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

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

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

Fig. 1

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.

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