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Supplementary data include Figure S1(Structural determinants of ESC inhibitors), Figure S2 (Validation and functional characterization of esMTi), Figure S3 (Generation and characterization of P4H deficient ESCs), Figure S4 (Budesonide activity on A549 lung tumor cells ); Figure S5 (Budesonide activity on SUM159 cells); Table S1 (The primer sequences and application); Table S2 (The Primary Antibodies and application); Table S3 (List of all ESC inhibitors), Table S4 (ESC inhibitors with anti-cancer activity and related references); Table S5 (List of esMT inhibitors), Table S6 (esMT inhibitors with anti-collagen/fibortic effects)

Collagen prolyl hydroxylation (CPH), which is catalyzed by prolyl 4-hydroxylase (P4H), is the most prevalent posttranslational modification in humans and requires vitamin C (VitC). Here, we demonstrate that CPH acts as an epigenetic modulator of cell plasticity. Increased CPH induced global DNA/histone methylation in pluripotent stem and tumor cells and promoted cell state transition (CST). Interfering with CPH by either genetic ablation of P4H subunit alpha-2 (P4HA2) or pharmacologic treatment reverted epigenetic changes and antagonized CST. Mechanistically, we suggest that CPH modifies the epigenetic landscape by reducing VitC for DNA and histone demethylases. Repurposed drugs targeting CPH-mediated metabolic perturbation, such as the antiasthmatic budesonide, blocked metastatic dissemination of breast cancer cells in vivo by preventing mesenchymal transition. Our study provides mechanistic insights into how metabolic cues and epigenetic factors integrate to control CST and paves the way for the development of novel antimetastatic strategies.Significance:A phenotype-based high-throughput screening reveals unforeseen metabolic control of cell plasticity and identifies budesonide as a drug candidate for metastatic cancer.

PDF file 1545 K. Methods and Materials Cell culture, Scratch Assay/Immunfluorescence, siRNA transfection Fig. S1: Archazolid inhibits the acidification of lysosomes. Fig. S2: Archazolid inhibits cancer cell migration. Fig. S3: EGF-R and Her2 expression on the cell surface after archazolid treatment. Fig. S4: EGF-R plays a role in cell polarization. Fig.S5: The downregulation of the V-ATPase subunit c affects the cell migration process also in L3.6pl cells. Fig. S6: Rac1-GFP is less localized in Rab5-induced enlarged endosomes of archazolid treated cells. Fig. S7: Dissemination of 4T1-Luc breast cancer cells is decreased in archazolid A (1mg/kg) pretreated mice. Fig. S8: Scheme proposing the impact of V-ATPase inhibition on cell migration. Fig.S9: Beside the localization in lysosomes the V-ATPase is also localized at the plasma membrane. Fig. S10: Archazolid affects cellular recycling.

AVI file - 4312K, Time-lapse video-recording of a representative JVM3 cell expressing GFP-CCR7 distribution and migrating in a CCL19 gradient (source of CCL19 towards the bottom).

AVI file - 503K, Time-lapse video-recording of a representative JVM3 cells exposed to a CCL19 gradient (source of CCL19 towards the bottom) over a period of 5 min.

AVI file - 1553K, Time-lapse video-recording of a representative JVM3 cell expressing GFP-actin and migrating in a CCL19 gradient (source of CCL19 towards the bottom).

PDF file - 57K, Accession numbers and amplified context sequences of the genes analyzed by quantitative RT-PCR.

PDF file - 54K, Additional information regarding CCR7 internalization.

AVI file - 1602K, Supplemental Movies 6-8 shows Time-lapse video-recording of zoomed sections of the lamellipodium of a representative JVM3 cell expressing GFP-CIP4 and migrating in a CCL19 gradient. The four panels show GFP-CIP4 (green), pseudo-colour scale indicating CIP4 level, transmission and merge.

