Cell lines and cell culture. Human embryonic kidney cell line 293, murine melanoma cell lines B16-F10 and B16BL6 (a metastatic variant of B16 melanoma cells), and mouse fibroblast cell line Mus dunni were purchased from the American Type Culture Collection (Manassas, VA). All cell lines were cultured at 37 °C in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) and antibiotics, except for B16BL6 cells, which were cultured in modified Eagle's medium (Gibco) supplemented with 5% fetal bovine serum, modified Eagle's medium vitamin solution (1 mmol/l, product 11120-052; Gibco), penicillin (500 IU/ml), and streptomycin (50 µg/ml). All have tested negative for Mycoplasma using Hoeschst dye (MP Biomedicals, Irvine, CA), cell culture, and PCR.
Mice. C57BL/6 mice were obtained from SLC (Japan SLC, Tokyo, Japan). Mice were used at 6–7 weeks of age, and all animal studies were performed according to institutionally approved protocols at Yonsei University College of Medicine.
Ad-ΔB7 is an Ad5-based E1B/E3-deleted oncolytic Ad with substitution in retinoblastoma binding sites of E1A (ref. 17
). To generate an oncolytic Ad that expresses IL-12 at the E1 region of Ad-ΔB7, pCA14/E1AE1B19-IL-12 (ref. 6
) E1 shuttle vector was linearized with Nde
I digestion and then homologous recombination was induced in Escherichia coli
BJ5183 with Bst
BI-digested Ad-ΔB7 to generate Ad-ΔB7-IL-12. To generate Ads that express 4-1BBL at the E3 region of Ad-ΔB7, the full-length mouse 4-1BBL complementary DNA was first cloned by reverse transcriptase-PCR using total RNA from bone marrow–derived activated DCs. The 4-1BBL complementary DNA (53–982 nucleotides of National Center for Biotechnology Information L15435) was generated using following primer pairs (sense: 5′-ATGGATCCACCATGGACCAGCACACACTTG-3′ antisense: 5′-AGATAAGC TTTCATTCCCATGGGTTGTC-3′). The resulting PCR product was digested with Bam
III and cloned into the Bam
III-digested pSP72ΔE3/CMV-polA (ref. 40
) Ad E3 shuttle vector, generating pSP72-E3/4-1BBL E3 shuttle vector. This newly constructed pSP72-E3/4-1BBL E3 shuttle vector was then recombined with Ad-ΔB7 and Ad-ΔB7-IL-12, generating Ad-ΔB7-4-1BBL and Ad-ΔB7-IL-12/4-1BBL, respectively. All viral vectors were produced and propagated following standard procedures.41
Viral particle numbers were calculated from measurements of absorbance at 260 nm (A260
), where 1 absorbency unit is equivalent to 1012
VP/ml, and infectious titers were determined by limiting dilution in 293 cells.
Expression of 4-1BBL and IL-12.
The expression of 4-1BBL was determined by western blot analysis. At 48 hours after infection of B16-F10 cells with Ad-ΔB7-4-1BBL or Ad-ΔB7-IL-12/4-1BBL at various MOIs, proteins in the cell extracts were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. The membrane was incubated with primary monoclonal goat anti-mouse 4-1BBL Ab (R&D Systems, Minneapolis, MN) and anti-β-actin Ab for 1 hour. After incubation with secondary rabbit anti-goat IgG (H+L)-horseradish peroxidase Ab (Southern Biotech, Birmingham, AL) for 45 minutes at room temperature, filters were developed with the enhanced chemiluminescence system (Santa Cruz Biotechnology, Santa Cruz, CA). The expression of IL-12 was determined by ELISA as previously described.7
Briefly, B16-F10 cells were infected with Ad-ΔB7-IL-12 or Ad-ΔB7-IL-12/4-1BBL at various MOIs. At 48 hours after infection, IL-12 in the supernatant was determined with ELISA using anti-mouse IL-12 purified monoclonal Ab (ENDOGEN, Woburn, MA) and biotin-conjugated anti-mouse IL-12 monoclonal Ab (ENDOGEN).
Generation of bone marrow–derived DC. Femur and tibia marrow cells from C57BL/6 mice were depleted of erythrocytes using red blood cell lysis buffer (Sigma, St Louis, MO). Cells were then cultured in complete RPMI 1640 media containing 10% fetal bovine serum, GM-CSF (10 ng/ml; ENDOGEN), and IL-4 (10 ng/ml; ENDOGEN). On day 2, floating cells were discarded and the adherent cells were replenished with fresh complete media. At day 4, culture supernatant was collected and centrifuged, and the cell pellet was resuspended in fresh RPMI 1640 containing cytokines and returned to the original plate. On day 6, adherent DCs were incubated with B16-F10 cell lysate (50 µg/ml) for 24 hours and then matured by addition of 1 µg/ml of lipopolysaccharide (Sigma) for 24 hours. Mature DCs were harvested and phenotypic markers of DC was confirmed by fluorescence-activated cell sorting analysis.
