Published online Jul 26, 2026. doi: 10.4252/wjsc.119892
Revised: February 27, 2026
Accepted: April 3, 2026
Published online: July 26, 2026
Processing time: 165 Days and 9.1 Hours
Regulatory T (Treg) cells have been demonstrated to be associated with poor prognosis in patients with hepatocellular carcinoma (HCC). In our previous research, we showed that Treg cells increase the number of cancer stem cells (CSCs) in HCC by inhibiting forkhead box P3 (FOXP3) and upregulating β-catenin expression.
To investigate whether the long non-coding RNA (lncRNA) Flicr, present in Treg-derived exosomes, promotes the stem-like properties of HCC.
GW4869 was applied during the co-culture of HCC cells with Treg cells or Treg cell-conditioned medium. LncRNA Flicr was manipulated to investigate whether FLICR suppresses FOXP3 and activates β-catenin, thereby promoting an increase in CSCs in HCC. The stem cell-like properties of HCC were evaluated using reverse transcription-quantitative polymerase chain reaction, immunofluorescence staining, fluorescence in situ hybridization, flow cytometry, tumor-sphere formation assays, and in vivo tumorigenicity experiments. Western blot analysis was performed to measure the protein levels of FOXP3, glycogen synthase kinase-3 beta, and β-catenin.
After co-culturing with Treg cells or their conditioned medium, HCC cells ex
These findings demonstrated that lncRNA Flicr was present in Treg-derived exosomes, leading to the inhibition of FOXP3, activation of β-catenin, and the subsequent promotion of stem-like characteristics in HCC cells.
Core Tip: In our previous study, we demonstrated that regulatory T (Treg) cells increase the population of tumor-initiating cells in hepatocellular carcinoma by suppressing forkhead box P3 and enhancing β-catenin expression. However, it remained unclear whether Treg cells exert these effects through exosomes and which specific components within Treg-derived exosomes are responsible. In this study, we revealed that exosomal long non-coding RNA Flicr, derived from Treg cells, suppresses forkhead box P3 and glycogen synthase kinase-3 beta expression and activates the Wnt/β-catenin signaling pathway, thereby promoting stem cell-like properties in hepatocellular carcinoma cells.
- Citation: Tu YJ, Liu YQ, Cai HY, Pan YY, Liu C. Regulatory T cell-derived exosomes regulate β-catenin expression to promote the stem cell-like properties of hepatocellular carcinoma. World J Stem Cells 2026; 18(7): 119892
- URL: https://www.wjgnet.com/1948-0210/full/v18/i7/119892.htm
- DOI: https://dx.doi.org/10.4252/wjsc.119892
Liver cancer, primarily hepatocellular carcinoma (HCC), which accounts for 75% to 85% of cases, ranks as the third leading cause of cancer-related deaths and is the sixth most commonly diagnosed cancer worldwide[1]. Although recent advancements in HCC treatment have been promising, the five-year overall survival rate remains low. The disease’s heterogeneity, tendency to recur, and resistance to therapy make HCC a significant public health challenge[2]. Strong evidence indicates that tumor heterogeneity is driven by a subset of cells with stem or progenitor-like characteristics, known as cancer stem cells (CSCs) or tumor-initiating cells[3]. These CSCs possess unique stem cell-like abilities to self-renew and differentiate, thereby regenerating all tumor properties[4]. The presence of CSCs in HCC leads to inevitable tumor recurrence after initially successful chemotherapy and/or radiotherapy and also contributes to tumor dormancy and treatment resistance[2,5].
Regulatory T (Treg) cells are a subset of CD4+CD127- T lymphocytes that consistently express the transcription factor forkhead box P3 (FOXP3) and the α chain of the interleukin-2 (IL-2) receptor, known as CD25[6]. Upon activation, Treg cells suppress various immune cells through direct contact and contribute to the formation of a strongly immunosuppressive tumor microenvironment by releasing inhibitory cytokines such as transforming growth factor-beta, IL-10, and IL-35[7]. Treg cells have been associated with poor outcomes in patients with HCC[8,9]. Our previous research de
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States), unless otherwise specified. GW4869 (HY-19363) was obtained from MedChemExpress (Monmouth Junction, NJ, United States).
