Cancer Gene Therapy (2010) 17, 315–324 r
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ORIGINAL ARTICLE
Using lentiviral vectors for efficient pancreatic cancer gene therapy E Ravet1, H Lulka2, F Gross3, L Casteilla1, L Buscail2,4 and P Cordelier2 1
UMR 5241 CNRS-UPS, Centre Hospitalier Universitaire Rangueil, Toulouse, France; 2Institut National de la Sante´ et de la Recherche Me´dicale U858, I2MR de´partement Cancer, and Institut Louis Bugnard IFR150, Toulouse, France; 3Clinical Investigation Center of Biothe´rapie (CIC-BT 511), Centre Hospitalier Universitaire Rangueil, Toulouse, France and 4Department of Gastroenterology, Centre Hospitalier Universitaire Rangueil, Toulouse, France Pancreatic cancer (PC) remains a life-threatening disease. Efficient therapeutic gene delivery to PC-derived cells continues to present challenges. We used self-inactivated lentiviral vectors to transduce PC-derived cells in vitro and in vivo. We showed that lentiviral vectors transduce PC-derived cell lines with high efficiency (490%), regardless of the differentiation state of the cell. Next, we transferred human interferon beta (hIFN-b) gene. Expression of hIFN-b in PC cells using lentiviral vectors resulted in the inhibition of cell proliferation and the induction of cell death by apoptosis. In vivo, lentiviral administration of hIFN-b prevented PC tumor progression for up to 15 days following gene therapy, and induced tumor regression/stabilization in 50% of the mice treated. Again, hIFN-b expression resulted in cancer cell proliferation inhibition and apoptosis induction. We provide evidence that human immunodeficiency virus (HIV)-1-based lentiviral vectors are very efficient for gene transfer in PC-derived cells in vitro and in vivo. As a consequence, delivery of hIFN-b stopped PC tumor progression. Thus, our approach could be applied to the 85% of PC patients with a locally advanced disease. Cancer Gene Therapy (2010) 17, 315–324; doi:10.1038/cgt.2009.79; published online 13 November 2009 Keywords: pancreatic adenocarcinoma; lentiviral vectors; gene therapy; hIFN-b apoptosis.
Introduction
Pancreatic cancer (PC) is a common malignancy with a 5-year survival rate of 0.4–4%.1 PC is highly resistant to conventional therapies, that is, chemotherapy and radiotherapy. Surgical resection improves the prognosis; however, only 15% of patients with PC are eligible for the procedure.2 Most treatment failures are due to local recurrence, hepatic metastases, or both, and occur within 1–2 years after surgery.3 Therefore, there is an urgent need to develop new therapeutic strategies for PC. Cancer gene therapy is very promising, and nearly 70% of gene therapy clinical trials have been devoted to cancer. Many gene delivery methods have been explored for PC gene therapy.4–7 We have experimented with nonviral vectors, adenoviral and SV40-based vectors, which showed gene delivery to PC cells, and provided evidence of therapeutic efficacy.8–11 However, the current low transduction efficiency of the synthetic nonviral vector and the transient gene expression using both viral vectors Correspondence: Dr P Cordelier, INSERM U858, I2MR, De´partement Cancer Equipe 12. BP 84225, Baˆtiment L3, Haute Garonne, 31432 Toulouse Cedex 4, France. E-mail:
[email protected] Received 22 March 2009; revised 16 June 2009; accepted 4 September 2009; published online 13 November 2009
