Tissue fluidification promotes a cGAS-STING cytosolic DNA response in invasive breast cancer

Abstract

The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.

Fig. 1. Tissue fluidification induces a CytoDR gene signature.

a, Volcano plot of differentially expressed genes (DEG) in control and RAB5A-expressing MCF10.DCIS monolayers. All significantly RAB5A-expressing deregulated genes are in red (upregulated) and blue (downregulated). Enrichment (log₂(fold change)) is plotted on the x-axis and significance (Wald test –log₁₀(p-value two-sided)) is plotted on the y-axis. Labels are the most upregulated genes. Outlined in red are interferon-stimulated genes. b, GSEA of DEGs in RAB5A-expressing MCF10.DCIS.com monolayer over control cells. GSEA was performed using the Hallmarks pathway gene sets in the GSEA Molecular Signatures Database. Moderated t-statistic was used to rank the genes. Significantly enriched pathways are reported (one-sided P < 0.05) with the bar outline colour corresponding to the BH-adjusted P value. P values were calculated as the number of random genes with the same or more extreme enrichment score (ES) value divided by the total number of generated gene sets. c, GSEA enrichment plot of differentially expressed genes in RAB5A-expressing MCF10.DCIS.com monolayer using the KEGG (Kyoto Encyclopedia of Genes and Genomes) cytosolic DNA-sensing pathway (hsa04623). The green curve corresponds to the ES curve, which is the running sum of the weighted ES obtained from GSEA software, while the normalized ES (NES) and the corresponding one-sided P value are reported within the graph. d, Scatter plots of mRNA expression levels of IFI27, IFI44, IFI44L, IFI6, IFIT1, IFIT3, ISG15, MX2 and OASL determined by qRT-PCR in RAB5A-expressing MCF10.DCIS monolayers relative to control cells. Data are expressed as log₁₀ values, horizontal lines represent group medians. Each dot represents an independent experiment. Values were normalized to the controls of each experiment. e, Immunoblots of lysates from control (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com monolayers with the indicated antibodies (n = 3 independent experiments). M_w is indicated on the left. Source data

Fig. 2. A cGAS–STING pathway mediates tissue fluidification-dependent CytoDR genes.

a, Heatmap of CytoDR DEGs in RAB5A-MCF10.DCIS.com monolayers silenced for the indicated genes (Supplementary Fig. 1a). Data are the ratio of gene expression of each condition relative to mock-scramble-oligos-treated RAB5A-cells. The mean ± s.d. (n = 3 experiments); each-pair two-tailed Student’s t-test are in Source Data Fig. 2a. b, Heatmap of CytoDR DEGs in RAB5A-MCF10.DCIS.com monolayers treated with cGAS inhibitor RU.521(7 μg ml^–1), or STING antagonist, H-151 (4 μg ml^–1), or TBK1/IKK inhibitor, MRT67307 (20 μM). Data are the ratio of gene expression in each condition relative to vehicle-treated RAB5A-cells. The mean ± s.d. (n = 3 independent experiments), each-pair two-tailed Student’s t-test are in Source Data Fig. 2b. c, Heatmap of CytoDR DEGs in RAB5A-MCF10.DCIS.com monolayers silenced for the indicated genes. The data are the ratio of gene expression in each of conditions relative mock-scramble-oligos-treated RAB5A-cells. The mean ± s.d. (n = 7 experiments), each-pair two-tailed Student’s t-test are in Source Data Fig. 2c. d, Immunoblots of control- (CTR) and RAB5A-MCF10.DCIS.com monolayers with the indicated antibodies (n = 3 independent experiments). e, Still images of cell contours are indicated by pseudo-colouring of EGFP-CDH1, control- (CTR) and RAB5A-MCF10A cells (Supplementary Video 4). Scale bar, 10 μm. f, Scheme depicting cell maximum positive deformation (MPD) and negative deformation (MND). g, Data are the mean ± s.d. of MPD and MND (80 cells per condition in n = 4 independent experiments). Two-tailed Mann–Whitney non-parametric test. h, Consecutive frames (top-left, top-right and bottom-left subpanels) of the same RAB5A-expressing MCF10A nucleus (Supplementary Video 5). Continuous lines with different shades of red represent the profiles obtained via nuclear segmentations. In the bottom-right supanel is reported a superposition of the three profiles shown in the other subpanels. Scale bar, 5 μm. i, Comparison of the nuclear mean square strain (MSS) of control and RAB5A-MCF10A (left panel) or MCF10.DCIS.com (right panel) cell monolayers. The MSS is obtained by tracking and segmenting N nuclei over the time window 4–20 h (N > 5,000 and N > 1,000 for control- and RAB5A-MCF10A monolayers, respectively; >N700 and >N400 for control- and RAB5A-MCF10.DCIS.com monolayers, respectively). Continuous lines are best fitting curves to the data with an exponential model. Insets report the nuclear strain rate as mean ± s.d. (n = 10 randomly populated subsets of cells), two-tailed t-test. j, Levels of LMNA, LMNB1 and RAB5A mRNA in RAB5A- and control-MCF10.DCIS.com monolayers. The data are the mean (n = 7 for LMNA, n = 25 for LMNB1 n = 25 for RAB5A). Two-tailed Mann–Whitney non-parametric test. k, Immunoblots of control- and RAB5A-MCF10.DCIS.com monolayers with the indicated antibodies (n = 3 independent experiments). l. Scatter plot of the expression level of Lamin A/C and Lamin B1 in control and RAB5A-expressing MCF10.DCIS.com monolayers. Data are mean ± s.d. of the integrated density/cell measured in different FOV in n = 3 independent experiments, unpaired two-tailed t-test with Welch’s correction. P values are indicated in each graph. Source data