Treg cells were isolated from the peripheral blood of a patient with HCC at Shenzhen People’s Hospital. The study was approved by the Ethics Committee of Shenzhen People’s Hospital, and written informed consent was obtained from the patient.
Treg cells were expanded in X-VIVO™ 15 medium (BE02-060F, Lonza, Basel, Switzerland) supplemented with 2% heat-inactivated patient serum, 500 U/mL recombinant human IL-2 (T&L Biological Technology, Beijing, China), MACSiBeads preloaded with CD3 and CD28 antibodies (130-095-353, Miltenyi Biotec, Bergisch Gladbach, Germany), and 10 ng/mL rapamycin (HY-10219, MedChemExpress, Monmouth Junction, NJ, United States). After 14 days of expansion, the CD3/CD28 MACSiBead particles were removed, and the conditioned medium was collected following an additional 3 days of continuous culture. The GW4869 was applied after the condition medium had been collected.
The human HCC cell line HCC-LM3 (RRID: CVCL_6832), obtained from Cellcook Biotech (Guangzhou, Guangdong Province, China), was cultured in high glucose Dulbecco’s Modified Eagle’s Medium (H-DMEM, Gibco, Carlsbad, CA, United States) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, United States) at 37 °C in an incubator with a 5% CO2 atmosphere. Single cells dissociated from monolayer cultures were counted and suspended in 2.5% (w/v) sodium alginate (ALG) at a concentration of 1 × 106/mL. The cell suspension was then extruded into a 100 mmol/L calcium chloride (CaCl2) solution. The gelation process to form calcium ALG beads took 30 minutes.
After 10 days of culture, HCC-LM3 cells formed tumor spheres within the ALG beads. These ALG-encapsulated HCC-LM3 cells were then co-cultured for 3 days with either Treg cells, Treg cell-conditioned medium, or exosomes derived from Treg cells, in H-DMEM medium supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 incubator. 10 μΜ GW4869 was added either during the co-culture of HCC cells with Treg cells or during the co-culture of HCC cells with Treg cell-conditioned medium. To retrieve the encapsulated HCC cells from the ALG beads, they were treated with 55 mmol/L sodium citrate before being used in subsequent experiments.
Treg cells were isolated using the procedure described in our previous study[10]. These cells were cultured in an exosome-free medium (C0922, Beyotime, Beijing, China) for 24 hours prior to exosome extraction. The conditioned medium was collected, and exosomes were isolated according to the manufacturer’s instructions with the BeyoExo™ Enhanced Exosome Isolation Kit (C3622M, Beyotime, Beijing, China). Specifically, 1.9 mL of the isolation reagent was added to every 10 mL of conditioned medium, mixed thoroughly, and the mixture was incubated overnight at 4 °C. Subsequently, the medium was centrifuged at 10000 × g for 30 minutes at 4 °C to pellet the exosomes. The exosome pellet was then resuspended in 100 μL of saline and stored at -20 °C.
To assess the protein content of the exosomes, an equal volume of lysis buffer for exosome (C3632, Beyotime, Beijing, China) was added to the exosome suspension, gently mixed, and incubated on ice for 10 minutes to ensure complete lysis. The lysate was then centrifuged at 10000 × g for 5 minutes at 4 °C, and the supernatant was collected. The protein concentration of the exosomes was measured using the BCA Protein Assay Kit (P0010S, Beyotime, Beijing, China).
Treg-derived exosomes were diluted with phosphate-buffered saline (PBS) to reach a total volume of 1 mL, and their size was determined using the Zetasizer Nano ZS device (Malvern Panalytical, United Kingdom).
The isolated exosomes were fixed with 2% paraformaldehyde in 0.1 mol/L of PBS. After 15 minutes, 5 μL of the exosome samples were placed onto carbon-coated Cu/Rh grids (Merck, Germany) and left at room temperature for 1 minute. Excess liquid was then blotted with filter paper and replaced three times with 5 μL drops of 1% uranyl acetate in double-distilled water. The stained grids were subsequently observed using a JEOL TEM-JEM1400 microscope (JEOL Ltd., Japan).