present major disadvantages in the cost of clinical application. An alternative approach is the development of more competitive expression vectors for PC gene delivery. Human immunodeficiency virus (HIV)-derived VSV-G-pseudotyped lentiviral vectors provide efficient gene transfer in proliferative and quiescent cells and mediate stable, high-level transgene expression both in vitro and in vivo.12–14 To date, the potential of lentiviral-mediated gene delivery to PC cells has been insufficiently explored. Treatment of PC with biological agents such as interferons (IFNs) may improve survival. IFNs elicit direct antiproliferative effects on tumor cells, including PC.15–17 In vitro, human IFN-beta (hIFN-b) has greater antiproliferative effect than hIFN-a on PC-derived cells.17 Human IFN-b mediates the innate immune responses against microbial infections and is a constituent of the host defence against oncogenesis. Systemic administration of hIFN protein therapy is approved in the United States and abroad for use in combination with chemotherapy in patients with nonresectable or metastatic PC.18–20 Although minimal success was achieved in these studies, Picozzi et al.21 reported a 5-year survival of 55% using an IFN-based chemoradiation protocol for patients with resectable PC. Taken together, IFN-based therapy efficacy for PC is limited and transient, and toxicities restrict further dose escalation. We hypothesize that gene
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therapy using lentiviral vectors may allow for the targeted and sustained expression of hIFN in PC tumor cells. In this study, we show that lentiviral vectors are very efficient for the transfer of genes in PC-derived cells, in vitro and in vivo. Delivery of the hIFN-b gene results in cancer cell proliferation and tumor progression inhibition. This information will be useful for the development of novel gene therapy approaches for PC treatment. Materials and methods
Cell lines and cell culture All the PC-derived cell lines used in this study were obtained from LGC Standards (Molshein, France), and were cultured as described elsewhere.22 HCT-116 were purchased from LGC Standards and maintained in Dulbecco’s modified Eagle’s medium (DMEM) þ 4.5 g l1 glucose (Invitrogen, Cergy Pontoise, France). The human embryonic kidney 293FT cell line used for production of the vectors was obtained from Invitrogen, and maintained in DMEM þ 4.5 g l1 glucose (Invitrogen). All media were supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units ml1 penicillin and 100 mg ml1 streptomycin (all reagents were from Invitrogen). Cell lines were grown in a humidified incubator at 37 1C in 5% CO2, and kept free from mycoplasma contamination using plasmocin (InvivoGen, Toulouse, France). DNA constructs and vector production The DNA vectors used in this study (TRIP-DU3-EF1ahuIFNb, encoding for hIFN-b, and TRIP-DU3-EF1aenhanced green fluorescent protein (EGFP), encoding for EGFP) contain the central HIV-1 DNA fragment flap encompassing the central polypurine tract (cPPT) and central termination sequence (CTS) cis-active sequences. cPPT and CTS cis-active sequences are responsible for the formation of the DNA flap during HIV-1 reverse transcription and strongly promote nuclear import in transduced cells.23 Plasmids pHCMV-G,24 encoding for VSV-G protein, and pCMVD8.91,25 encoding for HIV-1 accessory proteins, were kindly donated by Dr DubartKupperschmitt (Paris, France). Replication defective, self-inactivating lentiviral vectors were generated in a BSL-3 facility (BiviC core vector production, IFR 150, Toulouse, France) through transient transfection of 293FT cells with packaging and lentiviral vector plasmids using calcium phosphate precipitation as described elsewhere.26 Lentiviral vectors were concentrated by ultracentrifugation and stored in phosphate-buffered saline (PBS) at 80 1C. The batches of the different vectors used were prepared together to ensure similar transduction efficiencies.27 All batches were checked for replicative virus-free after transduction of 293FT cells and analysis of cellular extracts and culture supernatant for p24 presence, up to three passages (10 days) in culture. The viral titers were determined on HCT116 cells and expressed in transduction unit per ml (TU ml1) as described elsewhere.26 In addition, vector concentrations were quantified by p24 enzyme-linked immunosorbent assay (ELISA) (Innotest, Ingen, Paris).