AVI file - 1134K, Time-lapse video-recording of a representative CIP4 knocked-down JVM3 cells exposed to a CCL19 gradient (source of CCL19 towards the bottom) over a period of 12 min.

PDF file - 93K, CIP4 is dispensable for CCR7 internalization (S1), CIP4 knock-down impairs the polarized morphology of JVM3 cells (S2).

AVI file - 3816K, Time-lapse video-recording of a representative JVM3 cell expressing GFP-CIP4 and migrating over a period of 37 min in a CCL19 gradient (source of CCL19 towards the bottom). The four panels show GFP-CIP4 (green), pseudo-colour scale indicating CIP4 level, transmission and merge.

AVI file - 2015K, Supplemental Movies 6-8 shows Time-lapse video-recording of zoomed sections of the lamellipodium of a representative JVM3 cell expressing GFP-CIP4 and migrating in a CCL19 gradient. The four panels show GFP-CIP4 (green), pseudo-colour scale indicating CIP4 level, transmission and merge.

AVI file - 2141K, Supplemental Movies 6-8 shows Time-lapse video-recording of zoomed sections of the lamellipodium of a representative JVM3 cell expressing GFP-CIP4 and migrating in a CCL19 gradient. The four panels show GFP-CIP4 (green), pseudo-colour scale indicating CIP4 level, transmission and merge.

Endocytosis allows cells to transport particles and molecules across the plasma membrane. In addition, it is involved in the termination of signalling through receptor downmodulation and degradation. This traditional outlook has been substantially modified in recent years by discoveries that endocytosis and subsequent trafficking routes have a profound impact on the positive regulation and propagation of signals, being key for the spatiotemporal regulation of signal transmission in cells. Accordingly, endocytosis and membrane trafficking regulate virtually every aspect of cell physiology and are frequently subverted in pathological conditions. Two key aspects of endocytic control over signalling are coming into focus: context-dependency and long-range effects. First, endocytic-regulated outputs are not stereotyped but heavily dependent on the cell-specific regulation of endocytic networks. Second, endocytic regulation has an impact not only on individual cells but also on the behaviour of cellular collectives. Herein, we will discuss recent advancements in these areas, highlighting how endocytic trafficking impacts complex cell properties, including cell polarity and collective cell migration, and the relevance of these mechanisms to disease, in particular cancer. The canonical function of endocytosis is molecule internalization. Beyond this role, endocytic trafficking has emerged as a process central to the spatiotemporal regulation of cell signalling. Endocytic trafficking thus controls many cellular processes and tissue-wide properties, including cell migration and polarity, and its deregulation has been implicated in pathologies, particularly cancer.

Sheet-like membrane protrusions at the leading edge, termed lamellipodia, drive 2D-cell migration using active actin polymerization. Microspikes comprise actin-filament bundles embedded within lamellipodia, but the molecular mechanisms driving their formation and their potential functional relevance have remained elusive. Microspike formation requires the specific activity of clustered Ena/VASP proteins at their tips to enable processive actin assembly in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering are poorly understood. Systematic analyses of B16-F1 melanoma mutants lacking potential candidate proteins revealed that neither inverse BAR-domain proteins, nor lamellipodin or Abi are essential for clustering, although they differentially contribute to lamellipodial VASP accumulation. In contrast, unconventional myosin-X (MyoX) identified here as proximal to VASP was obligatory for Ena/VASP clustering and microspike formation. Interestingly, and despite the invariable distribution of other relevant marker proteins, the width of lamellipodia in MyoX-KO mutants was significantly reduced as compared to B16-F1 control, suggesting that microspikes contribute to lamellipodium stability. Consistently, MyoX removal caused marked defects in protrusion and random 2D-cell migration. Strikingly, Ena/VASP-deficiency also uncoupled MyoX cluster dynamics from actin assembly in lamellipodia, establishing their tight functional association in microspike formation.Significance StatementUnlike filopodia that protrude well beyond the cell periphery and are implicated in sensing, morphogenesis and cell-to-cell communication, the function of microspikes consisting of actin-filament bundles fully embedded within lamellipodia is less clear. Microspike formation involves specific clustering of Ena/VASP family members at filament-barbed ends to enable processive actin polymerization in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering have remained unknown. Here, we systematically analyzed these processes in genetic knockout mutants derived from B16-F1 cells and show that Ena/VASP clustering at microspike tips involves Lamellipodin, but not inverse BAR-domain proteins, and strictly requires unconventional Myosin-X. Complete loss of microspikes was confirmed with CRISPR/Cas9-mediated MyoX knockout in Rat2 fibroblasts, excluding cell type-specific effects.

