or in combination with Ad.COXbulletMK. B, Ad.Luc vector was administered alone or in combination with Ad.H/F. Twenty-four hours postinfection, bioluminescence was analyzed. One representative experiment out of three. Light intensity is presented as pseudocolors: blue, intense; red, most intense. In this xenograft model, as few as 2,500 luciferase-expressing tumor cells distributed throughout the peritoneal cavity could be detected externally.

 
Administration of the Ad.Luc vector with an equal infective dose of Ad.H/F resulted in a 61.2-fold enhanced photon emission when compared with the respective luciferase-encoding vector alone Fig. 5B. In both sets of experiments, the i.p. administered dose of luciferase encoding vector resulted in vitro in an ED10 of luciferase activity in the initial number of injected cfPac1 cells.

Treatment with FMG-Encoding Vector and Chemotherapy Results in Enhanced In vivo Treatment Efficacy
To determine whether the synergy observed in the in vitro cytotoxicity assays would also result in an enhanced in vivo treatment efficacy, we evaluated this treatment combination in an i.p. and s.c. cfPac1-Luc3 pancreatic xenograft model in nude mice.

In a pilot toxicity study, nude mice received one i.p. injection of 2 x 108 GTU Ad.H/F. One week after treatment, we did not observe clinical signs suggestive of bowel perforation or any other toxicity. When exploratory laparotomy was done, we did not observe peritoneal adhesions or macroscopic signs of hepatitis.

For the i.p. tumor model, animals were treated with the Ad.H/F vector alone or in combination with gemcitabine, and/or the replication-restricted oncolytic adenovirus Ad.COXbulletMK (Fig. 6A and B ). As shown in Table 3 , untreated animals had a median survival of 19 days. Animals receiving Ad.H/F or gemcitabine had an improved survival (42 and 31 days, respectively; log-rank test, P < 0.001). Administration of Ad.H/F and gemcitabine resulted in a significantly improved survival when compared with single-agent treatment (47 days, P < 0.001).


Figure 6
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  		Figure 6. I.p. pancreatic xenograft model (cfPac1-Luc3). Animals were treated with a replication-defective adenovirus encoding measles virus H and F, gemcitabine, and a replication-restricted oncolytic adenovirus Ad.COXbulletMK alone/or in combination. We monitored survival by Kaplan-Meier analysis of animals treated only with Ad.H/F and/or gemcitabine (A) and combination with Ad.COXbulletMK (B).

 

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  		Table 3. In vivo efficacy study

 
To improve the transduction efficiency of the replication-defective Ad.H/F vector, we transcomplemented this vector with the replication-restricted adenovirus Ad.COXbulletMK. Administration of this vector alone resulted in an enhanced survival (P < 0.001) due its oncolytic effect, which was further improved by combination with Ad.H/F (P ≤ 0.014). The best treatment outcome was observed with the triple therapy (P ≤ 0.041). At the end of the observation period (day 120), three of eight animals treated with this novel triple therapy, consisting of Ad.H/F, gemcitabine, and Ad.COXbulletMK, were alive and tumor-free. Treatment with Ad.H/F and Ad.COXbulletMK resulted in one long-term survivor. In all other treatment groups, there were no long-term survivors. The only sign of toxicity we observed was some weight loss in the group of animals that received the triple therapy. However, none of the animals died.

In addition, we evaluated the antineoplastic efficacy of this treatment regimen in a s.c. pancreatic xenograft model. As shown in Fig. 7 and Table 3, on day 28, the median tumor volume of untreated animals was 1,625 mm3. The median tumor volume of animals treated with gemcitabine, Ad.H/F, and Ad.COXbulletMK alone was 1,347, 1,009, and 1,187 mm3, respectively. When compared with untreated animals, the tumor volume was significantly reduced (P ≤ 0.046). The combination of Ad.H/F with gemcitabine showed a significantly reduced tumor volume compared with treatment with the individual compounds and untreated animals (P ≤ 0.001). The median tumor volume of animals treated with the triple therapy was 343 mm3.


