Animals
This study was approved by Ethics Committee of Harbin Medical University. Female wild-type BALB/c mice with 6-8 weeks old were used in the present study. Animals were fed under humidity-, temperature-, and light-controlled conditions with free water and food.
Cell Lines and Culture Conditions
The breast cancer cell line 4T-1 was obtained from the Harbin Medical University Cancer Hospital Lab, and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Thermo Fisher Scientific, USA), supplemented with 10% (v/v) fetal bovine serum (Gibco, Thermo Fisher Scientific, USA) at 37°C in incubator with 5% CO2. The cells were mycoplasma free and showed appropriate morphology during experiments.
Surgery and Hemorrhage Procedure
Mice were divided into three groups randomly: Control group, Surgery group and Surgery-hemorrhage group (n=7 in each group). All mice were deprived of food overnight but free access to water for 24 hr preoperatively. For surgery and hemorrhage, animals continuously inhalated isoflurane and fixed in a supine position on warm pat to maintain rectal temperature at 37 ± 0.5 °C throughout the operation. A length of 2.0-cm incision along the middle line of the abdominal wall were made under sterile conditions. The intestine was isolated outside for 30 mins. For hemorrhage, the 27-gauge needles (Becton Dickinson, MD) cannulated into the left lateral of femoral artery and vein of surgical mice. Blood pressure was measured via arterial catheter using a blood pressure analyzer (SurgiVet, USA). Nearly 20% of total blood volume was withdrawn in 30 mins through the vein to induce hemorrhage. Ringer’s lactate solution was infused slowly for resuscitation through the vein. After ensuring there was no bleeding, closed the incision aseptically and sterily. Animals in Surgery group accepted the same procedure but with no hemorrhagic shock. In Control group, mice received same anesthesia without any procedures else.
Bone metastasis model
In the present study, implantation of 4T-1 breast cancer into the mouse was used as bone metastasis model [2]. Briefly, 1×105 4T-1 cells injected orthotopically into the left mammary fat pad and the other 3×105 4T-1 cells mixed with matrigel (Sigma, USA) injected into the proximal end of the left tibia. The surgical procedure was performed at 24 hr post 4T1 cells challenge.
Histopathological analysis
For histopathological studies, the femoras and tibias implanted within tumor cells were fixed within 4% paraformaldehyde and embedded in a paraffin block. The sections with 5 um-thick were performed from each fragment. Histology slides were stained with hematoxylin and eosin to assess histological changes. Percent of bone resorption was calculated from the laser scanning confocal microscope images using Adobe Photoshop software. Then chose at least three images of each slides to get a mean value.
Tartrate-resistant Acid Phosphatase (TRAP) and Alkaline Phosphatase (ALP) Staining
Immunohistochemical (IHC) staining was used to assess the activity of osteoclasts. Briefly, slides were stained for rat anti-mouse TRAP (Abcam, UK), antibodies for 3 hr at 37°C, followed by blocked for 1 hr using the biotin-streptavidin per-oxidase kit, rat primary antibody. The stained slides were then developed using DAB kits (Vector Laboratories, Burlingame, CA, USA). To assess the activity of osteoblasts, ALP staining was carried out by ALP staining Kits according to manufacturer’s instruction (Nanjing Jiancheng Bioengineering Institute, Jiangsu Sheng, China). Images were captured with a Nikon Digital Sight DS-Fi1 camera and NIS-Element software (Nikon Instruments, Melville, NY, USA) and observed under an optical microscope at 200×.
Isolation of Bone Myeloid Cells (BMCs) and MDSCs
In mcie, the BMCs were collected from mice in Control group, Surgery group and Surgery-hemorrhage group at day 3 and day 7. In tumor bearing mice, BMCs in femurs and tibias were isolated at day 3 and day 7 after surgery. Then filter the cell suspension through a 70-μm cell strainer. The erythrocytes were lysed with lysis buffer (BD Biosciences, San Jose, CA), and then the cells were cultured in RPMI1640 medium with 10% FBS and 1% penicillin and streptomycin.
MDSCs from surgery-hemorrhage mice at day 7 were separated using Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi Biotec, Germany) according to the manufacturer’s instructions.
