ADMSCs isolation
To achieve enough number of cells from the best sources, adipose tissues were separately obtained from healthy donors (aged between 22 and 35 years) undergoing surgical procedures. They all signed an informed consent form approved by the ethics committee of Shahid Beheshti University of Medical Sciences (Ethical code: IR. SBMU.REC.1400.010).
Briefly, after washing adipose tissues from lipoaspirate samples with phosphate-buffered saline (PBS), they were digested with 0.1% collagenase I (Sigma, USA) for 40 min at 37°C with gentle agitation. At the end of the incubation time, collagenase was neutralized by adding FBS-containing medium and digested samples were centrifuged at 1200 RPM for 20 min (Hettich, Germany). The resultant cell pellet was resuspended in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 15% FBS and 1% penicillin and streptomycin, seeded in culture flasks and maintained in a humidified atmosphere at 37°C and 5% CO2. After 24 h, cells were washed to discard non-adherent cells and the fresh medium was replaced. During the expansion, half medium refreshment was done twice per week for optimal growth.
ADMSCs characterization
Immunophenotyping of ADMSCs was performed using flow cytometry. At the third passage, 1×106 ADMSCs were suspended in PBS and then incubated with primary antibodies including CD45-FITC, CD14-FITC, CD34-PE, CD90-FITC, CD73-PerCP, and CD105-PerCP (eBioscience, USA) for 30 min. Identification of ADMSCs’ surface markers was performed by FACSCalibur flow cytometer (BD Biosciences, USA).
The osteogenic and adipogenic differentiation potential were assessed using respective induction media and protocols [19]. ADMSCs (1×104 cells/well) at passage 3 were seeded into 24-well plates and cultured in DMEM/F12 with 10% FBS. After 24 h, the differentiation media were replaced and refreshed every three days. 21 days after osteogenic induction, cells were fixed with 10% neutral formaldehyde and stained with 0.1% Alizarin red S dye (Sigma-Aldrich, USA). For Adipogenic differentiation, after 14 days, cells were fixed and stained with 0.5% Oil Red O dye (Sigma-Aldrich, USA). The differentiated cells were observed by the light inverted microscope (Olympus, USA).
Cell lines and culture conditions
HUVEC (human umbilical vein endothelial cells), MDA-MB-231 cells (triple-negative breast cancer cell line), and MCF-10A (non-tumorigenic breast cell line) were obtained from the Pasteur Institute of Iran (Tehran, Iran). MCF-10A and MDA-MB-231 cells were grown in DMEM containing 10% horse serum and FBS, respectively. HUVECs were cultured in DMEM/F-12 supplemented with 10% FBS. All cells were maintained in a humidified atmosphere at 37°C and 5% CO2.
Preparation of ADMSC-conditioned media and exosome isolation
The ADMSCs at the 3rd passage were used for the collection of conditioned medium (CM). When cells reached 70-75% confluence, they were adapted to FBS-free medium containing 1% insulin-transferrin-selenium (ITS; Sigma, USA). Serum-free ADMSC-CM was collected after 72 h and used for exosomes isolation and characterization. AnnexinV/PI staining was performed to evaluate cell viability after 72h serum starvation.
ADMSC-exosomes were isolated from ADMSC-CM using EXOCIB exosome purification kit (Cibbiotech, Iran) according to the manufacturer’s instructions. Briefly, exosome precipitation solution was added to ADMSC-CM and incubated overnight at 4 °C. Then, the samples were centrifuged at 3000 RPM for 40 min at 4 °C. After removing the supernatant, exosomes were resuspended with PBS for the following experiments.
Exosome characterization
The size distribution of extracted exosomes was determined using dynamic light scattering (DLS) Zetasizer (Malvern, UK). The morphology and size of exosomes were observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For TEM imaging (Zeiss EM900), after processing of ADMSC-exosomes (fixation, dehydration, and sectioning), the ultrathin sections were prepared and stained using uranyl acetate and lead citrate and visualized under electron microscopy. For SEM (KYKY-EM3200, China), after fixation and dehydration of exosomes, they were left on the glass substrate to dry at room temperature and then were analyzed by scanning electron microscope.
Loading ADMSC- exosomes with miRNA mimic
To load miRNA-218-5p mimics (Bioneer, Korea) into ADMSC-exosomes, electroporation method was used. ADMSC-exosomes at a final concentration of 100 µg/µl protein (measured by BCA) were mixed with electroporation buffer (in a 1:1 ratio) and 100 pmol of synthetic miR-218 mimics or negative control (Scramble), and electroporated at 0.400 kV using an electroporation instrument (Eppendorf, Germany). To evaluate the efficiency of the loading protocol, MDA-MB-231 cells were incubated with 100 µg/ml of manipulated exosomes for 48 h and miR-218 encapsulation in exosomes was quantified using qRT-PCR.
