Fig 2: Immuno-magnetic bead assay detects GPC3 in HCC exosomes, not exosomes secreted by cholangiocarcinoma (HuCCA) or human embryonic kidney cells (HEK293). (A) GPC3 detection in exosomes isolated from two HCC cell lines. GPC3 was not detected in exosomes isolated from non-HCC cell lines (HuCCA or HEK293). (B) The bar graph represents the relative distribution of GPC3 and CD81 among HCC and non-HCC cultures. Data indicate mean ± standard deviation and are analyzed by the Mann–Whitney U-test. P values were displayed as **P<0.01, ***P<0.001. (C) Shows the concentration of EVs isolated from hepatic and non-hepatic culture by NanoSight. (D) The bar graph shows the mean and mode of EV sizes derived from hepatic and non-hepatic cell cultures. (E) Representative images of different cell culture-derived EVs were captured from actual videos of Nanoparticle tracking analysis.

Fig 3: Association between GPC3 expression and the differentiation of liver cancer. (A) Well-differentiated liver cancer tissue and the same field. (A1). The tumor cells demonstrated a thin trabecular pattern with mild atypical cells (HE at magnification, ×200). (A2) GPC3 was absent (GPC3 immunostaining at magnification, ×200). (A3) Positive cells were <5% (Ki-67 immunostaining at magnification, ×200). (B) Moderately-differentiated liver cancer. (B1) Tumor cells arranged in trabeculae measuring =3 cells-thick (HE at magnification, ×200). (B2) Tumor tissue indicated moderate staining with GPC3 (GPC3 immunostaining at magnification, ×200). (B3) Positive cells were ~30% (Ki-67 immunostaining, ×200). (C) Poorly differentiated liver cancer and same field. (C1) The tumor tissue exhibited a solid pattern with marked atypical cells (HE at magnification, ×200). (C2) Tumor tissue indicated strong staining with GPC3 (GPC3 immunostaining at magnification, ×200). (C3) Positive cells were >50% (Ki-67 immunostaining at magnification, ×200). HE, hematoxylin and eosin; GPC3, glypican 3; Ki-67, antigen Ki-67.

Fig 4: Show immune-magnetic affinity isolation detect GPC3 in HCC cell culture-derived exosomes, not in the microvesicles. (A) Detection of exosomes and microvesicles in the extracellular vesicles isolated from HCC cell culture. First, EVs were isolated from HCC cell culture using by total exosome isolation reagent. Second, exosomes and microvesicles were selected from total EVs using CD63 or Annexin A1 antibodies conjugated magnetic beads. Immuno-affinity capture exosomes or microvesicles were stained with Alexa Fluor 488 labeled GPC3. Approximately 53.4% of CD63 conjugated magnetic beads show positive for GPC3 as compared to 0.9% of Annexin A1 conjugated beads. About 92.8% of CD63 beads show positive for CD81, as compared to 45.7% of Annexin A1 beads. (B) Western blot analysis shows the distribution of GPC3 in the total non-selected and selected EVs. (C) The bar graph represents the relative intensity of GPC3 proteins according to particle-specific protein bands in Western blot. The results are expressed as the mean intensity ± standard deviation and analyzed by Mann Whitney U-test. *P < 0.05, **P < 0.01.

Fig 5: Impaired autophagy promotes GPC3 accumulation in exosomes. CD63-GFP stable cell line was treated with increasing concentrations of Torin 1 or Bafilomycin A1 for 72 hours. (A) Demonstrate the expression of CD63-GFP in Torin 1 treated and Bafilomycin A1 treated by fluorescence microscopy. (B) Quantification of CD63-GFP expression in Torin 1 and Bafilomycin A1 treated culture by Image J software. (C) Fluorescence microscopy images show the expressions of GPC3, and Tsg 101 in Torin 1 and Bafilomycin A1 treated cells. (D) Western blot analysis shows autophagy induction by Torin 1 leads to GPC3 and CD63 degradation. Data indicate mean ± standard deviation and are analyzed by the Mann–Whitney U-test. P values were displayed as **P<0.01, ***P<0.001.