Fig. 3. Nuclear envelope ruptures activate cGAS in fluidized monolayers.

a, Immunofluorescence images of doxycycline-treated, EGFP-cGAS control (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com cells (n = 4 experiments) seeded either sparsely or as confluent monolayers. Scale bar, 10 μm. Red arrow point to EGFP-cGAS perinuclear foci b, Scatter plot of the percentage of MCF10.DCIS.com cells with perinuclear cGAS enrichment is expressed as the mean ± s.d. (>15 FOV per experimental condition in n = 4 independent experiments), unpaired two-tailed t-test with Welch’s correction. c, Quantification of cGAMP levels by ELISA from control (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com cell extracts. Data are the cGAMP amounts (ng) per mg of total cell extract expressed as the mean ± s.d. (n = 8 experiments), two-tailed Mann–Whitney non-parametric test. d, Snapshot of time-lapse (Supplementary Video 6) EGFP-3NLS-expressing control (CTR) and RAB5A MCF10.DCIS.com monolayers (n = 2 independent experiments), displaying events of NE rupture and EGFP-3NLS leakage (red arrowheads). Scale bar, 10 μm. e, Scatter plot of the number of nuclear envelope rupture events per FOV reported as mean ± s.d. (10 FOV per experimental conditions in n = 2 independent experiments), two-tailed Mann–Whitney non-parametric test. f, CLEM analysis of cGAS perinuclear foci. RAB5A-expressing MCF10.DCIS.com monolayers transfected with EGFP-cGAS were plated on MaTek dishes with grids. Cells identified on grids by confocal microscopy were processed for electron microscopy and z-axis serial sections were stained with gold-labelled anti-GFP antibody to detect EGFP-cGAS (right). Dashed boxes indicate regions that were progressively magnified in EM. The bottom images show a 3D tomographic reconstruction (left) and 3D models of an NE rupture site (right panels). Arrowheads indicate the site of NE rupture (n = 2 independent experiments). Source data

Fig. 4. Tissue fluidification induces nuclear stiffness, heterochromatin reorganization and actin remodelling.

a, Left: map of the divergence of the velocity field from PIV in RAB5A-MCF10A monolayer. Cold (warm) colours indicate negative (positive) values of divergence. Right: magnified view of a smaller portion (70 × 70 μm) centred on a segmented nucleus (red outline). The velocity field (green arrows) converges to the central cell, corresponding to a local negative value of divergence and compressive deformation. Scale bar, 70 μm. b,c, Root mean square value σ_∇ of the divergence of the velocity field from PIV MCF10A (b) and MCF10.DCIS.com (c) monolayers. Different points correspond to different FOVs, each one corresponding to ~1.2 × 10⁴ and ~8 × 10³ cells for MCF10A and MCF10.DCIS.com samples respectively. Black lines are the averages ± s.d. two-tailed t-test. d,e, Nuclear strain rate ${\stackrel{°}{\gamma }}_N$ as a function of the corresponding cell strain rate ${\stackrel{°}{\gamma }}_C$ for MCF10A (d) and MCF10.DCIS.com (e) monolayers. ${\stackrel{°}{\gamma }}_N$ is obtained from nuclear segmentation, while ${\stackrel{°}{\gamma }}_C$ is estimated from the divergence of the velocity field. Data are grouped into evenly spaced bins along the horizontal axis. Symbols and error bars are the mean and standard deviation of the ${\stackrel{°}{\gamma }}_N$-values in each bin, respectively. Straight lines are best fitting curves with a linear model ${\stackrel{°}{\gamma }}_N=s{\stackrel{°}{\gamma }}_C$. Insets: the ratio between the effective elastic moduli E_N/E_CY of the nucleus and the cytoplasm reported as mean ± s.d. obtained as the slope of best fitting line to the data in the main panel (n = 10 randomly populated subsets of cells), two-tailed t-test. f, Immunofluorescence images of control (CTR) and RAB5A MCF10.DCIS.com monolayers (n = 3 experiments), stained with DAPI and anti-H3K27me3-antibody. Magnified images are shown. Scale bar, 10 μm. g, Relative H3K27me3 intensity of control (CTR) and RAB5A MCF10.DCIS.com monolayers silenced or not for EZH2 or SUZ12. Each dot represents a cell, and the median is indicated (>1,400 cells per experimental condition for CTR and RAB5A, >400 cells per experimental condition in n = 3 independent experiments for siRNA-treated conditions), Kruskal–Wallis/Dunn’s test. h, Ratio of H3K27me3 intensity of the nuclear central region over the periphery in control-(CTR) and RAB5A-(RAB5A)-MCF10.DCIS.com cells. Each dot is a cell and the mean ± s.d. is indicated. (>1,000 cells per experimental conditions in n = 3 experiments), two-tailed Mann–Whitney non-parametric test. i, Immunofluorescence images of control (CTR) and RAB5A MCF10.DCIS.com monolayers (n = 3 experiments), silenced for EZH2 or SUZ12 and stained with DAPI and anti-H3K27me3-antibody. Scale bar, 10 μm. j, Immunofluorescence images of control (CTR) and RAB5A MCF10.DCIS.com monolayers (n = 2 experiments), stained with phalloidin to detect F-actin. Scale bar, 20 μm. k, Percentage of cells with actin rings per FOV expressed as mean ± s.d (dots represent seven FOVs in n = 2 independent experiments), two-tailed Mann–Whitney non-parametric test. Source data