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Transduction of PC-derived cells with lentiviral vectors A total of 5 104 PC-derived cells were plated in 48-well clusters and transduced overnight in 250 ml of transduction medium (serum-free medium þ 15% BIT9500 þ 4 mg ml1 Protamine Choay) in the presence of purified lentiviral vectors. BIT9500 and Protamine Choay are from Stem cells Techonologies (Grenoble, France) and Sanofi Aventis France (Paris, France), respectively. Medium was changed, and cells were collected at 72 h after transduction. EGFP-positive cells were quantified by flow cytometry analysis (fluorescence-activated cell sorting (FACS)) on a FACScalibur (Beckton Dickinson France SAS, Le Pont-De-Claix, France). Human IFN-b production was measured using Biosource ELISA kit following the manufacturer’s recommendation (Invitrogen). Preparation of conditioned media from MS5-transduced cells MS5 cells (1 10e6) were seeded in 100-mm cell culture dishes. After 24 h, the culture medium was replaced by transduction medium containing purified LV(IFN). As control, MS5 cells were transduced with LV(GFP). Cells were incubated for 48 h and the culture media were collected, centrifuged for 5 min at 200 g, and filtrated using 0.45-mm Millipore Ultrafree centrifugal filters (Millipore, Molsheim, France). Conditioned media were stored at 80 1C. Determination of cell proliferation Cell proliferation assays were carried out in 35-mm dishes. In all, 50 103 PC-derived cells were cultured in complete medium for 24 h (2 ml per dish). On the next day, cells were transduced with LV(IFN). Control cells were transduced with LV(GFP). Cell growth was measured 4 days later by cell counting using a Coulter counter model ZM (Beckman Coulter, Roissy, France). All experiments were conducted with different batches of lentiviral vectors. Transduced cell were not selected in this study. Western blotting Proteins were extracted from transduced cells, resolved on SDS-polyacrylamide gels, and transferred to nitrocellulose membrane. After room-temperature blocking for 1 h, blots were incubated overnight at 4 1C with antibodies purchased from Cell signaling (Cell Signalling Technology, Ozyme, Saint Quentin Yvelines, France) diluted according to the manufacturer’s recommendations. Secondary horseradish peroxidase (HRP)-conjugated antibodies (dilution 1:10 000, Perbio Science, Brebie`res, France) were added, and blots were incubated for 1 h at room temperature. Immunoreactive proteins were visualized using ECL immunodetection (Immobilon, Millipore Corporation, Vandoeuvre, France). In vivo gene transfer in subcutaneous tumor models The protocol was approved by the IFR150 Institutional Animal Use Committee. Capan-1 cells (2 10e6) were harvested, collected in 100 ml sterile PBS and implanted subcutaneously into the left flanks of severe combined
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immunodeficient (SCID) CB 17 mice. After 10 days, when the tumors measured B100 mm3 in volume, the animals were administered a single intratumoral injection of LV(GFP) or LV(IFN) vectors in a level 2 animal safety facility. Control animals were injected with 10 ml of PBS. Six animals were used per group. The tumors were monitored every day and measured every 3–4 days. The animals were killed 2 weeks after the injection and the tumors were analyzed.
