Cell culture
The human NSCLC cell lines A549 and H1299 were cultured in DMEM medium (Gibco, USA), and the human acute monocytic leukemia cell line THP-1 was cultured in RIPA 1640 medium (Gibco, USA). Both medium contained with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). For macrophage polarization, THP-1 cells were treated with 100nM phorbol-12-myristate-13 acetate (PMA; Sigma-Aldrich) for 48 hours, after which the medium was discard and the cells were washed twice with pre-warmed phosphate buffered saline (PBS). The PMA-differentiated THP-1 macrophages were then cultured for another 24 h in the RPMI 1640 complete medium (without PMA) to obtain the resting state of macrophages (M0). For M1 or M2 macrophages polarization, M0 macrophages were cultured for 48 hours in the medium supplemented with 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) and 20 ng/mL IFN-γ (PeproTech) or 20 ng/mL IL-4 and IL-13 (PeproTech), respectively. All cells were cultured at 37°C in a 5% CO2 atmosphere.
Exosome isolation
THP-1-differentiated M2 macrophages were cultivated for 24 hours in FBS-free RIPA 1640 medium, and which the medium was collected and exosomes were extracted by differential ultracentrifugation as described previously [34]. Briefly, to remove cells and debris, the conditioned media was centrifuged at 300 g for 5 minutes and 2,000 g for 15 minutes. Then, the supernatant was harvested and centrifuged at 15,000 g for 30 min at 4 ℃ to eliminate large extracellular vesicles. The exosomes were isolated by centrifugation (Beckman Coulter Avanti J30I) at 100,000 g for 90 minutes. Finally, the isolated exosomes were re-suspended in 200µL PBS and used immediately or stored at -80 degrees Celsius.
Exosome identification
TSG101, CD63, and Alix were utilized as positive controls in Western blot analysis, whereas as a negative control, calnexin, an endoplasmic reticulum protein, was used. The Nanosight NS300 system (Nanosight Technology, Malvern, UK) was employed to directly monitor the number and size distribution of exosomes.
Exosome uptake assays
The extracted exosomes were treated with PKH26 Fluorescent Cell Linker Kits (Sigma-Aldrich) according to the manufacturer's protocol to visualize exosome internalization. Next, the tagged exosomes were cultured with H1299 cells for 6 h. The cells were fixed in 4% paraformaldehyde for 30 minutes before being stained using Abcam's CytoPainter Phalloidin iFluor 488 Reagent for 30 minutes. The nuclei were then stained with Hoechst 33342 (Cell Signaling Technology, Danvers, MA) for 10 minutes. A confocal microscope was used to look at how H1299 cells took in exosomes.
Transwell assay
For cell migration experiments, 2×104 NSCLC cells were resuspended in 200µL of FBS-free media and planted into the 24-well Transwell cell culturing chambers (8µm pore size, BD), and 650µl of medium containing 10% FBS was added into the lower chamber. For cell invasion assays, 4×104 NSCLC cells were resuspended in 200µL of FBS-free media and planted into the upper inserts with pre-coated Matrigel, and 650µl of media containing with 10% FBS were added to the lower chamber. For NSCLC cells and M2 macrophages indirect co-culture assays, 2×104 THP-1 cells were seeded into the lower chamber and they were induced to polarize towards M2 macrophages according to the above protocols. After then, NSCLC cells were harvested and suspended in 200µl of FBS-free DMEM before being transferred to the upper compartment. The cells in the upper chamber were wiped out after 24 hours, and the cells on the lower chamber were fixed with 4 percent paraformaldehyde and stained with 0.5 percent crystal violet. For identifying immune molecules in M2-exos that induce NSCLC cells migration and invasion, A549 and H1299 cells were cocultured with M2-exos, M2-exos + anti-IgG blocking antibody (M2-exos + IgG Ab), M2-exos + anti-ITG αvβ3 blocking antibody (M2-exos + ITG αvβ3 Ab, BioLegend, San Diego, CA) for 24 hours. Control group NSCLC cells were incubated with PBS. The results of NSCLC cells migration and invasion were photographed and counted. At least three random microscopic fields (magnification×100) were taken, and the cells were counted. All experiments were performed in triplicate.