Fig. 5. Endocytic-dependent tissue fluidification results in DNA damage.

a,b, Images of control and RAB5A-MCF10.DCIS.com monolayers (n = 3 experiments), stained with the indicated antibodies. Scale bar, 20 μm. c, The scatter plot shows the mean of 53BP1 foci per nuclei or γH2AX foci per nuclei (>150 cells in n = 3 experiments), two-tailed Mann–Whitney non-parametric test. d, Representative images of neutral comet assay in MCF10.DCIS.com monolayer cells expressing RAB5A. Control cells irradiated (IR 5 Gy) or not (CTR) are also shown. Scale bar, 100 μm. e, Quantification of DNA damage by tail moment analysis. Horizontal bars indicate the means and s.d. of from n = 2 independent experiments; >100 cells per sample were scored, one-way ANOVA. Source data

Fig. 6. Increased RAB5A-expression, cGAS perinuclear accumulation and DNA damage in human invasive ductal carcinoma.

a, Immunofluorescence images of control (CTR) and RAB5A MCF10.DCIS.com cells injected into mammary fat pads of immunocompromised mice. After one week, mice were fed doxycycline to induce RAB5A expression. Four weeks after doxycycline treatment, primary tumours were co-stained with DAPI and γH2AX, or DAPI and RAB5A. Scale bar, 100 μm. b, Percentage of γH2AX-positive cells per FOV. Data are mean ± s.d. (n = 2 experiments, with five mice per experiment), unpaired two-tailed t-test with Welch’s correction. c, Immunohistochemical analysis of cGAS in control (CTR) and RAB5A MCF10.DCIS.com cells injected into mammary fat pads of immunocompromised mice and treated as in a. Primary tumours were stained with an anti-cGAS antibody. Right panel: a higher magnification of the boxed region to highlight cGAS accumulation in perinuclear dots (black arrowheads) or crescent-like distribution (blue arrowhead). Scale bar 80 μm. d, The percentage of cells with perinuclear cGAS enrichment is expressed as the mean ± s.d. (each dot represents an FOV out of >5 FOVs per experimental condition in n = 2 independent experiments), two-tailed Mann–Whitney non-parametric test. e, Multiplex immunohistochemistry/Immunofluorescence (mIHC/IF) of RAB5A, γH2AX and DAPI in human DCIS. Magnified images from the selected yellow boxes are shown. Scale bar, 150 μm. f, The percentage of γH2AX-positive nuclei in cells that express high (>2 on a scale from 0,1,2,3) or low (<2 on a scale from 0,1,2,3) levels of RAB5A in the tumour bulk or margin (see Methods for details and Supplementary Video 13). The areas of n = 9 independent DCIS were analysed with more than 200 nuclei per area, two-tailed Mann–Whitney non-parametric test. g, Multiplex immunohistochemistry/immunofluorescence (mIHC/IF) of RAB5A, cGAS, and Dapi in human DCIS. Magnified images from the selected yellow boxes are shown. Scale bar, 300 μm. h, The percentage of cGAS-positive cells that express high (>2 on a scale from 0,1,2,3) or low (<2 on a scale from 0,1,2,3) levels of RAB5A in the tumour bulk, or the margin or the whole tumour (Methods). The areas of n = 9 independent DCIS were analysed with more than 200 nuclei per area, two-tailed Mann–Whitney non-parametric test and unpaired two-tailed t-test with Welch’s correction. Source data

Extended Data Fig. 1. Tissue fluidification induces a CytoDR gene signature in MCF10 monolayers and has marginal effect on Interferon and Interferon receptor genes.

