Six to eight week old female BALB/c mice (18–22 g) were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in the vivarium in filter top cages containing sterile bedding. After arrival, mice were quarantined for at least five days, and fed chow and water ad libitum. All animal experiments were in compliance with protocols approved by the University of North Carolina at Charlotte Animal Care and Use Committee.
Murine gammaherpesvirus-68 (HV-68)
Maintenance of viral stocks-Murine gammaherpesvirus-68 (HV-68; ATCC # VR-1465) stocks were prepared by infecting baby hamster kidney cells (BHK-21; ATCC # CCL-10) at a low multiplicity of infection (MOI), followed by preparation of cellular lysates, as described previously [9–12].
Infection of animals-Groups of mice were anesthetized with isoflurane and mock treated by intranasal instillation of saline, or infected intranasally with 6 × 104 plaque forming units of HV-68. Animals were housed for 6 months following infection before transplanting 4 T1 breast cancer cells.
Assay of plaque-forming units in cell media and lysates-Replicating HV-68 was quantified by adding 1:3 serial dilutions of cell media or lysates to BALB/3 T12-3 cell (ATCC # CCL-164) monolayers. After the monolayers were incubated with virus, cells were overlayed with 1% Plaque Assay Agarose (BD Biosciences, San Diego, CA) in medium with 30% fetal bovine serum. After 5 days in 5% CO2, overlays were removed and cell monolayers fixed and stained with crystal violet. All serial dilutions were performed in triplicate.
T1 breast cancer cells
Maintenance of 4 T1 cells-4 T1 cells (highly metastatic; ATCC# CRL-2539) were used as model breast cancer cells . Cells were cultured in ATCC complete growth medium (RPMI 1640 medium with 10% fetal bovine serum and 2 mM L-glutamine, adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and 0.05 mM 2-mercaptoethanol).
Injection and monitoring of animals-To produce tumors, 3.5×104 4 T1 cells in 50 ml of phosphate-buffered saline (PBS) were injected into the right abdominal mammary fat pad. Following injection, animals were monitored and weighed three times a week until the last week of the experiment, when they were monitored daily.
Bone marrow-derived macrophage and dendritic cell culture
Bone marrow derived macrophages and dendritic cells were isolated and characterized as previously described [10, 14]. Cells were grown in ATCC complete growth medium supplemented with M-CSF (macrophages) or GM-CSF (dendritic cells) and incubated in 6-well plates with HV-68 for varying lengths of time, and at various HV-68 to cell ratios. For some cultures, LPS (1 ng/ml) was added as a positive control for S100A8/A9 secretion. At the indicated times, cells or culture supernatants were taken for nucleic acid and protein analysis.
Nucleic acid analyses
Preparation of cDNA-Total RNA was isolated using Trizol (Invitrogen; Carlsbad, CA), as previously described [10, 15–17]. RNA samples were incubated with RNase-free pancreatic DNase (RQ1 DNase, Promega, Madison, WI) as per the manufacturer’s instructions, the RNA precipitated with EtOH and resuspended in 50 μl of nuclease-free H2O. RNA concentrations were determined with a Gene Spec III spectrophotometer (Naka Instruments, Japan) using a 10 μl cuvette. For cDNA synthesis, one μg of RNA was reverse-transcribed in the presence of random hexamers (50 ng/μl), 10 mM dNTPs, 2.5 mM MgCl2 using ImProm-II reverse transcriptase (Promega) in the buffer supplied by the manufacturer. cDNA was precipitated with one-tenth volume of 3 M sodium acetate (pH 5.2) and 3 volumes of EtOH, and resuspended in 50 μl of nuclease-free H2O.
Semiquantitative PCR-mRNA transcript (cDNA) levels were examined by PCR. 100 ng of cDNA was combined with 2.5 U of Taq polymerase (Promega), 0.2 mM each dNTP, 25 pmol of each primer and PCR buffer containing 2.5 mM MgCl2 as provided by the manufacturer. Samples were cycled using 95° denaturation for 35 seconds, 60°C annealing for 75 seconds and 72°C extension for 90 seconds, with the first three cycles using extended denaturation, annealing and extension times. PCR was for 35 cycles, except for GAPDH, which was for 28 cycles. The extension time of the last cycle was for 5 min at 72°C. Forty percent of each amplified PCR product was electrophoresed on an ethidium bromide-stained 2% agarose gel and photographed under UV illumination.
