Cell Culture
4T1 cell line (ATCC, CRL-2539) was purchased from National Collection of Authenticated Cell Cultures (Shanghai, China). The cell line was maintained in RPMI 1640 (Thermo Scientific-CN), supplemented with 10% FBS (HyClone, Beijing wobisen technology co., LTD, China) and antibiotic/antimycotic 10,000 U/ml.
Mouse Model
Female BALB/c mice (8-10-weeks old) were purchased from the Ex&InVivo Biotechnology company (Shijiazhuang, China). All mice were housed in a specific pathogen-free environment under protocols approved by the Animal Care Committee of Hebei Medical University, China, and all experiments related to the mice were performed in accordance with the approved guidelines.
All animal experiments were done according to a protocol approved by the Institutional Animal Care and Use Committee. 1×106 4T1 cells were inoculated into the fourth mammary fat pad on the right of the mice (50 µl/mouse). 10 days after tumor inoculation, when the tumor diameter was about 7mm, local radiotherapy treatment was performed with a dose of 0 or 20Gy. Radiotherapy was performed by using a 225 kVp cabinet X-ray system. Animals were anesthetized and placed under a 3.2-mm lead shield with a 1-centimeter round hole to focus the tumor.
Isolation Of M-mdscs And Pmn-mdscs
MDSCs were isolated from single cell suspensions of lung tissues of the right lobe of mice of 4T1 tumor-bearing mice. Firstly, single cells of lung tissue were isolated by mechanical method, and then Ficoll gradient centrifugation method was used to obtain the mononuclear cells. Then, the single mononuclear cells were labeled with anti-mouse CD16/CD32 antibody for 30 min, and subsequently followed staining by 488 conjugated anti-CD11b murine antibodies for another 30 min. After incubation, CD11b positive cells were isolated with a FACS Aria cell sorter. The CD11b+ cells were divided into two tubes and then the PE conjugated anti-Ly6C or Ly6G antibody was added for 30 min. Finally, the anti-PE microbeads were added and M-MDSCs or PMN-MDSCs were isolated per the manufacturer’s protocol. The purity of the M-MDSCs and PMN-MDSCs was assessed by flow cytometry and found to be ≥ 99.8%.
Flow Cytometry
To analyze the frequency of M-MDSCs, PMN-MDSCs or macrophages in lung of 4T1 tumor-bearing mice, the single mononuclear cells from lung tissue were labeled with CD32/16 antibodies for 30 min, and followed with 488-conjugated CD11b (dilution 1/400), APC-conjugated Gr-1 (dilution 1/100) and PE-conjugated F4/80 (dilution 1/200) antibodies for another 30 min. The flow cytometry data were acquired using BD LSRII flow cytometer and analyzed by FlowJo Software (Tree Star).
To analyze the CXCR2 or CCR2 expression in MDSCs, the single mononuclear cells from lung tissue were labeled with APC-conjugated CXCR2 or CCR2 antibody following CD32/16 antibodies blocking.
Cytokine Array And Elisa
The presence of cytokines in the exosomes released by 4T1 cells were detected using the Mouse Cytokine Array C3 by commissioning Shanghai Kangcheng Biotechnology Co., LTD. Mouse cytokines ELISA kits (CXCL1, G-CSF, GM-CSF and CCL2) were used per the manufacturer’s recommendations. For cytokine determination in mouse lung tissue, the left lower lobe of mice (about 100mg/ mouse) was taken, and 500µl PBS was added. The tissue was mechanically grinded in a high-speed low-temperature tissue grinder (Wuhan, China), followed by centrifuging at 10000 RMP for 10 min. The supernatant was taken and the protein concentration was determined by CBA method.
Mdscs Migration Assay
Migration assays utilized a 24-well plate Boyden chamber assay with 8µm pore insert. The purified MDSCs were seeded into the upper compartment of the insert (2×105/250µl/well). The inserts were placed in the lower chamber. Subsequently, 500 µl of the medium (RPMI 1640 medium supplemented with 0.5% FBS, 25 mM HEPES, pH 7.4) containing exosomes (50µg/ml) or lung tissue extract (50µg/ml) or BMDMs culture supernatant (1:1) were added to the lower chamber. For migration assays utilizing small molecule inhibition of CXCR2 or CCR2 signaling, SB265610 or RS504393, was added at a final concentration of 1µM or 1.5µM, respectively. The migrated cells in the lower chamber were counted after 16–18 h. Experiments were done in triplicates.
Histology And Lung Metastases Analysis
Lung tissues from the left upper lobe of mice were taken and fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometre sections were stained with hematoxylin & eosin (HE). Numbers of lung metastases were quantified on seven hematoxylin and eosin-stained representative sections. The area of each lung metastases was measured and divided into two groups according to their size: small metastases with an area less than 0.4 mm2 and large metastases with an area greater than 0.4 mm2, as described previously [31].
Western Blot
Western Blot
For protein detection, cells supernatant was separated with RIPA buffer (Sigma). 50 µg of protein was separated using a 10 or 12% SDS-PAGE gel. The proteins were transferred onto PVDF membrane (Bio Rad Laboratories) using semi dry Trans-Blot (Bio Rad Laboratories). The primary antibodies were incubated with the membrane overnight at 4°C. Subsequently, blots were washed and incubated with appropriate secondary antibodies (Beyotime, Haimen, China) for 1 h at room temperature. Images were obtained using a chemiluminescence (ECL) detection system (ProteinSimple, San Jose, CA). Quantified band intensities were normalized using β-actin protein. Blots were scanned with a Tanon imaging system (5200, Shanghai, China). Primary antibodies against Vimentin, N-cadherin, E-cadherin, CD9, TSG101, CXCR2, CCR2 were purchased from ABclonal (Wu Han, China).