a. Volcano plot of DEG between control empty vector and RAB5A-MCF10A monolayers. All significantly RAB5A-deregulated genes are indicated in red (upregulated) and blue (downregulated). The enrichment (log2Fold Change) is on the x-axis and the significance (Wald test -log10 p value two-sided) is on the y-axis. Labels show genes validated in Fig. 1d. b. Gene set enrichment analysis (GSEA) of DEG in RAB5A-MCF10A monolayer over control cells. Moderated t-statistic was used to rank the genes. Significantly enriched pathways (one-sided P-value < 0.05) are with the colour of the outline of the bar corresponding to the BH-adjusted P-value. P-value was calculated as the number of random genes with the same or more extreme ES value divided by the total number of generated gene sets. c. Immunoblots of lysates from doxycycline-treated control (CTR) and RAB5A-(RAB5A)-MCF10.DCIS.com monolayers in 1 experiment out of n = 4 independent ones with the indicated antibodies. Mw is on the left. d. Heatmap of the row-normalized expression level of leading-edge genes for the interferon alpha and gamma response pathways, the interferon and interferon receptor genes in each of the 3 replicates of control empty vector (EV) and RAB5A-expressing (RAB5A)-MCF10.DCIS and MCF10A monolayers. Magnified labels on the right indicate genes validated in Fig. 1d. Normalized expression has been calculated for each gene starting from DeSeq2 regularized log-transformed expression value and then normalized by row mean values. Source data

Extended Data Fig. 2. Tissues fluidification induces CytoDR gene expression in various normal and tumoral mammary epithelia.

a. mRNA expression levels of IFI27, IFI44, IFI44L, IFI6, IFIT1, IFIT3, ISG15, OASL and RAB5A in RAB5A-MCF10.DCIS.com spheroids over the respective control. Data are the mean (n = 3 independent experiments). b. mRNA expression levels of RAB5A, IFI44, IFI44L, IFI27, IFI6, IFIT1, IFIT3, ISG15 and OASL in RAB5A-MCF10.DCIS.com monolayers or sparsely seeded cells (sparse) over the corresponding control cells. Data are the mean ± s.d. (n = 3 independent experiments). In a. and b., Values were normalized to the controls of each experiment. c. Average migration speed, v_m, of control (CTR) or RAB5A-(RAB5A)-MCF10.DCIS.com cells seeded at various densities, and monitored by time lapse (Supplementary Video 1). The values were obtained by PIV analysis evaluated over the time window 8–40 h. Data are the mean ± s.d. (n = 3 independent experiments). d. Relative expression levels of the indicated ISG genes of control (CTR) or RAB5A- (RAB5A)-MCF10.DCIS.com cells seeded at various densities as described in c. Data are the mean (n = 3 independent experiments). Values were normalized to the controls at each dilution and expressed as a relative expression with respect to the maximal level for each ISG at the highest seeded density. e. mRNA expression levels of the indicated genes in control- and RAB5A-cells seeded at jamming density (fluidized monolayer RAB5A). The same cells were subsequently replated sparsely in the continuous presence of doxycycline to ensure the maintenance of the transgene expression before extracting RNA. Data are the mean ± s.d. (n = 3 independent experiments). Values were normalized to the controls of each experiment, unpaired two-tailed t-test with Welch’s correction. f. mRNA expression levels of IFI6, IFIT1, ISG15 and OASL in MCF10.DCIS.com monolayers seeded at jamming density incubated or not with hypotonic medium (composed of a 1:1 mix of media and H₂O + 1 mM CaCl₂ + 1 mM MgCl₂) at the indicated time points. Data are expressed as mean (n = 3 independent experiments). Values were normalized to the controls of each experiment. All values are significantly different with a p value less than 0.05, each-pair two tailed Student’s t-test. g. Migration parameters, obtained from PIV, of control (CTR) or RAB5A-(RAB5A)-MCF10.DCIS.com monolayers treated with hypotonic media for 48 h (Supplementary Video 2), as in 2f. Left panel: migration speed v_m. Central panel: spatial velocity correlation function C_vv evaluated over the time window 8–40 h. In the inset are reported the corresponding correlation lengths L_c obtained from stretched exponential fits on C_vv curves (black dashed lines in the main panel). Right panel: root mean square velocities in the center of mass reference frame. Velocity fields are computed over 4 FOVs per condition (each FOV including more than 2 × 10³ cells). Data are averages over different FOVs (right and left panel) and different time intervals (central panel). Shadowed error bars in left and right panel are evaluated as the standard deviation over the different FOVs. Error bars in central panel are the standard deviation of the mean over 5 different time intervals. h. mRNA expression levels of IFI27, IFI44, IFI44L, IFI6, IFIT1, IFIT3, ISG15, OASL and RAB5A in MCF10.DCIS.com monolayers subjected to different conditions (doxycycline-induction of RAB5A-expression, hypotonic treatment or the combination of both) over control cells. Data are the mean (n = 2 independent experiments). Values were normalized to the controls of each experiment. Source data

Extended Data Fig. 3. Tissue fluidification-dependent CytoDR is mediated by RAB5A in HaCat.

