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 was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Human sample

The use of human subjects was reviewed and approved by the Ethics Committees of Dalian Municipal Central Hospital and 2nd Clinical Medical College of Jinan University, and all work was conducted in accordance with the Declaration of Helsinki (1964). The experiment was conducted with the human subjects’ understanding and consent.

20 mL of peripheral blood was obtained from a 41-year-old male patient with advanced HCC. The serum was placed in a 56 ℃ water bath for 30 min for inactivation. The mononuclear cells were then isolated by gradient centrifugation with lymphocyte separation medium (Lymphoprep, 08751, STEMCELL Technology, Vancouver, BC, Canada).

Treg cell isolation and expansion

Tregs were isolated from peripheral blood mononuclear cells of the HCC patient using the 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/– Treg cells using MidiMACS™ Separator and Starting Kits (130-042-301, Miltenyi Biotec).

Tregs were expanded in X-VIVO™ 15 medium (BE02-060 F, Lonza, Basel, Switzerland) supplemented with 2% heat-inactivated patient serum, 500 U/mL recombinant human IL-2 (T&L Biological Technology, Beijing, China), MACSiBeads pre-loaded with CD3 and CD28 antibodies (130-095-353, Miltenyi Biotec), and 10 ng/mL rapamycin (HY-10,219, MedChemExpress).

HCC cell culture, encapsulation

Human HCC cell line HCC-LM3, purchased from Cellcook (Cellcook Biotech, Guangzhou, China), has recently been authenticated by karyotype analysis. HCC-LM3 cells were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (H-DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco) in a 37 °C incubator with an atmosphere of 5% CO2.

Single cells dissociated from monolayer cultures were counted and suspended in 2.5%, (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.

HCC cells and Tregs co-culture

HCC-LM3 cells formed tumor spheres in ALG beads after 10 days of culture. Then the ALG beads encapsulated HCC-LM3 cells were co-cultured with Tregs for 3 days in H-DMEM supplemented with 10% FBS in a 37 °C incubator with an atmosphere of 5% CO2, the ratio of HCC cells and Tregs were 10:1. Tregs and HCC cells were separated by sedimentation and filtration with 100 μm strainer to remove Tregs (352,360, 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’-ACACGCATGTTTGCCTTCT-3’, and 5’-TGGCAAATGGTGTCTGCAA-3’. A scrambled sequence (5’-TGACGCGATACGTATTGTA-3’) was used as a negative control. Transfection of HCC-LM3 cells were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). DNA-liposome complexes were prepared at 4˚C to a final volume of 1 µg/µl and added to HCC-LM3 cells (1 µg/ml). Transfection was performed for 6 h at 37 ℃.

Flow cytometry

Tregs were labeled with FITC Mouse Anti-Human CD4 (1:5) (561,005, BD Biosciences, Franklin Lakes, NJ, USA), PE Mouse Anti-Human CD25 (1:5) (555,432, BD Biosciences) and Alexa Fluor® 647 Mouse anti-Human FoxP3 (1:20) (561,184, BD Biosciences) antibodies for 30 min on ice, followed by washing with phosphate buffered saline (PBS) (Gibco), FITC Mouse IgG1 (555,748, BD Biosciences), PE Mouse IgG1 (555,749, BD Biosciences), Alexa Fluor® 647 Mouse IgG1 (557,732, BD Biosciences) were used as isotype controls. As for FoxP3 staining, Human FoxP3 Buffer Set (560,098, BD Biosciences) was used. Briefly, Tregs were fixed with Buffer A, incubated for 10 min at room temperature (RT), and permeabilized with buffer C, incubated for 30 min at RT. Flow cytometry was performed by FACSCanto II flow cytometer (BD Biosciences), and the data were analyzed and presented using Flowjo software version 10 (Flowjo, 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 supplementary Table 1. The results were presented as the calculated comparative expression ratios of the target sample to the control group for each sample using the Ct method (2−∆∆ Ct).

Immunofluorescence staining

HCC-LM3 cell spheres were fixed with 4% paraformaldehyde (PFA) and washed with PBS (Gibco) three times. After cytospin preparation, cells were treated with 0.05% Triton-X 100 (Sigma-Aldrich), then incubated with CD133 primary antibody (1:400) (64,326, Cell Signaling Technology, Danvers, MA, USA) in PBS containing 1% goat serum (16,210,064, 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 min at RT. The primary antibody was omitted for negative control. Nuclear staining was performed using Hoechst 33,342 (H3570, Thermal Fisher Scientific). The samples were observed using an inverted fluorescence microscope (DMI8, Leica, Solms, Germany).

Western blot

Cells were lysed in a lysis buffer containing protease and phosphatase inhibitors (Keygentec, Nanjing, China). Protein concentration was quantified by the BCA protein assay kit (Keygentec), and an 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). The PVDF membranes were blocked with 3% bovine serum albumin (BSA) (Sigma) and hybridized with anti-c-Myc antibody (1:100) (sc-373,712, Santa Cruz, Dallas, TX, USA), anti-FoxP3 antibody (1:200) (sc-166,212, Santa Cruz), anti-GSK3β antibody (1:200) (sc-71,186, Santa Cruz), anti-β-catenin antibody (1:200) (sc-7963, Santa Cruz), and anti-β-actin antibody (1:500) (sc-47,778, Santa Cruz) overnight at 4 ˚C. After washing with TBST (Tris-buffered saline and Tween 20) (Keygentec), the PVDF membranes were incubated with secondary antibodies (PV9000, ZSGB-bio, Beijing, China) at room temperature for 60 min, 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.

Sphere formation assay

HCC-LM3 cells from control and co-culture groups were trypsinized into single cells and resuspended in CSCs medium consisting of DMEM/F-12 (Invitrogen) supplemented with epidermal growth factor (PHG0311, Gibco), basic fibroblast growth factor (PHG0266, Gibco), insulin (41,400,045, Gibco), B27 (17,504,044, 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 counted.

In vivo tumorigenesis assay

All animal experiments were approved by the Jinan University Laboratory Animal Ethics Committee. Male BALB/c nude mice, 4–6 weeks of age, were used in this study. 5 × 106 HCC-LM3 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 six 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.

Statistical analysis

All individual in vitro experiments were performed with at least three replicates. Data were expressed as means ± standard deviation (SD). The significance of differences between the two groups was determined using unpaired Student’s t-tests. Differences were considered significant at P < 0.05.