Flow cytometry analysis (FACS)
Single cell suspensions from spleens and bone marrow were filtered through a 70 µm cell strainer (BD Bio sciences, USA) and resuspended in FACS buffer at a concerentration of 1×106 cell/100ul. Then cells were respectively stained with FITC conjugated anti-mouse CD11b, APC conjugated anti-mouse Gr-1 antibodies (BD Biosciences, USA) respectively at room temperature for 30 minutes. Finally cells were fixed in 2% paraformaldehyde until detected using the FACS Scan II (BD Biosciences, USA) and analyzed with FlowJo software (Treestar, USA).
Western blot
MDSCs were lysed in RIPA buffer and the protein concentration was analysed by BCA protein assay kit (Beyotime Biotechnology, China). Samples were separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked for 60 mins in TBS/5% no fat milk, and then incubated with the anti-PD-1, anti-PD-L1 (1:1000) and GAPDH (1:10000) antibodies (Abclonal, China) at 4°C temperature for 12 h. Then the HRP-conjugated secondary antibody was incubated for 30 mins at room temperature. At the end the membrane was exposed by ABI western blot imaging system.
Real time-polymerase chain reaction (RT-PCR)
RT-PCR analysis was used to detected the mRNA levels of inducible nitric oxide synthase (iNOS), arginine (Arg)-1 and indoleamine-2,3-dioxygenase (IDO) which is associated with the immunosuppressive activity of MDSCs. The procedures were performed as our previously described [2]. The primer sequences (InvitrogenTM, USA) were as previously described. iNOS forward, 5’-CCGAAGCAAACAT-CACATTCA-3’; reverse, 5’ -GGTCTAAAGGCTCCGGGCT-’3. IDO forward 5’-TGTG GCTAGAAATCTGCCTGT-3’, reverse 5’-CTGCGATTTCCACCAATAGAG-3’; Arg-1 forward 5’-CTCCAAGCCAAAGTCCTTAGAG-3’; reverse, 5’-AGGAGCT GTCATTAGGGACATC-3’. β-actin forward 5’-AGAGGGAAATCGTGCGTGAC-3’; reverse 5’-CAATAGTGAT GACCTGGCCGT-3.
Co-culture system in vitro
To determine the influences of MDSCs on the breast cancer cells, the MDSCs were added into 4T-1 tumor cells cultures at a ratio of 100 : 1 in RPMI 1640 medium with 10% FBS, 1% penicillin and streptomycin, as well as 50 ng/ml GM-CSF [2]. Anti-mouse PD-1 (2 ug/ml, eBioscience, USA), and isotype (5 ug/ml, Biolegend, USA) antibodies were added to detected in some cases.
Proliferation assay
Total 4×103 tumor cells were seeded in 96-well plates and allowed to attach for 24 hr. MDSCs or MDSCs-depleted BMCs were seeded into plate and co-cultured with 4T1 cells at a ratio of 100:1. The plates were washed three time to discard the BMCs and 10% CCK solution were added into plated at 12 hr, 24 hr, 48 hr, and 72 hr respectively. Then detected the OD value of each well The proliferation of breast cancer cells with or without BMCs was evaluated with. Each experiment repeated at least in triplicate.
Cell invasion and migration assays
The transwell assays with matrigel was used for cell invasion assays. Permeable supports (Corning Incorporated Life Sciences, USA) were precoated with 40 ul of 1 mg/ml matrigel. Then freshly isolated BMCs 4×106 cells suspended in 200 ul medium without FBS were placed on the top chamber of each well (Millipore, MA, USA). For migration assays, freshly isolated BMCs 5×106 and 4×104 4T1 cells suspended in 200 ul medium without FBS were placed on the upper chamber without matrigel (Millipore, MA, USA). The lower chamber was filled with 800 ul of medium with 10% FBS as the nutritional attractant.
Then the cells were fixed with 4% polyformaldehyde for 1 h and then stained with 0.1% crystal violet. Cleaned off the cells that not invaded to the lower surface of membrane. The membranes were then visualized under an optical microscope at 200 ×. Images were captured with a Nikon Digital Sight DS-Fi1 camera and NIS-Element software. Cells in three different visual fields were counted.
Statistics
Statistical analysis was carried out by SPSS 19.0 software. Results were represented as means ± standard deviations (SD). One-way ANOVA was used to assess the significance of intergroup differences. The two-tailed t test was performed to compare the difference between two groups. Statistical significance was accepted for p values < 0.05. In vitro, each experiment was repeated at least three times.