a–c. Migration parameters of HaCat monolayers, obtained from nuclear tracking. Doxycycline-treated control or RAB5-expressing HaCat monolayers were serum starved (SS) for 48 h before addition of 100 ng/ml of EGF and monitored by time-lapse microscopy (Supplementary Video 3). (a) Migration speed v_m. Data are averages over 5 FOVs for each condition. Shadowed error bars are evaluated as the standard deviation over the different FOVs. (b) Spatial velocity correlation functions C_vv evaluated over the time window 8–40 h. The inset report the corresponding correlation lengths L_c obtained from stretched exponential fits on C_vv curves (black dashed lines in the mail panel). Data are averages over 5 time intervals for each condition. Error bars are evaluated as the standard deviation of the mean over the different time intervals. (c) Root mean square velocities in the center of mass reference frame. Velocity fields are computed over different FOVs, encompassing in total about 10⁴ tracked cells per frame per condition. Data are averages over 5 FOVs for each condition. Shadowed error bars are evaluated as the standard deviation over the different FOVs.d. Box plot of the mRNA expression levels of RAB5A, IFI6, IFI27, IFI44, IFIT1, IFIT3, ISG15 and OASL in doxycycline-treated control (CTR) and RAB5A-HaCat monolayers treated exactly as described above with respect to control cells. Monolayers were serum starved mock-treated or stimulated with 100 ng/ml EGF: Hatched blue bar, serum starved HaCat CTR + EGF; Red bars, HaCat RAB5A- serum starved; hatched red bar, serum starved HaCat RAB5A stimulated with 100 ng/ml EGF (Serum Starved HaCat RAB5A + EGF) over control serum starved HaCat cells. Data are expressed as mean ± s.d. (n = 3 independent experiments). Values were normalized to the controls of each experiment. Source data

Extended Data Fig. 4. cGAS accumulation at nuclear envelope ruptures.

a. Immunofluorescence images of control and RAB5A-monolayers (n = 3 independent experiments), stained with Dapi (Cyan), Lamin A/C (Green) and Lamin B1 (red). Scale bar 15 μm. b. Sequential Z axis sections of EGFP-cGAS-expressing RAB5A-MCF10.DCIS.com monolayers used for 3D tomographic reconstruction shown in Fig. 3f (lower right image). The image outlined in red correspond the one shown in Fig. 3f (upper right image). Scale Bar, 500 nm. c. Immunofluorescence images of control (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com monolayers (n = 4 independent experiments), transiently transfected with EGFP-cGAS and stained with DAPI and anti-Lamin A/C antibody to detect nuclei and nuclear lamina, respectively. Magnified images of the selected boxed area are shown. Scale bar 20 μm.

Extended Data Fig. 5. Mechanical characterization of nuclei in control and fluidized tissues.

a. Nuclear elasticity (Young’s modulus) using atomic force microscopy (AFM)-based force indentation of the nuclear surface through the cell cortex of control and RAB5A-MCF10.DCIS.com monolayers seeded at jamming density. Data are the mean ± s.e.m. (100 cells/condition in n = 3 independent experiments), two-tailed Mann–Whitney non-parametric test. Scale bar, 200 µm. b, c. Quantification of cytoplasmic rheology using genetically encoded multimeric nanoparticle (GEM). b) Time- and ensemble- averaged Mean Square Displacement (MSD) plots for control (blue line) and RAB5A (red line) in MCF10.DCIS.com monolayers seeded at jamming density. c) Cumulative distribution function showing the coefficient of diffusion, CDF, (D_eff) data of for control (blue line) and RAB5A (red line) MCF10.DCIS.com monolayers seeded at jamming density. d. Nuclear deformation of control and RAB5A monolayers subjected to a 22% biaxial stretch. Control and RAB5A-MCF10.DCIS.com monolayers were seeded onto elastic PDMS membranes in a biaxial stretching device. Thenuclear area strain, Δa was measured in various, randomly picked fields of view. Bottom, middle and top lines of boxes show the 25th, 50th and 75th percentiles, respectively, and whiskers show 1.5 times the interquartile range from hinges (>29 FOV/experimental condition in n = 2 independent experiments, indicated by different size symbols, respectively), two tailed Mann–Whitney non-parametric test. e. The rates of aspect ratio (A/R) changes of mCherry-H2B control (CTR) and RAB5A-MCF10.DCIS.com cells that are passively going through a constricted 6 micrometre-wide channel schematically depicted on the left (Supplementary Video 7), were calculated for several cells in the time interval needed to pass from an undeformed (A/R₀, at t₀), just before entering into the constriction, to a completely squeezed configuration (A/R_f, at t_f). The rates of nuclear AR variation were extrapolated through a robust fit in R using the ‘robustbase’ package http://robustbase.r-forge.r-project.org/, where the rate of AR changes as function of time is the slope of the fitted curve. Scatter plot data of mean ± S.D. (114 cells/control and 61 cells/RAB5A of n = 3 independent experiments) of the rate of A/R changes, two-tailed Mann–Whitney non-parametric test. f. Images of the SUN2-stained NE of control and RAB5A-MCF10.DCIS.com cells (n = 3 experiments), seeded at jamming density. Scale bar 2 µm. g. Scatter plot of EOP quantifications expressed as mean ± s.d. (> 181 cells/condition in n = 3 independent experiments), two-tailed Mann–Whitney non-parametric test. h. Quantification of NE fluctuations in EGFP-Nesprin-expressing, control and RAB5A-MCF10.DCIS.com cells. Nuclei of live cells transfected with EGFP-Nesprin were recorded at 250 msec per frame for 5 min (Supplementary Video 8). NE fluctuations were obtained from the standard deviation of the NE from its mean position (see Methods and in the inset scheme). The box extends from the 25th to 75th percentile, the whiskers show minimum and maximum values, and the line within the box represents the median (n = 60 position along the NE/condition in >10 cells/ condition), two-tailed Mann–Whitney non-parametric test. i–k. Nuclear mechanics of live cells obtained by Brillouin micro-spectroscopy. i) Representative Brillouin spectra (anti-Stokes peaks) of nuclei of RAB5A (upper panel) and control cells (lower panel). j) Mean and median of Brillouin shifts (black line) measured in RAB5A (n = 9) and control cells (n = 11). The box extends from the 25th to 75th percentile, the whiskers show the mean ± s.d., and the line within the box represents the median, unpaired one-tailed t-test with Welch’s correction. k) From Brillouin shifts, we derived the apparent longitudinal elastic modulus. The box extends from the 25th to 75th percentile, the whiskers show the mean ± s.d., and the line within the box represents the median, unpaired one-tailed t-test with Welch’s correction. P values are indicated in each graph. Source data

