A glucose-enriched lung pre-metastatic niche triggered by matrix stiffness-tuned exosomal miRNAs in hepatocellular carcinoma

Abstract

Apart from the classic features, it is almost unknown whether there exist other new pathological features during pre-metastatic niche formation in hepatocellular carcinoma (HCC). Our previous works have highlighted the contribution of increased matrix stiffness to lung pre-metastatic niche formation and metastasis in HCC. However, whether increased matrix stiffness influences glucose metabolism and supply of lung pre-metastatic niche remains largely unclear. Here we uncover the underlying mechanism by which matrix stiffness-tuned exosomal miRNAs as the major contributor modulate glucose enrichment during lung pre-metastatic niche formation through decreasing the glucose uptake and consumption of lung fibroblasts and increasing angiogenesis and vascular permeability. Our findings suggest that glucose enrichment, a new characteristic of the lung pre-metastatic niche triggered by matrix stiffness-tuned exosomal miRNAs, is essential for the colonization and survival of metastatic tumor cells, as well as subsequent metastatic foci growth.

Fig. 1. Conditioned medium derived from HCC cells grown on the high-stiffness substrate accelerates the formation of the lung pre-metastatic niche.

a Schematic illustration of tumor-free mouse models with lung pre-metastatic niches induced by Hepa1-6-L/H-CM, created in BioRender. Zhao, Y. (2025) https://BioRender.com/d32b589. b, c Flow cytometry analysis (b) and quantification (c) of CD11b⁺CD45⁺ bone marrow-derived cells (BMDCs) in lung tissues (n = 2 mice per group on days 6, 14, 18, and 22; n = 4 mice per group on day 26). d qRT-PCR analysis of pre-metastatic niche-related genes in lung tissues on day 26 (n = 4 mice per group). Data were normalized to β-actin. e IHC staining of fibronectin in lung tissues on day 26 (n = 4 mice per group). f, g Percentage of CD11b⁺Gr-1⁺ myeloid-derived suppressor cells (MDSCs) (f) and CD8⁺ T cells (g) in lung tissues on day 26 (n = 4 mice per group). h IHC staining of CD31 in lung tissues on day 26 (n = 4 mice per group). Scale bars: black, 200 μm; red, 50 μm (e, h). i IF images for CD31 and VE-cadherin in lung tissues on day 26 (n = 4 mice per group). Scale bars: 20 μm. j Western blot analysis of fibronectin and MMP9 in lung fibroblasts treated with MHCC97H/Hep3B-L/H-CM grown on lung stiffness substrates. k Adherent HCC cells on lung fibroblast monolayer treated with MHCC97H/Hep3B-L/H-CM grown on lung stiffness substrates (n = 6 biological replicates). Scale bars: 200 μm. l Western blot analysis of ZO-1, VE-cadherin and VEGFR2 in HUVECs treated with MHCC97H/Hep3B-L/H-CM. The samples derive from the same experiment but different gels for ZO-1 and VE-cadherin, and another for VEGFR2 and β-actin were processed in parallel. m Permeability of HUVEC monolayer treated with MHCC97H-L/H-CM to FITC-dextran (n = 3 biological replicates). No monolayer cells, no HUVECs on the upper chamber. Representative images are presented from indicated biologically independent experiments (b, e–i, k). Representative blot (20 μg protein per group) was shown from 3 biologically independent experiments (j, l), 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 (c–m). L low-stiffness substrates, H high-stiffness substrates, CM conditioned medium, OD498 optical density (OD) measured at 498 nm. Source data are provided as a Source Data file.

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.

Fig. 3. Tumor-derived exosomes as the major contributor modulate glucose enrichment during H-CM-induced lung pre-metastatic niche.

