Targeting Nuclear Mechanics Mitigates the Fibroblast Invasiveness in Pathological Dermal Scars Induced by Matrix Stiffening

Abstract Pathological dermal scars such as keloids present significant clinical challenges lacking effective treatment options. Given the distinctive feature of highly stiffened scar tissues, deciphering how matrix mechanics regulate pathological progression can inform new therapeutic strategies. Here, it is shown that pathological dermal scar keloid fibroblasts display unique metamorphoses to stiffened matrix. Compared to normal fibroblasts, keloid fibroblasts show high sensitivity to stiffness rather than biochemical stimulation, activating cytoskeletal‐to‐nuclear mechanosensing molecules. Notably, keloid fibroblasts on stiff matrices exhibit nuclear softening, concomitant with reduced lamin A/C expression, and disrupted anchoring of lamina‐associated chromatin. This nuclear softening, combined with weak adhesion and high contractility, facilitates the invasive migration of keloid fibroblasts through confining matrices. Inhibiting lamin A/C‐driven nuclear softening, via lamin A/C overexpression or actin disruption, mitigates such invasiveness of keloid fibroblasts. These findings highlight the significance of the nuclear mechanics of keloid fibroblasts in scar pathogenesis and propose lamin A/C as a potential therapeutic target for managing pathological scars.

) Evaluation of the fibronectin immobilization on the substrates.Representative fluorescence images for substrates modified with HiLyte Fluor TM 488 labeled fibronectin (left) showing the homogenous distribution of fibronectin on the surface (right) (d).Yellow lines indicate the pixel regions used for intensity profiling.Quantification of the fibronectin density based on the fluorescence intensity showing the fibronectin immobilization amount is the same regardless of the substrate stiffness (e, n = 3).*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; two-tailed paired Student's t-test or one-way ANOVA followed by Tukey's post hoc tests.

Fig. S2 .
Fig. S2.Preparation of PDMS substrates.(a) The blending ratios of Sylgard 184 and 527 and corresponding stiffness of PDMS substrates (n = 3).(b) Schematic illustrating the surface modification of PDMS substrates with

Fig. S3 .
Fig. S3.Cell adhesion and spreading in response to the matrix stiffness after 2 days of culture on the substrates.(a, b) Quantitative analysis of cell adhesion number (n = 45 cells/cm 2 ) and spreading area (n = 50-70 cells/condition) of normal fibroblast (NF, a) and keloid fibroblast (KF, b) in response to the stiffness of PDMS substrates.(c) Representative images of F-actin staining (red) of NF and KF cultured either soft (10 kPa) or stiff (2MPa) (left) along with the quantification of cell spreading area (right, n = 50-70 cells/condition).Scale bar, 200 μm.Nuclei are stained with DAPI (blue).Data are representative of at least three independent experiments.*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; two-way ANOVA followed by Tukey's post hoc tests.

Fig. S6 .
Fig. S6.Focal adhesion formation in normal fibroblasts and keloid fibroblasts after 2 days of culture on the substrates.(a) Representative immunostaining for paxillin (green) and (b) quantitative analysis of focal adhesion (FA) formations including the quantification of the number (n = 30 FA number/cell), size (n = 100 FA/condition), and aspect ratio (AR, n = 100 FA/condition) of focal adhesions based on the immunostaining images.Data are representative of at least three independent experiments.Scale bar, 50 μm (main images), and 15 (inserts).Nuclei are stained with DAPI (blue).**p < 0.01 and ****p < 0.0001; two-way ANOVA followed by Tukey's post hoc tests.

Fig. S7 .
Fig. S7.Actin formation in normal fibroblasts and keloid fibroblasts after 2 days of culture on the substrates.(a) Representative images of F-actin staining (red) on the basal and apical planes and (b) quantitative analysis of the F-actin anisotropy, the number, and thickness of F-actin bundles (d, n = 50 -200 cells/condition from 3 independent experiments).Scale bar, 25 µm (main images), and 10 µm (inserts).*p < 0.05 and ****p < 0.0001; two-way ANOVA followed by Tukey's post hoc tests.

Fig. S10 .
Fig. S10.Evaluation of in vitro cell proliferation.NFs or KFs were cultured on either soft or stiff substrates with or without TGF-β1 treatment for 4 days.(a-d) Representative bright-field images of NFs (a) or KFs (c) cultured on soft or stiff substrates with or without TGF-β1 treatment after 1, 2, and 4 days of culture (left) and quantification of the proliferation rate based on CCK-8 assay (b, d, n = 3 replicates/condition from 3 independent experiments).Scale bar, 500 µm.(e, f) Bar graphs representing the proliferation rate at day 2 (e) or day 4 after (f) culture.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns: not significant; two-way ANOVA followed by Tukey's post hoc tests.

Fig. S14 .
Fig. S14.Lamin A/C immunostaining showing its distribution within the nucleus of normal fibroblasts (a) or keloid fibroblasts (b).XZ-and YZ-axes projections on the equatorial plane (EP) of the nucleus (top).Maximum intensity projection (MIP) of a series of z-stacks images covering the entire nuclear region

Fig. S16 .
Fig. S16.Lamin A/C expression in human keloid tissues from three independent donors.(a) Representative images of immunohistochemical staining for lamin A/C in human keloid tissues.Scale bar, 2 mm (0.2 X), 40 μm (10 X), and 10 μm (40 X).(b) The nuclear area (n = 100 -144 nuclei/condition), contour ratio (n = 100 -136 nuclei/condition), and lamin A/C intensity (n = 100 -135 nuclei/condition) of nuclei quantified based on the immunostaining.(c) Correlation plots depicting the relationship between lamin A/C intensity and contour ratio.The correlation coefficient (Pearson's r) was determined by linear fits as in (c, dash line for Soft, and solid line for Stiff).(d) Representative images of co-immunostaining for lamin A/C (green)

Fig
Fig. S18.Nuclear deformability analysis by the microfluidic micropipette aspiration assay.(a) Representative time-lapse image series of the deformation of fluorescently labeled cell nuclei in the microfluidic micropipette aspiration device.Scale bar, 10 µm.NFs, KFs, or KFs transfected with mCherry-LMNA plasmid (OE) were pre-cultured on either soft or stiff substrates for 2 days before the assay.(b) The relative proportions of populations depending on nuclear deformability.(c) The nuclear deformation rate was measured based on the protrusion length upon aspiration (n > 30 nuclei/condition).(d, e) Nuclear protrusion profiles of NF (d) and KF (e) for 180 sec of aspiration (d, n = 22 -45 nuclei/condition).The slope (m)

Fig. S20 .
Fig. S20.Scratch-based planar migration assays.Cells were seeded on either soft or stiff substrates and the migration was monitored with or without TGF-1 treatment for 24 hours.(a) Pseudo-colored cell migration tracking from a representative time-lapse movie and (b) quantification (n = 3 replicates from 3 independent experiments).Scale bar, 200 μm.*p < 0.05 and **p < 0.01; two-way ANOVA followed by Tukey's post hoc tests.