Figure 6.

EVs with low PKLR levels secreted by FBP1-depleted hepatocytes accelerate liver tumorigenesis in vivo

(A) Experimental scheme examining the effect of hepatocyte-derived EVs in a DEN-induced HCC model using C57BL/6 mice. (B) Western blot analysis of PKLR expression in EVs secreted by indicated clones. (C) Representative image of whole livers (left) and the number of surface tumor nodules (right) in DEN-induced HCC mice after injecting EVs secreted by indicated clones, n = 6. (D and E) Representative images (left) and quantifications (right) of Ki67 (D; scale bar, 100 μm) and TUNEL staining (E; scale bar, 100 μm) in DEN-induced HCC mouse livers treated with EVs secreted by indicated clones, n = 6. (F) Quantifications of NK and T cells in mouse livers, spleens, and periphery blood harvested from DEN-treated mice after injecting EVs secreted by indicated clones, n = 6. (G) Representative histograms (top) and quantifications (bottom) of activated NK cells (TNF-α positive) in aforementioned livers and spleens, n = 6. (H) Representative IHC staining (top; scale bar, 100 μm) and correlations (bottom) among FBP1, PKLR, and infiltrated NK cells in early-stage human HCC tissues. (I) Kaplan-Meier overall survival analysis of two HCC patient groups based on PKLR and FBP1 expression levels in their tumor tissues as sequenced by TCGA. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by unpaired Student’s t test. See also Figures S7–S9.