Adenoviruses expressing IL-12 (Ad.IL-12) and IL-23 (Ad.IL-23) have been described previously.13, 25
Ad.IL-12, Ad.IL-23 and Ad.Psi5 (empty vector) were prepared as follows: Viruses were propagated on HEK-293 cells and purified by CsCl banding, followed by dialysis in 3% sucrose solution. Particle titer of purified viruses was determined by spectroscopy using the equation, (OD260
, with the virus being diluted 1:50 prior to measuring OD. The particle titer was used to calculate MOI in all experiments. Infectious titers were determined using quantitative real-time PCR as previously described26
and were approximately 100-fold less than particle titers. Viruses were aliquoted and stored at −80° C until use.
Adenoviruses expressing single-chain (sc) versions of IL-12 and IL-23 were designed as follows: To construct Ad.scIL-12, the IL-12p40 precursor (Met1 to Ser335) was linked to the mature p35 subunit (Arg23 to Ala215) using the previously described 15 amino acid linker (Gly4
To construct Ad.scIL-23, the IL-12 p40 precursor (Met1 to Ser335) was linked to the mature p19 subunit (Leu20 – Ala196) using (Gly4
Relative cytokine expression of each adenoviral preparation was analyzed by infecting 4×104 MCA205 cells with a 500 MOI of Ad.IL-12 or Ad.IL-23 for 1 hour at 37°C/5% CO2 in serum free media. Complete media was added and cells were incubated for 72 hours, after which supernatants were harvested. IL-12 and IL-23 content was analyzed using the mouse IL-12 p70 and mouse IL-23 p19/p40 Ready-Set-Go IL-23 ELISA kits (eBioscience, San Diego, CA), respectively, following manufacturers’ instructions.
Biological activity of Ad.scIL-23 was assayed as follows: 4×104 MCA205 cells were infected with a MOI 1000 of Ad.scIL-23 or Ad.Psi5 and supernatants harvested 72 hours post-infection. Splenocytes were then harvested from C57BL/6 mice, mechanically dissociated and treated with Red Cell Lysis buffer (Invitrogen, Carlsbad, CA) to remove all red blood cells. Splenocytes were plated at a concentration of 2×106 cells per well in a 24 well plate and 24 hours later treated with supernatants from adenovirus-infected MCA205s. Forty-eight hours after treatment, splenocyte supernatants were collected and analyzed for induction of IL-17 expression using the Mouse IL-17 Immunoassay (R&D Systems, Minneapolis, MN).
Female C57BL/6 and p40-deficient mice on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME). IL-23 (p19) deficient mice have been previously described and were a kind gift of Dr. Jay Kolls.28
Mice were used at 6 to 7 weeks of age. Animals were maintained under pathogen free conditions at the Biotechnology Center Animal facility at the University of Pittsburgh. All procedures preformed were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
MCA205 fibrosarcoma cells were maintained in RPMI supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin (Gibco, Carlsbad, CA) and L-glutamine (Gibco, Carlsbad, CA). Cells were kept in a humidified chamber at 37°C/5% CO2 and passaged every 2–3 days.
In Vitro Transduction of MCA205 Cells
MCA205s were plated on 24-well plates at a concentration of 4×104 cells per well and infected with a MOI 500 of Ad.IL-12 and Ad.IL-23, either virus alone or Ad.Psi5. Seventy-two hours post-infection, supernatants were harvested and analyzed using Quantikine Mouse IL-23, IL-12 p70 and IL-12/IL-23p40 Immunoassays (R&D Systems, Minneapolis, MN).
In Vivo Tumor Studies
For use in in vivo tumor experiments, confluent layers of MCA205 cells were dissociated by trypsin, washed 3 times with Hanks Balanced Salt Solution (HBSS) (Gibco, Carlsbad, CA) and counted using trypan blue exclusion. Mice were inoculated with 1×105 MCA205 cells in 100uL HBSS subcutaneously in the abdomen. IL-23 (p19) and IL-12/23 (p40) deficient mice were treated on days 7, 9 and 11 post-tumor inoculation by intratumoral injection of 5×1010 particles (approximately 5×108 PFUs) of either Ad.IL-12, Ad.IL-23 or Ad.Psi5.
