Cell lines and cell culture. B16-F10 (murine melanoma) cells were maintained in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco BRL), 100 IU/ml penicillin, and 100 µg/ml streptomycin.
Mice. C57BL/6 mice were obtained from SLC (Tokyo, Japan). Mice were used at 6–7 week of age, and all animal studies were performed according to institutionally approved protocols at Yonsei University College of Medicine.
. To generate an Ad expressing IL-12 and GM-CSF at the E1 and E3 region, respectively, we first constructed an E3 shuttle vector expressing GM-CSF. The murine GM-CSF
gene was excised from pCDNA3.1/GMCSF using NheI and XhoI, and sub-cloned into the Ad E3 shuttle vector, pSP72-E3, generating a pSP72/E3/GMCSF E3 shuttle vector. The newly constructed pSP72/E3/GMCSF was then cotransformed with an Ad total vector (pvmdl324BstBI) into Escherichia coli
BJ5183, yielding a pdl324ΔE3/GMCSF Ad plasmid. Structure of the resultant recombinant vector was confirmed by restriction enzyme digestion and PCR analysis. To construct an Ad E1 shuttle vector expressing IL-12, murine IL-12
gene excised from pCA14/IL1249
was sub-cloned into pXC1ΔB7 E1 shuttle vector,25
generating a pXC1ΔB7/IL12 E1 shuttle vector. The newly constructed pXC1ΔB7/IL12 E1 shuttle vector was then cotransformed with pdl324ΔE3/GMCSF into E. coli
BJ5183 for homologous recombination, generating a pAd-ΔB7/IL12/GMCSF Ad vector (
). All viruses were propagated in 293 cells and purified by CsCl density purification, dissolved in storage buffer (10 mmol/l Tris, 4% sucrose, 2 mmol/l MgCl2
), and stored at −80 °C. Viral particle numbers were calculated from measurements of absorbance at 260 nm (A260
), where one absorbency unit is equivalent to 1012
viral particles/ml. The infectious titers (plaque-forming units per milliliter) were determined by limiting dilution assay on 293 cells. The particle/plaque-forming unit ratio for Ad-ΔB7 and Ad-ΔB7/IL12/GMCSF were 23.7:1 and 20.9:1, respectively. The MOI was calculated from infectious titers.
Generation of bone marrow-derived DC. Bone marrow cells were harvested from flushed marrow cavities of femurs and tibias of C57BL6 mice under aseptic conditions. The cells were depleted of erythrocytes using RBC lysis buffer (Sigma, St Louis, MO) and were cultured in complete RPMI 1640 media (Gibco BRL) supplemented with 10% fetal bovine serum, GM-CSF (10 ng/ml, ENDOGEN, Woburn, MA), and IL-4 (10 ng/ml, ENDOGEN), 2-mercaptoethanol 50 µmol, 100 IU/ml penicillin, and 100 µg/ml streptomycin. On day 2, the nonadherent cells were removed and the plates were replenished with fresh complete media containing GM-CSF and IL-4. On day 4, culture supernatant was collected and centrifuged, and the cell pellet was resuspended in fresh media containing cytokines and returned to the plate. On day 6, the DCs were incubated with the tumor lysate (50 µg/ml) for 24 hour lipopolysaccharide (1 µg/ml, Sigma) were added at day 7 for DCs maturation. After incubation for 24 hours, mature DCs were harvested and used in following studies. Immature DCs were cultured at same condition for 6 days as described above. But, the cell was not exposed to the tumor lysate and lipopolysaccharide.
Fluorescence-activated cell sorting (FACS) analysis. For the phenotypic analysis, the DCs were stained with surface molecules using immunofluorescence and analyzed by FACS analysis. Cells were stained with anti-mouse CD11c, CCR7, CD40, CD80, CD86, or MHC I/II (Pharmingen, San Diego, CA) Ab at 4 °C for 45 minutes. After twice of PBS washing, the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-rat IgG secondary Ab at 4 °C for 15 minutes. For the assessment of CD4+CD25+ T-cell population in the DLN, single cells were obtained from DLN after mechanical dissociation. The cells were stained with anti-mouse CD4 (phycoerythrin) and anti-mouse CD25 (fluorescein isothiocyanate) Ab at 4 °C for 45 minutes. All samples were analyzed on a BD Biosciences BD-LSR II Analytic Flow Cytometer, using FACSDiva software (BD Biosciences, San Jose, CA).
In vivo antitumor effect. B16-F10 cells (5 × 105) were injected subcutaneously into the right abdomen of 6–7 week-old male C57BL/6 mice. When the tumor volumes reached of around 120–130 mm3, mice were sorted into groups with similar means of tumor volumes. Treatment groups included PBS-only control, Ad-ΔB7/IL12/GMCSF only (5 × 109 VP/injection), DCs only (1 × 106 cells/injection), or a combination of DCs and Ad-ΔB7/IL12/GMCSF). In parallel, one group of mice was given a treatment regimen of a high dose of Ad-ΔB7/IL12/GMCSF (5 × 1010 VP/injection) in combination with DCs (1 × 106/injection). Tumor-bearing mice were intratumorally injected with three doses of Ad on days 0–2, followed by three injections of DCs on days 3–5. Tumor growth was monitored every other day using a caliper, and tumor volume was calculated by the following formula: volume = 0.523 LW2, where L is length and W is width. Animals with tumors that were >3,000 mm3 were killed for ethical reasons.