The expression of CD63 and CD81 on Treg-derived exosomes was examined using an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, United States). Briefly, the Treg-derived exosomes were diluted in PBS to a final volume of 100 μL and incubated with FITC-conjugated Mouse Anti-Human CD63 (1:5 dilution, 557288, BD Biosciences, Franklin Lakes, NJ, United States) and FITC-conjugated Mouse Anti-Human CD81 (1:5 dilution, 551108, BD Biosciences, Franklin Lakes, NJ, United States) antibodies for 30 minutes on ice, followed by washing with PBS. For CD133 staining, 100000 HCC-LM3 cells were incubated with Alexa 488-conjugated CD133 antibody (1:50 dilution, 53-1331-80, Thermo Fisher Scientific, Pittsburgh, PA, United States) for 30 minutes on ice, then washed with PBS. Flow cytometry was conducted using a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, United States), and the data were analyzed and presented using FlowJo software version 10 (FlowJo, Ashland, OR, United States).
The plasmids used to construct FLICR vectors were derived from pcDNA3.1-GreenZeo (Genechem, Shanghai, China). An antisense oligonucleotide (ASO) (RIBOBIO, Guangzhou, Guangdong Province, China) with the targeting sequence 5’-GAGTGGTTGTTGGACGACTA-3’ was employed to inhibit lncRNA Flicr, while a lncRNA ASO negative control (lnc6N0000002-1-10, RIBOBIO, Guangzhou, Guangdong Province, China) was used as the negative control. Transfection of Treg cells with plasmids was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States). DNA-liposome complexes were prepared at 4 °C to a final concentration of 1 μg/μL and then added to Treg cells at 1 μg/mL. The transfection process lasted for 6 hours at 37 °C. For ASO transfection, the riboFECT CP Transfection Kit (C10511-05, RIBOBIO, Guangzhou, Guangdong Province, China) was used, with both ASO and ASO negative control at a concentration of 50 nM. This transfection was conducted for 24 hours at 37 °C.
Quantitative reverse transcription polymerase chain reaction (RT-qPCR, two-step method) was employed to assess the relative expression levels of genes, using GAPDH or β-actin as internal reference controls. Total RNA was extracted using TRIzol® reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s protocol. Reverse transcription (RT) was conducted using the PrimeScript RT Reagent Kit (RR036A, Takara, Shiga, Japan). Real-time PCR amplification was carried out with SYBR Premix Ex Taq (Perfect Real Time, RR820A, Takara, Japan). Both PCR amplification and fluorescence detection were carried out on a 7500 Real-Time PCR System (Thermo Fisher Scientific, Pittsburgh, PA, United States). The primers utilized in this study are detailed in Supplementary Table 1. Data were expressed as com
HCC-LM3 cells from different experimental groups were trypsinized into single-cell suspensions and resuspended in CSC medium composed of DMEM/F-12 (Thermo Fisher Scientific, Pittsburgh, PA, United States) supplemented with epidermal growth factor (PHG0311, Gibco, Carlsbad, CA, United States), basic fibroblast growth factor (PHG0266, Gibco, Carlsbad, CA, United States), insulin (41400045, Gibco, Carlsbad, CA, United States), and B27 supplement (17504044, Gibco, Carlsbad, CA, United States). The cells were plated at a density of 1 × 104 cells per well in ultra-low attachment 6-well plates. After 21 days of culture, with half of the medium replaced every 3 days, tumor spheres were observed and counted.
All animal experiments were approved by the Laboratory Animal Ethics Committee of Central Hospital of Dalian University of Technology. Seventy-five BALB/c nude mice, aged 4 weeks to 6 weeks, including 35 females and 40 males, were obtained from Beijing Vital River Laboratory Animal Technology Company. The animal protocol was designed to minimize pain or discomfort to the animals. The mice were acclimated to laboratory conditions (23 °C, 12 hours/12 hours light/dark cycle, 50% humidity, ad libitum access to food and water), for two weeks prior to experimentation. The mice were randomly assigned to fifteen experimental groups, each consisting of five animals. In five of these groups, there were three female mice and two male mice, and in the remaining ten groups, there were two female mice and three male mice. A total of 2 × 106 HCC-LM3 cells from different experimental groups were suspended in 80 μL of saline supplemented with 20 μL of Matrigel (BD Biosciences, Franklin Lakes, NJ, United States) and then injected subcutaneously into the dorsal flanks of the mice. Tumor volume (in cm3) was measured weekly using electronic calipers and calculated using the formula: (length × width × width) × π/6. After 4 weeks or 5 weeks, the animals were euthanized by intravenous administration of pentobarbital sodium at a dose of 150 mg/kg.
