The adenovirus vectors used for this study are based on the Ad5 backbone with deletions of E1 and E3 and the expression cassette in the E1 region (39
). The AdMIP-3α vector expresses the human MIP-3α cDNA under control of the constitutive cytomegalovirus early/immediate promotor/enhancer (41
). The AdNull vector, serving as a negative control, is identical to the AdMIP-3α vector but contains no transgene in the expression cassette (41
). The Ad vectors were purified by cesium chloride density gradient ultracentrifugation, titered by plaque-forming assay on 293 cells, and demonstrated to be free of replication competent Ad (39
Female C57Bl/6 (H-2b), Balb/c (H-2d), and CD4- or CD8-knockout (C57Bl/6) mice, 6–8 weeks old, were obtained from The Jackson Laboratories (Bar Harbor, Maine, USA). Animals were housed under specific pathogen-free conditions and treated according to National Institutes of Health guidelines.
CT26 is an undifferentiated colon adenocarcinoma cell line (H-2d) derived by intrarectal injections of N
-methylurethane in a female Balb/c mouse (provided by N.P. Restifo, National Cancer Institute, Bethesda, Maryland, USA) (43
). CT26.CL25 is derived from CT26 cells modified to express the Escherichia coli
β-galactosidase (βgal) gene (43
). The SVBalb fibroblast cell line is syngeneic to Balb/c mice (provided by L. Gooding, Emory University, Atlanta, Georgia, USA) (44
). Both the B16 murine melanoma cell line and Lewis lung cell carcinoma (LLC) cell line are syngeneic for C57Bl/6 mice (H-2b) (obtained from the American Type Culture Collection, Manassas, Virginia, USA). The CT26 cell line was maintained in complete RPMI-1640 media (10% FBS, 100 μg/mL streptomycin, 100 U/mL penicillin) (GIBCO BRL, Gaithersburg, Maryland, USA). The CT26.CL25 cell line was maintained in complete RPMI-1640 media containing 400 μg/mL G418 (GIBCO BRL). All other cell lines were maintained in complete DMEM.
Generation of DCs from bone marrow.
Primary bone marrow DCs were obtained from mouse bone marrow precursors as described by Inaba et al. (45
). In brief, erythrocyte-depleted murine bone marrow cells harvested from femurs were plated in complete RPMI media supplemented with recombinant murine GM-CSF (100 U/mL) and recombinant murine IL-4 (20 ng/mL; Genzyme, Farmington, Massachusetts, USA). On days 2 and 4, nonadherent granulocytes were gently removed and fresh media were added. On day 6, loosely adherent proliferating DC aggregates were dislodged and replated. On day 6 of culture, nonadherent cells with the typical morphological features of DCs were used for the in vitro migration assay to test the function of the AdMIP-3α vector (see later here).
Function of AdMIP-3α in vitro.
To evaluate the MIP-3α mRNA expressed from AdMIP-3α vector, the A549 lung carcinoma cell line was infected with AdMIP-3α or the AdNull control vector (each, 10 moi). After 3 days, total RNA was extracted using Trizol reagents (GIBCO BRL). RNA samples were separated by electrophoresis on a 1% agarose gel containing 0.66 M formaldehyde, blotted onto a filter membrane (Duralon-UV; Stratagene, La Jolla, California, USA). Hybridization was carried out with 32P-labeled probes (Prime It kit; Stratagene) at 65°C in Quick-Hyb solution (Stratagene). Membranes were washed twice for 5 minutes at room temperature in 2 × SSC and 0.1% SDS and once at 65°C in 0.1× SSC and 0.1% SDS and were exposed with x-ray film at –80°C with an intensifying screen.
To demonstrate that production of the MIP-3α protein was directed by the AdMIP-3α vector, cells and culture supernatants of A549 cells infected with AdMIP-3α or AdNull (each, 10 moi) for 3 days were prepared and used for Western analysis. The A549 cells were lysed in extraction buffer (4% SDS, 250 mM Tris-HCl [pH 6.8], 10% glycerol, 1% β-mercaptoethanol) for 10 minutes at 95°C. The cell lysates were centrifuged at 12,000 g for 10 minutes at 4°C, and 5 μL of the lysate supernatants was separated in an 18% Tris-HCl gel (Bio-Rad Laboratories, Hercules, California, USA) by SDS-polyacrylamide gel electrophoresis and electrotransferred onto supported nitrocellulose membrane (0.45 μm) (Bio-Rad Laboratories). Immunological detection using anti-human MIP-3α polyclonal antibody (R&D Systems Inc., Minneapolis, Minnesota, USA) was performed by the enhanced chemiluminescence method according to the instructions of the manufacturer (Amersham Life Sciences Inc., Arlington Heights, Illinois, USA).
To evaluate the function of the MIP-3α protein directed by the AdMIP-3α vector, directed migration of DCs induced by supernatants of A549 cells infected with AdMIP-3α was assayed by a modification of Boyden’s chamber method using microchemotaxis chambers and filters (5-μm diameter) (46
). Murine DCs were suspended at a concentration of 106
cells/mL in RPMI medium 1640 supplemented with 1% FBS. Fifty microliters of suspension was placed in the upper chamber, and 25 μL of supernatant of A549 cells infected for 3 days with AdMIP-3α, or AdNull, or naive, uninfected cells was placed in the lower chamber. The chamber was incubated for 90 minutes at 37°C. Directed migration was expressed as the number of cells that had migrated to the lower chamber, seen in 5 high power fields. Checkerboard analysis of the supernatants of AdMIP-3α–infected A549 cells was carried out to distinguish chemotaxis from chemokinesis. Different dilutions of supernatants were added to upper and lower chambers, and the apparatus was incubated for 90 minutes at 37°C. Directed migration was expressed as the number of cells that had migrated to the lower chamber, seen in 5 high power fields. The data are presented as mean ± SE.