Established tumor models for in vivo antitumor effect. B16-F10 cells (5 × 105) were injected subcutaneously into the right abdomen of 6- to 7-week-old male C57BL/6 mice. When the tumor volume reached around 100 mm3, animals were sorted into groups with similar mean tumor volumes and different doses of oncolytic Ads (Ad-ΔB7 or Ad-ΔB7/4-1BBL at 1 × 1010 VP; Ad-ΔB7/IL-12 or Ad-ΔB7/IL-12/4-1BBL at 5 × 109 VP) were injected intratumorally three times every other day in a volume of 20 µl diluted in PBS. Tumor growth was monitored everyday by measuring two perpendicular tumor diameters using calipers. 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.
IFN-γ detection in tissue homogenates. Tumor tissues were obtained from intratumoral treated mice by presented various viruses. After 5 days of final injection of viruses, tumors were grinded and liquefied in the PBS with protease inhibitor cocktail (cat no. P8340; Sigma). IFN-γ expression of treated tumors was determined by IFN-γ ELISA kit (ENDOGEN) according to the manufacturer's instructions. Each experiment was carried out three to four times with three replicates in each group.
Tumor-specific IFN-γ ELISpot assay.
The ELISpot assay was used to determine the B16-F10 tumor-specific IFN-γ-secreting T cells. As responder cells, splenocytes from mice in all groups on day 5 after final treatment were stimulated with irradiated B16-F10 in medium supplemented with recombinant human IL-2 (100 units/ml) for 5 days. Splenocytes were then serially diluted from 2 × 104
to 8 × 105
cells per well and incubated in anti-IFN-γ monocolonal Ab–coated plates. After 12 hours of incubation, plates were developed as previously described.7
After overnight drying at room temperature in the dark, plates were evaluated. Each experiment was carried out three to four times with three replicates in each group.
Viable splenocyte samples were isolated from Ad-ΔB7/IL-12/4-1BBL and/or DCs treatment animals, and the cytolytic activity of CTL was determined as described previously.7
Percent-specific cytotoxicity was determined from the formula: % Specific cytotoxicity = [(experimental release − spontaneous release)/(maximum release − spontaneous release) × 100].
Histological and IHC staining. After treatment by Ad and/or DCs, tumors were harvested, snap-frozen, and 10-µm sections were prepared in Tissue Tek (Sakura Finetec, Torrance, CA). Tumor sections were blocked with 4% PBS–bovine serum albumin (Sigma) for 1 hour and incubated for 2 hours with appropriate dilution of biotin-labeled CD4 (purified rat anti-mouse CD4 monoclonal Ab; Pharmingen, San Diego, CA), CD8 (purified rat anti-mouse CD8 monoclonal Ab; Pharmingen), CD86 (purified rat anti-mouse CD86 monoclonal Ab; Pharmingen), or CD11c (purified hamster anti-mouse CD11c monoclonal Ab; Pharmingen) in 1% PBS–bovine serum albumin. Diaminobenzidine/hydrogen peroxidase (Dako, Carpinteria, CA) was used as the chromogen substrate. All slides were counterstained with Meyer's hematoxylin.
Analysis of DC migration in vivo. To investigate the ability of DCs to migrate into regional lymph nodes in vivo, activated DCs were first transduced with GFP-expressing Ad at an MOI of 300. After 48 hours of transduction, 1 × 106 DCs alone or in combination with Ad-ΔB7/IL-12/4-1BBL oncolytic Ad (5 × 109 VP) were intratumorally injected into established B16-F10 tumors three times every other day, in a sequence of Ad followed by DCs. At 48 hours after final intratumoral DC injection, the DLNs were harvested and stained with GFP-specific Ab for the analysis of the presence of DCs.
Quantification of spontaneous pulmonary metastasis. The B16BL6 spontaneous metastasis model was initiated by subcutaneously inoculation of 1.5 × 105 tumor cells in 50 µl of Hank's buffered salt solution into the right footpad. When tumors reached a volume of 200 mm3, the primary tumors were treated with PBS, DCs (1 × 106 cells), Ad-ΔB7/IL-12/4-1BBL (5 × 109 VP), or Ad-ΔB7/IL-12/4-1BBL plus DCs. Ad-ΔB7/IL-12/4-1BBL oncolytic Ad was intratumorally injected on days 1, 3, and 5, and DCs were injected into the intrapleural cavity on days 2, 4, and 6. On day 9, the primary tumor was then surgically excised by amputating the right hind leg below the knee under mild anesthesia. At 3 weeks after final treatment, lungs were harvested for assessment of metastatic tumor lesions.
Statistical analysis. The data were expressed as mean ± SE. Statistical analyses of the data were performed using the two-tailed Student's t-test (SPSS 10.0 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.