HCC-LM3 cell spheres were fixed with 4% paraformaldehyde and rinsed three times with PBS. Following cytospin preparation, the cells were incubated overnight at 4 °C with an Alexa 488-conjugated CD133 antibody (1:500 dilution, 53-1331-80, Thermo Fisher Scientific, Pittsburgh, PA, United States) diluted in PBS containing 1% goat serum (16210064, Thermo Fisher Scientific, Pittsburgh, PA, United States). Following incubation, the cells were washed three times with PBS. Nuclear staining was performed using DAPI (C1006, Beyotime, Beijing, China). The samples were then examined using a laser scanning confocal microscope (STELLARIS 5, Leica, Germany).
Fluorescence in situ hybridization (FISH) was carried out using the RiboTM Fluorescent in Situ Hybridization Kit (C10910, RIBOBIO, Guangzhou, Guangdong Province, China). In brief, HCC-LM3 cells were fixed with 4% paraformaldehyde and then rinsed with PBS. The cells were permeabilized by adding 1 mL of pre-chilled permeabilization solution and incubated at 4 °C for 5 minutes. Subsequently, the cells were washed three times with PBS for 5 minutes. Next,
Cells were lysed in a lysis buffer containing protease and phosphatase inhibitors (Keygentec, Nanjing, Jiangsu Province, China). Protein concentrations were measured with the BCA protein assay kit (Keygentec, Nanjing, Jiangsu Province, China), and equal amounts of protein were loaded into each lane. Electrophoresis was performed at a constant voltage on 10% polyacrylamide gels. Proteins were then transferred onto polyvinylidene fluoride membranes (Merck, Germany). The membranes were blocked with 3% bovine serum albumin and incubated overnight at 4 °C with the following primary antibodies: Anti-FOXP3 (1:500 dilution, 22228-1-AP, Proteintech, Wuhan, Hubei Province, China), anti-glycogen synthase kinase-3 beta (anti-GSK3β, 1:1000 dilution, 22104-1-AP, Proteintech, Wuhan, Hubei Province, China), anti-β-catenin (1:500 dilution, 51067-2-AP, Proteintech, Wuhan, Hubei Province, China), and anti-GAPDH (1:10000 dilution, 60004-1, Proteintech, Wuhan, Hubei Province, China). After washing with Tris-buffered saline with Tween 20 (Keygentec, Nanjing, Jiangsu Province, China), the membranes were incubated at room temperature for 90 minutes with either HRP-conjugated goat anti-rabbit IgG (H + L) antibody (1:5000 dilution, SA00001-2, Proteintech, Wuhan, Hubei Province, China) or HRP-conjugated goat anti-mouse IgG (H + L) antibody (1:10000 dilution, SA00001-1, Proteintech, Wuhan, Hubei Province, China). Following additional washes with Tris-buffered saline with Tween 20, protein bands were detected using an enhanced chemiluminescent detection kit (Seven Biotech, Beijing, China).
All experiments were conducted at least three times. Data are presented as means ± SD. Differences between two groups were assessed using unpaired Student’s t-tests. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was applied. A P < 0.05 was considered statistically significant.