a Transmission electron microscope (TEM) images of exosomes purified from MHCC97H/Hep3B-L/H-CM (n = 3 biological replicates). Scale bars: 50 μm. b Molecule markers of exosomes detected by western blot (n = 3 biological replicates). The samples derive from the same experiment but different gels for TSG101, CD63, and Hsp70, another for ALIX, and another for Albumin and Cytochrome C were processed in parallel. c Internalization of DIO-labeled exosomes (green) by lung fibroblasts and vascular endothelial cells (n = 3 biological replicates). Scale bars: 5 μm. d Schematic illustration of a co-culture system in vitro simulating pre-metastatic niche environment (upper panel) and western blot analysis of fibronectin and MMP9 in lung fibroblasts treated with MHCC97H-L/H-Exo in this system (lower panel). Upper panel was created in BioRender. Zhao, Y. (2025) https://BioRender.com/k02k700. e Flow cytometry analysis of CD11b⁺Gr-1⁺ MDSCs in differentiated bone marrow cells (BMCs) co-cultured with L/H-Exo-treated lung fibroblasts (n = 3 biological replicates). f Schematic illustration of tumor-free mouse models with lung pre-metastatic niches induced by Hepa1-6-L/H-Exo, created in BioRender. Zhao, Y. (2025) https://BioRender.com/k94x898. g Flow cytometry analysis of CD11b⁺CD45⁺ BMDCs in the pre-metastatic lung tissues on day 26 (n = 5 mice per group). h Levels of glucose in the cell-free interstitial fraction fluid from the pre-metastatic lung tissues on day 26 (n = 5 mice per group). i Multiplex immunofluorescence of TSG101, CD31, and VE-cadherin in the pre-metastatic lung tissues on day 26 (n = 5 mice per group). Scale bars: 20 μm. j Bioluminescence imaging (BLI) of lung metastasis lesions in lung tissues on day 54 and day 61 (n = 5 mice per group). k HE staining and quantification of lung metastasis lesions in lung tissues on day 61 (n = 5 mice per group). Scale bars: red, 2 mm; blue, 500 μm; black, 100 μm. Representative images are presented from indicated biologically independent experiments (a–c, e, g, i–k). Representative blot (20 μg protein per group) was shown from 3 biologically independent experiments (d), and β-actin was used to normalize protein quantification. Data are presented as mean ± SD, and P values were calculated using one-way ANOVA (d) or two-tailed unpaired Student’s t-test (e, g–i, k). L low-stiffness substrates, H high-stiffness substrates, Exo exosome. Source data are provided as a Source Data file.

Fig. 4. Matrix stiffness-tuned exosomal let-7d-5p promotes glucose enrichment during lung pre-metastatic niche formation.

a Heatmap of the known differentially expressed miRNAs between MHCC97H-L-Exo and MHCC97H-H-Exo (n = 3 biological replicates). b miRNA qRT-PCR analysis of let-7d-5p, miR-365a-5p, miR-194-5p, and miR-125a-5p in MHCC97H/Hep3B-L/H-Exo (n = 3 biological replicates). Data were normalized to U6. c qRT-PCR analysis of glycolysis-related genes in lung fibroblasts transfected with indicated miRNA mimics or mimic NC (n = 4 biological replicates). Data were normalized to β-actin. d Western blot analysis of glucose transporters and glycolytic enzymes in lung fibroblasts with let-7d-5p overexpression or knockdown (n = 3 biological replicates). The samples derive from the same experiment but different gels for GLUT1 and PFKP, and another for PKM2, HK2, and β-actin were processed in parallel. e 2-NBDG uptake in lung fibroblasts with let-7d-5p overexpression or knockdown (n = 3 biological replicates). f Flow cytometry analysis of CD11b⁺CD45⁺ BMDCs recruitment in the pre-metastatic niche lung tissues induced by Hepa1-6-Mock/Exo or Hepa1-6-let-7d-5p-OE/Exo (n = 4 mice per group). g 2-NBDG uptake in lung fibroblasts (CD45^-CD31^-CD140a⁺ cells) in the pre-metastatic niche lung tissues induced by Hepa1-6-Mock/Exo or Hepa1-6-let-7d-5p-OE/Exo (n = 4 mice per group). h IHC staining of GLUT1, PFKP, and HK2 expressions in the pre-metastatic lung tissues induced by Hepa1-6-Mock/Exo or Hepa1-6-let-7d-5p-OE/Exo (n = 4 mice per group). Scale bars: black, 200 μm; red, 50 μm. i HE staining and quantification of lung metastasis lesions in lung tissues on day 61 (n = 5 mice per group). Scale bars: red, 2 mm; blue, 500 μm; black, 100 μm. Representative images are presented from indicated biologically independent experiments (e–i). Representative blot (20 μg protein per group) was shown from 3 biologically independent experiments (d), 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 (b, c, f–i) or one-way ANOVA (d, e). L low-stiffness substrates, H high-stiffness substrates, MFI mean fluorescence intensity, Exo exosome, WT wild type, OE overexpression. Source data are provided as a Source Data file.

