Fig. 1: TYK2 blockade in mammary acini promotes EMT and invasion under normal mammary tissue stiffness.

a–c Human MCF10A acini grown in three-dimensional polyacrylamide (3D-PA) hydrogels were treated for 5 days with momelotinib (JAK1/JAK2/TYK2 inhibitor), filgotinib (JAK1/2 inhibitor), or vehicle (DMSO) at the indicated concentrations. Representative bright-field images (a) and quantification of invasive structures (fraction of total) after momelotinib (20 nM, 100 nM) (b) or filgotinib (20 nM, 100 nM) (c) treatment (n = 6 wells per group, three independent experiments). d–g Control or TYK2-silenced MCF10A acini grown on 3D-PA gels for 5 days. Representative bright-field images (d), quantification of invasive structure (e) (n ≥ 6 wells per group, three independent experiments), immunofluorescence staining for E-cadherin (green), fibronectin (red), and DAPI (blue), representative images from 3 independent experiments (f), and immunoblot analysis of E-cadherin, vimentin, and TYK2 with GAPDH as loading control, representative of two independent experiments (g). h, i Control or TYK2-silenced mouse Eph4Ras acini grown on 3D-PA gels for 5 days. Representative bright-field images (h) and quantification of invasive structures (i) (n = 6 wells per group, three independent experiments). j Schematic of patient-derived organoid isolation and three-dimensional culture of PDX-derived organoids in PA hydrogels of defined stiffness (Created in BioRender. https://BioRender.com/dqo40p6). k–m Control or TYK2-silenced PDX-derived organoids (PIM025) grown on 3D-PA gels for 5 days: qPCR analysis of TYK2 mRNA expression, n = 5 wells per group, three independent experiments (k), representative bright-field images after 5 days on 3D-PA gels (l), and quantification of invasive structures (m) (n = 5 wells per group, three independent experiments). Invasive structures were scored using predefined morphological criteria (protrusive, stellate/branching outgrowth) and expressed as a fraction of total acini/organoids per sample. Data are mean ± SEM, dots represent independent wells. Where indicated, quantification is shown for different wells from one representative experiment, and similar results were obtained in independent replicate experiments (see n and number of independent experiments stated for each panel), ****p < 0.0001; ***p < 0.001; *p < 0.05; ns, not significant. Two-group comparisons used unpaired two-tailed Student’s t-test; multiple comparisons used one-way ANOVA with Dunnett’s multiple-comparisons test. Scale bars: 100 μm (a, d, h, l), 25 μm (f). Exact P values and source data are provided in the Source Data file.