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

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.