PCR primer sets were designed by using IDT SciTools and purchased from IDT (Integrated DNA Technologies, Coralville, IA). Primer sets used for amplification are as follows:
ORF65 (open reading frame-65-murid herpesvirus 4; accession no. NC_001826; 221 bp product).
Forward: 5’-ATG CTC CAG AAG AGG AAG GGA CAC-3’.
Reverse: 5’-TTG GCA AAG ACC CAG AAG AAG CC-3’.
GAPDH (glyceraldehyde-3-phosphate dehydrogenase; accession no. NM_
008084; 346 bp-spans exons 3 to 5).
Forward: 5’-CCA TCA CCA TCT TCC AGG AGC GAG-3’.
Reverse: 5’-CAC AGT CTT CTG GGT GGC AGT GAT-3’.
S100A8 (calgranulin A; accession no. NM_013650; 249 bp-spans exons 2 and 3):
Forward: 5’-GAG AAG GCC TTG AGC AAC CTC ATT G-3’.
Reverse: 5’-CCT TGT GGC TGT CTT TGT GAG ATG-3’.
S100A9 (calgranulin B; accession no. NM_009114; 230 bp-spans exons 2 and 3):
Forward: 5’-GCA AGA AGA TGG CCA ACA AAG CAC-3’.
Reverse: 5’-TCA AAG CTC AGC TGA TTG TCC TGG-3’.
Western blot analysis-Protein in Laemmli sample buffer was electrophoresed on SDS-polyacrylamide gels and transferred to Immobilon P. Filters were blocked for 2 hr with 5% instant nonfat dried milk in TBS, incubated for 2 hr with primary antibody, washed for 30 min with TBS + 0.05% Tween 20, further incubated with HRP-conjugated secondary antibody for 1 hr, washed and developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher, Rockford, IL). Protein bands were visualized with X-ray film. Antibodies were from R&D Systems (Minneapolis, MN).
ELISA-S100A8/S100A9 serum levels were measured using an Immundiagnostik S100A8/S100A9 (calprotectin) ELISA kit (Alpco; Salem, NH) designed to measure the concentration of the S100A8/S100A9 heterodimer.
Fluorescence-activated cell sorting (FACS) analysis
44 days following the injection of 4 T1 tumor cells, mice were euthanized and tissues excised. Bone marrow cells were isolated from the femurs of individual mice as previously described [10, 15]. Splenocyte single cell suspensions from individual mice were made by passing tissue through a 30-gauge wire mesh. Cells were washed with sterile PBS (300 × g for 10 min), resuspended in PBS containing 10% non-immune rabbit serum (Invitrogen, Camarillo, CA) and incubated on ice for 45 min with fluor-conjugated antibodies (eFluor 450-anti-CD11b and PE anti-Gr-1, eBioscience, San Diego, CA). After washing, cells were resuspended, analyzed, and then sorted using the FACSAria II cell sorter (BD Biosciences, San Diego, CA). Analyses were performed on>20,000 cells per individual spleen or bone marrow isolate.
T-cell suppression assay
Single cell suspensions of splenocytes were prepared as described above, and red blood cells were removed using a lysing reagent (Sigma-Aldrich, St. Louis, MO). Splenic leukocytes were washed and resuspended in RPMI-1640 containing 10% fetal bovine serum. CD11b+Gr-1+ cells were then isolated using a mouse myeloid-derived suppressor cell (MDSC) isolation kit (Miltenyi Biotec, Auburn, CA).
To isolate T lymphocytes, spleens were removed from normal, uninfected mice, and splenic leukocytes prepared as described above. Total T lymphocytes were isolated using magnetic separation (Pan T cell Isolation Kit; Miltenyi Biotec).
For co-cultures, the indicated numbers of MDSC were added to 2 × 105 T lymphocytes in anti-CD3 coated microtiter wells (T cell activation plates, BD Biosciences, Bedford, MA). After 72 hours of co-incubation, the amount of IFN-γ present in the culture supernatants was determined using an ELISA (DuoSet mouse IFN-γ; R&D Systems, Minneapolis, MN) as an indication of T lymphocyte activation.
For statistical analysis, data were analyzed using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA). Analyses were performed using Student’s t-test, or by one-way analysis of variance (ANOVA) with Tukey’s Multiple Comparison Test as post-test. Mean values are presented in the figures +/− the Standard Error of the Mean (SEM). Results marked with an (*) were determined to be statistically significant at P<0.05.