Immunostaining for Ki67 in pancreatic tumors Capan-1 tumors were harvested and fixed in formalin. Sections (4-mm thick) were prepared from paraffinembedded sections and rehydrated. Following antigen retrieval, sections were incubated for 10 min in Protein Block, Serum-free reagent to reduce background staining. All reagents were from DakoCytomation, Trappes, France. Next, slides were incubated overnight at 4 1C with anti-Ki67 antibody, (dilution: 1:100). Antibody incubations were carried out in antibody diluent. After three washes in PBS, slides were incubated in 3% H2O2 for 30 min at room temperature for endogenous peroxidase inhibition. Slides were quickly rinsed in distilled water, washed twice in PBS and incubated for 30 min at room temperature with Envision þ system-HRP (DakoCytomation, Trappes, France). After washing in distilled water, slides were incubated in AEC þ reagent (DakoCytomation) and counterstained with Mayer’s hematoxylin. Immunostaining was observed with an optical microscope, and quantified using VisioLab2000 image analyzer (Biocom, Les Ulis, France). Cell-death assays For Annexin-V and propidium iodide detection, Capan-1 cells were collected following treatment and labeled with either Annexin-fluorescein isothiocyanate (DakoCytomation) or propidium iodide (Invitrogen) as per the manufacturer’s recommendation. Apoptotic and necrotic cells were quantified using FACScalibur and Cell quest pro software (Becton Dickinson). Detection of chromosome breakdown in PC tumors was performed by TdTmediated dUTP nick end labeling (TUNEL) assay using ApopDETEK and Simply sensitive kits, following the manufacturer’s recommendations (ApopDETEK, Enzo Life Sciences, Farmingdale, NY). Statistical analysis Results are expressed as mean ± s.e. Data were compared by one-way analysis of variance (with post hoc Turkey’s HSD test) or unpaired t-tests using Graphpad Instat software (Graphpad Software, La Jolla, CA) (*Po0.05, **Po0.01; ***Po0.001). Po0.05 was considered significant.
Results
HIV-1-derived lentiviral vectors transduction of PC cell lines To document the efficacy of lentiviral gene delivery in PC, we used four different cell lines as models of different
stages of PC differentiation.28 Three different multiplicity of infections of LV(GFP) (ranging from 0.8 to 3.2) were tested. The results presented Figure 1a show that lentiviral vectors successfully transduce all cell lines tested with high efficacy (from 68 to 98% GFP-positive cells following a single transduction, without selection), regardless of their differentiation state. We asked whether GFP expression was stable over time. As shown in Figure 1b, Capan-1 and Panc-1 cells retain lentiviral-mediated GFP expression up to 10 passages in culture, without selection.
Delivery of hIFN-b gene in PC-derived cells using lentiviral vectors Owing to the toxicity of hIFN-b in human cell lines, we used mouse fibroblasts to titer hIFN-b production following LV(IFN) transduction. As shown in Figure 2a, transduction of MS5 cells with increasing amount of LV(IFN) results in the dose-dependent secretion of hIFN-b. We next transduced PC-derived cells with the same dose of LV(IFN). Cell proliferation was measured by cell counting 4 days later. Control cells were transduced with LV(GFP) to measure intrinsic lentiviral vector toxicity. Figure 2b shows that LV(IFN) transduction of PC cells induces a strong antiproliferative effect. Inhibition of cell proliferation reaches 72±8.5% following the administration of the highest dose of LV(IFN). Of importance, PC-derived cells transduced with LV(IFN) died within 7 days following gene transfer, regardless of the multiplicity of infection used (data not shown). It is noteworthy that LV(GFP) transduction did not alter cell proliferation as compared with mocktransduced cells. Despite recent progress, efficient gene delivery to the entire tumor cell population in vivo remains elusive. Thus, we tested whether hIFN-b produced from cells receiving LV(IFN) gene transfer could alter the cell proliferation of untransduced PC cells. We transduced MS5 cells with LV(IFN) and collected their supernatant. As a control, MS5 cells were transduced by LV(GFP). We show that secreted hIFN-b strongly impairs the proliferation of untransduced cancer cells in a dose-dependent manner, as compared with control supernatant (Figure 2c). Thus, IFN-b elicits a strong antiproliferative bystander effect of PC cells. Characterization of the molecular mechanisms involved in LV(IFN)-mediated antiproliferative effect After transduction with LV(IFN), both Capan-1 and BxPC-3 cells round up and detach from the culture plate (Figure 3a). This change in cell morphology is strongly suggestive of apoptosis. We next measured the changes in membrane symmetry by Annexin-V labeling. As shown in Figure 3b, IFN-b gene transfer induces a 4.5-fold increase in Annexin-V-positive cells, as compared with control LV(GFP)-transduced cells. Using western blotting, we show that lentiviral delivery of IFN results in caspase activation and Poly(ADP-ribose) Polymerase cleavage, as compared with LV(GFP)-transduced cells (Figure 3c). Taken together, we show that delivering hIFN-b in vitro using lentiviral vectors strongly inhibits PC cell proliferation and induces cell death by apoptosis.
