Regulatory T cell-derived exosomes regulate β-catenin expression to promote the stem cell-like properties of hepatocellular carcinoma

Figure 1 Co-culturing hepatocellular carcinoma cells with regulatory T cells and their conditioned medium enhanced the stem-like properties of hepatocellular carcinoma cells. A: Expression of stem cell-related genes in hepatocellular carcinoma (HCC) cells was measured by quantitative real-time polymerase chain reaction (n = 6). ^aP < 0.05, compared to the control group; ^bP < 0.01, compared to the control group; ^dP < 0.01, compared to the co-culture group (n = 3); B: Immunofluorescence staining was used to detect CD133+ cancer stem cells in HCC-LM3 cells. Scale bar = 100 μm, n = 3; C: Flow cytometry analysis of the proportion of CD133+ cancer stem cells in HCC-LM3 cells (n = 3); D: Sphere-formation assay assessing the in vitro tumor sphere formation capability of HCC-LM3 cells. Scale bar = 200 μm, n = 3; E: In vivo tumorigenicity of HCC-LM3 cells and tumor volume changes in nude mice after subcutaneous injection of 2 × 10⁶ HCC-LM3 cells over 5 weeks. ^aP < 0.05, compared to the control group; ^cP < 0.05 compared to the co-culture + GW4869 group (n = 5); F: Western blot analysis of forkhead box P3, glycogen synthase kinase-3 beta, and β-catenin expression in HCC-LM3 cells (n = 3). Biological replicates were used in all experiments. Control group: HCC-LM3 cells cultured alone. Co-culture group: HCC-LM3 cells co-cultured with regulatory T (Treg) cells. CM group: HCC-LM3 cells cultured with conditioned medium from Treg cells. Co-culture + GW4869 group: HCC-LM3 cells co-cultured with Treg cells in the presence of the exosome inhibitor GW4869. CM + GW4869 group: HCC-LM3 cells cultured with conditioned medium from Treg cells in the presence of GW4869. OCT4: Octamer-binding transcription factor 4; SOX2: Sex-determining region Y-box 2; KLF4: Kruppel-like factor 4; MYC: Myelocytomatosis oncogene; FOXP3: Forkhead box P3; GSK3β: Glycogen synthase kinase-3 beta.

Figure 2 Characterization of regulatory T cell-derived exosomes. A: The size distribution of regulatory T (Treg) cell-derived exosomes; B: Morphological observation of Treg-derived exosomes using transmission electron microscopy; C: Flow cytometry analysis showing the expression of CD63 and CD81 on Treg-derived exosomes.

Figure 3 Regulatory T cell-derived exosomes enhanced the stem-like properties of hepatocellular carcinoma cells in a dose-dependent manner. A: Immunofluorescence staining was used to identify CD133+ cancer stem cells in hepatocellular carcinoma (HCC)-LM3 cells. Scale bar = 100 μm, n = 3; B: Flow cytometry analysis of the proportion of CD133+ cancer stem cells in HCC-LM3 cells, n = 3; C: Sphere-formation assay assessing the in vitro tumor sphere formation capability of HCC-LM3 cells. Scale bar = 200 μm, n = 3; D: In vivo tumorigenicity was assessed by monitoring tumor volume changes in nude mice over 5 weeks after subcutaneous injection of 2 × 10⁶ HCC-LM3 cells. ^aP < 0.05, compared to the control group; ^bP < 0.01, compared to the control group; ^cP < 0.05, compared with the 5 μg group; ^dP < 0.01, compared with the 25 μg group, n = 5; E: Western blot analysis of forkhead box P3, glycogen synthase kinase-3 beta, and β-catenin expression in HCC-LM3 cells, n = 3. Biological replicates were used in all experiments. 0 μg group: HCC cells co-cultured without regulatory T (Treg) cell-derived exosomes; 5 μg group: HCC cells co-cultured with 5 μg of Treg-derived exosomes; 25 μg group: HCC cells co-cultured with 25 μg of Treg-derived exosomes; 50 μg group: HCC cells co-cultured with 50 μg of Treg-derived exosomes. FOXP3: Forkhead box P3; GSK3β: Glycogen synthase kinase-3 beta.

