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

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.