Escape from cell-cell and cell-matrix adhesion dependence underscores disease progression in gastric cancer organoid models.

OBJECTIVE: Cell-cell (CC) and cell-matrix (CM) adhesions are essential for epithelial cell survival, yet dissociation-induced apoptosis is frequently circumvented in malignant cells. DESIGN: We explored CC and CM dependence in 58 gastric cancer (GC) organoids by withdrawing either ROCK inhibitor, matrix or both to evaluate their tumorigenic potential in terms of apoptosis resistance, correlation with oncogenic driver mutations and clinical behaviour. We performed mechanistic studies to determine the role of diffuse-type GC drivers: ARHGAP fusions, RHOA and CDH1, in modulating CC (CCi) or CM (CMi) adhesion independence. RESULTS: 97% of the tumour organoids were CMi, 66% were CCi and 52% were resistant to double withdrawal (CCi/CMi), while normal organoids were neither CMi nor CCi. Clinically, the CCi/CMi phenotype was associated with an infiltrative tumour edge and advanced tumour stage. Moreover, the CCi/CMi transcriptome signature was associated with poor patient survival when applied to three public GC datasets. CCi/CMi and CCi phenotypes were enriched in diffuse-type GC organoids, especially in those with oncogenic driver perturbation of RHO signalling via RHOA mutation or ARHGAP fusions. Inducible knockout of ARHGAP fusions in CCi/CMi tumour organoids led to resensitisation to CC/CM dissociation-induced apoptosis, upregulation of focal adhesion and tight junction genes, partial reversion to a more normal cystic phenotype and inhibited xenograft formation. Normal gastric organoids engineered with CDH1 or RHOA mutations became CMi or CCi, respectively. CONCLUSIONS: The CCi/CMi phenotype has a critical role in malignant transformation and tumour progression, offering new mechanistic information on RHO-ROCK pathway inhibition that contributes to GC pathogenicity.

Mitochondrial fission links ECM mechanotransduction to metabolic redox homeostasis and metastatic chemotherapy resistance.

Metastatic breast cancer cells disseminate to organs with a soft microenvironment. Whether and how the mechanical properties of the local tissue influence their response to treatment remains unclear. Here we found that a soft extracellular matrix empowers redox homeostasis. Cells cultured on a soft extracellular matrix display increased peri-mitochondrial F-actin, promoted by Spire1C and Arp2/3 nucleation factors, and increased DRP1- and MIEF1/2-dependent mitochondrial fission. Changes in mitochondrial dynamics lead to increased production of mitochondrial reactive oxygen species and activate the NRF2 antioxidant transcriptional response, including increased cystine uptake and glutathione metabolism. This retrograde response endows cells with resistance to oxidative stress and reactive oxygen species-dependent chemotherapy drugs. This is relevant in a mouse model of metastatic breast cancer cells dormant in the lung soft tissue, where inhibition of DRP1 and NRF2 restored cisplatin sensitivity and prevented disseminated cancer-cell awakening. We propose that targeting this mitochondrial dynamics- and redox-based mechanotransduction pathway could open avenues to prevent metastatic relapse.

Comprehensive understanding of anchorage-independent survival and its implication in cancer metastasis.

Detachment is the initial and critical step for cancer metastasis. Only the cells that survive from detachment can develop metastases. Following the disruption of cell-extracellular matrix (ECM) interactions, cells are exposed to a totally different chemical and mechanical environment. During which, cells inevitably suffer from multiple stresses, including loss of growth stimuli from ECM, altered mechanical force, cytoskeletal reorganization, reduced nutrient uptake, and increased reactive oxygen species generation. Here we review the impact of these stresses on the anchorage-independent survival and the underlying molecular signaling pathways. Furthermore, its implications in cancer metastasis and treatment are also discussed.

Matrix stiffness and its influence on pancreatic diseases.

