Fig. 5: Collagen I accumulation and radial alignment support Myosin II driven invasive and metastatic behaviour.
a Schematic showing how MgCl2 affects collagen matrix alignment, illustrated in side and top views. b Collagen reflectance images comparing H2O and MgCl2 treatment, showing reflectance and CurveAlign matrix alignment maps. Scale bar: 20 µm. c Matrix analysis quantifying fibre parameters (length, width, straightness, distance, alignment score, fibre number/mm²). n = 150 and 132 images, three independent experiments. d Angle deviation histogram comparing control versus MgCl2-treated collagen fibres (n = 30 samples per group). e AFM elasticity (Pa) measurements of control and MgCl2-treated collagen. n = 15 per group, two independent experiments. f Reflectance images with gap analysis maps. Scale bar: 20 µm. g Gap analysis quantification comparing control and MgCl2 collagen fibres. n = 6941 and 6125 gaps analysed, three independent experiments. h Immunofluorescence of F-actin (magenta) and pMLC2 (green) in 4599 cells ±MgCl2. Nuclei in blue. Scale bar: 20 µm. i Cell rounding index and pMLC2/cell area quantification in 4599 cells ±MgCl2. n = 140 and 160 cells, three independent experiments. j Spheroid assay showing 4599 cell invasion length in non-aligned (H2O) /aligned (MgCl2) matrices. Scale bar: 200 µm. k Area coverage quantification from 4599 tumour spheroids at 900 µm from spheroid core comparing non-aligned (H2O) /aligned (MgCl2) matrices. n = 4 independent experiments. l Diagram of 3D invasion assay schematic using educated 4599 cells in non-aligned (H2O) /aligned (MgCl2) matrices. m Western blot of ppMLC2 and MLC2 in primed cells. n = 3 independent experiments. The samples derive from the same experiment but different gels for pp-MLC2T19/S19 and GAPDH, and another for MLC2 were processed in parallel. n 3D invasion images at 0 µm and 50 µm with quantification. Scale bar: 100 µm. n = 3 independent experiments. o Diagram of in vivo lung invasion assay schematic using pre-educated 4599 cells. p Lung parenchyma representative image at 48 h post-injection. Scale bar: 100 µm. ROI quantification at 30 min and 48 h. n = 14 mice, 2 independent experiments. Unpaired t-test (c, i, k, n) and Mann Whitney test (e, f) between -/+ conditions. One-way ANOVA with Šídák post-hoc test (m). Data is displayed as violin plots showing individual data points and medians (c, e, i), boxplots showing median, interquartile range and individual data points (k, n), Bar plot with Mean and SD (m, p), and histogram in (d). Cartoons in Fig. 5(a, h, l, o) created in BioRender. Maiques, O. (2025) https://BioRender.com/z35l297.