Extended Data Fig. 6. Fluidized MCF10.DCIS.com monolayers display an increase nuclear size and enrichment in PRC2-targets.

a. Immunofluorescence images of nuclei in control (CTR) and RAB5A-(RAB5A)-MCF10.DCIS.com monolayers (n = 3 experiments). Scale bar 20 μm. b. Scatter Plot of the nuclear area per field of view is the mean ± s.d. (> 100 FOV/experimental conditions in n = 3 independent experiments), two-tailed Mann–Whitney non-parametric test. c. Transcription factor enrichment analysis of DEG in MCF10.DCIS.com over control monolayers and entries of the ChEA and ENCODE databases. In the Bars are one-sided P-value (combination of Fisher’s exact test and deviation from expected rank for random input gene-set). Bars are coloured according to the transcription factor/target-gene interactions from ChIP-seq/chip experiments database where they have been found enriched. d. GSEA Enrichment plot of the PRC2 targets gene set in the CGP collection of the GSEA Molecular Signatures Database using DEG in RAB5A-MCF10.DCIS.com monolayers. The green curve is the ES (enrichment score) curve, which is the running sum of the weighted enrichment score obtained from GSEA software, while the normalized enrichment score (NES) and the corresponding one-sided test is shown. e, f. Box plot of the mRNA expression levels of EZH2 and SUZ12 in RAB5A-MCF10.DCIS.com monolayer over control cells silenced with the indicated oligos. Data are the mean ± s.d. (n = 3 independent experiments). Values were normalized to the controls of each experiment. g. Visual representation of genomic tracks for ChIP-seq signal enrichment and SAMMY-seq fractions comparison for a representative chromosome (chr 16). For each position along the chromosome (x axis) the enrichment signal (y axis) is the normalized log2 ratio of ChIP over input control samples sequencing reads for ChIP-seq and the normalized log2 ratio of S4 over S3 sequencing (S4/S3) for SAMMY-seq. The data shown in the genomic tracks (from top to bottom) are: three SAMMY-seq replicates on CTR and RAB5A samples; two H3K9me3 ChIP-seq replicates (performed with two alternative antibodies - see methods) on CTR and RAB5A. h. Average SAMMY-seq enrichment signal over heterochromatic regions (metaprofile). SAMMY-seq experiment profiles computed on CTR and on RAB5A-expressing samples are reported (3 replicates for each condition depicted with lines of different colors - see colour legend). The heterochromatic regions were selected based on H3K9me3 ChIP-seq (with ab-176916-Abcam antibody) enriched domains (n = 78 domains). The x-axis reports the relative position with respect to the start and end borders of the considered domains (domain bodies rescaled) in addition to 1Mb flanking regions with absolute (unscaled) coordinates. The y-axis reports the average SAMMY-seq enrichment across all the considered domains. Similar results are obtained by considering either the S4/S2 or the S4/S3 SAMMY-seq fractions comparisons (log2 ratios) to highlight the location of the less accessible and soluble chromatin fraction. i. mRNA expression levels of IFI27, IFI44, IFI44L, IFI6, OASL and RAB5A in RAB5A- MCF10.DCIS.com cells over control cells injected into mammary fat pads of mice. After one week, mice were fed with doxycycline to induce RAB5A expression and the primary tumours were isolated 4 weeks after doxycycline treatment. Data are the mean (n = 2 independent experiments). Values were normalized to the controls of each experiment. P values are indicated in each graph. Source data

