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.