Cell lines and Cell culture
The A549 and THP-1 cells were obtained from Chinese Academy of Sciences, Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). A549/Luc cells (A549 cells stably express luciferase) were constructed by Synthgene (Nanjing, China). THP-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and A549 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) (Gibco, Rockville, USA) supplemented with 10% FBS (fetal bovine serum) (Gibco, Rockville, USA) and 100 µg/ml streptomycin and penicillin (Gibco, Rockville, USA) in a humidified atmosphere at 37˚C. After 7 days in culture, THP-1 cells were treated with 100 ng/mL phorbol 12-myristate-13-acetate (PMA) to differentiate into macrophages for 48 h. Next, 100 ng/mL lipopolysaccharide (LPS) and 20 ng/mL interferon-γ (IFN-γ) were adopted to treat THP-1 cells for 24 h, polarizing them into M1 macrophages. Following treatment with 20 ng/mL interleukin-4 (IL-4) for 72 h, the cells were polarized into M2 macrophages.
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 the 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 analyzed by quantitative real-time PCR with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene or U6 as a standard control. Primers of TNF-α, IRF5, IRF4, Arg-1, miR-155, miR-19b-1-5p, miR-12206-5p, miR-3091-3p, miR-12180-3p, U6 and GAPDH 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-3; Reverse: 5’-GTGCAGGGTCCGAGGT-3’); miR-19b-1-5p (Forward: 5’-GCGAGTTTTGCAGGTTTGCA-3; Reverse: 5’-AGTGCAGGGTCCGAGGTATT-3’); miR-12206-5p (Forward: 5’-GCGCGTACTATGCCTGGAAG-3; Reverse: 5’-AGTGCAGGGTCCGAGGTATT-3’); miR-3091-3p (Forward: 5’-GCGGGCCTGACCAGTCT-3; Reverse: 5’-AGTGCAGGGTCCGAGGTATT-3’); miR-12180-3p (Forward: 5’-GCGCGAGGAGCTGTGGA-3; Reverse: 5’-AGTGCAGGGTCCGAGGTATT-3’); U6 (Forward: 5’-TCGGCAGCACATATACTAA-3’; Reverse: 5’-CGCTTCACGAATTTGCGTGT-3’); GAPDH (Forward: 5’-GACCTCAACTACATGGTT-3’; Reverse: 5’-AACCATGTAGTTGAGG-3’). These primers were synthesized and purified by RiboBio (Guangzhou, China).
Cell migration and invasion assays
The cell invasion and migration assays were performed by 24-well Transwell cell culture chambers with 8-μm sized pores with or without precoated Matrigel (BD Biosciences, San Jose, CA). Specifically, A549 cells, A549 cells co-cultured with M2 macrophages treated with or without GW4869 and A549 cells co-cultured with exosomes from M1/M2 macrophages or M2 macrophages transfected with different plasmids, at a density of 5 × 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 DMEM medium (10% FBS). After incubation for 24 h, the cells remaining in the upper chamber were removed, 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 from M1 or M2 macrophages were isolated by density gradient ultracentrifugation according to previously reported protocol . Briefly, cell culture medium was collected and centrifuged at 1,000g for 10 min, 2,000g for 20 min, 4,000g for 30 min and 10,000g for 1 h to obtain the supernatant. The exosomes were collected by centrifuging the supernatant at 100,000g for 2 h at 4 °C. The size stribution and concentration of exosomes were analyzed 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.
The aberrant miRNAs expressions of M1-exosome and M2-exosome were analyzed by miRNAs sequencing. Briefly, total RNA was isolated from M1-exosome and M2-exosome using TRIzol reagent (Invitrogen, MA, USA). 250 ng total RNA of M1-exosome and M2-exosome were extracted to prepare the small RNA sequencing library by using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, USA). The libraries were finally sequenced and the Solexa CHASTITY quantity filtered reads were harvested as Clean Reads. For data analysis, differentially expressed miRNA profiles between M1-exosome and M2-exosome groups were compared, fold changes were calculated to identify significant differentially expressed miRNAs and hierarchical clustering was performed. The selected miRNAs were verified by qRT-PCR.
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 exosomes, cells or tissues. 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); 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
Cy3-labeled miR-155 mimics were purchased (GenePharma, China). The A549 cells were co-cultured with exosomes isolated from M2 macrophages transfected with Cy3-labeled miR-155 to further observe the transfer of miRNA. Then, the cells were washed with PBS and 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 purchased. For the in vivo lung metastases model, exosomes purifified from M1/M2 macrophages or M2 macrophages transfected with 1×109 ifu of miR-155 inhibitor lentivirus were respectively injected into the peritoneum. Four days post-injection, 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.