The matrix stiffness of the extracellular matrix(ECM), which is the slow elastic force on cells, has gradually become investigated. And a higher stiffness could induce changes in cell biological behaviors and activation of internal signaling pathways. Imbalanced stiffness of ECM is associated with a number of diseases, including pancreatic disease. In this review, we discuss the components of the ECM and the increased stiffness caused by unbalanced ECM changes. Next, we describe how matrix stiffness transmits mechanical signals and what signaling pathways are altered within the cell in detail. Finally, we discuss the effect of ECM on the behavior of pancreatic diseases from the perspective of matrix stiffness.

Plexin-B2 facilitates glioblastoma infiltration by modulating cell biomechanics.

Infiltrative growth is a major cause of high lethality of malignant brain tumors such as glioblastoma (GBM). We show here that GBM cells upregulate guidance receptor Plexin-B2 to gain invasiveness. Deletion of Plexin-B2 in GBM stem cells limited tumor spread and shifted invasion paths from axon fiber tracts to perivascular routes. On a cellular level, Plexin-B2 adjusts cell adhesiveness, migratory responses to different matrix stiffness, and actomyosin dynamics, thus empowering GBM cells to leave stiff tumor bulk and infiltrate softer brain parenchyma. Correspondingly, gene signatures affected by Plexin-B2 were associated with locomotor regulation, matrix interactions, and cellular biomechanics. On a molecular level, the intracellular Ras-GAP domain contributed to Plexin-B2 function, while the signaling relationship with downstream effectors Rap1/2 appeared variable between GBM stem cell lines, reflecting intertumoral heterogeneity. Our studies establish Plexin-B2 as a modulator of cell biomechanics that is usurped by GBM cells to gain invasiveness.

CASP4 gene silencing in epithelial cancer cells leads to impairment of cell migration, cell-matrix adhesion and tissue invasion.

Inflammatory caspases, including human caspase-4 (CASP4), play key roles in innate immune responses to promote fusion of phagosomes harboring pathogenic bacteria with lysosomes, halt intracellular replication of pathogens, maturation and secretion of pro-inflammatory cytokines. The role of inflammatory caspases in cancer cells remains poorly investigated. Here, we explored the consequences of modulating CASP4 expression levels on the migratory behavior of epithelial cancer cell lines. By a gene silencing approach and in vitro and in vivo studies we show that down-regulation of CASP4 leads to impaired cell migration and cell-matrix adhesion. This phenotype is accompanied by an increased actin cytoskeleton polymerization, changes in the overall organization of adherens junctions (AJs) and number and size of focal adhesions. Interestingly, the cell migration deficit could be reversed by epithelial growth factor treatment, and depletion of calcium ions unveiled a role of CASP4 in the novo assembly of AJs, suggesting that the role of CASP4 is not cell-autonomous. Finally, CASP4-silenced A431 cells exhibited a severe reduction in their ability to invade lung tissue, when injected into nude mice. Overall, our data support the emerging evidence that inflammatory caspases can regulate cell migration through actin remodeling and uncover a novel role of CASP4 in cancer cell behavior.

Membrane-Tethered Metalloproteinase Expressed by Vascular Smooth Muscle Cells Limits the Progression of Proliferative Atherosclerotic Lesions.

BACKGROUND: The MMP (matrix metalloproteinase) family plays diverse and critical roles in directing vascular wall remodeling in atherosclerosis. Unlike secreted-type MMPs, a member of the membrane-type MMP family, MT1-MMP (membrane-type 1 MMP; MMP14), mediates pericellular extracellular matrix degradation that is indispensable for maintaining physiological extracellular matrix homeostasis. However, given the premature mortality exhibited by MT1-MMP-null mice, the potential role of the proteinase in atherogenesis remains elusive. We sought to determine the effects of both MT1-MMP heterozygosity and tissue-specific gene targeting on atherogenesis in APOE (apolipoprotein E)-null mice. METHODS AND RESULTS: MT1-MMP heterozygosity in the APOE-null background (Mmp14+/-Apoe-/- ) significantly promoted atherogenesis relative to Mmp14+/+Apoe-/- mice. Furthermore, the tissue-specific deletion of MT1-MMP from vascular smooth muscle cells (VSMCs) in SM22α-Cre(+)Mmp14F/FApoe-/- (VSMC-knockout) mice likewise increased the severity of atherosclerotic lesions. Although VSMC-knockout mice also developed progressive atherosclerotic aneurysms in their iliac arteries, macrophage- and adipose-specific MT1-MMP-knockout mice did not display this sensitized phenotype. In VSMC-knockout mice, atherosclerotic lesions were populated by hyperproliferating VSMCs (smooth muscle actin- and Ki67-double-positive cells) that were characterized by a proinflammatory gene expression profile. Finally, MT1-MMP-null VSMCs cultured in a 3-dimensional spheroid model system designed to mimic in vivo-like cell-cell and cell-extracellular matrix interactions, likewise displayed markedly increased proliferative potential. CONCLUSIONS: MT1-MMP expressed by VSMCs plays a key role in limiting the progression of atherosclerosis in APOE-null mice by regulating proliferative responses and inhibiting the deterioration of VSMC function in atherogenic vascular walls.