To investigate possible synergy by delivering Ad.IL-12 and Ad.IL-23 at separate time points, mice bearing two tumors were treated in one with 5×108 particles of Ad.IL-12 or 2.5×1010 particles of Ad.IL-23 as follows: Ad.IL-12 on day 7, followed by Ad.IL-23 on day 11; Ad.IL-23 on day 7 followed by Ad.IL-12 on day 11; Ad.IL-12 on days 7 and 11; Ad.IL-23 on days 7 and 11; or Ad.Psi5 on days 7 and 11. Alternatively, to investigate possible anti-tumor synergy between co-delivered Ad.IL-12 and Ad.IL-23, mice bearing two, day 7 tumors were treated simultaneously with 1×109 particles of Ad.IL-12 and 5×1010 particles of Ad.IL-23 or either virus alone.
To investigate the anti-tumor activity of Ad.scIL-23, mice were injected intratumorally on days 7, 9 and 11 post-tumor inoculation with 5×1010 particles of Ad.scIL-12, Ad.scIL-23 or Ad.Psi5. To explore possible synergy between Ad.IL-12 and Ad.scIL-23, mice bearing day 7 MCA205 tumors were treated once intratumorally with 1×108 particles of Ad.IL-12 and 1×109 particles of Ad.scIL-23, either virus alone or Ad.Psi5. In all synergy experiments, total quantity of virus injected was kept constant between groups by co-delivery of Ad.Psi5. In all experiments, tumor volume was monitored using a metric caliper until mice were sacrificed due to excessive tumor size or tumor ulceration. Tumor-free or “cured” mice were subject to tumor challenge 1–2 months after initial tumor resolution with 1×105 MCA205 cells subcutaneously in the abdomen.
Tumors of treated mice were harvested on day 15 post-tumor inoculation (day 8 post-treatment), snap frozen in 2-methylbutane and stored at −80°C. Tumors were then cut by cryostat, ten micron sections placed onto charged slides and stained for CD8 and CD31 as follows: For analysis of intratumoral CD8+ T-cell infiltrate from mice treated with Ad.IL-12 and Ad.IL-23, sections were fixed in −20°C acetone and slides blocked in Exogenous Peroxidase Block (DAKO, Carpinteria, CA), followed by block in 10% goat serum. CD8 antibody (1:150 dilution)(BD Biosciences, San Jose, CA) was then added, followed by incubation with biotinylated goat anti-rat secondary antibody (1:250)(BD Biosciences, San Jose, CA) in DAKO Antibody Diluent (DAKO, Carpinteria, CA). Slides were treated with ABC Vectastain kit (Vector Laboratories, Burlingame, CA) and developed using DAB Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA) following manufacturers’ instructions. Slides were then counterstained with eosin, dehydrated in increasing concentrations of ethanol, followed by xylene, and coverslipped using Permount Media (Fisher, Pittsburgh PA).
Alternatively, for analysis of tumors from mice treated with Ad.IL-12 and Ad.scIL-23, sections were permeabilized using 10% Triton-X (Sigma, St. Louis, MO), blocked in 2% BSA (Sigma, St. Louis, MO) and incubated with rat anti-mouse CD8 antibody (1:100 dilution)(BD Biosciences, San Jose, CA) overnight at room temperature. Slides were then incubated with Alexa-Flour goat anti-rat secondary (1:500 dilution)(Invitrogen, Eugene, OR) and coverslipped using Flourmount media (SouthernBiotech, Birmingham, AL).
To investigate tumor angiogenesis, sections were permeabilized with 10% Triton-X (Sigma, St. Louis, MO) followed by block in 2% BSA (Sigma, St. Louis, MO). Sections were then incubated in rat anti-mouse CD31 primary antibody (1:100) (BD Biosciences, San Jose, CA), followed by Alexa-Flour goat anti-rat secondary (1:500 dilution)(Invitrogen, Eugene, OR).
Kaplan-Meier survival curves were plotted using SPSS version 16.0. Mice were monitored until excessive tumor volume or tumor ulceration, at which time they were sacrificed and recorded as occurrence of an event (death). Cured mice or those with tumors that did not warrant sacrifice by the end of the experiment were censored. Log-rank tests of the survival curves provided p-values. An unpaired T-test was used to analyze immunohistochemistry results if variances between data were equal; if variances were unequal, the Mann-Whitney U test was performed. Statistical analyses were 2-tailed, with a p value less than 0.05 considered statistically significant.