Cell lines and Cell culture
The A549 cells were obtained from Chinese Academy of Sciences, Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). A549/Luc cells (A549 cells stably expressing luciferase) were constructed by Synthgene (Nanjing, China). Cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) (Gibco, Rockville, USA) supplemented with 10% fetal bovine serum (FBS) and 100 µg/ml streptomycin and penicillin (Gibco, Rockville, USA) in a humidified atmosphere at 37˚C.
The immunohistochemistry assay was performed according to Yin et al . The sections were incubated with the primary antibody of CD68 (Ab955, 1: 100, Abcam, Cambridge, UK) at 4 °C overnight and horseradish peroxidase labeled goat anti-mouse IgG antibody (A205719, 1: 200, Abcam, Cambridge, UK) at room temperature for 1 h. The Color reaction was performed with diaminobenzidine chromogen solution (Dako, Carpinteria, USA). Brown-yellow particles represented the positive expression of CD68 protein and blue particles represented the nucleus stained by hematoxylin (Sigma, USA).
Rna Extraction And Quantitative Real-time Pcr Analysis
The extraction and reverse transcription of total RNA were performed according to the previous report . The expression levels of TNF-α, IRF5, IRF4, Arg-1 and miR-155 were analysed by quantitative real-time PCR with the GAPDH gene as a standard control. Primers of TNF-α, IRF5, IRF4, Arg-1 and miR-155 were as follows: TNF-α (Forward: 5’- CCTCTCTCTAATCAGCCCTCTG-3’; Reverse: 5’-GAGGACCTGGGAGTAGATGAG-3’); IRF5 (Forward: 5’-GGGCTTCAATGGGTCAACG-3’; Reverse: 5’-GCCTTCGGTGTATTTCCCTG-3’); IRF4 (Forward: 5’-GCTGATCGACCAGATCGACAG-3’; Reverse: 5’-CGGTTGTAGTCCTGCTTGC-3’ ); Arg-1 (Forward: 5’-GTGGAAACTTGCATGGACAAC-3’; Reverse: 5’-AATCCTGGCACATCGGGAATC-3’); miR-155 (Forward: 5’-GGAGGTTAATGCTAATCGTGATAG; Reverse: 5’- GTGCAGGGTCCGAGGT-3’ )
Cell Migration And Invasion Assays
The cell invasion and migration assays were performed by Chamber matrigel invasion 24-well DI kit (BD Biosciences, San Jose, CA). Specifically, A549 cells as the control, A549 cells co-cultured with M2 macrophages treated with or without 5 µM GW4869 and A549 cells co-cultured with exosome from M0/M2 macrophages or M2 macrophages transfected with different plasmids, at a density of 2 × 104 cells/ml, were re-suspended with 200 µl DMEM medium (serum-free) and seeded into the upper chamber, while the lower chamber was placed with 600 µl complete DMEM medium (10% FBS). For cell invasion, A549 cells cocultured with exosome of M0 or M2 macrophages were added to the lower chamber, and the medium with 10% FBS was placed in the upper chamber. After incubation for 24 h, the invaded or migrated A549 cells were fixed with the methanol (100%), stained with crystal violet (0.1 mg/mL) and counted under a microscope.
Isolation, Identification And Labeling Of Exosomes
The exosomes were isolated by density gradient ultracentrifugation according to previously reported protocol . Briefly, cell culture medium was collected and centrifuged at 1,000 g for 10 min, 2,000 g for 20 min, 4,000 g for 30 min and 10,000 g for 1 h, with the supernatant being retained each time. The exosomes were collected by centrifuging the samples at 100,000 g for at least 2 h at 4 °C. Size distribution and concentration of exosomes were analysed at a flow rate of 0.03 ml per min using a Zetasizer Nano ZS (Malvern Instrument, UK) and NanoSight NS300 (Westborough, MA, USA), respectively. Purified exosomes were labeled with the PKH-67 green fluorescent linker Mini Kit (Sigma, USA) according to the manufacturer’s instructions.
Western Blot Analysis
The radio-immunoprecipitation assay (RIPA) lysis buffer (Solarbio, Shanghai, China) with 0.5% phenylmethanesulfonyl fluoride (PMSF) (Solarbio, Shanghai, China) was used to extract the total protein of exosome, cell or tissue. The protein concentration was quantified by the BCA protein quantification Kit (Sigma, USA). The primary antibodies in this study were purchased from Abcam: rabbit anti-CD63 (ab68418, 1: 500), CD81 (ab109201, 1: 200), RASSF4 (ab243709, 1: 1000) and TSG101 (ab30871, 1: 500) and mouse anti-E-cadherin (ab11512, 1: 1000), vimentin (ab8978, 1: 1000) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, 1: 5000). The horseradish peroxidase labeled goat anti-rabbit IgG antibody (ab205718, 1: 10000) and goat anti-mouse antibody (ab6789, 1: 10000) were available as the secondary antibodies. Image J software was used to quantify each protein band.
Detection Of Cy3-labeled Mir-155 Exosome Transfer
The A549 cells were co-cultured with Cy3-labeled miR-155 exosome or free Cy3-labeled miR-155 for 24 h to confirm the transfer of miR-155 by exosomes. Then, the cells were washed with PBS and were incubated with Hoechest33342 at room temperature. Images were obtained by a confocal microscope.
Luciferase Reporter Assay
The 3'-UTR segments of RASSF4 in wild type and mutant were synthesized and inserted into a firefly luciferase reporter construct. Luciferase activity in this study was measured by the Dual Luciferase Reporter Assay System (Promega, USA) according to the protocol.
6-week-old male athymic BALB/c nude mice were used. For the in vivo lung metastases model, A549/Luc cells were injected into the tail vein of representative mice (n = 5 per group). The luciferase signal intensity from days 0 to 28 is on equivalent scales in the models. Bioluminescent flux (photons/s/cm2/steradian) was determined for the lung metastases. Metastatic progression was monitored and imaged using an IVIS-100 system (Caliper Life Sciences, MA, USA) 10 min after intraperitoneal injection of luciferin (300 mg/kg i.v.) in 80 µl of saline. After 28 days, mice were sacrificed and tissues were separated for further experiments. Animal care and euthanasia were carried out with the approval of the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University.
Hematoxylin & eosin (HE) staining
The dewaxed sections were firstly incubated with hematoxylin to stain the nucleus for 5 min, then 1% ethanol-hydrochloric acid for 30 s and eosin solution for 3 min. Finally, the sections were dehydrated in graded alcohol following by clearing in xylene.
All statistical analyses were performed by GraphPad Prism 6.0 software. The Student’s t-test was used to analyze significant differences in this study. The error bars indicate the standard deviation from the mean of triplicate measurements. Asterisks indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001) compared with the corresponding control.