Fig. 5. A pathway of matrix stiffness-tuned exosomal let-7d-5p/HMGA2/E2F1 acetylation/GLUT1, PFKP, and HK2 in lung fibroblasts participates in modulating glucose enrichment.

a Predicted target proteins of let-7d-5p using three publicly available bioinformatic tools. b Western blot analysis of HMGA2 in lung fibroblasts with let-7d-5p overexpression or knockdown. c Schematic illustration of luciferase reporter plasmids for HMGA2 3’UTR (left panel) and relative luciferase activity determined after co-transfection of miRNA mimic and plasmids (right panel) (n = 3 biological replicates). d Effects of HMGA2 overexpression (HMGA2-OE) on GLUT1, PFKP, and HK2 expressions in lung fibroblasts with let-7d-5p overexpression. The samples derive from the same experiment but different gels for GLUT1 and PFKP, and another for HK2, HMGA2, and β-actin were processed in parallel. e Immunoprecipitation assay of interaction between HMGA2 and Rb in the nuclear protein of lung fibroblasts. f Immunoprecipitation assay of interaction between HMGA2 or HDAC1 and E2F1-Rb complex in the nuclear protein of lung fibroblasts with let-7d-5p overexpression or downregulation. g E2F1 acetylation level (Pan Ac-Lys levels of E2F1-captured proteins) and HDAC1 interacted with E2F1 in the nuclear protein of lung fibroblasts with let-7d-5p overexpression or downregulation determined by immunoprecipitation assay. h ChIP-qPCR analysis of E2F1 occupancy on SLC2A1, PFKP, and HK2 promotors in lung fibroblasts (relative to input) (n = 3 biological replicates). i Effects of TSA intervention on E2F1 acetylation levels in the nuclear protein (left panel) and GLUT1, PFKP, and HK2 expression in the total protein (right panel) of lung fibroblasts with let-7d-5p overexpression. The samples derive from the same experiment but different gels for GLUT1 and HK2, and another for PFKP and β-actin were processed in parallel. j, k Effects of E2F1 mutation (K117/120/125 R) on E2F1 acetylation levels in the nuclear protein (j) and GLUT1, PFKP, and HK2 expressions in the total protein (k) of lung fibroblasts with let-7d-5p downregulation. The samples derive from the same experiment but different gels for GLUT1 and PFKP, and another for HK2 and β-actin were processed in parallel. Representative blot was shown from 3 biologically independent experiments (b, d–g, i–k). Protein loading was 20 μg in each group and β-actin was used to normalize total protein quantification (b, d, i, k), and Histone H3 was used as a loading control in the nuclear protein (f, g, i, j). Data are presented as mean ± SD, and P-values were calculated using one-way ANOVA (b–d, i, k) or two-tailed unpaired Student’s t-test (h). Ac-Lys acetylated-Lysine, WT wild type, mut mutant, WCL whole cell lysate. Source data are provided as a Source Data file.

Fig. 6. Matrix stiffness-tuned exosomal miR-365a-5p effectively regulates angiogenesis and vascular permeability to influence glucose enrichment in the lung pre-metastatic niche.

a qRT-PCR analysis of tight junction-related genes in HUVECs transfected with indicated mimics or mimic NC (n = 3 biological replicates). Data were normalized to β-actin. b Western blot analysis of ZO-1, VE-cadherin, and VEGFR2 in vascular endothelial cells transfected with miR-365a-5p mimic or inhibitor (n = 3 biological replicates). The samples derive from the same experiment but different gels for ZO-1 and β-actin, and another for VE-cadherin and VEGFR2 were processed in parallel. c Schematic illustration of FITC-dextran leakiness assay (left panel) and trans-endothelial invasion assay (right panel), created in BioRender. Zhao, Y. (2025) https://BioRender.com/d12t168. d Permeability of the HUVEC monolayer (transfected with miR-365a-5p mimic or inhibitor) to FITC-dextran (n = 3 biological replicates). e Invaded HCC cells that passed through the HUVEC monolayer transfected with miR-365a-5p mimic or inhibitor (n = 6 biological replicates). Scale bars: 200 μm. f Chicken embryo chorioallantoic membrane (CAM) incubated with Hepa1-6-Mock/CM or Hepa1-6-miR-365-1-5p-OE/CM (n = 3 biological replicates). g Tube formation assay of HUVECs transfected with miR-365a-5p mimic or inhibitor (n = 6 biological replicates). Scale bars: 200 μm. h, i Immunofluorescence images for concanavalin A co-localization with FITC-dextran (h) and quantification of extravasated dextran (i) in lung tissues induced by PBS, Hepa1-6-Mock/Exo, or Hepa1-6-miR-365-1-5p-OE/Exo (n = 5 mice per group). Scale bars: white, 100 μm; yellow, 20 μm. j, k IHC staining and quantification of CD31 in lung tissues induced by PBS, Hepa1-6-Mock/Exo, or Hepa1-6-miR-365-1-5p-OE/Exo (n = 5 mice per group). Scale bars: black, 200 μm; red, 50 μm. Representative images are presented from indicated biologically independent experiments (e–h, j). Representative blot (20 μg protein per group) was shown from 3 biologically independent experiments (b), 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) or one-way ANOVA (b, d, e, g, i, k). WT wild type, OD498, optical density (OD) measured at 498 nm, CM conditioned medium; Exo, exosome. Source data are provided as a Source Data file.

