Regulatory T cells enhance stem-like characteristics of hepatocellular carcinoma cells through Wnt/β-catenin pathway

Background: Tumor initiating cells (TICs) were conrmed to drive the therapeutic resistance of hepatocellular carcinoma (HCC), but the mechanism by which tumor microenvironment maintains the HCC stemness is not fully understood. This study aims to investigate the effect of regulatory T cells (Tregs) on the TICs characteristics of HCC. Methods: Immunocytochemistry, ow cytometry, real-time PCR, western blot, in vitro sphere-formation, and in vivo tumorigenesis assay were used to detect HCC TIC characteristics. Additionally, the association between FoxP3 expression, Wnt/β-catenin pathway activation, and HCC stemness were analyzed by forced expression or inhibition of FoxP3. Results: It was showed Tregs enhanced the stemness of HCC cells by upregulation of TIC-related markers CD133, Oct3/4, Sox2, c-Myc, Klf4, Nanog, CD13, EpCAM, and induction of epithelial to mesenchymal transition (EMT), increase of TICs ratio, as well as promotion of tumorigenic ability. Moreover, β-catenin and c-Myc were upregulated in HCC cells when co-cultured with Tregs. After Wnt/β-catenin pathway inhibition, HCC stemness was also inhibited. In addition, Tregs-derived exosomes played the same role as Tregs in enhancing HCC TIC properties, and exosome inhibition led to decreased TIC ratio as well as TIC markers expression. Furthermore, Tregs-derived exosomes down-regulate FoxP3 and GSK3β of HCC cells. Forced expression of FoxP3 resulted in increased GSK3β, decreased β-catenin and TIC ratio of HCC. In contrast, FoxP3 interference reduced GSK3β, increased β-catenin and TIC ratio. Conclusions: This study, for the rst time, demonstrated Tregs enhanced HCC stemness through Wnt//β-catenin pathway by down-regulating FoxP3.

Tregs are a subset of CD4 + CD25 + CD127 − T lymphocytes which constitutively express the transcription factor FoxP3 (forkhead box P3). Activated Tregs inhibit different subsets of immune cells via contactdependent ways between checkpoint molecules and their ligands involving PD-1, PD-L1, CTLA-4, GITR, Tim-3, and galectin-9 4 . Tregs have been shown to increase with tumor stage and correlate with poor prognosis in HCC 5,6 . Nevertheless, the role of Tregs in regulation of HCC cellular behavior, including proliferation, metastasis, especially TICs characteristics, was undetermined. In the current study, we showed that Tregs promoted the stemness of HCC by down-regulation of FoxP3, and activation of Wnt/βcatenin pathway.

Materials
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise speci ed.
Sodium alginate (Qingdao Jingyan Bio-Tech, Qingdao, China) was puri ed 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 + CD127 dim/-Regulatory T Cell Isolation Kit (130-094-775, Miltenyi Biotec, Bergisch Gladbach, Germany). Brie y, the isolation of CD4 + CD25 + CD127 dim/regulatory T cells was performed with a cocktail of biotinylated antibodies and anti-Biotin microbeads for the depletion of non-CD4 + and CD127 high cells. Then the ow-through fraction of pre-enriched CD4 + CD127 dim/-T cells is labeled with CD25 microbeads for subsequent positive selection of CD4 + CD25 + CD127 dim/regulatory T cells with MidiMACS™ Starting Kit (Miltenyi Biotec).
Tregs were cultured in X-VIVO TM  Single cells dissociated from monolayer cultures were counted and suspended in 1.5%, (w/v) sodium alginate at a cell density of 1×10 6 /ml. The cell suspension was extruded into 100 mM CaCl 2 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 ltration 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.
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. 5×10 6 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 anks of mice. Each experimental group included ve mice. Animals were sacri ced after 6 weeks, and tumor volume (cm 3 ) was measured weekly using electronic calipers and calculated with the formula (length × width ×height) × Π/2.

Tregs-derived exosomes isolation and identi cation
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

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 signi cant at P < 0.05.
Tumor sphere formation and tumorigenesis assay were performed to further con rm the enhancement of HCC stemness. After co-cultured with Tregs, HCC cells formed more compact spheres (Figure 2a) as well as larger tumors in nude mice (P=0.0386) (Figure 2b-c).The above results veri ed that Tregs enhanced the stemness of HCC cells.

