Fig. 2: Glucose enrichment occurs during H-CM-induced lung pre-metastatic niche.

a–c Western blot analysis of glucose transporters and glycolytic enzymes (a), 2-NBDG uptake (b), and glucose consumption (c) in lung fibroblasts treated with MHCC97H/Hep3B-L/H-CM grown on lung stiffness substrates (a, b n = 3 biological replicates; c n = 4 biological replicates). a The samples derive from the same experiment but different gels for GLUT1 and PFKP, another for SGLT2 and β-actin, and another for PKM2 and HK2 were processed in parallel. d Untargeted metabolomic analysis of differential intracellular glycolytic metabolites in lung fibroblasts treated with MHCC97H-L/H-CM grown on lung stiffness substrates (n = 6 biological replicates). e qRT-PCR analysis of glucose transporters and glycolytic enzymes in pre-metastatic lung tissues (n = 4 mice per group). Data were normalized to β-actin. f IHC staining of GLUT1 in pre-metastatic lung tissues (n = 4 mice per group). Scale bars: black, 200 μm; red, 50 μm. g Adherent HCC cells on lung fibroblast monolayer treated with MHCC97H-L-CM, H-CM, or L-CM + HG grown on lung stiffness substrate (n = 6 biological replicates). h Adherent HCC cells pre-treated with function-blocking antibody of integrin β1 on lung fibroblast monolayer grown on lung stiffness substrates (n = 6 biological replicates). Scale bars: 200 μm (g, h). i, j Flow cytometry analysis of CD11b⁺Gr-1⁺ MDSCs (i) in differentiated bone marrow cells (BMCs) treated with NG, HG, or HG + 2-DG and their p-mTOR (S2448) expression (j) (n = 3 biological replicates). k-m Western blot analysis of glucose transporters and glycolytic enzymes (k), 2-NBDG uptake (l) and glucose consumption (m) in lung fibroblasts treated with MHCC97H/Hep3B-H-CM-Exo-free grown on lung stiffness substrates (k, l n = 3 biological replicates; m n = 4 biological replicates). k The samples derive from the same experiment but different gels for GLUT1, another for PFKP and β-actin, and another for PKM2 and HK2 were processed in parallel. Representative images are presented from indicated biologically independent experiments (b, f–j, l). Representative blot (20 μg protein per group) was shown from 3 biologically independent experiments (a, k), and β-actin was used to normalize protein quantification. Data are presented as mean ± SD, and P values were calculated using two-tailed unpaired Student’s t-test (a–f, k–m) or one-way ANOVA (g–j). L low-stiffness substrates; H high-stiffness substrates; CM conditioned medium, MFI mean fluorescence intensity, NG normal glucose concentration, 5.5 mM, HG high glucose concentration, 25 mM, Ab antibody, Exo exosome. Source data are provided as a Source Data file.