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Figure 1 Efficient gene transfer into pancreatic cancer (PC) cells using lentiviral vectors. (a) PC cell lines are incubated with LV(GFP) at specified multiplicity of infections (MOIs) or ng p24 per ml. Fluorescence intensity is measured by fluorescence-activated cell sorting (FACS) analysis 48 h later. The percentage of GFP-positive cells is indicated. (b) Analysis of GFP expression in Capan-1 and Panc-1 cell lines 10 weeks (10 passages) after transduction with LV(GFP).
In vivo gene transfer studies in tumor models We next assessed the ability of hIFN-b gene transfer to alter PC tumor growth in vivo. We first established PC tumors into the flanks of SCID mice. Next, we injected a
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single intratumoral dose of LV(IFN) into exponentially growing tumors. PBS and LV(GFP) were used as control. LV(GFP) transfer results in 72 þ 3.5% cells positive for GFP (Figure 4a). As shown in Figure 4b,
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LV(IFN)-treated mice show a significant inhibition of tumor progression (80±17%). On the other hand, PBS and LV(GFP)-injected tumors progressed by 210±31 and 168±34%, respectively, during the same period of time. Remarkably, LV(IFN) delivery induced stabilization and/ or regression of 66% of the tumors (Figure 4c). We next investigated the molecular mechanisms involved in the LV(IFN)-mediated antitumoral effect. As
shown in Figure 5a (left panel), tumors from animal receiving the control vector LV(GFP) show intense staining for KI67, indicating the presence of proliferating cells. On the other hand, few cells are apoptotic in these tumors (Figure 5b, left panel). It is noteworthy that tumors from LV(IFN)-treated mice showed a marked reduction in cancer cells proliferation (Figure 5a, right), and strong TUNEL (Figure 5b, right), indicative of massive cancer cell apoptosis. Quantitative analysis showed an 84±3% (Po0.001) decrease in the number of proliferating cancer cells following LV(IFN) gene transfer, whereas the number of apoptotic cells was increased 10.5±2.5-fold (Po0.001). We further characterized the molecular mechanisms involved in hIFN-b antitumoral effect on pancreatic tumors. As shown in Figure 5c, LV(IFN) gene transfer induces the sequential activation of caspases followed by PARP cleavage to provoke PC cell death by apoptosis, in vivo. Treatment with the control vector LV(GFP) did not alter caspase activation pathway, nor PARP cleavage. Altogether, we showed here for the first time that a single administration of hIFN-b using lentiviral vectors in vivo strongly impedes tumor growth in a very aggressive model of PC.
Discussion
In this study, we show that lentiviral gene delivery of hIFN-b strongly inhibits PC cell proliferation both in vitro and in vivo. This antiproliferative effect is associated with induction of apoptosis. Gene delivery to PC cells may allow for new therapeutic approaches to this life-threatening disease. In recent years, much effort has been devoted to the development of appropriate viral delivery vehicles for the treatment of PC. Since 1980s, the use of gene delivery vectors based on retroviruses has attracted much attention. These vectors present unique advantages, such as lack of immunogenicity due to the removal of most genes encoding viral
Figure 2 Human interferon beta (hIFN-b) production following lentiviral vectors transduction inhibits pancreatic cancer (PC) cell proliferation. (a) Enzyme-linked immunosorbent assay (ELISA) detection of hIFN-b production in MS5 cells after transduction with LV(IFN) at the indicated doses. Results are mean±s.e.m. of three experiments carried out with two different batches of lentiviral vectors. (b) PC-derived cell lines were transduced with LV(IFN) at the indicated doses. As a control, Capan-1 cells were transduced with 1000 ng equivalent of p24 per ml of LV(GFP) (control). After 4 days, cell proliferation was measured by cell counting. Results are mean±s.e.m. of three experiments carried out with two different batches of lentiviral vectors (***Po0.001 vs control (LV(GFP))transduced cells). (c) Capan-1 cells were incubated with the culture medium of MS5 cells transduced with 1000 ng p24 per ml LV(GFP) or with LV(IFN) at the indicated doses. After 4 days, cell proliferation was measured by cell counting. Results are mean±s.e.m. of three experiments carried out with two different batches of MS5 cells culture supernatant and compared with medium collected following LV(GFP) transduction of MS5 cells (***Po0.001).
