Regulatory T cells inhibit FoxP3 to increase the population of tumor initiating cells in hepatocellular carcinoma

Purpose Tumor initiating cells (TICs) or cancer stem cells (CSCs) are considered to be the main culprit of hepatocellular carcinoma (HCC) initiation and progression, nevertheless 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, flow cytometry, real-time PCR, western blot, in vitro sphere-formation, and in vivo tumorigenesis assay were used to detect HCC ‘stemness’. Additionally, after forced expression or inhibition of FoxP3, β-catenin expression and HCC ‘stemness’ were investigated. Results Tregs enhanced the ‘stemness’ of HCC cells by upregulating TIC-related markers CD133, Oct3/4, Sox2, c-Myc, Klf4, Nanog, CD13, EpCAM, and inducting epithelial to mesenchymal transition (EMT), increasing TICs ratio, as well as promoting tumorigenic ability. Moreover, β-catenin and c-Myc were upregulated in HCC cells after co-cultured with Tregs. HCC ‘stemness’ was inhibited after treatment with Wnt/β-catenin pathway inhibitor. Furthermore, forced expression of FoxP3 resulted in increased GSK3β, decreased β-catenin and TIC ratio in HCC. In contrast, FoxP3 interference reduced GSK3β, enhanced β-catenin and TIC ratio of HCC. Conclusion This study, for the first time, demonstrated that Tregs increased the population of TICs in HCC by inhibiting FoxP3 as well as promoting β-catenin expression. Supplementary Information The online version contains supplementary material available at 10.1007/s00432-024-05892-2.


Introduction
HCC is the most prevalent primary liver cancer, which ranks as the sixth most common cancer and the third most leading cause of cancer-related death worldwide.A higher incidence rate of hepatitis B virus-related HCC occurs in China, and over 50% of HCC-related deaths are in China (Sung et al. 2021).Late diagnosis, frequent relapse, and the refractory nature to chemotherapy render HCC an intractable disease.
Due to the inherently high genetic instability, a small population within the HCC has evolved with the ability of to initiate and maintain cancer growth.Rapidly growing evidence has demonstrated that some HCCs, if not all, are been reported to be one of the most active signaling pathways indispensable to self-renewal and drug resistance of HCC TICs (Kang et al. 2019).
It was found that Foxp3 + Treg cells were capable of inducing colorectal cancer cells to become cancer-initiating cells (Yang et al. 2011).Additionally, Tregs could induce the expression of core cancer stem cell-related genes and spheres formation adbility, resulting in increased cancer stemness and tumorigenic potential of glioma cancer cell (Liu et al. 2021).Nevertheless, the role of Tregs, which reside in the HCC tumor environment, in the regulation of HCC cellular behavior, including proliferation, metastasis, and especially TICs characteristics, was undetermined.In this study, it was found for the first time that Tregs enhanced the 'stemness' of HCC by inhibiting FoxP3 and up-regulating β-catenin.

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-yearold 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 + CD127 dim/− Regulatory T Cell Isolation Kit Miltenyi Biotec,Bergisch Gladbach,Germany).Briefly, 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 flow-through fraction of pre-enriched CD4 + CD127 dim/-T cells is labeled with CD25 microbeads for subsequent positive selection of CD4 + CD25 + CD127d im/- Treg cells using MidiMACS™ Separator and Starting Kits Miltenyi Biotec).
Single cells dissociated from monolayer cultures were counted and suspended in 2.5%, (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.

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% CO 2 , 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).Shorthairpin small interfering RNA sequences were 5' and 5 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 ℃.

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 Prime-Script 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 Light-Cycler ® 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 ).

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 × 10 4 cells/well in ultra-low In vitro tumor sphere formation assay and in vivo tumorigenesis assay were performed to further confirm the enhancement of HCC cell 'stemness'.After co-cultured with Tregs, HCC-LM3 cells formed more compact spheres (Fig. 2a) and also larger tumors in nude mice (Fig. 2b-c).The above-mentioned results verified that Tregs enhanced the 'stemness' of HCC cells.

Tregs inhibited FoxP3 of HCC cells to increase the TIC population
It was found that FoxP3 was lower expressed in HCC tissue compared to that in para-tumor tissue, in contrast, CD133 level was higher in tumor tissue (HLivH180Su10, Xinchao Biotech, Shanghai, China) (supplementary Fig. 1).After cocultured with Tregs, FoxP3, and GSK3β were both significantly down-regulated in HCC-LM3 cells (Fig. 4).