Tregs enhanced HCC stemness through exosomes
Exosomes isolated from the conditioned medium of Tregs had typical morphology and size (Figure 4a), which were veri ed to express the exosomal markers CD63 and CD81 (Figure 4b).
After co-cultured with Tregs-derived exosomes (stained with Dio) (Figure 4c Lower expression of FoxP3 promoted Wnt/β-catenin pathway to enhance HCC stemness It was found that FoxP3 was lower expressed in HCC tissue compared to that in paratumor tissue, in contrast, CD133 level was higher in tumor tissue (Fig. S1). After co-cultured with Tregs-derived exosomes, FoxP3 and GSK3β were both signi cantly down-regulated in HCC cells (Fig. S2). Furthermore, forced expression of FoxP3 (P=0.0209) led to higher expression GSK3β while lower expression of β-catenin in HCC cells (Figure 5a-b). In addition, CD133 + TICs ratio decreased from 3.863 ± 1.730 to 1.145 ± 0.423 (P=0.0421) after forced expression of FoxP3 (Figure 5c-d).

Discussion
Accumulating evidence indicates that HCC therapeutic resistance and recurrence are closely associated with cancer stem cells (CSCs), or TICs 7, 8 . Tregs function as dominant inhibitory components in the immune microenvironment of HCC, which are undisputed to be associated with the invasiveness of HCC, and are a promising independent predictor of recurrence and survival in HCC patients 9,10 .
A few studies have reported the ability of Tregs to drive the tumor cells to be TICs. Yang 20 . In this study, we found FoxP3 expression in tumor tissue was signi cantly lower than that in paratumor tissue, while CD133 was signi cantly higher in tumor tissue compared to paratumor tissue. In addition, forced expression of FoxP3 led to signi cantly lower number of HCC TICs, and in contrast, FoxP3 inhibition signi cantly increased the HCC TICs. These results were in accordance with Liu et al., which showed FoxP3 was signi cantly down-regulated in cancer stem cell-like cells of colorectal cancer, and forced expression of FoxP3 signi cantly decreased self-renewal ability of cancer stem cells including the side population, cancer stem cell marker CD133, colonosphere formation ability in vitro, and tumor formation ability in vivo 21 .
Abnormal initiation of Wnt/β-catenin pathway has been recognized in HCC TICs 22 . After co-cultured with Tregs, GSK3β was signi cantly down-regulated and β-catenin and c-Myc were signi cantly upregulated in HCC cells. Moreover, after Wnt/β-catenin pathway was inhibited, TIC related genes as well as TICs ratio were signi cantly decreased.
Exosomes are secreted extracellular membrane vesicles, serve as vehicles for transfer cytosolic proteins, lipids, and nucleotides including non-coding RNAs between cells 23 . It was reported Tregs-derived exosomes could suppress immune cell proliferation and cytokine secretion to assist Tregs-mediated immune suppression 24 . Nonetheless, the role Tregs-derived exosomes play in regulating tumor cell behavior remains poorly understood. This study, for the rst time, showed Tregs-derived exosomes signi cantly up-regulated TICs related genes, and increased CD133 + TICs ratio in HCC cells. Furthermore, lower FoxP3, GSK3β and higher β-catenin, c-Myc were found in HCC cells after co-cultured with Tregsderived exosomes. And which were reversible by inhibition of exosomes release.
Though the experimental design of this study was formulated and executed after thorough consideration and literature search, there exist several limitations that need to be mentioned. First of all, weather GSK3β was the direct target of FoxP3 was unrevealed. Secondly, the correlation of FoxP3 expression with the metastasis, relapse, and overall survival of HCC patients should be investigated to further con rm the tumor suppressor role of FoxP3 in HCC. Thirdly, the detailed regulatory mechanism by which Tregsderived exosomes enhance HCC cell stemness was unde ned. LncRNAs (long non-coding RNAs) or miRNAs or other components in Tregs-derived exosomes responsible for affecting HCC stemness should be identi ed in further studies.

Conclusions
In summary, this study reported Tregs, through their exosomes, suppressed FoxP3, GSK3β and activated β-catenin to enhance the stemness of HCC. It was the rst study to show that Tregs regulated HCC TIC characteristics.