The experimental groups included MDA-MB-231 cells treated with miR-218 or scramble containing exosomes, cells treated with unmodified exosomes, and untreated cells.
RNA extraction, cDNA synthesis, and qPCR
Total RNA from MDA-MB-231 cells treated with modified-exosomes and their controls was extracted after 48 hours using Hybrid-RTM (GeneAll, Korea) according to the manufacturer’s instructions. Using RT-Stem loop [20] and random hexamer primers, miRNA and target mRNAs were respectively transcribed to complementary DNA (cDNA).
Relative expression of miR-218 and target mRNAs was evaluated using TaqMan® probe and SYBR Green I Master Mix (Amplicon, Germany), respectively, in a StepOne instrument (Applied Biosystems, USA). SNORD47 and β-actin were considered as the internal references for miR-218 and mRNAs, respectively. The 2 –ΔΔCt method was employed to compute the relative expression of miRNA and mRNAs.
MTT assay
MDA-MB-231 cells were plated in a 96-well plate (3×103 cells/well) and incubated in serum-containing DMEM. After overnight incubation, cells were treated with modified exosomes (100 µg/ml) and their controls in a serum-free medium. At defined time points, the medium was removed and cells were incubated with 100 µL of MTT solution in PBS (0.5 mg/mL) for 3 h in the cell culture incubator. After removing the supernatant, 100 µL of dimethyl sulfoxide (DMSO) was added to each well for 2 h to dissolve formazan crystals. The optical density was read at 570 nm using a microplate reader (BioTek, USA).
Annexin V/PI assay
MDA-MB-231 cells were plated in a 24-well plate (5× 104 cells/well). 48 and 72 h after their incubation with exosomes (100 µg/ml) in respective groups, cells were trypsinized and stained with annexin V/ fluorescein isothiocyanate (FITC) / propidium iodide (PI) kit according to the manufacturer’s instructions (Abcam, US). FACSCalibur flow cytometer was employed for cell analysis (BD Biosciences, USA). The annexin V + / PI - and annexin V + / PI + cells were considered as early and late apoptotic cells, respectively.
Scratch assay
MDA-MB-231 cells (12 × 104 cells/well) were plated in a 24-well plate. On reaching 90-95% confluence, the scratch was made across each well using a sterile 100 µl tip. After washing with DMEM to remove cell debris, cells were treated with 100 µg/ml modified exosomes in serum-free medium. Plates were photographed by an inverted microscope for 48 h and the images were processed and quantified using the ImageJ software (NIH, USA).
Cell migration assay
5×104 MDA-MB-231 cells suspended in serum-free medium with modified exosomes (100 µg/ml) were added into the upper chamber of transwell inserts (24-well insert; pore size 8µm; SPL) and exposed to FBS-containing medium (as a chemoattractant) in the bottom chamber for 48 h. After incubation time, non-migrated cells were removed by scraping the upper surface of the chamber, and inserts were fixed and then stained with crystal violet. Five random fields were selected for counting the number of migrated cells.
Cell invasion assay
5×104 MDA-MB-231 cells were seeded to matrigel (Corning, USA) coated inserts. Matrigel, being prepared by mixing with serum-free DMEM at a ratio of 1:2, was added to inserts and maintained at 37 ̊C for 2h to solidify. The next steps were similar to those described for the migration assay.
In vitro angiogenesis assays
HUVECs were plated in a 96-well plate (3 × 103 cells /well) and incubated in DMEM-F12 overnight. Then, the culture medium was replaced with conditioned media of breast cancer cells treated with modified ADMSC-exosomes and their controls. After 24, 48, and 72 h, viable cells were evaluated by MTT assay.
In vitro migration assay was performed as described above. 4 × 104 HUVECs were added in the upper chamber of transwell inserts and cultured in serum-free DMEM-F12. The conditioned media of breast cancer cells treated with exosomes-encapsulated miR-218 and their controls were added to the lower chamber. After 24 h, migrated cells were stained and counted in five randomly selected microscopic fields.
In vitro capillary network formation was evaluated by tube formation assay. 3 × 104 cells were seeded into matrigel- coated 48-well plate. Next, the cells were incubated with conditioned media collected from breast cancer cells treated with miR-218 containing exosomes and their controls for 24 h. The number of meshes and total branching length were quantified by randomly selecting five fields per well by using angiogenesis analyzer ImageJ plugin (NIH, USA).
Statistical analysis
Statistical analysis was conducted using GraphPad Prism (GraphPad, San Diego, CA). Student’s t-test was used for comparison between two groups while data among multiple groups were compared by one-way ANOVA. All experiments were performed in triplicate. The data was finally presented as mean ± SD and the asterisks show significant p-value: * p.value < 0.05; ** p value < 0.001; *** p value < 0.0001; and **** p.value < 0.00001).