Figure 7
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  		Figure 7. S.c. pancreatic cancer tumor model. cfPac1-Luc3 xenografts were grown s.c. to volumes of ~200 mm3. Groups of mice (n = 6) were treated with a replication-defective adenovirus encoding measles virus H and F, gemcitabine, and a replication-restricted oncolytic adenovirus Ad.COXbulletMK alone/or in combination. Tumors were measured at day 28. Points, tumors. Horizontal lines, median values.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we analyzed whether the efficacy of chemotherapy can be enhanced by expression of measles virus FMGs (H and F).

Because this strategy relies on the efficient transduction of tumor cells, we analyzed adenoviral transduction efficiency. We were able to show in vitro that short-term cultures of pancreatic cancer cells can be sufficiently transduced with an Ad5-based vector. This is important for the clinical applicability of this strategy because short-term cultures of human melanoma cells for instance are difficult to transduce with an Ad5-based vector in contrast to long-term cultures (52). Yet, the transduction efficiency of short-term cultures of pancreatic cancer cells when compared with established cell lines (cfPac1 and Panc1) was significantly reduced by up to 2.7-fold, respectively.

Furthermore, synergistic cell killing of pancreatic cancer cells expressing the measles virus FMGs, in combination with chemotherapeutic drugs used clinically for the treatment of pancreatic cancer, was observed in vitro over most of the cytotoxic dose range by the median-effect principle described by Chou and Talalay (9). Permanent cell lines are often in continuous culture for several years and thus likely to have genetic aberrations. Additionally, the pancreatic cancer cell lines cfPac1 (53) and Panc1 (54) were established before gemcitabine was available. Thus, the drug resistance pattern differed significantly between the permanent and the short-term pancreatic cell cultures. Nevertheless, we confirmed in two of the three short-term cultures of pancreatic cancer cells synergistic toxicity of cell fusion induced by measles virus FMGs and chemotherapy.

In cells treated with gemcitabine, the Annexin V binding was similar to those expressing FMG. However, when cells were treated with Ad.H/F and gemcitabine, the Annexin V binding rose by ~5-fold. One conceivable explanation for the synergistic toxicity observed in vitro is the proapoptotic effect of the measles virus FMGs. This observation might be supported by another study demonstrating that expression of gibbon ape leukemia virus FMGs causes syncytia formation with bioenergetic cell death (55). However, another study using gibbon ape leukemia virus reported a nonapoptotic mechanism of syncytial-mediated cell killing (21).

We assessed in an i.p. and s.c. pancreatic xenograft model, using a constitutively luciferase-expressing human tumor cell line cfPac1-Luc3, whether the in vitro findings could be confirmed in vivo. In the i.p. model, we used survival as the primary therapeutic end point. One day before treatment, bioluminescence imaging was carried out to assure peritoneal carcinomatosis. Despite the moderate sensitivity of the permanent pancreatic cancer cell line cfPac1 to gemcitabine, we used this drug as the chemotherapeutic agent because gemcitabine is used as first-line therapy for patients with advanced pancreatic cancer.

Previously, it has been shown by several groups in s.c. tumor models that the fraction of transduced tumor cells will be greatly enhanced when replication-defective viral vectors will be rendered replication competent in cells transduced with viruses or mutants that transcomplement each other for missing gene products pivotal for vector replication and thus allowing in situ vector production and transduction of neighboring tumor cells (12–16).