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proteins, thus allowing repeated use. However, these factors face critical limitations, such as instability of viral particles, low viral titers and the inability to transduce nondividing cells.29–31 Other gene delivery systems, such
as complexed DNA (PEI), adenovirus and SV40-derived vectors showed poor efficiencies or transient expression of the transgene, respectively.8,9 Lentiviral vectors were developed to overcome these limitations. The lentiviral vector-mediated gene therapy has great promise in cancer treatment, because of its long-term expression and high efficiency in transducing dividing and nondividing cells, including the chemoresistant population located in the hypoxic cores of tumors.32,33 However, lentiviral vectormediated gene therapy for PC still lags behind other viral vector delivery system. Two studies described the use of lentiviral vectors to drive transgene expression in PCderived cells, but each study had several shortcomings.34,35 Owing to the limited transduction efficiencies (from 17 to 56%), cell populations needed to be selected in vitro after transduction in both studies. In vivo, Hase et al.34 did not show intratumoral gene transfer, whereas Segara et al.35 reported short-term efficient EGFP delivery to Miapaca-2 tumor xenografts. These results do not reflect the progress represented by the lentiviral system. In this study, we found that HIV-based vectors were very efficient in transducing PC-derived cells. In vitro, 70–90% of the cells express EGFP at the lowest multiplicity of infection used (0.8), regardless of their state of differentiation. In addition, EGFP expression is stable up to 10 weeks in culture. In vivo, EGFP is expressed in a majority of the tumor cells, up to 14 days following intratumoral administration. In this study, lentiviral-based vectors surpass nonviral vectors (PEI), adenoviral or SV40-based vectors in their ability to transduce PC-derived cells.8,9 Understanding the molecular basis of PC has provided a wide range of potential intracellular targets for gene therapy approaches.36 Targeting PC by molecular abnormality remains elusive because of the accumulation of multiple genetic changes during its multistep carcinogenesis. During the last decade, numerous studies illustrated the gene transfer or systemic delivery of IFN-b to inhibit tumor growth in multiple animal models from gliomas,37 to hepatocellular carcinoma,38 metastatic breast cancer,39 prostate cancer,40–42 rectum, colon and endometrial tumors,43–45 lung tumors,46,47 renal cell carcinomas and melanoma,48,49 neuroblastoma,50,51 disseminated peritoneal cancer,52 bladder cancer,53 malignant mesothelia54 and fibrosarcomas.55 Recently, a cancer gene therapy trial for gliomas based on the intratumoral administration of
Figure 3 Lentiviral-mediated delivery of human interferon beta (hIFN-b) induces pancreatic cancer (PC) cell death by apoptosis. (a) Representative photomicrographs showing the effect of hIFN-b 4 days after transduction with 250 ng p24 per ml of LV(GFP) (left), or LV(IFN) (right). Capan-1 cells (top panel), BxPC-3 cells (bottom panel). (b) Capan-1 cells were transduced with 250 ng p24 per ml of LV(IFN). As control, Capan-1 cells were transduced with the same dose of LV(GFP). Annexin-V binding was quantified by fluorescenceactivated cell sorting (FACS) at 48 h after transduction. Proteins were extracted and subjected for western blotting using the indicated antibodies (c) Results are representative of three experiments carried out with two different batches of lentiviral vectors. (***Po0.001).