Expression of IL-12 and GM-CSF. IL-12 and GM-CSF expression were determined using an ELISA according to the manufacturer's instructions. B16-F10 melanoma cells were plated onto six-well plates at 1 × 104 per well and then infected with Ad-ΔB7/IL12/GMCSF at MOIs of 50, 100, and 500. At 48 hour after infection, supernatants were harvested and the level of IL-12 and GM-CSF was determined with conventional IL-12 ELISA kit (ENDOGEN) and GM-CSF ELISA kit (R&D systems, Minneapolis, MN), respectively. For the assessment of cytokine expression in tumor tissue, tumor tissues were removed from mice treated with Ad and/or DCs at 3 days after final treatment. Tissues were homogenized and liquefied in PBS containing protease inhibitor cocktail (Sigma). IL-12, GM-CSF, VEGF, and TNF-α level were measured by conventional ELISA kits (ENDOGEN and R&D systems). Each experiment was carried out three to four times with three replicates in each group.
Histology and immunohistochemistry. Tumor tissues were harvested from mice after 3 days of final treatment, and embedded in paraffin and sectioned at a thickness of 4 µm for hematoxylin and eosin (H&E) staining. For immunohistochemical staining, tumor tissues were snap-frozen and sectioned at a thickness of 7 µm. Tumor sections were blocked with 4% PBS–bovine serum albumin (Sigma) for 1 hour and incubated over night with appropriate dilution of anti-CD4 (purified rat anti-mouse CD4 monoclonal Ab; Pharmingen), anti-CD8 (purified rat anti-mouse CD8 monoclonal Ab; Pharmingen), anti-CD86 (purified rat anti-mouse CD86 monoclonal Ab; Pharmingen), anti-CD11c (purified hamster anti-mouse CD11c monoclonal Ab; Pharmingen), or anti-CCL21 (purified rat anti-mouse CCL21 monoclonal Ab; R&D systems) in Ab diluents (DAKO, Glostrup, Denmark). After overnight incubation, the sections were washed twice in PBS and incubated with horseradish peroxidase-conjugated goat anti-rat or mouse anti-hamster Ab (Southern Biotechnology, Birmingham, AL) for 1 hour Diaminobenzidine/hydrogen peroxidase (DAKO) was used as the chromogen substrate. All slides were counterstained with Meyer's hematoxylin.
Evaluation of DC migration in vivo. DCs were labeled with CellTracker Red CMTPX (Invitrogen, Carlsbad, CA) on day 6 of DC culture and were harvested on day 8. The tumor-bearing mice were intratumorally injected with DCs (1 × 106/time) alone three times every day or intratumorally injected with Ad-ΔB7/IL12/GMCSF (5 × 109 VP/time or 5 × 1010 VP/time) three times every day prior to DC injection. At 48 hour after final treatment, the DLNs were harvested and dissociated into single cells for FACS analysis. The number of CMTPX+ DCs and CD11c+ DCs was quantified on a fluorescence-activated cell sorter (Becton Dickinson, Sunnyvale, CA) and data from 50,000 events were collected for further analysis.
Statistical analysis. The data was expressed as mean ± SE. Statistical analyses of the data were performed using the two-tailed Student's t-test (SPSS 13.0 software; SPSS, Chicago, IL). P values of <0.05 were considered statistically significant (*P < 0.05; **P < 0.01). Analysis of variance was used for multiple group comparison on antitumor effect examination.
Figure S1. Characterization of bone marrow-derived DCs. DCs were cultured in the presence of IL-4 and GM-CSF, and were matured by tumor lysate pulsing and lipopolysaccharide stimulation. (a) The purity of DCs culture was evaluated by fluorescence-activated cell sorting analysis using PE-conjugated CD11c-specific antibody. (b, c) Co-stimulator molecules were significantly upregulated in mature DCs. Similar results were obtained in three independent experiments.
Figure S2. DC migration assay in vivo. (a) DCs were labeled with CMTPX in vitro. The CMTPX labeled DCs in fluorescence microscopic field (× 200 & × 400). The percentage of CMTPX+ DCs (b) and CD11c+ DCs (c) in the draining lymph nodes (DLNs) from mice treated with Ad-ΔB7/IL12/GMCSF and/or DC.
Figure S3. Fluorescence-activated cell sorting analysis of CD4+CD25+ T-cells in draining lymph nodes (DLNs) from mice treated with Ad-ΔB7/IL12/GMCSF and/or DCs. The tumor-bearing mice were intra-tumorally injected with DCs (1 × 106/time) alone three times on days 0-2 (DC alone group) or intra-tumorally injected with Ad-ΔB7/IL12/GMCSF (5 × 109 VP/time) three times on days 0-2 prior to DC injection on days 3-5 (Ad-ΔB7/IL12/GMCSF and/or DC group). At 48 hr after final treatment, the DLNs were harvested and dissociated into single cells for FACS analysis.