Cell culture
As previously described, from healthy adult donors who underwent posterior lumbar discectomy, human epidural ADSCs were obtained so as to treat or lumbar disc herniation or lumbar fracture6. According to the ethical guidelines of the Qingdao University Affiliated Hospital in Qingdao, China, all the procedures were performed. In this study, the human B cell non-Hodgkin lymphoma cell lines SU-DHL-10 and SU-DHL-4 for Type Culture Collection from the China Centre were applied. Both lymphoma cell lines were cultured in culture medium (DMEM/F12, 10% foetal bovine serum (FBS)) under the condition of a humidified 5% CO2 chamber at 37°C.
Exosome isolation
Exosomes were obtained from epidural ADSC supernatants by differential centrifugation. The medium was discarded when ADSCs reached 70% confluence. Then, in serum-free DMEM/F12, the cells were cultured for another 24 h. The supernatants were collected and then cleared by sequential centrifugation at 3,000 x g for 30 min or 15,000 x g for 30 min. After filtration through 0.22-mm filters (Millipore, Billerica, MA), the supernatants were ultracentrifuged at 120,000 x g for 2 h. With sterile PBS, the exosomes were washed and collected several times. Exosome concentrations were determined with a Pierce BCA protein assay kit (Thermo Fisher Scientific).
Electron microscopy
Nearly 50 μl of prepared exosomes were adsorbed and put onto formvar carbon-coated 300-mesh copper grids in approximately 10 min. Then, at room temperature for 30 min, the adsorbed exosomes were dried and with 3% phosphotungstic acid, they were negatively dyed. Later, the exosomes were studied through taking advantage of a transmission electron microscope (Olympus Software Imaging Solutions) at 120.0 kV. Moreover, to capture images of the exosomes, a digital camera was used.
Immunohistochemistry
Tumour tissue samples were split into 4-μm sections and embedded in paraffin. With 100 μl of a solution, antigen retrieval was conducted at 4°C including antibodies against Ki67 and S100A4 (1:200 dilution, Abcam MA, USA) after dehydrating the tissue samples in a graded alcohol series. Moreover, with the sections for 20 min at 37°C, a diluted biotinylated secondary antibody was incubated. To visualize target proteins, haematoxylin was used as a tissue counterstain, and a fresh 3,3-diaminobenzidine (DAB) solution was also used. Two observers assessed the expression of target proteins independently by taking advantage of an Olympus FV500 optical microscope (Olympus, Tokyo, Japan) . In addition, to investigate the intensity and area of staining in five random regions (200× magnification) and evaluate the protein expression level, Image-Pro Plus 5.1was applied.
Immunofluorescence
For 24 h, B cell non-Hodgkin lymphoma cells were cultured with or without purified ADSC exosomes (10 μg/ml). Then, all the cells were gathered and separated, and later for 60 min, they were fixed with 4% paraformaldehyde. The fixed cells were cut into 4-μm sections and embedded in paraffin. Later, in PBS they were washed for three times and blocked with 10% goat serum for 1 h. After that in 0.2% Triton X-100 the sections were washed twice. Next, they were incubated with primary antibodies (anti-S100A4 and anti-vimentin were purchased from Abcam, MA, USA), secondary antibodies (Invitrogen) and DAPI (Guangzhou RiboBio, Guangzhou, China). Using a fluorescence microscope, images were captured.
Proliferation and invasion assays
For the following tests, B cell non-Hodgkin lymphoma cells were applied after an incubation with purified exosomes (10 μg/ml) for 48 h. In previous studies, cell invasion assays and growth assays were conducted according to the manufacturer’s instructions described.
EdU/PI assay
According to the manufacturer’s instructions (Guangzhou RiboBio, Guangzhou, China), 5-Ethynyl-2’-deoxyuridine (EdU)/propidium iodide (PI) assays were conducted by taking advantage of a Cell-Light EdU in vitro klow cytometry kit. In brief, in a 6-well plate cells were cultured overnight, with a 20-min incubation with EdU followed. Then, they were fixed with 70% ethanol at -20°C overnight and washed twice with PBS. Later, the cells were stained with a FITC-conjugated secondary antibody for 1 h at room temperature and denatured in 2 N HCl for 45 min. Moreover, with 40 g/ml RNase A and 200 g/ml PI, the cells were incubated for 30 min and finally analysed by flow cytometry.