Discussion
Accumulating evidence indicates that therapeutic resistance and recurrence of HCC are closely associated with CSCs or TICs (Chen et al. 2015;Ishiguro et al. 2020).Nevertheless, how HCC CSCs or TICs characteristics were maintained by tumor microenvironment remains unclear.
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 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 × 10 6 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 (cm 3 ) 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.
FoxP3 was initially identified as a "switch" for the development and function of Tregs and thought to be restricted to hematopoietic tissues.Recently, reports have demonstrated that FoxP3 was also expressed in tumor cells, suggesting that FoxP3 might have a broader role in cancers.However, the biological function and clinical relevance of FoxP3 in tumor cells remain controversial.Some studies found that FoxP3 levels elevated in several tumor cell types, and indicated tumor progression (Grimmig et al. 2013;Merlo et al. 2009;Zeng et al. 2013).While others reported FoxP3 was a cancer suppressor promising independent predictor of recurrence and survival in HCC patients (Hassan et al. 2019;Liu et al. 2019).A few studies have reported the ability of Tregs to drive tumor cells to become TICs.Yang et al. found Foxp3 + IL-17 + Tregs induced colorectal cancer cells to up-regulated TICs-related markers including CD133, CD44s, CD166, EpCAM, and ALDH1 (Yang et al. 2011).Xu et al. disclosed that Tregs upregulated the 'stemness' property of breast cancer cells by increasing the side-population, promoting tumor sphere formation, and enhancing the expression of 'stemness'-related genes including Sox2, Nanog, Oct3/4 (Xu et al. 2017).
In this study, for the first time, we found that Tregs enhanced the 'stemness' of HCC cells, demonstrated by increased TICs ratio, upregulated expression of TICs-related ratio before and after co-culture, student's t-test, * P < 0.05, compared with the control group.(f) RT-qPCR analysis of HCC TICs-related genes and (g) EMT-related genes, n = 3, student's t-test, * P < 0.05, compared with the control group.(h) Western blot analysis of β-catenin and c-Myc in HCC cells before and after co-culture HCC TICs, and in contrast, FoxP3 inhibition significantly increased HCC TICs.These results were in accordance with Liu et al., which showed FoxP3 was significantly down-regulated in cancer stem cell-like cells of colorectal cancer, and forced expression of FoxP3 significantly decreased self-renewal ability of cancer stem cells including reduced side population, cancer stem cell marker CD133 expression, colonosphere formation ability in vitro, as well as tumor formation ability in vivo (Liu et al. 2017).
Abnormal initiation of the Wnt/β-catenin pathway has been recognized in HCC TICs (Guo et al. 2019).APC, Axin, CKIα, and GSK3β formed the "β-catenin destruction complex", which connects to β catenin molecules, phosphorylates it, and promote ubiquitylation and degradation.GSK3β functions as a switch in regulating β-catenin stability (Wu and Pan 2010).Cytoplasmic gene in breast cancer (Zou et al. 2007a(Zou et al. , 2007b)), prostate cancer (Wang et al. 2009), gastric cancer (Ma et al. 2013), as well as HCC (Shi et al. 2017).Shi et al. found that higher expression of FoxP3 significantly correlated with early TNM stage, better survival, and reduced recurrence.Additionally, they demonstrated FoxP3 suppressed the proliferation and invasion of HCC cells in vitro and reduced tumor growth in vivo (Shi et al. 2017).Liu et al. reported that FoxP3 underexpression was closely related to a decreased overall survival (OS), and low FoxP3 expression was an independent risk factor for predicting OS prognosis of HCC patients (Liu et al. 2023).In this study, we found FoxP3 expression in tumor tissue was significantly lower than that in para-tumor tissue, while CD133 was significantly higher in tumor tissue compared to para-tumor tissue.Furthermore, forced expression of FoxP3 led to the significantly lower number of This study, for the first time, showed Tregs enhanced the 'stemness' of HCC cells, nevertheless, there are some limitations.Firstly, Tregs in this study were isolated from peripheral blood, but the use of intratumoral Tregs might be closer to the real tumor microenvironment.It was demonstrated a great heterogeneity in Tregs.Tumorinfiltrating Tregs and Tregs from peripheral blood have different gene signatures.Tregs from peripheral blood levels of β catenin are tightly controlled by GSK3β and the degradation of β catenin in cytoplasm could inhibit the Wnt pathway.Once GSK3β is suppressed, β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it binds to the LEF/TCF complex and activates the downstream genes such as CD44, EpCAM, c-Myc, cyclin D1, among others (Vilchez et al. 2016).We found that after co-culturing with Tregs, GSK3β was significantly down-regulated and β-catenin as well as c-Myc was significantly upregulated in HCC cells.Moreover,

Conclusions
In summary, this study revealed Tregs increased the HCC TIC population through the suppression of FoxP3 and GSK3β and the upregulation of β-catenin.It was the first study to show that Tregs in liver tumor microenvironment regulated HCC TIC characteristics.
showed little clonal enrichment, while much more tumorinfiltrating Tregs were clonally enriched (Zheng et al. 2017).Secondly, whether GSK3β was the direct target of FoxP3 was unrevealed in this study.Thirdly, the correlation of FoxP3 expression with the metastasis, relapse, and overall survival of HCC patients should be investigated to further confirm the tumor suppressor role of FoxP3 in HCC.Fourthly, we have evaluated if the Treg-derived exosomes played the same role as Treg cells in increasing HCC TICs (data not shown), lncRNAs (long non-coding RNAs) or miRNAs or other components in Tregs-derived exosomes responsible for increasing HCC TIC population

Fig. 2
Fig. 2 Tregs enhanced the in vitro sphere formation and in vivo tumorigenic ability of HCC-LM3 cells.(a) Sphere formation assay of HCC-LM3 cells before and after co-cultured with Tregs, n = 3, bar: 200 μm.(b) In vivo tumorigenicity of HCC cells before and after co-cultured