When HCC cells were co-cultured with Treg cells, there was a significant increase in the expression of stem cell-related genes octamer-binding transcription factor 4 (OCT4), NANOG, CD133, sex-determining region Y-box 2 (SOX2), myelocytomatosis oncogene (MYC), and Kruppel-like factor 4 (KLF4), showing fold increases of 1.59 ± 0.17 (P < 0.001), 2.45 ± 0.25 (P < 0.001), 4.15 ± 1.38 (P < 0.001), 2.18 ± 0.43 (P < 0.001), 3.23 ± 0.82 (P < 0.001), and 2.65 ± 0.80 (P < 0.001) respectively, compared to HCC cells cultured alone (control group) (Figure 1A). A similar pattern was observed when HCC cells were cultured with the conditioned medium from Treg cells, with gene expression increases of 1.72 ± 0.22 (P < 0.001), 2.52 ± 0.35 (P < 0.001), 3.87 ± 0.67 (P < 0.001), 2.33 ± 0.51 (P < 0.001), 2.99 ± 0.32 (P < 0.001), and 2.29 ± 0.48 (P < 0.001) relative to control (Figure 1A). The addition of GW4869-a known inhibitor of exosome biogenesis-during co-culture with Treg cells markedly attenuated the upregulation of these genes, reducing their expression levels to 1.08 ± 0.13 (P < 0.001), 1.49 ± 0.23 (P < 0.001), 1.57 ± 0.28 (P < 0.001), 1.29 ± 0.25 (P < 0.001), 1.61 ± 0.20 (P < 0.001), and 1.50 ± 0.20-fold (P < 0.001), respectively (Figure 1A). However, GW4869 did not decrease these gene expressions when HCC cells were cultured with the conditioned medium from Treg cells (Figure 1A). Consistent with the gene expression data, immunofluorescence staining (Figure 1B) and flow cytometry analysis (Figure 1C) showed that the proportion of CD133-positive CSCs in HCC cells increased after co-culture with Treg cells or their conditioned medium. GW4869 reduced the number of CSCs when HCC cells were co-cultured with Treg cells but had no effect when co-cultured with Treg-derived conditioned medium (Figure 1B and C). Sphere-formation assays further confirmed that co-culture with Treg cells or their conditioned medium enhanced the sphere-forming capacity of HCC cells, resulting in larger and more compact spheres (Figure 1D). GW4869 inhibited the sphere-formation ability when HCC cells were co-cultured with Treg cells but not with the conditioned medium (Figure 1D), suggesting that direct cell-cell contact or exosome secretion is required for this effect. In vivo tumorigenesis assays in nude mice demonstrated that co-culture with Treg cells or their conditioned medium increased the tumorigenic potential of HCC cells, leading to larger tumors (Figure 1E). GW4869 suppressed the oncogenic ability of HCC cells co-cultured with Treg cells but had no effect when cells were cultured with the conditioned medium (Figure 1E). Additionally, Treg cells and their conditioned medium decreased the levels of FOXP3 and GSK3β and increased β-catenin expression in HCC cells (Figure 1F). The presence of GW4869 during co-culture with Treg cells reversed these changes. Overall, these findings indicate that Treg cells and their conditioned medium promote CSC characteristics in HCC cells, suggesting that exosomes derived from Treg cells play a key role in this process.
Exosomes were isolated from the conditioned medium of Treg cells. Their size distribution was analyzed (Figure 2A), and the characteristic bilayer lipid membrane structure was confirmed by transmission electron microscopy (Figure 2B). Flow cytometry analysis demonstrated that the exosomal markers CD63 and CD81 were highly expressed on Treg-derived exosomes, with positive rates of 74.4% and 88.2%, respectively (Figure 2C).
To determine whether exosomes present in the conditioned medium from Treg cells were primarily responsible for enhancing CSC-like properties in HCC cells, varying concentrations of Treg-derived exosomes were added to HCC cell cultures. Flow cytometry analysis revealed a dose-dependent increase in the proportion of CD133+ CSCs following exosome treatment (Figure 3A and B). Consistently, sphere-formation assays showed that HCC cells exposed to higher doses of Treg-derived exosomes formed larger and more numerous tumor spheres (Figure 3C). In vivo tumorigenicity was also enhanced, with xenograft models exhibiting increased tumor growth (Figure 3D). Moreover, escalating doses of Treg-derived exosomes led to upregulation of β-catenin and downregulation of FOXP3 and GSK3β in HCC cells (Figure 3E). These results suggested that Treg-derived exosomes played a pivotal role in promoting CSC-like properties in HCC cells, likely through activation of the Wnt/β-catenin signaling pathway.