RNA-seq and GSEA
SU-DHL-10 cells treated with or without ADSC exosomes were stored at -80°C after being collected in 1 ml of TRIzol reagent (Thermo Fisher Scientific). According to the instructions of the Illumina TruSeq RNA Sample Prep Kit, libraries were prepared. And on a MiSeq instrument, sequencing was conducted. At Annoroad Gene Technology Co., Ltd. (Beijing, China), the experiments were conducted. And with RSEM software, the data were analysed. All RNA-seq data can be obtained in the Sequence Read Archive (SRA) under accession nos. PRJNA65327.
Through putting the fold change data from differential expression analysis into GSEA software (Broad Institute) by taking advantage of the gene set database c2.cp.v5.1.symbols, GSEA of the RNA-seq data was conducted.
shRNA transfection
For 24 h, B cell non-Hodgkin lymphoma cells (5×105) were seeded and cultured in 10-cm plates. Then, through making use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA), they were transfected with a scrambled shRNA control (GeneChem, Shanghai, China) or S100A4-specific shRNA.
qRT-PCR assay and RNA isolation
B cell non-Hodgkin lymphoma cells were gathered. Additionally, total RNA was isolated through taking advantage of TRIzol reagent (Invitrogen, Carlsbad, USA). Later, 2 μg of RNA was applied for first-strand cDNA synthesis. By using a reverse transcription kit (Toyobo, Osaka, Japan), first-strand cDNA synthesis was conducted. According to the instructions of manufacturer, quantitative real-time PCR was conducted with gene-specific TaqMan probes (Applied Biosystems) and master mix (Thermo Fisher Scientific). Gene expression was normalized to GAPDH expression. The used TaqMan probes included S100A4, Hs00656410_ce; and GAPDH, Hs02786624_g1.
Western blotting
To remove cell debris, B cell non-Hodgkin lymphoma cells were centrifuged at 10,000 x g and lysed for 10 min on ice in NP40 buffer (Beyotime, Shanghai, China). SDS-PAGE was used to separate the same amout (30 μg) of cell extracts. And then they were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA, Hercules). Then, primary antibodies against β-actin (1:2,000 dilution; Abcam, MA, USA) and human S100A4, TSG101, CD63, E-cadherin and vimentin (1:1,000; Cell Signaling Technology) cultivates the PVDF membranes. Then, with the membranes, peroxidase-conjugated AffiniPure secondary IgG antibodies (H+L) (1:2,000; R&D Systems) were incubated. On a chemiluminescence detection system, the protein-antibody complexes were detected and quantitated by using Image Lab™ version 5.1 software (Bio-Rad, Hercules, CA).
In vivo metastasis assays and tumour growth
The Animal Ethics Committee of the Affiliated Hospital of Qingdao University approved all experimental protocols involving animals. And from the Shanghai Animal Centre (Shanghai, China), four-week-old female BALB/c nude mice were purchased. Moreover, S100A4 shRNA/shNC SU-DHL-10 cells (5×107 cells/mouse) were injected subcutaneously to establish a xenograft model. After SU-DHL-10 cell injection, ADSC exosomes (30 μg, twice per week) were injected into the tumours twice per week. Six mice were included in each of the four study groups. The mice were injected with SU-DHL-10 cellsshNC + exosomes, SU-DHL-10 cellsshNC + PBS, SU-DHL-10 cellsshRNA + exosomes or SU-DHL-10 cellsshRNA + PBS. After injecting cells for 7 weeks, the animals were sacrificed.
As described before, the luminescence of SU-DHL-10 cells was evaluated14.
Statistical analysis
In our study, the data are expressed as the mean ± standard deviation. We also calculated the means after conducting no less than three independent experiments. , The significance of differences was analysed with a two-tailed Student’s t-test or one-way analysis of variance, and P<0.05 was regarded as significant.