Using bioluminescence imaging, we showed that rendering the replication-defective Ad.Luc vector replication competent by transcomplementation resulted in enhanced i.p. tumor transduction efficiency. This strategy of transcomplementation is important for adenoviral vectors because most of the conditionally replication-competent vectors cannot carry a therapeutic gene due to size constraints. In our Ad.COXbulletMK vector, the ~6.4 kb FMG expression cassette would have clearly exceeded viral packaging capacity. Thus, we transcomplemented the FMG-encoding vectors with the oncolytic replication-restricted adenovirus Ad.COXbulletMK. However, because oncolytic adenoviruses can enhance the efficacy of chemotherapeutic agents (20), as also observed in both of our tumor models, this transcomplementation approach might improve chemotherapy by itself independently of the FMG expression. On the other hand, chemotherapeutic agents will also inhibit viral replication (56, 57). Furthermore, without using chemotherapy, several groups reported previously that the intratumoral expression of FMGs enhances the efficacy of virotherapy (24, 58). We were able to confirm these findings in both of our tumor models. However, although the replication-defective Ad.H/F vector can only transduce a small fraction of cells in a given tumor, the administration of Ad.H/F in combination with gemcitabine was significantly more efficacious in both tumor models than treatment with the individual compounds. Thus, we were able to support our in vitro data in the adenoviral system. In the i.p. tumor model, the survival of animals treated with Ad.H/F in combination with the oncolytic adenovirus was significantly enhanced when compared with animals treated with the individual compounds, confirming previous results (24). When the treatment with Ad.H/F and Ad.COXbulletMK was combined with gemcitabine, the number of tumor-free, long-term survivors was thrice higher.

Furthermore, we observed by bioluminescence imaging an enhanced photon emission when the Ad.Luc vector was coadministered with the replication-defective Ad.H/F vector. However, due to the syncytia formation, the relationship of photon emission and transduction efficiency might be separated. A conceivable explanation for this might be that 24 hours after transduction, fused cells can produce higher luciferase amounts than single cells. Similarly, this might be true for therapeutic genes. Furthermore, due to the syncytia formation, fewer cells within the tumor have to be genetically modified. This is an extension of previous findings demonstrating enhanced viral spread and oncolytic efficacy when FMGs are expressed (24, 58).

Thus, the significantly improved therapeutic outcome of animals receiving the triple therapy was attributed to multiple factors, including, most likely, improved FMG expression throughout the tumor, enhanced sensitivity of the tumor cells to gemcitabine by adenoviral gene products, and FMG expression. Qualitatively similar results were obtained in a s.c. pancreatic xenograft model.

In summary, intratumoral expression of measles virus FMGs can enhance the efficacy of chemotherapy. This effect can be enhanced by the multimodal process of combination with oncolytic viruses. Because it has been previously reported that fusogenic membrane proteins can enhance the immunogenicity of tumor cells (21), this approach might be even more effective in an immunocompetent host.


   Acknowledgments
 
We thank Klaus Überla for creating an inspiring environment and providing constant support; Andrea Frilling (Klinik für Allgemeine und Transplantationschirurgie, Essen, Germany); Cathrin Walter (Innere Klinik-Tumorforschung, Universitätsklinikum, Essen, Germany) and Wibke Bayer for critical review of the manuscript; and Matthias Dobbelstein and German P. Horn for helpful discussions and advice.


   Footnotes
 
Grant support: Deutsche Forschungsgemeinschaft and Wilhelm Sander-Stiftung (O. Wildner).

