Animals and cell lines
BALB/c mice (specific pathogen free [SPF], female, 6-8 weeks old) were purchased from Harlan (Zeist, The Netherlands) and were housed under pathogen-free conditions at the animal care facility of Erasmus MC. All experiments were approved by the local ethical committee for animal welfare (Erasmus University Committee of Animal Experts, Rotterdam, the Netherlands) and complied with the guidelines for the welfare of animals in experimental neoplasia by the United Kingdom Coordinating Committee on Cancer Research (UKCCCR), and by the Code of Practice of the Dutch Veterinarian Inspection. The mesothelioma AB1 cell line was kindly provided by Professor B.W.S. Robinson (School of Medicine and Pharmacology, Sir Charles Gairdner Hospital Unit, The University of Western Australia, Perth, Australia).
Tumour growth of murine mesothelioma in BALB/c mice
BALB/c mice were divided into 4 groups. Each group consisted of 6 mice. On day 0, all mice were injected with a lethal dose of 0.5 × 106
AB1 tumour cells. From day 0 onwards, group 1 and 3 received a control diet while group 2 and 4 received celecoxib diet (500 mg celecoxib/kg [Celebrex®
; Pfizer, New York, NY, USA]). Both were starch-based diets manufactured by Harlan Teklad (Madison, WI, USA) as described by T. Hahn et al
]. Mice in group 3 and 4 received DC-based immunotherapy consisting of 1 × 106
tumour-lysate loaded dendritic cells at day 10.
Dendritic cells were culture from bone marrow using RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 5% heat inactivated fetal bovine serum (FBS [Hyclone, Waltham, MA, USA]), 20 ng/ml GM-CSF (kindly provided by Kris Thielemans, VU Brussels, Belgium), and 50 μM β-mercaptoethanol (Sigma-Aldrich BV, Zwijndrecht, The Netherlands) for 9 days. At day 8, cells were pulsed with tumour-lysate (to the equivalent of three AB1 cells per DC) and overnight matured with 100 ng/ml LPS E. Coli 026:136 (Sigma-Aldrich BV). On the day of vaccination, DCs were harvested and purified by Lympholyte-Mammal (Cedarlane, Hornby, ON, Canada) density gradient centrifugation, and washed three times in phosphate-buffered saline (PBS [Gibco]). Cells were resuspended at a concentration of 1 × 106 viable cells in 500 μl PBS and intraperitoneally injected.
The occurrence of tumour growth, body weight, physical well-being and survival were measured for 2 months, as described previously [28
Immunohistochemistry on tumour cells and biopsies
Tumour cells were cultured in RPMI supplemented with 5% FBS on two chambers Falcon culture slides (BD biosciences, Erebodegem, Belgium) starting with 5 × 104 AB1 cells per well. Celecoxib was added to the culture when cells reached a confluence of 60% for 24 hours in a concentration of 10 μg/ml and 100 μg/ml. Resected tumour material was obtained from the peritoneal cavity of AB1 inoculated BALB/c mice at day 15. Tumour biopsies were embedded in Tissue-Tek II optimum cutting temperature (OCT) medium (Miles, Naperville, IL, USA), snap-frozen in liquid nitrogen and stored at -80°C. Tissue sections (6 μm) were cut on a HM-560 cryostat (Microm, Heidelberg, Germany).
For the COX-2 expression a rabbit affinity-purified IgG against murine COX-2 was used (Cayman chemical, Montigny-le-Bretonneux, France) (this antibody shows no cross-reactivity with COX-1). For the COX-1 expression a rabbit affinity-purified IgG against murine COX-1 was used. These primary antibodies were incubated for 1 hour at room temperature. Binding of antibodies was detected using the goat anti-rabbit alkaline phosphatase (AP) (Sigma-Aldrich). Naphtol-AS-MX-phosphate (0.30 mg/ml [Sigma-Aldrich]) and new fuchsine (160 mg/ml in 2 M HCl [Chroma-Gesellschaft, Köngen, Germany]) were used as substrate. Hematoxylene was used as counterstaining. The specificity of the antibodies was checked using a protein concentration-matched non-relevant monoclonal antibody and PBS.
Mouse mesothelioma biopsies were double stained for Gr-1-FITC (clone RB6.8C5) and COX-2. As secondary antibodies horseradish peroxidase (HRP) conjugated goat anti-FITC (Rockland, Gilbertsville, PA, USA) and AP-conjugated goat anti-rabbit (Sigma-Aldrich) were used. Naphtol-AS-MX-phosphate and 1 mM Fast Blue (Sigma-Aldrich) were used as substrate for AP and NovaRed was used as substrate for HRP, according to the manufacturer's instructions (Vector, Burlingame, CA, USA).