Extended Data Fig. 7. IHC characterization of RAB5A, pCHK1 and cGAS in human DCIS and breast cancer organoids.

a. Representative (1 out of more than 20 tested) multiplex immunohistochemistry/immunofluorescence (mIHC/IF) of RAB5A, pCHK1 and DAPI in human Ductal breast Carcinoma in Situ (DCIS). A magnified image from the selected yellow boxes is shown. Scale bar 250 μm. b. Representative (1 out of more than 20 tested) multiplex OPAL immunohistochemistry/immunofluorescence (mIHC/IF) of cGAS and γH2AX in 2 distinct human DCIS. Scale bar 100 μm. c. Representative immunohistochemistry analysis of cGAS in human DCIS with invasive buds in n = 5 distinct human DCIS. A magnified image from the selected boxed area is shown. Red Arrow indicates the crescent-like, perinuclear accumulation of cGAS signal. Scale bar 80 μm. d. Representative immunohistochemistry analysis of cGAS in locally invasive human DCIS (n = 9 distinct human DCIS). Magnified images from the selected boxed area are shown. Red arrow indicates the crescent-like, perinuclear accumulation of cGAS signal, black arrows indicate the perinuclear dotted distribution of cGAS. Scale bar 40 μm. e. Representative multiplex immunohistochemistry/Immunofluorescence (mIHC/IF) of RAB5A, γH2AX, cGAS and DAPI in n = 5 patient-derived breast cancer organoids. Magnified images from the selected yellow boxes are shown. Scale bar 150 μm.

Extended Data Fig. 8. Human breast cancer organoids that persistently rotate display elevated RAB5A, and enrichment in ISG and PRC2-dependent genes.

a. 3D rendering of 5 living breast cancer organoids. The size of each box along the x- and y-directions corresponds to 200 μm. b. Thin blue, orange and yellow curves: temporal evolution of the x, y and z components of the angular velocity associated with the rotation of the organoid depicted in the box above each panel, respectively. Thick black curves represent the angular speed, that is, the modulus of the angular velocity. Organoids are categorized as ‘rotating’ if their average angular speed is larger than 0.03 cycles/h. c. Symbols: overlap parameter Q(τ) obtained from 3D-DVA analysis, capturing the internal rearrangement dynamics of the organoid depicted in the box above each panel. Continuous thin lines are best fitting curves to the data with an exponential model $Q\left(\tau \right)=e^{-\tau /{\tau }^{*}}$. Vertical thick lines are drawn in correspondence with the characteristic relaxation time τ^* obtained from the fitting model. d. Expression levels of RAB5A isoform in n = 3 patients-derived rotating (red) and n = 2 patients-derived non-rotating (blue) organoids. Bottom, middle and top lines of boxes show the 25th, 50th and 75th percentiles, respectively, and whiskers show 1.5 times the interquartile range from hinges. e. Heatmap showing the top 100 deregulated genes (top 50 up and top 50 down) in each of the 3 replicates of the rotating and non-rotating organoids. f. GSEA of DEG in rotating versus non-rotating organoids. Moderated t-statistic was used to rank the genes. Reported are significantly enriched pathways (one-sided P-value < 0.05; number of random genes with the same or more extreme ES value divided by the total number of generated gene sets) with the colour of the outline of the bar corresponding to the BH-adjusted P-value. g. GSEA Enrichment plot of DEG in rotating versus non-rotating organoids. The green curve corresponds to the ES (enrichment score) curve, which is the running sum of the weighted enrichment score obtained from GSEA software, while the normalized enrichment score (NES) and the corresponding one-sided P-value are reported within the graph. h. Quantitative changes in the expression of the INF-related DNA damage resistance signature genes in rotating versus non-rotating organoids. The log2Fold Change is plotted on the x-axis and the significance (Wald test p value two-sided) is defined by the colour code of the outline. i. Transcription factor enrichment analysis for overlap between the input set of differentially expressed genes in rotating versus non-rotating organoids and entries of the ChEA and ENCODE databases In the bars is the one-sided P-value (combination of Fisher’s exact test and deviation from expected rank for random input gene-set). Source data

Extended Data Fig. 9. YAP1/TAZ and cGAS–STING-dependency of EMP gene activation, collective migration and invasion in RAB5A fluidized monolayers.

