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

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.