Abstract The connection between the properties of a cell tissue and those of the single constituent cells remains to be elucidated. At the purely mechanical level, the degree of rigidity of different cellular components, such as the nucleus and the cytoplasm, modulates the interplay between the cell inner processes and the external environment, while simultaneously mediating the mechanical interactions between neighboring cells. Being able to quantify the correlation between single-cell and tissue properties would improve our mechanobiological understanding of cell tissues. Here we develop a methodology to quantitatively extract a set of structural and motility parameters from the analysis of time-lapse movies of nuclei belonging to jammed and flocking cell monolayers. We then study in detail the correlation between the dynamical state of the tissue and the deformation of the nuclei. We observe that the nuclear deformation rate linearly correlates with the local divergence of the velocity field, which leads to a non-invasive estimate of the elastic modulus of the nucleus relative to the one of the cytoplasm. We also find that nuclei belonging to flocking monolayers, subjected to larger mechanical perturbations, are about two time stiffer than nuclei belonging to dynamically arrested monolayers, in agreement with atomic force microscopy results. Our results demonstrate a non-invasive route to the determination of nuclear relative stiffness for cells in a monolayer. Graphic abstract

Circulating tumor cell (CTC) clusters are associated with increased metastatic potential and worse patient prognosis, but are rare, difficult to count, and poorly characterized biophysically. The PillarX device described here is a bimodular microfluidic device (Pillar-device and an X-magnetic device) to profile single CTCs and clusters from whole blood based on their size, deformability, and epithelial marker expression. Larger, less deformable clusters and large single cells are captured in the Pillar-device and sorted according to pillar gap sizes. Smaller, deformable clusters and single cells are subsequently captured in the X-device and separated based on epithelial marker expression using functionalized magnetic nanoparticles. Clusters of established and primary breast cancer cells with variable degrees of cohesion driven by different cell-cell adhesion protein expression are profiled in the device. Cohesive clusters exhibit a lower deformability as they travel through the pillar array, relative to less cohesive clusters, and have greater collective invasive behavior. The ability of the PillarX device to capture clusters is validated in mouse models and patients of metastatic breast cancer. Thus, this device effectively enumerates and profiles CTC clusters based on their unique geometrical, physical, and biochemical properties, and could form the basis of a novel prognostic clinical tool.

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 that occurs in various tissues. Whether this tissue-level mechanical transition impact phenotypes during carcinoma progression remains unclear. We show, here that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. Cells react to these protracted mechanical stresses by mounting a mechano-protective response that includes enlarged nuclear size and rigidity, altered heterochromatin distribution, and the remodeling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with 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-dependent cytosolic DNA response gene program. This mechanically-driven transcriptional rewiring ultimately results in a change in cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemo-resistance in invasive breast carcinoma. One-Sentence Summary: A solid-to-fluid phase transition promotes a pro-inflammatory transcriptional response in invasive breast carcinoma

The original version of this Article contains an error in Fig. 1e, in which the representative image panel was inadvertently duplicated from Fig. 1d by the publisher. The correct representative image version of Fig. 1e is

As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nano-scale topography. Here we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nano-scale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by the I-BAR protein IRSp53, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios.TeaserCell stretch cycles generate PM evaginations of ≈100 nm which are sensed by IRSp53, triggering a local event of actin polymerization that flattens and recovers PM shape.