Flow cytometry staining and analysis
Flow cytometric assays were used to evaluate the expression of CD206 and HLA-DR as previously described [35]. Briefly, 5×105 M1 and M2 macrophages were harvested and stained with PE-CD206 or FITC-HLA-DRα antibody (BioLegend, San Diego, CA) for 15–20 minutes, and subsequently analysis using flow cytometry. Flow cytometry data were analyzed by the Flowjo (Treestar, USA) software.
Western blot analysis
Briefly, Whole cell lysates were electrophoresed in an 8 percent SDS-PAGE gel and then transferred to 0.22 m PVDF membranes (Millipore, USA) after being lysed in RIPA buffer with protease inhibitors. The membranes were blocked for 1h at 37°C in TBST with 5% skimmed milk powder before being probed with the specific antibody (1:1000) overnight at 4°C. Then, the membranes were incubated with secondary antibody (1:5000) for 1h at 37°C. The protein bands were identified using an ECL detection system (Bio-Rad, USA).
Reverse transcription and quantitative real-time PCR
Total cellular RNA was extracted using TRIzol reagent (Invitrogen, USA), and 1µg total RNA was reverse transcribed into first-strand complementary DNA (cDNA) using cDNA Synthesis Kit (EZBioscience, USA) according to protocols. Afterwards, the cDNA was performed to measure the relative gene expression level using real-time PCR. The expression of target genes was normalized to GAPDH levels in the samples in triplicates. The 2−ΔΔCT method was used to calculate the relative variation in gene expression. Additional file: Table S1 contains a list of primers.
Animals
Male Balb/c nude mice aged 4 to 6 weeks were purchased from Guangdong Medical Laboratory Animal Center in China. For establishing human NSCLC lung metastasis model in nude mice, 3×106 A549luc cells were resuspended in 200µl FBS-free DMEM and injected intravenously into Balb/c nude mice. To study the blockade effects of ITG αvβ3, 10µg M2-exos, M2-exo + ITG αvβ3 Ab and M2-exos + IgG Ab were administered to Balb/c nude mice every four days, respectively. A similar volume of PBS was injected into the control group. Mice were sacrificed after 50 days, and the lungs were assessed for metastatic lesions by comparing biofluorescence signal intensities. Tissue morphology was identified by hematoxylin and eosin (H&E) staining.
Construction and transfection of ITG αvβ3 shRNA and overexpression plasmids
As previously described, reliable knockdown and overexpression cell lines were established[36]. lentiviral vectors were used to create ITG αvβ3 shRNA and overexpressed plasmid. The generated plasmid was co-transfected into 293T cells for 48 hours with the viral packaging plasmids psPAX2 and pMD.2G. Lentiviral supernatants were harvested and filtered through 0.45µm filter before being cultivated for 24 hours with H1299 cells. Puromycin selection (2 g/ml) was applied to the cells. Additional file: Table S2 shows the targeting sequences for specific genes. Table S3 shows the primers of overexpressed genes.
Immunohistochemistry
Patients’ clinical tumour specimens were gathered at the Sun Yat-sen University Cancer Center in Guangzhou, China, who had been diagnosed with NSCLC. For patient specimens, all patients gave their agreement and enrolled in an IRB approved protocols at Sun Yat-sen University Cancer Center, which allowed the collecting and analysis of clinical data, archival, and paraffin specimens in compliance with ethical principles (Ethics Document No. SL-B2022-139-01). Tumour specimens were formalin-fixed and paraffin-embedded, as is standard laboratory pathology technique, and stored at the Sun Yat-sen University Cancer Center’s pathology department. The paraffin slices from patients' tissues were treated with primary anti-human antibodies at various dilutions (ITG αv, 1:400, ITG β3, 1:200) overnight at 4°C. They were then treated for 60 minutes at room temperature with the second antibody. The staining was identified by using DAB Kit (Zisbio) as directed by the manufacturer. Hematoxylin staining was measured using at least 5 randomly selected 200 or 400 fields of view after slides were stained for 6 minutes. Two pathologists independently evaluated the protein expression.
Statistical analysis
Unless otherwise specified, the results were presented as means SEM and analyzed using one-way ANOVA or Student's t-test analysis. The statistical significance level was set at p < 0.05. SPSS 22.0 or GraphPad Prism 7 were used for all statistical analyses.