Spleens were aseptically removed, and mechanically dispersed in cold HBSS (Invitrogen). Cell suspensions were filtered through a 100 μm nylon cell strainer (BD Biosciences), depleted of erythrocytes by osmotic lysis, washed twice in RPMI medium containing 5% FBS, and adjusted to a concentration of 1 × 106 cells/ml in FACs-buffer.
Splenocytes were stained with the following optimally diluted mAbs: Ly6c (FITC conjugated), MHCII (PE conjugated), CD11b (PercP-Cy5.5 conjugated) (all BD bioscience), CD8 (FITC conjugated), F4/80 (FITC conjugated), CD4 (PE conjugated), CD31 (PE-Cy7 conjugated), B220 (Alexa fluor 700 conjugated), Ly6g (APC-Cy7 conjugated) (all eBioscience), CD11c (PE-Texas red conjugated [Caltag, Burlingame, CA, USA]), and a live/dead marker (DAPI [Invitrogen]). Splenocytes were restimulated in the presence of GolgiStop (BD biosciences) for 4 hours using anti-CD3 and intracellular stained for Granzyme B (PE conjugated [Caltag]) and IFN-γ (APC conjugated [BD Biosciences]). Viability was determined by live dead aqua (Invitrogen). Acquisition of eight to nine colour samples was performed on a FACs LSR II cytometer (BD Biosciences). The analysis and graphical output were performed using FlowJo software (Tree Star Inc., Costa Mesa, CA, USA).
PGE2 levels in the peritoneal washes were determined using a specific ELISA assay for PGE2 (R&D systems, Abingdon, UK). Manufacturer's recommended protocols were followed. Serum was diluted appropriately to ensure that readings were within the limits of accurate detection.
Nitric oxide and its reactive products (NO)
Equal volumes of peritoneal wash (150 μl) were mixed with Greiss reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-1-naphthyl-ethylenediamine dihydrochloride in double-distilled water). After 30 min incubation at room temperature, the absorbance was measured at 548 nm using a microplate reader (Bio-Rad). Nitrite concentrations were determined by comparing the absorbance values for the test samples to a standard curve generated by serial dilution of a stock solution of sodium nitrite.
Reactive oxygen species (ROS)
The oxidation-sensitive dye dichlorodihydrofluorescein diacetate (DCFDA, Sigma-Aldrich) was used for the measurement of ROS production by splenocytes. Cells were incubated at 37°C in DMEM (Invitrogen) in the presence of 1 μM DCFDA for 60 min, and washed twice with cold PBS. Cells were stained with Abs directed against Gr-1 (PE conjugated [BD biosciences]) and CD11b (PercP-Cy5.5 conjugated [BD biosciences]) as previously described. Cells were washed with cold PBS after 20 min incubation. Acquisition of the samples was performed on a flowcytometer.
Tumour-specific lysis assay
Spleens were aseptically removed, and mechanically dispersed in cold PBS. Cell suspensions were filtered through a 100 μm nylon cell strainer (BD Biosciences), depleted of erythrocytes by osmotic lysis, washed twice in RPMI, and adjusted to a concentration of 4 × 106 cells/ml in RPMI medium supplemented with 5% FBS. After 48 h of culture, spleen cells were washed extensively. Mouse AB1 cells were incubated with 100 μCi of Na2 51CrO4 (ICN Biomedicals) for 2 hours at 37°C, washed three times, resuspended in culture medium at a concentration of 5 × 104 cells/ml. Splenocytes (150.000 cells per well) from either naïve mice, untreated mice, or mice treated with DC-based immunotherapy were mixed with 5 × 103 radiolabeled AB1 target cells and added to wells of a 96-well round-bottom microtiter plate (0.2 ml final volume) to achieve the desired effector:target (E:T) ratios. To determine the suppressive capacity of splenocytes from mice treated with control diet or celecoxib diet, 150.000 splenocytes from DC treated mice were mixed with 150.000 splenocytes from mice treated with either control diet or celecoxib diet. After 24 hour incubation, cells were mixed with radiolabeled AB1 target cells and added to wells of a 96-well round-bottom microtiter plate (0.2 ml final volume) to achieve the desired effector:target (E:T) ratios.
Plates were incubated for 4 hours at 37°C in a humidified atmosphere containing 5% CO2, and cell-free supernatants were collected from each well. The amount of 51Cr released from lysed AB1 target cells was determined by γ scintillation counting. Percent lysis was calculated using the formula: corrected % lysis = 100×(experimental release - spontaneous release [target cells incubated in medium alone])/(maximum release[2% Triton X-100 as lysing agent]-spontaneous release).
Data are expressed as mean ± SD. Comparisons between groups were made using the t-test. A two-tailed p-value < 0.05 was considered significant. Data presented as a percentage of tumour-free animals were analysed with Kaplan-Meier survival-curves, using the log-rank test to determine significance.