Cell culture and hypoxia treatment
A549 and RAW264.7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM medium with 10% fetal calf serum (FBS) and 1% penicillin/streptomycin at 37˚C in an atmosphere containing 5% CO2. Hypoxia environment was established with a hypoxia cell incubator of 1% O2.
Exos isolation and identification
A549 cells were cultured under normoxic (21% CO2) or hypoxic (1% O2) conditions for 3 days. The medium was replaced with 10% exos-depleted FBS at 80-90% confluence. Then the medium was collected and centrifuged at 300g for 10 min, 3000g for 20 min, 10,000g for 30 min, and ultra-centrifuged at 100,000 × g for 2 h at 4 °C to precipitate exos pellets. After the pellets were resuspended in PBS and stained with the phosphotungstic acid for 10 min, transmission electron microscopy experiment was performed for exos identification (13).
Nanoparticle tracking analysis
The nanoparticle relative concentrations were analyzed using the Nanosight (Malvern, Worcestershire, UK) and NTA analytical software (version 2.3, Nanosight) (14).
Western blot analysis
Cells were harvested and ultra-centrifuged at 100,000 × g for 30 min at 4°C to collect proteins. The concentration of proteins was determined with the BCA protein assay kit (Thermo Fisher; Waltham, MA, USA). 20 μg protein was loaded on an SDS–PAGE, separated by electrophoresis, and transferred to PVDF membrane. The membranes were blocked with 5% skimmed milk for 1 h and incubated with specific antibodies overnight at 4°C subsequently. Monoclonal antibodies (Abcam; Cambridge, UK) used in this study were as follows: CD9 (1:1000), CD81 (1:1000), TSG101 (1:1000), HSP70 (1:1000), ALIX (1:1000), Cleaved-caspase3 (1:1000), bcl-2 (1:1000) ,bax (1:1000), PKM2 (1:1000), p-P38 (1:1000), p38 (1:1000), p-AMPK (1:1000), AMPK (1:1000), CD206 (1:1000), Arginase-1 (1:1000), E-cadherin (1:1000), N-cadherin (1:1000), Vimentin (1:1000) and GAPDH (1:10,000). The membranes were incubated with HRP-conjugated secondary antibodies before being visualized using a Chemiluminescent HRP Substrate Kit (Millipore; Billerica, MA, USA).
A549 cells (1×104 /well) were seeded into 96-well plates and treated with normoxia/exos or hypoxia/exos for 24 h, 48 h, and 72 h. Then 10 μl CCK-8 solution (Dojindo; Kumamoto, Japan) was added to each well and incubated at 37°C for 2 h. The optical density (OD) value at 450 nm was measured with an Epoch microplate spectrophotometer (BioTek; Winooski, VT, USA) and cell viability was calculated.
A549 cells were seeded into 6-well plates. Then cells were scratched with a 200-µl sterile pipette tip at the confluence of 90%. The wounded monolayers were washed with PBS to remove cell debris and A549 cells were cultured with fresh serum-free medium with normoxia/exos or hypoxia/exos. The distance between the two edges of the wound was calculated at 0 h, 24 h, 48 h to evaluate the migration capacity.
Cell apoptosis analysis
Cell apoptosis was examined with the Annexin V-FITC apoptosis detection kit (BD Biosciences; New Jersey, USA). After treatment with normoxia/exos or hypoxia/exos, A549 cells were collected and resuspended in 1×binding buffer, followed by staining with Annexin V-FITC and PI in the dark at room temperature. Then the percentage of apoptotic cells was immediately analyzed by a flow cytometer (BD, Biosciences; New Jersey, USA).
Exo uptake assay
After being labeled with the PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich; St. Louis, MO, USA), exos were added to RAW264.7 cells. Then, RAW264.7 cells were fixed with 4% paraformaldehyde and stained with DAPI prior to its observation by a fluorescence microscopy.
Real-Time quantitative PCR (RT-qPCR)
Total RNA was isolated with the TRIzol reagent (Invitrogen; Carlsbad, CA, USA) according to the manufacturer's instructions. Reverse transcription reaction was performed by using reverse transcriptase kit (Transgen Biotech; Beijing, China). Subsequently, PCR was performed with a standard SYBR Green PCR kit (Transgen Biotech). GAPDH expression was regarded as the normalization control.
Migration and invasion assays
Migration and invasion assays were performed using transwell chambers (24 wells) (Corning; NY, USA) coated without or with Matrigel (BD Biosciences), respectively. 5 × 104 A549 cells were re-suspended in serum-free medium and placed in the top chamber. The lower chamber was filled with 1x104 macrophages induced by hypoxic/exos and normoxia/exos in 600 μl medium containing 10% FBS. After incubation for 24 h, the migrating or invading cells were fixed with methyl alcohol and subsequently stained with 0.1% crystal violet. The number of migrating or invading cells was counted using an Inverted microscope.
Hematoxylin-eosin (HE) staining
Suspicious lung metastasis tissues were assessed by HE staining. The tissues were placed in 10% formalin overnight, and then dehydrated and embedded in paraffin. Then the tissue samples were sliced to 4 μm thick sections, fixed on a glass slide, dried and stained. The sections were successively immersed in xylene, ethanol with gradient concentrations, and hematoxylin. After being mounted with resin, the sections were dried naturally, and observed and photographed with a light microscope.
All animal experiments were approved by the Guangzhou Medical University. Nude mice (male, 4-6 weeks of age, 18–20 g) were purchased from Guangdong Animal Experiment Center. Luciferase-labelled A549 cells were subcutaneously injected with into the oxter of nude mice. After the tumor grew to 50-100 mm3, mice were randomly divided into 3 groups: control, normoxia/exos, and hypoxia/exos (n=5). Exos derived from A549 cells under normoxia or hypoxia conditions were injected into mice via the tail vein for the treatment. In vivo fluorescence imaging was performed to visualize the tumor growth. Tumors size was measured using a caliper weekly. Four weeks after injection, mice were sacrificed under general anesthesia. For metastasis, A549 cells were injected into nude mice via the tail vein. 24 h after injection, mice were treated with exos via tail vein injection. Suspicious lung metastasis tissues were analyzed by HE staining.
All in vitro results were derived from at least three independent experiments. Statistical analyses, histograms drawing, and scatter plots were performed with SPSS 22.0 software (SPSS Inc. USA) and GraphPad Prism 6.0 software (GraphPad Software, Inc.) The significance of differences between two groups was determined using Student’s t-test. Comparations among more than two groups were performed with one-way analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant difference.