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Figure 4 In vivo gene delivery of human interferon beta (hIFN-b) using lentiviral vectors in pancreatic tumor xenograft. (a) GFP expression 14 days after transduction of pancreatic tumors with 250 ng p24 per ml of LV(GFP). Results are representative of 15 highpower fields from 6 different tumors per group. (b) Mean tumor progression±s.e.m of six different animals receiving either LV(GFP) or LV(IFN). *Po0.05, **Po0.01, ***Po0.001. (c) Individual pancreatic cancer (PC) tumor progressions after transduction of pancreatic tumors with 250 ng p24 per ml of LV(IFN).
adenoviral vectors encoding for hIFN-b (Ad.IFN-b) was shown to be feasible and associated with apoptosis induction.56 Intrapleural instillation of Ad.IFN-b gener-
Figure 5 In vivo gene delivery of human interferon beta (hIFN-b) using lentiviral vectors inhibits cell proliferation and induces cell death by apoptosis in pancreatic tumor xenograft. Representative photomicrographs of KI67 (a) and TUNEL (b) labeling 14 days after transduction of pancreatic tumors with 250 ng p24 per ml of either LV(GFP) and LV(IFN). Results are representative of 15 high-power fields from 6 different tumors per group. (c) Western blotting on protein tumors transduced either with LV(GFP) or LV(IFN) using the indicated antibodies. Results are representative of three different tumors for each group.
ated powerful antitumor immune responses in malignant pleural mesothelioma and metastatic pleural effusions patients.57 In addition, the combined effect of hIFN-b
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gene entrapped in liposomes and gemcitabine was evaluated in vitro on PC-derived cells.58 Thus, we elected hIFN-b as our reference gene to determine the therapeutic interest of using lentiviral vectors to drive antioncogenic gene expression into PC-derived cells. We show in this study that the antiproliferative properties of hIFN-b could be exploited to inhibit tumor growth in PC using lentiviral vectors. In vitro, we found that LV(IFN) dramatically inhibited PC cell proliferation, whereas in vivo, tumor progression was stopped following intratumoral gene transfer. In 66% of the case, we showed tumor stabilization and/or regression. In addition, we illustrated a potent antiproliferative bystander effect of hIFN-b. Interestingly, LV(IFN)-treated mouse remained free of tumor progression for 1 month, whereas control mice had to be killed because of prominent tumors. Hase et al.34 recently reported the in vivo delivery of pigment epithelium-derived factor using lentiviral vectors to inhibit PC in mice. However, tumor growth was only partially slowed following in vivo gene transfer. Here, we provide evidence, for the first time, of the notable therapeutic efficacy of lentiviral delivered therapeutic genes for established tumors. It is interesting that the antitumoral effect was measured with very low quantities of LV(IFN) particles. Using low, therapeutic dose of lentiviral vectors may prevent unwanted side effects. We next addressed the mechanism of the antitumoral effect of hIFN-b, both in vitro and in vivo. Activation of programmed cell death is an additional method to facilitate antitumor activity. Apoptosis has a critical role in differentiation and in the elimination of cells that sustain genetic damage or undergo uncontrolled cell proliferation.59 Direct cytotoxic effects mediated by IFNs have been extensively reported elsewhere.60 Here, we show that hIFN-b gene delivery activated caspase-8 cleavage to potentiate caspase-3 activation followed by changes in plasma membrane symmetry, cleavage of PARP, chromatin condensation, DNA fragmentation and cell death in PC cells, both in vitro and in vivo following LV(IFN) gene transfer. This is of particular importance because it has become evident that malignant cells have defects in cell death control and apoptosis, leading to resistance to radiotherapy and chemotherapy.59 Thus, activation of the caspase cascade by IFN gene delivery may hold promise in sensitizing pancreatic tumors to chemotherapeutic agents such as gemcitabine. In summary, in view of the efficacy shown in this study, clinical grade lentiviral vectors may be selected in the future to drive the expression of antioncogenic genes in pancreatic tumors. In particular, LV(IFN) could be injected by mean of endoscopic ultrasound61 in forthcoming clinical trials in combination with gemcitabine administration, for the treatment of the 85% of PC patients that cannot be operated in a curative manner due to a locally advanced disease. Conflict of interest
The authors declare no conflict of interest.
