Fig 2: Increased ENO1 expression is correlated with poor prognosis of HCC patients.A The expression level of ENO1 in LO2exo, HepG2exo, MHCC97Lexo, and HCCLM3exo determined by proteomics analysis. B Western blot analysis of ENO1 levels in LO2, HepG2, MHCC97L, and HCCLM3 cells. A representative of three independent experiments is shown. C Differential expression of ENO1 in 94 pairs of HCC and adjacent non-tumor tissues. D IHC staining of ENO1 expression in normal liver tissues, cirrhosis tissues, paired of HCC and non-tumor tissues and metastasis tissues. Representative photographs of ENO1 staining in different tissues are shown. Scale bar represent 800 µm and 100 µm. E Kaplan–Meier analysis of overall survival of 94 HCC patients with different ENO1 expression levels. F Kaplan–Meier analysis of overall survival of 365 HCC patients in the TCGA cohort. G Forest plot showing the association between ENO1 expression and HCC survival as revealed by univariate and multivariate analyses (HR, hazard ratio; CI, confidence interval). All data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 according to two-tailed Student’s t-test.

Fig 3: Exosome-derived ENO1 regulates integrin a6ß4 expression and activates the FAK/Src-p38MAPK pathway in HCC cells.A Immunofluorescence quantification of integrin a6 and ß4 expression in arbitrary units (a.u.) in MHCC97L-NC and HepG2-NC cells educated with MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 µg/ml). Scale bar represent 25 µm. B Western blot analysis of the impact of exosome-derived ENO1 on the expression of integrin a6ß4 and the activation of integrin-mediated FAK/Src-p38MAPK pathway in MHCC97L-NC and HepG2-NC cells educated with MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 µg/ml). A representative of three independent experiments is shown. All data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 according to two-tailed Student’s t-test.

Fig 4: Schematic diagram of the role of exosome-derived ENO1 in HCC growth and metastasis.Exosome-derived ENO1 facilitates the expression of integrin a6ß4 and the activation of the FAK/Src pathway, supporting downstream signaling via p38MAPK in HCC cells.

Fig 5: Exosome-derived ENO1 promotes HCC growth and metastasis.A Western blot analysis of ENO1 level in exosomes derived from HCCLM3-NC, HCCLM3-shENO1, MHCC97L-NC, MHCC97L-ENO1, HepG2-NC, and HepG2-ENO1 cells, and HA protein expression in MHCC97L-ENO1exos and HepG2-ENO1exos. B NTA analysis of the impact of altered ENO1 expression in HCC cells on the number of exosomes released. C Fluorescence microscopy analysis of PKH67-labeled exosomes with high ENO1 expression incorporation (green) by HCCLM3-shENO1, MHCC97L-NC, and HepG2-NC cells with relatively low ENO1 expression. Exosomes were isolated from HCCLM3-NC, MHCC97L-ENO1 and HepG2-ENO1 cells. Scale bar represent 10 µm. D Cell CCK-8 assays, E transwell migration and invasion assays of HCCLM3-shENO1, MHCC97L-NC, and HepG2-NC cells educated with HCCLM3-NCexos, MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 µg/ml). F Western blot analysis of ENO1, HA, E-cadherin, N-cadherin and Vimentin levels in HCCLM3-shENO1, MHCC97L-NC, and HepG2-NC cells educated with HCCLM3-NCexos, MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 µg/ml). G Evaluation of HCC metastasis by tumor size and number of nude mice with lung metastasis in mice injected in the tail vein with HCCLM3-shENO1 and MHCC97L-NC cells following education with HCCLM3-NCexos, MHCC97L-ENO1exos or self-secreted exosomes. For the in vivo experiments, four nude mice per group were used. A representative of three independent experiments is shown. All data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 according to two-tailed Student’s t-test.