After co-culture with Treg-derived exosomes, the expression of lncRNA Flicr in HCC cells was significantly upregulated, showing a 6.37 ± 3.52-fold increase (P < 0.001) compared to the control (Figure 4A). Furthermore, FISH analysis revealed that lncRNA Flicr was undetectable in HCC cells, but became clearly detectable following exposure to Treg-derived exosomes (Figure 4B), indicating that lncRNA Flicr was contained within Treg-derive exosomes and may be transferred to HCC cells. Notably, a portion of lncRNA Flicr was observed to localize in the CD133+ HCC cells (Figure 4B).
To investigate whether lncRNA Flicr transferred from Treg cells contributes to the expansion of the CSC population in HCC, we manipulated FLICR expression in Treg cells by either overexpressing it using a FLICR-expressing vector or suppressing it using FLICR-ASO. Introducing the FLICR vector resulted in a significant 6.53 ± 2.13-fold increase in lncRNA Flicr expression in Treg cells compared to control (P < 0.001) and empty vector (P < 0.001) groups (Supple
In contrast, FLICR interference in Treg cells using ASO efficiently suppressed lncRNA Flicr expression (Supple
Several biomarkers of CSC subsets in HCC have now been extensively identified, with CD133 recognized as one of the most well-characterized functional and phenotypic markers[11,12]. Transcription factors such as NANOG, OCT4, SOX2, KLF4, and MYC also play pivotal roles in maintaining the self-renewal capacity of CSCs[13,14]. In HCC CSCs, one or more highly conserved stemness-related signaling pathways-particularly the Wnt/β-catenin pathway-are aberrantly overactivated, contributing to the maintenance of stem-like properties in HCC cells[12,15]. In this study, we found that the expression of CSC-related genes, including NANOG, OCT4, SOX2, MYC, and KLF4, increased alongside with β-catenin activation, accompanied by an increase in the proportion of CD133+ HCC CSCs.
In our previous study, we demonstrated that Treg cells suppressed FOXP3 expression and upregulated β-catenin levels to enhance the stemness of HCC cells[10]. In the current study, we further showed that lncRNA Flicr, within the exosomes derived from Treg cells, was delivered to HCC cells, where it inhibited FOXP3 and activated β-catenin si
GW4869, an inhibitor of exosome biogenesis and secretion[16], was used during the co-culture of Treg cells and HCC cells. Treatment with GW4869 suppressed the acquisition of CSC-like traits in HCC cells, confirming the critical role of Treg-derived exosomes in mediating these effects. Exosomes are a major class of extracellular vesicles that transport bioactive molecules-including nucleic acids (such as mRNA, microRNA, and lncRNA), proteins, and lipids to neigh
In this study, co-culturing with Treg-derived exosomes resulted in increased levels of lncRNA Flicr in HCC cells. Furthermore, overexpression of lncRNA Flicr led to a decrease in FOXP3 expression, whereas inhibition of lncRNA Flicr caused an increase in FOXP3 levels, indicating that lncRNA Flicr regulates FOXP3 either directly or indirectly in HCC cells. Notably, previous studies have demonstrated that in Treg cells, lncRNA Flicr acts as a negative regulator of FOXP3 expression by modulation its transcription through a local cis-acting mechanism[20]. In the present study, we found that lncRNA Flicr was transferred from Treg cells to HCC cells via exosomes and similarly suppressed FOXP3 protein expression in HCC cells.
The cytoplasmic stability of β-catenin is tightly regulated by GSK3β, which facilitates the phosphorylation of β-catenin, thereby targeting it for ubiquitination and subsequent degradation by the proteasome[21]. In contrast, when GSK3β itself is ubiquitinated and degraded, β-catenin accumulates and triggers activation of the Wnt/β-catenin signaling pathway[22,23]. Consistent with our earlier findings, the downregulation of FOXP3 observed in this study was associated with decreased GSK3β levels and increased β-catenin accumulation[10]. These results suggested that lncRNA Flicr suppressed FOXP3 and consequently reduced GSK3β expression, thereby relieving the inhibitory control on β-catenin and promoting its nuclear translocation. This ultimately led to activation of the Wnt/β-catenin signaling pathway, enhancing the self-renewal and maintenance of stem-like characteristics in HCC.