Inhibition Of T Cell Function By Mdscs
For T cell proliferation assay, splenocytes from normal BALB/c mice were placed in triplicates into a U-bottom 96-well plates (1×105) with or without the presence of 10 µg/ml ConA and 10 µg/ml mrIL-2. The splenocytes were co-cultured with purified MDSCs for 68 h and then MTS (15 µl/well) was added for further culture for 4h. The OD570 value of cell culture supernatant was detected by microplate analyzer.
For CTL killing assay, splenocytes from 4T1 tumor-bearing mice were seeded in 24-well plates with complete RPMI 1640 medium containing recombinant mIL-2 at 10µg/ml and antigen (from 4T1 cell lysate, 50µg/ml), and the cultures were incubated for 6 d at 37℃ to induce CTL differentiation. During the incubation period, fresh medium was changed every 48 hours. Subsequently, the CTL cells, the purified MDSCs and 4T1 cells were plated in complete RPMI 1640 medium in round bottom 96-well plates in the ratio of 5:1:1. The cultures were incubated for 16 hours at 37℃. Then cultured supernatant and suspended cells were removed, the cells were digested with trypsin, and the number of dead cells was detected by flow cytometry after 7-AAD labeling.
4T1 cells co-cultured with MDSCs at the ratio of 10:1 and the representative pictures were shown at 0 and 24 hours after wound scratches were made. Migration rates were calculated by measuring the width of the wound scratches, the experiment was repeated three times.
Cell Scratch Test
When 4T1 cells seeded in 12-well plate reached a confluent state, a single scratch was made using a sterile 10 µl pipette tip. The cells were then incubated with FBS-free culture medium alone or containing M-MDSCs or PMN-MDSCs (105/well). Images of the scratches were captured at 0, 24 h with Olympus inverted microscope. The width of the scratch was analyzed using the Olympus CellSens Dimension software. Cell mobility was calculated according to the following formula. Mobility = (initial scratch width-current scratch width)/initial scratch width /2×100%.
Differentially Expressed Genes And Enrichment Analyses
NCBI GEO database (GSE62817) was used to screen out the differentially expressed genes (DEGs) between normal lung tissues and lung tissues from 4T1 mice with lung metastasis. To identify DEGs associated pathways and function annotations, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted by DAVID online database (Huang, Sherman & Lempicki, 2009a; Huang, Sherman & Lempicki, 2009b) (DAVID; https://david.ncifcrf.gov).
Exosome Isolation, Characterization, And Analyses
Isolation of exosomes for antibody array and all other experiments was done by ultracentrifugation, as described previously [25]. 4T1 cells were cultured in complete RPMI 1640 medium for 24 h and at which time the cells confluences rate was about 90%. The cells were irradiated at the dose of 0Gy or 20Gy. Then, the cells were cultured in serum-free RPMI medium for 72h and the culture medium was collected and centrifuged at 800g for 15 min to remove lifted cells and additional 10,000g for 30 min to remove cell debris. Exosomes were then harvested by centrifugation at 100,000g for 120 min (Beckman Ti70). The exosome pellet was resuspended in 100 µl of PBS. The morphology and particle size of exosomes were verified by electron microscopy. Western blotting analysis detected rich expression of exosomal specific markers (CD9 and TSG101).
Exosome Labeling, Uptaking And Treatment
The purified 50 µg exosomes were labeled using PKH26 membrane fluorescently dye (Sigma) for 20 min at 37°C. Labeled exosomes were washed in 20 ml of PBS, collected by ultracentrifugation to remove excessive PKH26, and resuspended in PBS. In experiments of exosomes uptake, the BMDMs were cultured with complete RPMI 1640 medium containing labeled exosomes (20 µg/ml) for 7 h and then the cells were harvested, washed with PBS, and analyzed by fluorescence microscope (Nikon, Japan).
For the experiment of stimulating BMDMs to produce cytokines by exosomes, BMDMs were treated with exosomes at a final concentration of 50ug/mL for 24h. Then, culture supernatant was removed, washed with PBS, and fresh RPMI 1640 complete medium was added and additional culture for 48h. The supernatant was collected for ELISA experiment.
For the experiment of treating normal mice with exosomes, 5µg exosomes in 50 µl PBS were injected into the tail vein every other day for 3 weeks. PBS was used as a control. On the second day after the end of exosome injection, half of the mice were sacrificed after anesthesia, and the frequency of MDSCs was analyzed by flow cytometry. The other half of the mice were injected with 1×105 4T1 cells through the tail vein and sacrificed 10 days later to observe lung tumor nodules through pathological sections.
Statistical Analysis
Error bars in graphical data represent means ± s.e.m. Statistical significance was determined using a two-tailed Student’s t-test or ANOVA. Statistical analyses were performed using GraphPad Prism software. P values were considered statistically significant when p < 0.05 (*p < 0.05; **p < 0.01; and ***p < 0.001).