2-1. Isolation of MSC-derived exosomes and ASCs characterization
In order to isolate the MSC-derived exosomes, a Total Exosome Isolation kit (EXOCIB, Iran) was used. The resulting cell suspension was centrifuged at 3000 RPM for 20 min to remove cellular debris. The supernatant was transferred to a new 1.5 ml tube and the pre-heated reagent-A was added as a 1:5 ratio. Following the incubation overnight at 4°C, the mixture was centrifugated at 3000 RPM for 45 min and the supernatant was removed after centrifuging and the pellet obtained was resuspended in DMEMc (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. The total protein concentration was measured for exosome quantification using the Bradford method. The phenotype of the ASCs was characterized by flow cytometry (BD FACSCalibur™ Flow Cytometer - BD Biosciences, USA) using a panel of antibodies against CD34, CD45, CD73, and CD90 (Thermo Fisher Scientific, USA).
2.2. Cell culture and preparation of ASC -conditioned media
Adipose-derived mesenchymal stem cells (Ad-MSCs) were purchased from the GenIran research & education center (Tehran, Iran). Colorectal cancer cell line (HCT-116) was purchased from Pasture Institute (Tehran, Iran); Then, Ad-MSCs at passage 3 were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin/ 100 Ug/ml streptomycin (P/S) and 1% L-glutamine until 70% confluence. The FBS was gradually removed and cells adapted to the FBS-free medium. After 4 hours of incubation in an FBS-free medium, the supernatant was collected and concentrated using 0.22 μm filters.
2.3. Characterizationof MSC-exosomes
10 μL of the isolated exosomes was loaded on 10% polyacrylamide gel. The electrophoresis procedure was run at 120 V for 3 h. The gel was transferred to nitrocellulose membrane, then membrane was blocked with 5% skim milk overnight. After three times washing with PBS-Tween20 the membrane incubated with a primary antibody (mouse anti-CD63 antibody, the concentration of 100µg/mL, (Abcam, USA). After washing with PBS-T, the membrane incubated with secondary HRP conjugated anti-mouse IgG, the concentration of 200µg/mL (Santa Cruz, USA). Hydrogen peroxide (H2O2) and 3, 3, 5, 5-Tetra-Methyl Benzidine (TMB) solution was added. The stop solution (1M H2SO4) was dispensed after color development. The signals were seen using an ECL detection kit (Amersham, London, UK) in accordance with the manufacturer's instructions. In addition, transmission electron microscopy (TEM) (ZeissEM10C) was done to confirm the shape of the isolated exosomes.
The purified exosomes were also labeled with the anti-human antibodies, including anti-CD81 and anti-CD63 antibodies, for flow cytometric analysis (both antibodies were purchased from eBioscience). The analysis was carried out, using a FACSCalibur flow cytometer. To analyze the size distribution of the exosomes, they were diluted in PBS and Tween-20. The size of them was measured using dynamic light scattering (DLS) Zetasizer Nano ZS (Malvern Instruments, UK).
2.4. HCT-116 cell lines co-culture
We seeded HCT-116 colon cancer cells (106 cells per well) into 6-well plates. MSC-conditioned medium (MSC-CM) in a 1:1 ratio or 100 mg/ml of MSC-derived exosomes were added to HCT-116 cells and cultured for 48 hours either alone (not treated), with MSC-CM, or with 100 ug/ml. All experiments were carried out in triplicate.
2.5. RNA Extraction and Real-Time PCR
In accordance with the manufacturer's instructions, RNA was extracted from cells using the Ana Cell Super RNA extraction kit (Ana Cell tec., Iran). By using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA), we measured the amount and quality of extracted RNA using an optical density 260/280 absorption ratio of >1.8. We generated cDNA using a cDNA Synthesis Kit (Ana Cell Tec, Iran) in the thermal cycler according to the manufacturer's instructions to evaluate MAGE-A6 and MAGE-A11 mRNA expression. Primers designed by NCBI Primer Blast Software and synthesized by Pishgam Biotech Co. (Tehran, Iran). The qPCR analysis was conducted on Rotor-Gene Q (QIAGEN, Hilden, Germany) using qPCR SYBER Green Master Mix (BioFact, Daejeon, Korea) according to the previous protocol (5). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was considered as a housekeeping gene and the relative expression levels were determined by the 2−ΔΔCt formula.
2.6. Statistical analysis
Statistical analysis was performed using Prism (GraphPad) Software (version 8.3.0). Data are presented as Mean ± SEM and p values<0.05 were statistically considered as the significance level. The normal distribution of data was examined by the Kolmogorov-Smirnov test. ANOVA test was used to compare the groups and analysis of the data. The Friedman test is used to evaluate the clinical score and body weight of all groups.