Epithelial monolayers exhibit higher cell extrusion rates in negatively curved valleys of hemi-cylindrical wave substrates.

(A-C) SEM images of cylindrical wave structures with halfperiods 200, 100 and 50 μm. Scale-bar: 100 μm. (D-F) Phase contrast images of confluent MDCK monolayers on 200, 100 and 50 μm waves, 24 hours after seeding. (G) Time-lapse excerpts demonstrating extrusion event registration (cyan objects) using our trained neural network. (H) Boxplot of extrusion events registered over 24 hours, on 200, 100 and 50 μm cylindrical waves, and on 200, 100 and 50 μm rectangular waves for comparison. Box shows the interquartile range (IQR) while whiskers are 1.5×IQR. Detailed statistics can be found in table S1. (I) Multi-channel time-lapse excerpts of cells incubated with activated caspase 3/7 reporter (false green over phase contrast), on flat, hill and valley regions of a 100 μm wave: fluorescence indicate dying cells. Scale-bars (unless otherwise stated): 50 μm.

Osmosis induced basal hydraulic stress is linked to cell extrusions.

(A) Confluent MDCK II monolayers form fluid-filled domes (arrows) in wave valleys when cultured for over 48 hours. Scale-bar: 100 μm. (B) Boxplot of extrusions per cell over 24 hours on 100 μm cylindrical waves, with monolayers subjected to osmolarity perturbations that included the addition of 4.1 wt. % sucrose, 1 % DMSO, 0.4 wt. % NaCl, 25 % water, and 25 % PBS. Box shows the interquartile range (IQR) while whiskers are 1.5×IQR. Detailed statistics can be found in table S3. (C) Bright-field/RICM time-lapse excepts showing dynamic basal fluid spaces whose motion (direction encoded colored paths) corresponded with the direction of focal adhesion (dark streaks e.g. indicated by white arrow) disassembly. (D) Bright-field/RICM time-lapse excepts showing the accumulation of basal fluid (asterisks) when iso-osmolarity was reinstated (at 180 min) in hyper-osmotically pre-conditioned monolayers. (E) Plot of the image histograms against time showing an increase in grey values as iso-osmolarity was restored, indicating a general increase in basal-to-substrate separations with decreasing apical media osmolarity. (F) Boxplot of histogram median grey values from (E) averaged separately over the duration of hyper- and iso-osmolarity treatments. Detailed statistics can be found in table S4. (G) Representative max-projected RICM z-stacks of flat, hill and valley regions. (H) Boxplot of histogram median grey values from images such as in (G). Detailed statistics can be found in table S4. Scale-bars in (C), (D), (G): 20 μm.

Solute permeable hydrogel substrates reduce epithelial cell extrusions, and surface curvature induces symmetry breaking in collective cellular forces.

(A) Time-lapse excerpts showing cell extrusion accumulation on stiff PDMS (control), and on soft silicone and PAM hydrogel of similar stiffness (scale bar: 50 μm). (B) Boxplot showing the cell extrusion rates from the 3 substrates in (A). Box shows the interquartile range (IQR) while whiskers are 1.5 ×IQR. Detailed statistics can be found in table S6. (C) Nuclei fluorescence cross-section shows deformation against surface on wave hills (scale bar: 10 μm). (D) 3D force microscopy reconstructed monolayer normal force distribution on a 100 μm wave, with wave profile shown below. (E) Graph showing the bootstrapped magnitude of calculated forces along the curved profile (purple shaded region). (F) Mean bootstrapped normal force vectors along the curved profile (purple shaded region). In (E) and (F), bootstrapping was performed with 10000 re-samples and the 95 % confidence interval is indicated in the shaded region for each respective color.

Modulation of basal hydraulic stress through media osmolarity and substrate solute permeability regulate cell extrusion via FAK-Akt pathway.

(A) Cell extrusion rates on 100 μm waves in normal, sucrose, and sucrose + 3 μM FAKI14 media. (B) Adding 6 μM FAKI14 leads to cell death that compromised monolayers. Scale-bar: 50 μm. (C) Immunoblots of FAK and Akt proteins in MDCK cells subjected to treatments that lead to varying basal hydraulic stresses for 24 hours; namely, iso-osmotic (control), hyper- (4.1 wt. % sucrose), hypo- (25 % water), iso-osmotic with soft PDMS and iso-osmotic with water/solute permeable PAM hydrogel. (D) and (E) Quantification of the relative expression levels of phosphorylated-FAK (tyr397) as a ratio of p-FAK to total FAK and phosphorylated-Akt (Ser473) as a ratio of p-Akt to total Akt, respectively. GAPDH was used as loading control (M ± SD, n = 5). (F) fluorescence images of cell nuclei (false blue), FAK (false red), and p-FAK at tyr397 (false green) on flat, hill and valley; the hill and valley images were unwrapped from 3D stacks. Scale-bar: 20 μm. (G) normalized p-FAK/FAK intensities from hill and valley cells. Statistical analysis in table S8. (H) and (I) Schematic of how osmolarity affect basal hydraulic stress and cell survival. (J) Schematic of how curvature induced force differences promote cell survival and death.