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

Received 3/ 8/06; revised 5/11/06; accepted 5/31/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics, 1999. CA Cancer J Clin 1999;49:8–31, 1.[Abstract/Free Full Text]
  2. Fernandez E, La Vecchia C, Porta M, Negri E, Lucchini F, Levi F. Trends in pancreatic cancer mortality in Europe, 1955-1989. Int J Cancer 1994;57:786–92.[Medline]
  3. Casper ES, Green MR, Kelsen DP, et al. Phase II trial of gemcitabine (2,2'-difluorodeoxycytidine) in patients with adenocarcinoma of the pancreas. Invest New Drugs 1994;12:29–34.[CrossRef][Medline]
  4. Burris HA III, Moore MJ, Andersen J, et al. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol 1997;15:2403–13.[Abstract/Free Full Text]
  5. Waxman S, Bruckner H. The enhancement of 5-fluorouracil anti-metabolic activity by leucovorin, menadione and {alpha}-tocopherol. Eur J Cancer Clin Oncol 1982;18:685–92.[CrossRef][Medline]
  6. Rothenberg ML, Moore MJ, Cripps MC, et al. A phase II trial of gemcitabine in patients with 5-FU-refractory pancreas cancer. Ann Oncol 1996;7:347–53.[Abstract/Free Full Text]
  7. Neoptolemos JP, Cunningham D, Friess H, et al. Adjuvant therapy in pancreatic cancer: historical and current perspectives. Ann Oncol 2003;14:675–92.[Abstract/Free Full Text]
  8. Goldberg RM, Sargent DJ, Morton RF, et al. A randomized controlled trial of fluorouracil plus leucovorin, irinotecan, and oxaliplatin combinations in patients with previously untreated metastatic colorectal cancer. J Clin Oncol 2004;22:23–30.[Abstract/Free Full Text]
  9. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27–55.[CrossRef][Medline]
  10. Ram Z, Culver KW, Oshiro EM, et al. Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells. Nat Med 1997;3:1354–61.[CrossRef][Medline]
  11. Jain RK. Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors. Cancer Res 1990;50:814–9s.
  12. Dion LD, Goldsmith KT, Strong TV, Bilbao G, Curiel DT, Garver RIJ. E1A RNA transcripts amplify adenovirus-mediated tumor reduction. Gene Ther 1996;3:1021–5.[Medline]
  13. Alemany R, Lai S, Lou YC, Jan HY, Fang X, Zhang WW. Complementary adenoviral vectors for oncolysis. Cancer Gene Ther 1999;6:21–5.[CrossRef][Medline]
  14. Wildner O, Morris JC, Vahanian NN, Ford HJ, Ramsey WJ, Blaese RM. Adenoviral vectors capable of replication improve the efficacy of HSVtk/GCV suicide gene therapy of cancer. Gene Ther 1999;6:57–62.[CrossRef][Medline]
  15. Wolkersdorfer GW, Morris JC, Ehninger G, Ramsey WJ. Trans-complementing adenoviral vectors for oncolytic therapy of malignant melanoma. J Gene Med 2004;6:652–62.[CrossRef][Medline]
  16. Lee CT, Park KH, Yanagisawa K, et al. Combination therapy with conditionally replicating adenovirus and replication defective adenovirus. Cancer Res 2004;64:6660–5.[Abstract/Free Full Text]
  17. Hoffmann D, Wildner O. Restriction of adenoviral replication to the transcriptional intersection of two different promoters for colorectal and pancreatic cancer treatment. Mol Cancer Ther 2006;5:1–8.[Abstract/Free Full Text]
  18. Freytag SO, Rogulski KR, Paielli DL, Gilbert JD, Kim JH. A novel three-pronged approach to kill cancer cells selectively: concomitant viral, double suicide gene, and radiotherapy. Hum Gene Ther 1998;9:1323–33.[Medline]
  19. Wildner O, Blaese RM, Candotti F. Enzyme prodrug gene therapy: synergistic use of the herpes simplex virus-cellular thymidine kinase/ganciclovir system and thymidylate synthase inhibitors for the treatment of colon cancer. Cancer Res 1999;59:5233–8.[Abstract/Free Full Text]
  20. Khuri FR, Nemunaitis J, Ganly I, et al. A controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat Med 2000;6:879–85.[CrossRef][Medline]
