Fig 2: The dysadherin/MMP9 axis facilitates metastasis potential by shaping the protumor microenvironment.a Representative in vivo bioluminescence images of mice injected with luciferase-labeled wild-type or dysadherin KO SW480 cells with/without MMP9 OE, accompanied by a corresponding graph showing the quantitative analysis of the region of interest. Middle: Representative hematoxylin and eosin-stained livers with metastasis (n = 5/group). b In situ zymography analysis and IF analysis of dysadherin, α-SMA, and collagen I expression in mouse liver tissue from metastatic tumors. c Heatmap comparing the relative expression of ECM deposition factors in metastatic tumors in the mouse liver (n = 5/group). d Heatmap comparing the relative expression of macrophage polarization and T-cell related markers in metastatic tumors in the mouse liver (n = 5/group). e Levels of IL-4 and IFN-γ in metastatic tumors, from ELISA data (n = 3/group). f Quantitative analysis of CD8 IF data from mouse livers with metastatic tumors (n = 5/group). g Heatmap comparing the relative expression of angiogenesis-related factors in metastatic tumors in the mouse liver (n = 5/group). h Quantitative analysis of CD31 IF data from mouse livers with metastatic tumors (n = 5/group). i Schematic summary of the study findings indicating the potential role of dysadherin in cancer cells in promoting ECM remodeling and CAF activation, which contributes to the formation of a malignant TME. BioRender software was used to create the figure under an academic license. The data are presented as means ± SEMs. *, **, and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test for comparisons among four groups. Source data are provided as a Source Data file.

Fig 3: Dysadherin enhances MMP9 expression via the FAK/c-JUN axis.a GSEA was performed using the mRNA-sequencing profiles of dysadherin KO and WT SW480 cells, which was performed in previous study24. b Three candidate genes related to three gene signatures were identified. Heatmap indicated FC and p-value (unpaired two-tailed Student’s t tests) from mRNA-sequencing data. c Downstream analysis indicating the potential link between dysadherin/FAK pathway and MMP9, leading to ECM remodeling, malignancy, and metastasis. d Kaplan-Meier analysis of CRC patients by dividing into three groups according to dysadherin and MMP9 expression. Statistical significance was determined by log-rank tests. e Immunoblotting for dysadherin, E-cadherin, and MMP9 expression and gel zymography in human CRC and normal colon cell lines. f Immunoblotting for dysadherin and MMP9 and gel zymography in dysadherin KO or OE CRC cells. g Venn diagram showing overlapping transcription factors that are positively correlated with MMP9 expression. h Immunoblotting for p-FAK, total FAK, p-c-JUN, total c-JUN, and MMP9 in EV and dysadherin OE HCT116 cells treated with or without 1 µM PND-1186 or 20 µM T-5224. i Promoter activity of MMP9 was tested via luciferase reporter assay. MMP9 promoter region-containing vector was transfected into EV and dysadherin OE HCT116 cells, and cells were treated with or without PND-1186 or T-5224. Total transcription was normalized to β-galactosidase transcription and is presented as fold change with respect to nontreated HCT116 cells. j Binding affinity of c-JUN on MMP9 promoter with or without PND-1186 or T-5224 in SW480 cells was tested via ChIP assay. i, j n = 3 biological replicates, representative of three independent experiments with similar results. Immunoblot assays were independently repeated three times with similar results. The data are presented as the means ± SEMs. *, **, and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test for comparisons among three groups. Source data are provided as a Source Data file. FC fold change, P paired normal, T tumor, WT wild-type, KO knockout, EV empty vector, OE overexpression, Ct threshold of cycle, dCt delta Ct, difference of Ct value between target and housekeeping gene.

Fig 4: The dysadherin/MMP9 axis increases proteolytic activity, invasive potential and CAF activation in the ECM. In situ zymography analysis (a), 3D invasion assay (b), and anchorage-independent growth assay (c) of Tet-pLKO-shMMP9 transfected EV and dysadherin OE HCT116 cells with or without doxycycline (n = 3 biological replicates, representative of three independent experiments with similar results). d Heatmap comparing the relative expression of factors related to collagens, ECM modification, and tumor promotion in MRC-5 cells cultured with culture medium (CM) from control or dysadherin OE HCT116 cells transfected with Tet-shMMP9 (n = 3 biological replicates). e IF analysis of α-SMA and collagen I expression in MRC-5 cells cultured with CM. The corresponding quantitation of 5 replicates is shown below (n = 5 biological replicates, representative of three independent experiments with similar results). f Immunoblot analyses of the expression of the α-SMA and fibroblast activation protein (FAP) as CAF markers, collagen I, p-SMAD2/3 and total SMAD2/3 in MRC-5 cells cultured with CM. Immunoblot assay was independently repeated three times with similar results. The data are presented as means ± SEMs. *, **, and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test for comparisons among six or four groups. Source data are provided as a Source Data file.