Figure 4 The level of long non-coding RNA FLICR in hepatocellular carcinoma cells increased after co-culturing with regulatory T cell-derived exosomes. A: Quantitative real-time polymerase chain reaction was used to measure long non-coding RNA FLICR levels in hepatocellular carcinoma (HCC) cells before and after co-culture with regulatory T-derived exosomes. ^aP < 0.05, compared to the control group, n = 3; B: Immunofluorescence staining was performed to detect CD133+ cancer stem cells, and fluorescence in situ hybridization was used to visualize long non-coding RNA FLICR expression in HCC cells. Scale bar = 100 μm, n = 3. Biological replicates were used in all experiments. Control group: HCC-LM3 cells cultured alone. Exosome group: HCC-LM3 cells co-cultured with regulatory T cell-derived exosomes.

Figure 5 Overexpression of FLICR in regulatory T cells promotes the stem-like characteristics of hepatocellular carcinoma cells. A: Immunofluorescence staining was used to identify CD133+ cancer stem cells, and fluorescence in situ hybridization was performed to visualize long non-coding RNA FLICR in hepatocellular carcinoma (HCC) cells following co-culture with FLICR-overexpressing regulatory T (Treg) cells. Scale bar = 100 μm, n = 3; B: Flow cytometry analysis of the proportion of CD133+ cancer stem cells in HCC-LM3 cells co-cultured with FLICR-overexpressing Treg cells, n = 3; C: Sphere-formation assay assessing the in vitro tumor sphere formation capability of HCC-LM3 cells co-cultured with FLICR-overexpressing Treg cells. Scale bar = 200 μm, n = 3; D: The tumorigenic potential of HCC-LM3 cells co-cultured with FLICR-overexpressing Treg cells was assessed in vivo by monitoring tumor volume changes in nude mice injected subcutaneously with 2 × 10⁶ HCC-LM3 cells over four weeks. ^bP < 0.01, compared to the control group; ^dP < 0.01, compared to the vector group, n = 5; E: Western blot analysis of forkhead box P3, glycogen synthase kinase-3 beta, and β-catenin expression in HCC-LM3 cells co-cultured with FLICR-overexpressing Treg cells, n = 3. Biological replicates were used in all experiments. Control group: HCC-LM3 cells co-cultured with Treg cells. Vector group: HCC-LM3 cells co-cultured with Treg cells transfected with an empty vector. OE group: HCC-LM3 cells co-cultured with Treg cells overexpressing long non-coding RNA FLICR. FOXP3: Forkhead box P3; GSK3β: Glycogen synthase kinase-3 beta.

Figure 6 Inhibiting FLICR in regulatory T cells reduced the stem-like characteristics of hepatocellular carcinoma cells. A: Immunofluorescence staining was used to detect CD133+ cancer stem cells, and fluorescence in situ hybridization visualized long non-coding RNA Flicr in hepatocellular carcinoma (HCC) cells after co-culture with regulatory T (Treg) cells in which FLICR was suppressed. Scale bar = 100 μm, n = 3; B: Flow cytometry analysis of the proportion of CD133+ cancer stem cells in HCC-LM3 cells co-cultured with FLICR-inhibited Treg cells, n = 3; C: Sphere-formation assays evaluated the ability of HCC-LM3 cells to form tumor spheres in vitro after co-culture with FLICR-inhibited Treg cells. Scale bar = 200 μm, n = 3; D: The tumorigenic potential of HCC-LM3 cells co-cultured with FLICR-inhibited Treg cells was assessed in vivo by monitoring tumor volume changes in nude mice injected subcutaneously with 2 × 10⁶ HCC-LM3 cells over 5 weeks. ^bP < 0.01, compared to the control group; ^dP < 0.01, compared to the antisense oligonucleotide (ASO) negative control (NC) group, n = 5; E: Western blot analysis of forkhead box P3, glycogen synthase kinase-3 beta, and β-catenin expression in HCC-LM3 cells co-cultured with FLICR-inhibited Treg cells, n = 3. Biological replicates were used in all experiments. Control group: HCC-LM3 cells co-cultured with untreated Treg cells. ASO-NC group: HCC-LM3 cells co-cultured with Treg cells transfected with an ASO negative control. ASO group: HCC-LM3 cells co-cultured with Treg cells in which long non-coding RNA Flicr was silenced using ASO. FOXP3: Forkhead box P3; GSK3β: Glycogen synthase kinase-3 beta.