  21. Bateman A, Bullough F, Murphy S, et al. Fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth. Cancer Res 2000;60:1492–7.[Abstract/Free Full Text]
  22. Galanis E, Bateman A, Johnson K, et al. Use of viral fusogenic membrane glycoproteins as novel therapeutic transgenes in gliomas. Hum Gene Ther 2001;12:811–21.[CrossRef][Medline]
  23. Li H, Haviv YS, Derdeyn CA, et al. Human immunodeficiency virus type 1-mediated syncytium formation is compatible with adenovirus replication and facilitates efficient dispersion of viral gene products and de novo-synthesized virus particles. Hum Gene Ther 2001;12:2155–65.[CrossRef][Medline]
  24. Ahmed A, Jevremovic D, Suzuki K, et al. Intratumoral expression of a fusogenic membrane glycoprotein enhances the efficacy of replicating adenovirus therapy. Gene Ther 2003;10:1663–71.[CrossRef][Medline]
  25. Nakamura T, Peng KW, Vongpunsawad S, et al. Antibody-targeted cell fusion. Nat Biotechnol 2004;22:331–6.[CrossRef][Medline]
  26. Dorig RE, Marcil A, Chopra A, Richardson CD. The human CD46 molecule is a receptor for measles virus (Edmonston strain). Cell 1993;75:295–305.[CrossRef][Medline]
  27. Erlenhoefer C, Wurzer WJ, Loffler S, Schneider-Schaulies S, ter MV, Schneider-Schaulies J. CD150 (SLAM) is a receptor for measles virus but is not involved in viral contact-mediated proliferation inhibition. J Virol 2001;75:4499–505.[Abstract/Free Full Text]
  28. Wild TF, Fayolle J, Beauverger P, Buckland R. Measles virus fusion: role of the cysteine-rich region of the fusion glycoprotein. J Virol 1994;68:7546–8.[Abstract/Free Full Text]
  29. Caballero M, Carabana J, Ortego J, Fernandez-Munoz R, Celma ML. Measles virus fusion protein is palmitoylated on transmembrane-intracytoplasmic cysteine residues which participate in cell fusion. J Virol 1998;72:8198–204.[Abstract/Free Full Text]
  30. Rodriguez R, Schuur ER, Lim HY, Henderson GA, Simons JW, Henderson DR. Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res 1997;57:2559–63.[Abstract/Free Full Text]
  31. Hallenbeck PL, Chang YN, Hay C, et al. A novel tumor-specific replication-restricted adenoviral vector for gene therapy of hepatocellular carcinoma. Hum Gene Ther 1999;10:1721–33.[CrossRef][Medline]
  32. Kurihara T, Brough DE, Kovesdi I, Kufe DW. Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen. J Clin Invest 2000;106:763–71.[Medline]
  33. Brunori M, Malerba M, Kashiwazaki H, Iggo R. Replicating adenoviruses that target tumors with constitutive activation of the wnt signaling pathway. J Virol 2001;75:2857–65.[Abstract/Free Full Text]
  34. Hernandez-Alcoceba R, Pihalja M, Qian D, Clarke MF. New oncolytic adenoviruses with hypoxia- and estrogen receptor-regulated replication. Hum Gene Ther 2002;13:1737–50.[CrossRef][Medline]
  35. Johnson L, Shen A, Boyle L, et al. Selectively replicating adenoviruses targeting deregulated E2F activity are potent, systemic antitumor agents. Cancer Cell 2002;1:325–37.[CrossRef][Medline]
  36. Banerjee NS, Rivera AA, Wang M, et al. Analyses of melanoma-targeted oncolytic adenoviruses with tyrosinase enhancer/promoter-driven E1A, E4, or both in submerged cells and organotypic cultures. Mol Cancer Ther 2004;3:437–49.[Abstract/Free Full Text]
  37. Esimone CO, Grunwald T, Wildner O, et al. In vitro pharmacodynamic evaluation of antiviral medicinal plants using a vector-based assay technique. J Appl Microbiol 2005;99:1346–55.
  38. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 1998;95:2509–14.[Abstract/Free Full Text]
  39. Inoue H, Tanabe T, Umesono K. Feedback control of cyclooxygenase-2 expression through PPAR{gamma}. J Biol Chem 2000;275:28028–32.[Abstract/Free Full Text]
