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.