Fig. 7: Matrix organisation drives a pro-metastatic transcriptional program.

a Mouse melanoma model showing intradermal injection of 4599 cells leading to lung metastasis. Cells isolated from different tumour areas (TB, PIF, DIF, Metastasis) for ex vivo experiments and RNA-seq. b Principal component analysis of 4599 cells’ transcriptome from different tumour regions. n = 3 independent experiments. c Gene overrepresentation analysis comparing PIF, DIF, and MET versus TB, grouped by ECM, Mechano-sensing, Cytoskeleton, and Angiogenesis signatures. d Volcano plots of transcription factors in PIF versus TB and DIF versus TB. Threshold: -0.5<Log2FC < 0.5, -Log10 p-adjusted value. e GSEA analysis of inflammatory pathways, comparing PIF, DIF, and Met to TB. Dotplot shows normalized enrichment scores (FDR < 0.1). f–h Immunoblots showing YAP, p-p65, p65, p-STAT3, STAT3, pp-MLC2, and MLC2 protein levels in 4599 cells from different tumour areas (n = 5 independent experiments), collagen concentrations (n = 4), and aligned/non-aligned matrix (n = 3). GAPDH is used as loading control. Quantification in Supplementary Fig. 7g. The samples for each panel derive from the same experiment but different gels for STAT3, MLC2 and GAPDH, another for p-STAT3^Y705, pp-MLC2^T19/S19, another for p65, another for p-p65^s536, and another YAP were processed in parallel. i Top, diagram of experimental setting showing priming in ± MgCl₂ (7 days), and further culture in plastic (6 days). Bottom, corresponding p-STAT3/STAT3 protein levels in 4599 cells and quantification. GAPDH is used as loading control. n = 3 independent experiments. The samples derive from the same experiment but different gels for p-STAT3^Y705 and GAPDH, and another for STAT3 were processed in parallel. j Left, protein levels of p-STAT3/STAT3 in HT1080 cells after YAP or NF-κB knockdown with PI treatment (24 h). GAPDH is used as loading control. Corresponding quantification of p-STAT3/STAT3. n = 3 independent experiments. The samples derive from the same experiment but different gels for p-STAT3^Y705 and GAPDH, another for STAT3, another for YAP1 and another for NF-Ƙβ1 were processed in parallel. k Top, confocal images showing F-actin, pMLC2, and nuclei after PI treatment with siYAP1, siNF-κB1, or siSTAT3. Bottom, corresponding cell rounding index and pMLC2/cell area quantification. Scale bar: 10 µm. n = 91, 97, 91, 91 and 92 cells, three independent experiments. l Multiplex immunohistochemistry of YAP (red), p65 (green), p-STAT3 (magenta) in 4599 primary tumours and lung metastases. Scale bars: 200 µm (primary, macro), 50 µm (micro, insets). m Nuclear H-score quantification (0–300) of transcription factors across tumour regions from (l). n = 11/group. One-way ANOVA with Šídák (I, j, k) and Tukey (m) post-hoc test. Data displayed as violin plots showing individual data points and medians (k, m), bar plot with mean and SD (I, j). Cartoons in Fig. 7 (a, f, h, i, j, l) created with BioRender.com. created with BioRender Maiques, O. (2025) https://BioRender.com/z57v040https://BioRender.com/f02u038.