Fig. 7. Exosomal miR-365a-5p increases angiogenesis and vascular permeability via inactivating TRPC4AP/Ca²⁺/CaMKII/ERK5/KLF2/4 pathway.

a Predicted target proteins of miR-365a-5p using three publicly available bioinformatic tools. b Western blot analysis of TRPC4AP in vascular endothelial cells transfected with miR-365a-5p mimic or inhibitor. c Schematic illustration of luciferase reporter plasmids for TRPC4AP 3’UTR (upper panel) and relative luciferase activity determined after co-transfection of miRNA mimic and plasmids (lower panel) (n = 3 biological replicates). d Intracellular Ca²⁺ fluorescence signals in vascular endothelial cells transfected with miR-365a-5p mimic or inhibitor (n = 5 biological replicates). Scale bars: 100 μm. e Effects of miR-365a-5p mimic or inhibitor on the activation state of CaMKII/ERK5/KLF2/4 pathway in vascular endothelial cells detected by western blot. The samples derive from the same experiment but different gels for p-CaMKII, another for CaMKII and KLF2, another for p-ERK5 and β-actin, and another for ERK5 and KLF4 were processed in parallel. f Effects of TRPC4AP overexpression on the activation state of CaMKII/ERK5/KLF2/4 pathway and the expression levels of ZO-1, VE-cadherin, and VEGFR2 in vascular endothelial cells transfected with miR-365a-5p mimic detected by western blot. The samples derive from the same experiment but different gels for p-CaMKII and p-ERK5, another for CaMKII, ERK5, and β-actin (left), another for TRPC4AP and VEGFR2, another for ZO-1, VE-cadherin and KLF2, and another for KLF4 and β-actin (right) were processed in parallel. g Effects of KLF4 silence on ZO-1 and VE-cadherin expressions in vascular endothelial cells transfected with miR-365a-5p inhibitor detected by western blot. The samples derive from the same experiment but different gels for ZO-1 and KLF4, and another for VE-cadherin and β-actin were processed in parallel. h Effects of KLF2 silence on VEGFR2 expression in vascular endothelial cells transfected with miR-365a-5p inhibitor detected by western blot. The samples derive from the same experiment but different gels for VEGFR2 and β-actin, and another for KLF2 were processed in parallel. Representative images are presented from indicated biologically independent experiments (d). Representative blot (20 μg protein per group) was shown from 3 biologically independent experiments (b, e–h), and β-actin was used to normalize protein quantification. Data are presented as mean ± SD, and P values were calculated using one-way ANOVA (b–c, e–h). WT, wild type; mut, mutant; Ca²⁺, calcium ions. Source data are provided as a Source Data file.

Fig. 8. Association of COL1^high/LOX^high with let-7d-5p and miR-365a-5p in HCC tissues and their clinical significance.

a FISH images of let-7d-5p and miR-365a-5p expressions in human HCC tissues with COL1^low/LOX^low (low-stiffness group, n = 24 patients) and COL1^high/LOX^high (high-stiffness group, n = 24 patients). Scale bars: white, 200 μm; red, 50 μm. b Quantification of let-7d-5p or miR-365a-5p expression in (a). c Survival curve analysis of HCC patients in the COL1^low/LOX^low group (n = 24 patients) and COL1^high/LOX^high group (n = 24 patients). d Survival curve analysis of HCC patients in the let-7d-5p^low/miR-365a-5p^low group (n = 16 patients) and let-7d-5p^high/miR-365a-5p^high group (n = 16 patients). e Mechanism by which matrix stiffness-tuned exosomal miRNAs modulate glucose enrichment during the formation of the lung pre-metastatic niche in HCC through inhibiting glucose uptake and consumption of lung fibroblasts and enhancing angiogenesis and vascular permeability. Schematic illustration was created in BioRender. Zhao, Y. (2025) https://BioRender.com/s25i400. Specifically, two pathways including matrix stiffness-tuned exosomal let-7d-5p/HMGA2/E2F1 acetylation/GLUT1, PFKP, and HK2 in lung fibroblasts and matrix stiffness-tuned exosomal miR-365a-5p/TRPC4AP/Ca²⁺/CaMKII/ERK5/KLF2/4 in vascular endothelial cells synergistically promote glucose enrichment during lung pre-metastatic niche formation. Representative images are presented from indicated biologically independent experiments (a). Data are presented as mean ± SD (b), and P values were calculated using two-tailed unpaired Student’s t-test (b) or log-rank test (c, d). COL1 collagen I, LOX lysyloxidase, BMDCs bone marrow-derived cells, CTCs circulating tumor cells. Source data are provided as a Source Data file.