Exosomal lncRNAs have emerged as critical regulators in modulating the immune microenvironment of HCC. Exosomes containing high levels of FAL1 can induce M2 polarization in macrophages, which in turn promotes the proliferation, invasion, and colony-forming ability of HCC cells, while reducing apoptosis and resistance to sorafenib. Blocking FAL1 can counteract these effects[24]. Additionally, HEIH is abundant in exosomes present in plasma and cell culture supernatants and is delivered to macrophages via HCC-derived exosomes, leading to M2 polarization and enhancing the proliferation, migration, and invasion of HCC cells[25]. Exosomes significantly contribute to Treg development, home
It has been reported that Treg-associated lncRNAs might contribute to the pathogenesis of breast cancer[28]. The researchers examined the expression of Treg-associated lncRNAs in breast tumor tissues and adjacent non-tumor tissues. They found that the levels of RMRP, TH2-LCR, MAFTRR, and GATA3-AS1 were significantly elevated in breast cancer samples compared to non-tumorous tissues. Additionally, significant positive correlations were observed between the expression of RMRP and MAFTRR in tumor tissues and factors such as nuclear grade, tubule formation, and tumor size. Furthermore, the expression of FLICR varied among tumors with different HER2/neu receptor statuses. Other studies on lung cancer revealed dysregulation of three Treg-related lncRNAs in lung cancer tissues, specifically, increased expression of TH2-LCR, IFNG-AS1, and MAFTRR was observed in lung cancer samples compared to normal tissues adjacent to the tumor, suggesting that these lncRNAs may influence the tumor microenvironment to promote cancer progression[29].
Treg have even become a target of anti-cancer therapies in clinical settings, various therapeutic approaches targeting Treg have been developed and advanced to clinical trials. These include antibodies that block CD25[30], low-dose metronomic cyclophosphamide[31], metabolic regulation of Tregs[32], and disruption of pathways that stabilize Treg[33]. Although these strategies have significantly reduced Treg populations, the depletion has not been sustained successfully, often resulting in a rebound that surpasses their previous numbers and suppressive activity. Effective therapies that deplete Treg or Treg-derived exosomes could potentially help control CSCs characteristics in HCC. However, because Treg cells and their exosomes play a crucial role in maintaining the balance of adaptive immune responses, strategies targeting Treg exosome secretion must carefully consider that their removal or imbalance could disrupt essential survival mechanisms[34].
This study has several unavoidable limitations. First, the Treg cells were isolated from the peripheral blood of an HCC patient, whereas intratumoral Treg cells within the tumor microenvironment may have a more significant impact on HCC progression. Treg cells are heterogeneous in phenotype and function, with three distinct subpopulations identified within human peripheral blood, which are CD45RA+FoxP3Lo, CD45RA-FoxP3hi and CD45RA-FoxP3Lo Treg cells[35]. Wang et al[36] compared Treg cell subpopulations in peripheral blood and those within tumors by analyzing paired samples from breast cancer patients. Their study included detailed assessments of key immune-regulating proteins, chemokine expression patterns, and T cell receptor repertoires. They found that CD45RA-FoxP3hi Treg cells in peripheral blood exhibit a similar immune phenotype and chemokine receptor expression pattern to intratumoral Treg cells, along with a higher degree of T cell receptor clonal overlap. This suggests that the CD45RA-FoxP3hi Treg cell subset in peripheral blood serves as a major source of intratumoral Treg cells in human breast tumors. Additionally, their responsiveness to cytokine signaling reflects the immunosuppressive potential within tumors and can predict clinical outcomes. Although intratumoral Treg cells were not successfully isolated from HCC patients in this study, the FOXP3hi Treg cells from peripheral blood displayed some shared characteristics, suggesting that observations made in peripheral blood Treg cells may also reflect the behavior of intratumoral Treg cells. Second, we did not determine whether GSK3β is directly regulated by FOXP3 at the transcriptional level, leaving some details of the molecular mechanism unclear. Third, although lncRNA Flicr was not detectable in HCC-LM3 cells, its expression level should be further validated in clinical HCC samples to better assess its potential relevance in human HCC.
In summary, this study revealed that exosomal lncRNA Flicr, derived from Treg cells, inhibits the expression of FOXP3 and GSK3β while activating the Wnt/β-catenin signaling pathway, thereby promoting stem cell-like properties in HCC cells.
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