Materials
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Sodium alginate (Qingdao Jingyan Bio-Tech, Qingdao, China) was purified by removing protein and endotoxin, according to the protocol used in our laboratory. XAV-939 and GW4869 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).
Human sample
The use of human subjects was reviewed and approved by the Ethics Committee of Dalian Municipal Central Hospital. Mononuclear cells were isolated from the peripheral blood of a HCC patient by gradient centrifugation with lymphocytes separation medium (Lymphoprep, 08751, STEMCELL Techbology, Vancouver, BC, Canada).
Tregs isolation
Tregs were isolated from peripheral blood of a HCC patient, by CD4+ CD25+CD127dim/- Regulatory T Cell Isolation Kit (130-094-775, Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, the isolation of CD4+ CD25+ CD127dim/– regulatory T cells was performed with a cocktail of biotinylated antibodies and anti-Biotin microbeads for the depletion of non-CD4+ and CD127high cells. Then the flow-through fraction of pre-enriched CD4+ CD127dim/– T cells is labeled with CD25 microbeads for subsequent positive selection of CD4+CD25+CD127dim/– regulatory T cells with MidiMACS™ Starting Kit (Miltenyi Biotec).
Tregs were cultured in X-VIVOTM 15 medium (BE02-060F, Lonza, Basel, Switzerland) supplemented with 2% heat-activated patient serum, 500 U/mL recombinant human IL-2 (T&L Biological Technology, Beijing, China), MACSiBead pre-loaded with CD3 and CD28 antibodies (130-095-353, Miltenyi Biotec), and 10 ng/ml rapamycin (HY-10219, MedChemExpress).
HCC cell culture, encapsulation and co-culture
Human HCC cell line MHCC-LM3, purchased from Cellcook (Cellcook biotech company, Guangzhou, China), were cultured in high glucose Dulbecco's Modified Eagle's Medium (H-DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Invitrogen) in a 37 °C incubator with an atmosphere of 5% CO2.
Single cells dissociated from monolayer cultures were counted and suspended in 1.5%, (w/v) sodium alginate at a cell density of 1×106/ml. The cell suspension was extruded into 100 mM CaCl2 solution. The gelation time to produce calcium alginate gel (ALG) beads was 30 min. The encapsulated MHCC-LM3 cells were co-cultured with Tregs for 3 days in H-DMEM supplemented with 10 % FBS. Then Tregs were removed by sedimentation and filtration with 100 μm strainer (352360, Corning, NY, USA). The encapsulated HCC cells were harvested from ALG beads by treating with 55 mM sodium citrate, and then used for further experiments.
Plasmid, shRNAs and cell transfection
The plasmids for generating vectors were prepared from p-CMV-GreenZeo (Genechem, Shanghai, China). Short-hairpin small interfering RNA sequences were 5’-GAAGCAGCGGACACTCAAT-3’, 5’-ACACGCATGTTTGCCTTC’.T-3’, and 5’-TGGCAAATGGTGTCTGCAA-3’. A scrambled sequence (5’-TGACGCGATACGTATTGTA-3’) was used as a negative control. Transfection of MHCC-LM3 cells were performed using Lipofectamine 3000 (Invitrogen). DNA‑liposome complexes were prepared at 4˚C to a final volume of 1 µg/µl and added to MHCC-LM3 cells (1 µg/ml). Transfection was performed for 6 h at 37 ℃.
Flow cytometry
Tregs were labeled with FITC Mouse Anti-Human CD4 (1:100), PE Mouse Anti-Human CD25 (1:200) and Alexa Fluor® 647 Mouse anti-Human FoxP3 (1:250) antibodies for 30 minutes on ice, followed by washing with phosphate buffered saline (PBS) (Gibco, Grand Island, NY, USA), FITC Mouse IgG1 (556649, BD Biosciences), PE Mouse IgG1 (5557449, BD Biosciences), Alexa Fluor® 647 Mouse IgG1 (557732, BD Biosciences) were used as isotype controls. As for FoxP3 staining, Human FoxP3 Buffer Set (560098, BD Biosciences) was used. Briefly, Tregs were fixed with Buffer A, incubated for 10 minutes at RT, and permeabilized with buffer C, incubated for 30 minutes at RT. Flow cytometry was performed by FACSCanto II flow cytometer, and the data were analyzed and presented using Flowjo software version 10 (Flowjo LLC, Ashland, OR, USA).
Quantitative reverse transcription polymerase chain reaction (RT-qPCR)
RT-qPCR (two-step method) was applied to examine the relative levels of the genes, using GAPDH as an internal control. The total RNA was isolated using TRIzol® reagent (Invitrogen), according to the manufacturer’s instructions. Reverse transcription (RT) was performed using a PrimeScript RT Reagent Kit (RR036A, TaKaRa, Shiga, Japan). Real-time PCR was carried out with SYBR Premix Ex Taq (Perfect Real Time) (RR820A, Takara). PCR amplification and fluorescence detection were performed using a LightCycler® 96 System (Roche, Basel, Swiss). The primers used in this study were listed in Table S. The results were presented as the calculated comparative expression ratios of target sample to control group for each sample using the Ct method (2-∆∆ Ct).