  40. Uehara K, Matsubara S, Kadomatsu K, Tsutsui J, Muramatsu T. Genomic structure of human midkine (MK), a retinoic acid-responsive growth/differentiation factor. J Biochem (Tokyo) 1992;111:563–7.[Abstract/Free Full Text]
  41. Horikawa I, Cable PL, Afshari C, Barrett JC. Cloning and characterization of the promoter region of human telomerase reverse transcriptase gene. Cancer Res 1999;59:826–30.[Abstract/Free Full Text]
  42. Horn GP, Vongpunsawad S, Kornmann E, et al. Enhanced cytotoxicity without internuclear spread of adenovirus upon cell fusion by measles virus glycoproteins. J Virol 2005;79:1911–7.[Abstract/Free Full Text]
  43. Graham FL, Prevec L. Methods for construction of adenovirus vectors. Mol Biotechnol 1995;3:207–20.[Medline]
  44. Richardson CD, Scheid A, Choppin PW. Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F1 or HA2 viral polypeptides. Virology 1980;105:205–22.[CrossRef][Medline]
  45. Mittereder N, March KL, Trapnell BC. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol 1996;70:7498–509.[Abstract]
  46. Hitt DC, Booth JL, Dandapani V, Pennington LR, Gimble JM, Metcalf J. A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein. Mol Biotechnol 2000;14:197–203.[CrossRef][Medline]
  47. Chou TC, Hayball MP. CalcuSyn: Windows software for dose effect analysis. Cambridge (England): AnonymousBiosoft; 1996.
  48. Chou TC. The median-effect principle and the combination index for quantitation of synergism and antagonism. In: Chou TC, Rideout DC, editors. Synergism and antagonism in chemotherapy. New York: Academic Press; 1991. p. 61–102.
  49. Chou J. Quantitation of synergism and antagonism of two or more drugs by computerized analysis. In: Chou TC, Rideout DC, editors. Synergism and antagonism in chemotherapy. New York: Academic Press; 1991. p. 223–44.
  50. Wirth T, Zender L, Schulte B, et al. A telomerase-dependent conditionally replicating adenovirus for selective treatment of cancer. Cancer Res 2003;63:3181–8.[Abstract/Free Full Text]
  51. Jacob D, Davis JJ, Zhang L, Zhu H, Teraishi F, Fang B. Suppression of pancreatic tumor growth in the liver by systemic administration of the TRAIL gene driven by the hTERT promoter. Cancer Gene Ther 2005;12:109–15.[CrossRef][Medline]
  52. Volk AL, Rivera AA, Kanerva A, et al. Enhanced adenovirus infection of melanoma cells by fiber-modification: incorporation of RGD peptide or Ad5/3 chimerism. Cancer Biol Ther 2003;2:511–5.[Medline]
  53. McIntosh JC, Schoumacher RA, Tiller RE. Pancreatic adenocarcinoma in a patient with cystic fibrosis. Am J Med 1988;85:592.[Medline]
  54. Lieber M, Mazzetta J, Nelson-Rees W, Kaplan M, Todaro G. Establishment of a continuous tumor-cell line (panc-1) from a human carcinoma of the exocrine pancreas. Int J Cancer 1975;15:741–7.[Medline]
  55. Higuchi H, Bronk SF, Bateman A, Harrington K, Vile RG, Gores GJ. Viral fusogenic membrane glycoprotein expression causes syncytia formation with bioenergetic cell death: implications for gene therapy. Cancer Res 2000;60:6396–402.[Abstract/Free Full Text]
  56. Zalmanzon ES, Mikhailova LN. The effect of pre-inhibition of DNA synthesis in continuous human amniotic cell culture (strain FL) on adenovirus type 5 reproduction. Acta Virol 1969;13:353–62.[Medline]
  57. Wildner O, Hoffmann D, Jogler C, Uberla K. Comparison of HSV-1 thymidine kinase-dependent and -independent inhibition of replication-competent adenoviral vectors by a panel of drugs. Cancer Gene Ther 2003;10:791–802.[CrossRef][Medline]
  58. Fu X, Tao L, Jin A, Vile R, Brenner MK, Zhang X. Expression of a fusogenic membrane glycoprotein by an oncolytic herpes simplex virus potentiates the viral antitumor effect. Mol Ther 2003;7:748–54.[CrossRef][Medline]




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