Therapeutic options for patients with advanced-stage hepatocellular carcinoma (HCC) are very limited. The only approved first-line treatment is the multi-tyrosine kinase inhibitor sorafenib, which shows low response rates and severe side effects. In particular, the compensatory activation of growth factor receptors leads to chemoresistance and limits the clinical impact of sorafenib. However, combination approaches to improve sorafenib have failed. Here we investigate the inhibition of cyclin-dependent kinase 5 (Cdk5) as a promising combination strategy to improve sorafenib response in HCC. Combination of sorafenib with Cdk5 inhibition (genetic knockdown by short hairpin RNA or CRISPR/Cas9 and pharmacologic inhibition) synergistically impaired HCC progression in vitro and in vivo by inhibiting both tumor cell proliferation and migration. Importantly, these effects were mediated by a mechanism for Cdk5: A liquid chromatography-tandem mass spectrometry-based proteomic approach revealed that Cdk5 inhibition interferes with intracellular trafficking, a process crucial for cellular homeostasis and growth factor receptor signaling. Cdk5 inhibition resulted in an accumulation of enlarged vesicles and respective cargos in the perinuclear region, considerably impairing the extent and quality of growth factor receptor signaling. Thereby, Cdk5 inhibition offers a comprehensive approach to globally disturb growth factor receptor signaling that is superior to specific inhibition of individual growth factor receptors. Conclusion: Cdk5 inhibition represents an effective approach to improve sorafenib response and to prevent sorafenib treatment escape in HCC. Notably, Cdk5 is an addressable target frequently overexpressed in HCC, and with Dinaciclib, a clinically tested Cdk5 inhibitor is readily available. Thus, our study provides evidence for clinically evaluating the combination of sorafenib and Dinaciclib to improve the therapeutic situation for patients with advanced-stage HCC.

Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

The accurate quantification of cellular motility and of the structural changes occurring in multicellular aggregates is critical in describing and understanding key biological processes, such as wound repair, embryogenesis and cancer invasion. Current methods based on cell tracking or velocimetry either suffer from limited spatial resolution or are challenging and time-consuming, especially for three-dimensional (3D) cell assemblies. Here we propose a conceptually simple, robust and tracking-free approach for the quantification of the dynamical activity of cells via a two-step procedure. We first characterise the global features of the collective cell migration by registering the temporal stack of the acquired images. As a second step, a map of the local cell motility is obtained by performing a mean squared amplitude analysis of the intensity fluctuations occurring when two registered image frames acquired at different times are subtracted. We successfully apply our approach to cell monolayers undergoing a jamming transition, as well as to monolayers and 3D aggregates that exhibit a cooperative unjamming-via-flocking transition. Our approach is capable of disentangling very efficiently and of assessing accurately the global and local contributions to cell motility.

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis, and cancer invasion and is often hijacked by viral infections 1. Unlike clathrin-mediated endocytosis, which exploits cargo-specific adaptors for selective protein internalization, the clathrin and dynamin-independent endocytic pathway (CLIC-GEEC, CG-pathway) has until now been considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids 2,3. Although the core molecular players of CG endocytosis have been recently defined, no cargo-specific adaptors are known and evidence of selective protein uptake into the pathway is lacking 3. Here, we identify the first cargo-specific adaptor for CG-endocytosis and demonstrate its clinical relevance in breast cancer progression. By combining unbiased molecular characterization and super-resolution imaging, we identified the actin-binding protein swiprosin-1 (EFHD2) as a cargo-specific adaptor regulating integrin internalization via the CG-pathway. Swiprosin-1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery, IRSp53 and actin. Swiprosin-1 is critical for integrin endocytosis, but not for other CG-cargo and supports integrin-dependent cancer cell migration and invasion, with clinically relevant implications for breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG-pathway and opens the possibility to discover more adaptors regulating it.