Animals, pancreatic cell lines and antibodies
The NOD/Shi-SCID IL2Rγnull mice were produced and housed in the University Bordeaux Segalen animal facility, according to the rules and regulations governed and enforced by the Institutional Animal Care and Use Committee. The animal facility institutional agreement number is A33063916. Animals were included in protocols between 6 and 8 weeks old. Mice were monitored weekly for body weights and were also examined for aspect and behavior during the time-course of the experiments. No changes were noticed except otherwise indicated. The PDAC PANC1 and MIAPACA2 cell lines were purchased from the ATCC (American Type Culture Collection, Molsheim, France). CAPAN2 and BXPC3 were kindly provided by Joel Tardive-Lacombe (INSERM U624, Marseille, France). The CAPAN2 and BXPC3 cells were maintained in RPMI (Invitrogen) with 10% Fetal Calf Serum (FCS, Invitrogen) and Penicillin/Streptomycin 1/100 (Invitrogen), the PANC1 and MIAPACA2 were cultured in DMEM with 10% FCS and Penicillin/Streptomycin 1/100.
The tdTomato-MIAPACA2 cell line was produced by transduction of the MIAPACA2 cells with a lentivirus carrying the tdTomato reporter gene (PGK-tdTomato, see below). Transduced cells were sorted by a BD FACS ARIA cell sorter (BD Biosciences, France).
The antibodies used in this study were purchased as follows: anti-HLA, anti-MUC4, rabbit IgGs (SIGMA ALDRICH, Lyon, France), anti-CLDN18 (GenWay, San Diego, CA-USA), anti-PSCA (Abcam, Paris, France), anti-PSCA (SIGMA ALDRICH, Lyon, France).
Vector construction, production and transduction of cells
pPGK-tdTomato lentiviral plasmid was constructed by transferring the tdTomato gene from p-tdTomato (Clontech, Saint Germain en Laye, France) into pRRL-Sin-cPPT.PGK.WPRE lentiviral plasmid (gift from Dr. Trono, Lausanne, Switzerland). LUCIFERASE-IRES-TK lentivirus plasmid was obtained by replacing GFP from pLOXgfp-IresTK (Addgene Plasmid # 12243, Cambridge, MA-USA) with the firefly-Luciferase gene (Clontech). Cloning details can be provided upon request. A LUCIFERASE-IRES-ZsGreen lentivirus plasmid was obtained as follows: the fireflyLuciferase gene was cloned into the pIRES2-ZsGreen1 vector (Clontech, Saint Germain en Laye, France). All lentiviral vectors were produced by calcium phosphate mediated triple transient transfection of 293 T cells with one of the vector transfer constructs, the packaging construct pCMVΔ8.91 (gift from Dr. Verma, La Jolla, CA-USA) and either VSV-G construct psPAX2 (gift from Dr. Trono) or 2.2 plasmid (a gift from Drs. Chen and Morizono, coding for a modified Sindbis virus glycoprotein envelope, Los Angeles, CA-USA). The viruses produced were concentrated by ultracentrifugation (through a 10% sucrose cushion). The capside protein p24 titrations were determined as already described .
Analysis of lentiviral transduction by flow cytometry
FACS analyses were performed on a FACScalibur flow cytometer (BD biosciences, Le Pont de Claix, France) on trypsinized cells 3–5 days after transduction. Transductions were carried out on 5.104 cells in 48-well plates. Virus mixes containing 100 ng of p24 were prepared in 250 μl of serum-free medium with antibodies at 0.5 μg/ml. Percentages of GFP-positive cells were determined using CellQuest software (Becton Dickinson, Le Pont de Claix, France) in comparison with non transduced cells, after counting of cells in the FL-1 channel.
Protein extracts were prepared in RIPA buffer and processed for western blotting. Membranes were incubated with the targeting antibodies or a rabbit anti-luciferase antibody (AbCam, Paris, France). Rabbit anti-GAPDH antibody (Cell Signaling Technologies, Saint-Quentin-en-Yvelines, France) was used to assess equal loading of the samples. Primary antibodies were detected with specific anti-rabbit- or anti-mouse-IgG-HRP (Cell Signaling Technologies). Proteins were visualized using the ECL detection system (Amersham Pharmacia Biotech, Orsay, France). Quantification by densitometry was performed with the ImageJ software.
Tumors were fixed in 10% NBF, embedded in paraffin and processed by routine histology procedures. The proportion of mitosis/total cells or apoptosis/total cells was evaluated after Hematoxilin staining by direct counting on pictures taken at X40 magnification.
In vitro cell proliferation assay
To test the effect of Herpes Simplex virus thymidine kinase on the tdTomato-MIAPACA2 cell viability in the presence of ganciclovir (GCV, SIGMA ALDRICH, Lyon, France), cells were first transduced with a lentivirus bearing the LUCIFERASE-IRES-TK transgene (see above). Cells were plated at 3.103 cells per well in 96-well plates. The day after, increasing doses of GCV (0-100 μM) were applied and cells were kept in culture for 10 days. Each point was done in quadruplet. The experiments were carried out 2 times. Cells were washed with PBS and treated with MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) - 2H-tetrazolium) solution (Promega, Charbonnières, France) for 1.5 h to determine cell viability by reading optical densities (OD) at 490 nm. Results are expressed as cell viability:
Bioluminescent imaging of pancreatic adenocarcinoma xenografts
For subcutaneous xenografts, groups of 3–5 mice were anesthetized with isoflurane. 8.105 to 106 cells in 100 μl medium were injected in the right flanks. When tumors reached about 100–150 mm3, diverse combinations of viruses (described in the result section) were injected in the tumors or directly in tumor-free pancreases in single injections. Briefly, 7 μg p24 of lentivirus were mixed, when necessary, with 5 μg of antibody and incubated 5 min at room temperature in 60 μl serum free-medium. Tumors were monitored for luciferase expression on a weekly basis as follows. 150 mg/kg of D-Luciferin (Interchim, Montluçon, France) was injected intraperitonally. After 10 min, mice were anesthetized by isoflurane and placed into a photon bioimager (BIOSPACE LAB, Paris, France) for about 20 min to acquire luminescence images. Signals were quantified with the M3Vision software (BIOSPACE LAB).
For orthotopic xenografts, groups of 4–6 mice were anesthetized with isoflurane. Pancreases were exposed and 8.105 of tdTomato-MIAPACA2 cells in 40 μl medium were injected directly in the pancreas. Tumor growth was monitored weekly with the bioimager using the fluorescence detection setting (Acquisition mode: FLI integration at 22 ms per frame, Excitation = 520 nm, Background = 480 nm, Emission = 570 nm, Filter cut off = 570 nm Illumination: 100%). When signals were easily detectable (after 21 days), mice were anesthetized with isoflurane and injected with the lentiviruses. Ten μg p24 of lentiviruses were incubated 5 min at room temperature in 60 μl serum free-medium with 5 μg of antibody and injected intra-tumorally as described above. Luciferase expression and fluorescence signal were monitored weekly. Two weeks after virus injections, mice were treated daily with GCV (1 mg/mouse).
Transduction efficiencies in vitro are expressed as mean% of transduced cells ± SD. MTS assay results and in vitro transduction results are expressed as mean ± SD. Luminescence quantifications performed in vivo are expressed as mean ± SEM. Statistical tests were performed with Student’s t tests or with a 2-sided Mann Whitney test for intra-pancreatic luciferase detections and tumor growth.