Immunofluorescence staining
MHCC-LM3 cell spheres were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich, Munich, Germany) and washed with PBS (Gibco) for three times. After cytospining, cells were treated with 0.05% Triton-X 100 (Sigma-Aldrich), then incubated with CD133 primary antibody (1:400) (64326, Cell Signaling Technology, Danvers, MA, USA) in PBS containing 1% goat serum (16210064, Thermal Fisher Scientific, Waltham, MA) at 4 °C overnight. Then the cells were treated with Alexa Fluor® 555 conjugated anti-rabbit IgG antibody (1:1000) (4413, Cell Signaling Technology) for 60 minutes at room temperature. The primary antibody was omitted for negative control. Nuclear staining was performed using Hoechst 33342 (H3570, Thermal Fisher Scientific). The samples were observed using an inverted fluorescence microscope (DMI8, Leica, Solms, Germany).
Western blot
Cells were lysed in lysis buffer containing protease and phosphatase inhibitors (Keygentec, Nanjing, China). Protein concentration was quantified by BCA protein assay kit (Keygentec), and equal amount of protein was loaded in each lane. Constant voltage electrophoresis was carried out with 10% polyacrylamide gels. Then the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merk, Massachusetts, USA). PVDF membranes were blocked with 3% bovine serum albumin (BSA) (Sigma) and hybridized with anti-FoxP3 antibody (1:1000) (ab450, Abcam, Cambridge, UK), anti-GSK3β antibody (1:1000) (ab93926, Abcam), anti-β-catenin antibody (1:1000) (ab6302, Abcam), and anti-β-actin antibody (1:2000) (ab8226, Abcam) overnight at 4 ˚C. After washed with TBST (Tris-buffered saline and Tween 20) (Keygentec), the PVDF membrane were incubated with secondary antibodies (PV9000, ZSGB-bio, Beijing, China) at room temperature for 60 minutes, followed by washing with TBST. Finally, PVDF membranes were covered with 3, 3-Diaminobenzidine (DAB) (ZLI-9017, ZSGB-bio) for the display of specific protein bands.
Tumor-sphere formation assay
MHCC-LM3 cells from control and co-culture groups were trypsinized into single cells and resuspended in TICs medium consisting of DMEM/F-12 (Invitrogen) supplemented with epidermal growth factor (PHG0311, Gibco), basic fibroblast growth factor (PHG0266, Gibco), insulin (41400045, Gibco), B27 (17504044, Gibco). The cells were seeded at a density of 1 × 104 cells/well in ultra-low attachment 6-well plates. After 21 days of culture with replenishment of one half of the medium every 3 days, tumor spheres were observed and count.
In vivo tumorigenesis assay
All animal experiments were approved by the Institutional Animal Care and Use Committee of Dalian Medical University. Male BALB/c nude mice, 4–6 weeks of age, were used in this study. 5×106 cells harvested from ALG beads before and after co-cultured with Tregs were suspended in 100 μl saline supplement with 50% Matrigel (BD Biosciences) respectively, and then injected subcutaneously into the dorsal flanks of mice. Each experimental group included five mice. Animals were sacrificed after 6 weeks, and tumor volume (cm3) was measured weekly using electronic calipers and calculated with the formula (length × width ×height) × Π/2.
Tregs-derived exosomes isolation and identification
Exosomes were isolated from the culture supernatant of Tregs by using a Total Exosome Isolation Reagent (4478359, Invitrogen), followed the manufacturer’s instructions. The exosome morphology was observed by transmission electron microscopy (TEM) (JEM1230, JEOL, Japan). The particle size and exosome concentration were determined by nanoparticle tracking analysis (NTA) (NanoSight NS300, Malvern, UK). Then characterized by flow cytometry analysis of exosome surface markers CD63 (10606D, Thermo Fisher Scientific) and CD81 (10622D, Thermo Fisher Scientific). Mouse IgG1 was used as an isotype control.
DiO staining of Tregs-derived exosomes
10 μg of exosomes were incubated with lipophilic tracer DiO solution (Thermo Fisher Scientific) for 20 min at 37 °C. Excessive DiO was removed with Exosome Spin Columns (MW 3000) (Thermo Fisher Scientific). DiO-labeled exosomes (5 μg) were added to the culture media of the ALG-MHCC-LM3 cells.
Statistical analysis
All individual experiments were performed at least three times, with three replicates. Data were expressed as means ± standard deviation (SD). The significance of differences between two groups was determined using unpaired Student’s t-tests. Differences were considered significant at P < 0.05.