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Acknowledgements
The authors thank the BSL-3 laboratory (plate-forme BiVIC IFR150) for vector production. In addition, the authors greatly thank Pr Jan Hoek for careful reading and editing of the paper. This work was supported by grants from INSERM, Re´gion Midi-Pyre´ne´es, and Cance´ropoˆle Grand Sud-Ouest. References 1 Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007; 57: 43–66. 2 Delpero JR. Surgical resection of pancreatic adenocarcinoma: indications and contraindications, pronostic factors and survival, recent advances. Cancer Radiother 2004; 8(Suppl 1): S73–S79. 3 Griffin JF, Smalley SR, Jewell W, Paradelo JC, Reymond RD, Hassanein RE et al. Patterns of failure after curative resection of pancreatic carcinoma. Cancer 1990; 66: 56–61. 4 Bhattacharyya M, Lemoine NR. Gene therapy developments for pancreatic cancer. Best Pract Res Clin Gastroenterol 2006; 20: 285–298. 5 Cordelier P, Buscail L. Gene therapy: a reality for tomorrow? Gastroenterol Clin Biol 2005; 29: 724–731. 6 Halloran CM, Ghaneh P, Costello E, Neoptolemos JP. Trials of gene therapy for pancreatic carcinoma. Curr Gastroenterol Rep 2005; 7: 165–169. 7 MacKenzie MJ. Molecular therapy in pancreatic adenocarcinoma. Lancet Oncol 2004; 5: 541–549. 8 Carrere N, Vernejoul F, Souque A, Asnacios A, Vaysse N, Pradayrol L et al. Characterization of the bystander effect of somatostatin receptor sst2 after in vivo gene transfer into human pancreatic cancer cells. Hum Gene Ther 2005; 16: 1175–1193. 9 Cordelier P, Bienvenu C, Lulka H, Marrache F, Bouisson M, Openheim A et al. Replication-deficient rSV40 mediate pancreatic gene transfer and long-term inhibition of tumor growth. Cancer Gene Ther 2007; 14: 19–29. 10 Vernejoul F, Faure P, Benali N, Calise D, Tiraby G, Pradayrol L et al. Antitumor effect of in vivo somatostatin receptor subtype 2 gene transfer in primary and metastatic pancreatic cancer models. Cancer Res 2002; 62: 6124–6131. 11 Vernejoul F, Ghenassia L, Souque A, Lulka H, Drocourt D, Cordelier P et al. Gene therapy based on gemcitabine chemosensitization suppresses pancreatic tumor growth. Mol Ther 2006; 14: 758–767. 12 Trono D. Lentiviral vectors: turning a deadly foe into a therapeutic agent. Gene Ther 2000; 7: 20–23. 13 Wiznerowicz M, Trono D. Harnessing HIV for therapy, basic research and biotechnology. Trends Biotechnol 2005; 23: 42–47. 14 Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat Med 2001; 7: 33–40. 15 Saidi RF, Williams F, Ng J, Danquah G, Mittal VK, ReMine SG et al. Interferon receptors and the caspase cascade regulate the antitumor effects of interferons on human pancreatic cancer cell lines. Am J Surg 2006; 191: 358–363. 16 Saidi RF, Williams F, Silberberg B, Mittal VK, ReMine SG, Jacobs MJ. Expression of interferon receptors in pancreatic cancer: identification of a novel prognostic factor. Surgery 2006; 139: 743–748.
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