Schematic diagram showing the steps for microfabricating smooth periodic hemi-cylindrical wave substrates using glass rods and iterative molding.

Demonstration of achieving dimensions smaller than commercially available glass rods using an iterative stretching and molding method.

a, a stretchy silicone with wave pattern was held stretched for optical curable resin casting. b, a subsequent stretchy silicone was molded against the new resin template created from a; the whole process was repeated until the desired reduction in dimension was reached.

Dimensional characterization of cylindrical and rectangular waves through fluorescent collagen I z-stacks.

a-c, 50, 100, and 200 μm cylindrical waves. d-f, 50, 100, 200 μm rectangular waves. Scale-bars = 50 μm. g and h, dimensions measured: curvature (Xp and Vp); width (Xw and Vw); and height (H). Values tabulated in table S1.

Analysis of cell density 24 hours post seeding.

a-c, surface unwrapped fluorescent z-stacks of Hoechst stained MDCKs on 200, 100 and 50 μm waves. Hills: cyan boxes and Valleys: magenta boxes. Scale-bars: 50 μm. d, boxplot of overall cell densities, determined from nuclei staining, across wave dimensions at the initial time point. e, cell densities in d plotted according to curvature type.

Calculation of the normalized extrusion rate.

a, architecture of the attention-gated residual U-Net. b, example of a manually annotated extrusion event using the sequence of 5 timeframes above and the ground truth binary mask below. c confusion matrix of the machine learning network. d, Left: bright-field image; Center: prediction from StarDist; Right: overlay of the prediction outline on the bright-field image. Scale-bars: 50 μm.

3D force microscopy.

a-c, schematic for the preparation of the gel as well as the cell seeding process. d, 3D-view showing the even distribution of fluorescent beads in the polyacrylamide gel. e, bright field image showing the cut get and the monolayer of cells on the curved substrate. Scale-bar: 500 μm. f, bright field images showing the removal of cells through the addition of 1 % SDS. g, maximum projected side profile of the polyacrylamide gel before (red) and after SDS treatment (green). h, Qualitative validation of the calculated displacements. Left: original fluorescent image showing the bead locations with cells (red) and without cells (green). Right: overlay of the mapped image (green) and the bead image with cells (red). i, 3D-view of the calculated displacements obtain using the two-frame 3D Farneback optical flow method. j, Representative 3D structure and mesh used in the finite element modelling of the polyacrylamide structure. Image used courtesy of ANSYS, Inc. k, 3D-view of the average nodal force obtain by solving the inverse problem. Scale-bars: 50 μm unless otherwise stated.

Table of cylindrical and rectangular wave dimensions.

Tables of statistical analysis on varying structure sizes and curvatures.

a, 2-way ANOVA analysis to determine if curvature and size of structure have an effect on the normalized extrusion rates. b, Post hoc t-test statistics for the comparisons made with regard to the different sizes of structures.

Tables of statistical analysis on varying media osmolarities.

a, 2-way ANOVA analysis to determine if osmolarity and curvature have an effect on the normalized extrusion rates. b, 1-way ANOVA of the simple effects on the normalized extrusion rates. c, Post hoc t-test statistics for the comparisons made with regard to the various experimental conditions.

Tables of statistical analysis of median RICM intensities subjected to osmolarity perturbations and over curvature types.

a, Two-tailed paired student’s t-test and effect size between the median RICM intensities over hyperand iso-osmotic treatment timeframes. b, 1-way ANOVA of median RICM intensities over different curvature types, and Tukey HSD post hoc test statistics for the curvature type comparisons along with the effect size calculated for the significant pair (hill-valley).

Tables of statistical analysis on varying the substrate permeability.

a, 1-way ANOVA of varying the substrate on the normalized extrusion rates. b, Post hoc t-test statistics for the comparisons made.

Tables of statistical analysis on the addition of FAKI14 inhibitor.

a, 2-way ANOVA analysis to determine if addition of FAKI14 and curvature have an effect on the normalized extrusion rates. b, 1-way ANOVA of the simple effects on the normalized extrusion rates. c, Post hoc t-test statistics for the comparisons made with regard to the various experimental conditions.

Dilution factors, incubation duration and manufacturer’s catalogue numbers for immunoblotting.

Statistical analysis of p-FAK/FAK fluorescence intensity ratios over hills and valleys.