In vivo function of the AdMIP-3α vector in tumors.
To demonstrate functional expression of MIP-3α after AdMIP-3α administration to tumors, B16 tumor cells (3 × 105 cells) were administered subcutaneously to C57Bl/6 mice. After 8 days, the tumors were injected with AdMIP-3α (5 × 108 pfu in 100 μL), AdNull (5 × 108 pfu in 100 μL), or PBS (100 μL). To demonstrate expression of the AdMIP-3α in the tumors, Northern analysis was carried out as already described here. RNA was extracted from tumors 3 days after intratumoral administration and hybridized (20 μg/lane) with a human MIP-3α probe or a GAPDH probe.
The ability of the AdMIP-3α vector to produce a protein that functioned in vivo to attract DCs was evaluated in tumors 3 days after administration of the AdMIP-3α, AdNull, PBS, or controls. Three days after intratumoral injection (5 × 108 pfu in 100 μL), mice were sacrificed and the tumor was harvested. Cryostat sections (8 μm) were placed on the slides, air dried, fixed in acetone for 10 minutes, and air dried for at least 30 minutes. After washing in PBS/0.01% Triton X100, the slides were incubated with PBS/0.01% Triton X 100/5% normal goat serum for 60 minutes, then incubated overnight at 4°C with a 1:25 dilution of rat anti-mouse DC antibody (anti-DEC205, NLDC145; Serotec, Washington, DC, USA), 1:50 dilution of hamster anti-mouse CD11c mAb (PharMingen, San Diego, California, USA), rat control IgG2a (Serotec), or hamster IgG, group 1.λ (Pharmingen). To identify T cells, anti-mouse CD8b.2 mAb (Ly-3.2; PharMingen), anti-mouse CD4 (L3T4; PharMingen), control rat IgG1, κ isotype standard (PharMingen), and rat IgG2a were used. After washing in PBS/0.01% Triton X 100, the slides were incubated with a 1:100 dilution of horseradish peroxidase–conjugated monoclonal anti-rat κ and λ light chains (Sigma Chemical Co., St. Louis, Missouri, USA) or horseradish peroxidase–conjugated anti-hamster IgG (Serotec), and the slides were examined using microscope.
To demonstrate AdMIP-3α modification of tumor growth in vivo, mice were injected subcutaneously on day 0 with tumor cells (3 × 105) including CT26.CL25 (n = 30 mice), CT26 (n = 30), B16 (n = 30), and LLC (n = 30). All injections were performed into the shaved right (or bilateral) flank(s) in a total volume of 100 μL. When the tumors had grown to 15–25 mm2 (day 6 for CT26.CL25; day 8 for CT26, B16, and LLC), mice were inoculated into the tumors with 100 μL of the AdMIP-3α or AdNull vectors (5 × 108 pfu) in PBS. The size of each tumor was monitored three times weekly. The tumor area was calculated and expressed as the average tumor area (mm2) ± SE. If animals appeared moribund or the diameter of the tumors reached 20 mm, the mice were sacrificed, and this was recorded as the date of death for survival studies. Survival of the animals was assessed using standard methodology. To evaluate the inflammation of lymph nodes after intratumoral injection of AdMIP-3α, ipsilateral, and contralateral inguinal lymph nodes were isolated 3 days after Ad vector injection into tumors (see later discussion here), and wet weight was measured. DCs attracted in inguinal lymph nodes 3 days after intratumoral administration of the AdMIP-3α, AdNull, or PBS were assessed by immunohistochemistry as already described here.
Tumor-specific cytotoxic T lymphocytes.
To assess the ability of intratumoral injection of AdMIP-3α to induce tumor-specific cytotoxic T lymphocytes (CTLs), splenocytes were isolated 12 days after Ad vector injection into the tumors (see earlier here) and restimulated at 3 × 106 cells/mL with 106 cells/mL irradiated (50 Gy) tumor cells. After 5 days of culture, the in vitro restimulated splenocytes were quantified using a 51Cr-release assay for their ability to lyse tumor cells. The percentage of specific a 51Cr release was expressed as follows: 100 × (experimental release – spontaneous release) / (maximal release – spontaneous release). SVBalb and C3 cells were used as control for Balb/c- and C57Bl/6-syngeneic tumors, respectively.
Adoptive transfer of splenocytes.
To demonstrate that in vivo administration of the AdMIP-3α vector sensitized the cellular host defense system against the relevant tumor, 10 days after the inoculation of the four types of tumors with Ad vectors (see earlier discussion here), the spleens were removed. Splenocytes (3 × 107 cells per mouse) were injected into recipient animals by tail vein. Seven days later (day 0), recipient animals were challenged by subcutaneous injection in the right flank with 3 × 105 relevant tumor cells. Survival was assessed as already described here.
The data are presented as mean ± SE. Statistical analysis was performed using two-way ANOVA. Statistical significance was determined at P < 0.05. Survival estimates and median survivals were determined using the method of Kaplan and Meier.