a. mRNA expression levels of CDH1, AXIN2, CDH2, EGF, MMP13, and ZEB1 in RAB5A-MCF10.DCIS.com monolayers over control cells. Data are the mean ± s.d. (each dot represents an independent experiment). Values were normalized to the controls of each experiment. b. Phase-contrast images of control empty vector (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com monolayers (n = 5 independent experiments). Scale bar, 20 μm. c. Immunofluorescence images of doxycycline-treated control (CTR) and RAB5A-(RAB5A) MCF10.DCIS.com spheroids (a representative image from 5 independent experiments) stained as indicated. Scale bar 20 μm. d. Heatmap representing color-coded expression levels of differentially expressed EMT genes in RAB5A-MCF10.DCIS.com monolayers silenced for YAP and TAZ. The data are the ratio between the level of gene expression in each of the conditions tested relative to those of mock-treated RAB5A-cells. The mean ± s.d. (at least n = 3 independent experiments), two tailed Student’s t-test (siRNA versus RAB5A-expressing cells) are reported in Source data_Figure 9D. e. mRNA expression levels of YAP and TAZ in RAB5A-MCF10.DCIS.com over control cells silenced with the indicated oligos. Data are expressed as mean ± s.d. (n = 3 independent experiments). Values were normalized to the controls of each experiment. f. Heatmap representing color-coded expression levels of differentially expressed EMT genes in RAB5A-expressing MCF10.DCIS.com monolayers silenced for the indicated genes. This analysis was carried out on the same total mRNA from the experiment shown in Fig. 2a. The effectiveness of silencing is reported in Supplementary Fig. 4a. The mean ± s.d. (at least n = 3 independent experiments), two tailed Student’s t-test (siRNA versus RAB5A-expressing cells) are reported in Source data_Figure 9F. g. Heatmap representing color-coded expression levels of differentially expressed EMT genes in RAB5A-MCF10.DCIS.com monolayers treated with the following small molecules: cGAS inhibitor, 7 μg/ ml RU.521, or the STING antagonist, 4 μg/ ml H-151. This analysis was carried out on the same total mRNA from the experiment shown in Fig. 2b. The data are the ratio between the level of gene expression in each of the conditions tested relative to vehicle-treated RAB5A-expressing cells. The mean ± s.d. (at least n = 3 independent experiments) with two tailed Student’s t-test are reported in Source data_Figure 9G. h. Scatter plot of the speed of wound closure of control (CTR) and RAB5A-expressing MCF10.DCIS.com monolayers treated with vehicle or the cGAS inhibitor, 7 μg/ ml RU.521, or the STING antagonist, 4 μg/ ml H-151 (Supplementary Video 11). Data are the mean ± s.d. (n = 3 independent experiments, in which 5 FOV/condition/experiment were analysed), two-tailed Mann–Whitney non-parametric test. i. Scatter plot of control (CTR) and RAB5A-expressing MCF10.DCIS.com monolayers invading into a Matrigel coated wound and treated with vehicle or the cGAS inhibitor, 7 μg/ ml RU.521, or the STING antagonist, 4 μg/ ml H-151 (Supplementary Video 12). Data are the mean ± s.d. (n = 3 independent experiments, in which 5 FOV/condition/experiment were analysed), two-tailed Mann–Whitney non-parametric test, is indicated in the figure. j. Heatmap representing color-coded expression levels of differentially expressed CytoDR genes in RAB5A-MCF10.DCIS.com monolayers shown in Supplementary Fig. 10f treated with the indicated small molecules: cGAS inhibitor 7 μg/ ml RU.521 or the STING antagonist 4 μg/ ml H-151. The data are the ratio between the level of gene expression in each of the conditions tested relative to vehicle-treated RAB5A-expressing cells. The mean ± s.d. (n> 3 independent experiments) and two tailed Student’s t-test are reported in Source data_Figure 9J. P values are indicated in each graph. Source data

Extended Data Fig. 10. Tissue fluidification enhances chemoresistance.

a. Quantitative changes in the expression of the INF-related DNA damage resistance signature genes in RAB5A-expressing MCF10.DCIS monolayers with respect to their respective controls. The log2Fold Change is plotted on the x-axis and the significance (Wald test p-value two-sided) is defined by the colour code of the outline. b. Dose-dependent analysis of cell proliferation of control (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com treated with the indicated concentrations of Docetaxel. Data are the mean ± s.d. (n = 3 independent experiments). c. Percentage of intact nuclei after treatment with the indicated doses of docetaxel in control (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com cells. Data are the mean ± s.d. (n = 3 independent experiments). Scale Bar is 20 μm d. Representative images of control empty vector (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com nuclei in the presence of 10nM Docetaxel. Intact and aberrant polynucleated cell nuclei are indicated by green and red contours. Scale bar 10 μm. e. Dose-dependent analysis of cell proliferation of control (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com treated with the indicated concentrations of Etoposide. Data are the mean ± s.d. (n = 3 independent experiments). f. Representative images of control empty vector (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com nuclei in the presence of 0.5 μM Etoposide. Scale bar 30 μm. g, h. Box Plot of the cell viability (by counting intact nuclei) and cell proliferation (measured as relative volume of spheroids) of control (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com spheroid grown over a Matrigel layer treated with the indicated concentrations of Docetaxel or Etoposide. Data are the mean ± s.d. (n = 3 independent experiments), unpaired one-tailed t-test with Welch’s correction. P values are indicated in each graph. Source data