Talin and vinculin are downregulated in atherosclerotic plaque; Tampere Vascular Study.

BACKGROUND AND AIMS: Focal adhesions (FA) play an important role in the tissue remodeling and in the maintenance of tissue integrity and homeostasis. Talin and vinculin proteins are among the major constituents of FAs contributing to cellular well-being and intercellular communication. METHODS: Microarray analysis (MA) and qRT-PCR low-density array were implemented to analyze talin-1, talin-2, meta-vinculin and vinculin gene expression in circulating blood and arterial plaque. RESULTS: All analyzed genes were significantly and consistently downregulated in plaques (carotid, abdominal aortic and femoral regions) compared to left internal thoracic artery (LITA) control. The use of LITA samples as controls for arterial plaque samples was validated using immunohistochemistry by comparing LITA samples with healthy arterial samples from a cadaver. Even though the differences in expression levels between stable and unstable plaques were not statistically significant, we observed further negative tendency in the expression in unstable atherosclerotic plaques. The confocal tissue imaging revealed gradient of talin-1 expression in plaque with reduction close to the vessel lumen. Similar gradient was observed for talin-2 expression in LITA controls but was not detected in plaques. This suggests that impaired tissue mechanostability affects the tissue remodeling and healing capabilities leading to development of unstable plaques. CONCLUSIONS: The central role of talin and vinculin in cell adhesions suggests that the disintegration of the tissue in atherosclerosis could be partially driven by downregulation of these genes, leading to loosening of cell-ECM interactions and remodeling of the tissue.

High-throughput proteomics reveal alarmins as amplifiers of tissue pathology and inflammation after spinal cord injury.

Spinal cord injury is characterized by acute cellular and axonal damage followed by aggressive inflammation and pathological tissue remodelling. The biological mediators underlying these processes are still largely unknown. Here we apply an innovative proteomics approach targeting the enriched extracellular proteome after spinal cord injury for the first time. Proteomics revealed multiple matrix proteins not previously associated with injured spinal tissue, including small proteoglycans involved in cell-matrix adhesion and collagen fibrillogenesis. Network analysis of transcriptomics and proteomics datasets uncovered persistent overexpression of extracellular alarmins that can trigger inflammation via pattern recognition receptors. In mechanistic experiments, inhibition of toll-like receptor-4 (TLR4) and the receptor for advanced glycation end-products (RAGE) revealed the involvement of alarmins in inflammatory gene expression, which was found to be dominated by IL1 and NFκΒ signalling. Extracellular high-mobility group box-1 (HMGB1) was identified as the likely endogenous regulator of IL1 expression after injury. These data reveal a novel tissue remodelling signature and identify endogenous alarmins as amplifiers of the inflammatory response that promotes tissue pathology and impedes neuronal repair after spinal cord injury.

Matrix geometry determines optimal cancer cell migration strategy and modulates response to interventions.

The molecular requirements and morphology of migrating cells can vary depending on matrix geometry; therefore, predicting the optimal migration strategy or the effect of experimental perturbation is difficult. We present a model of cell motility that encompasses actin-polymerization-based protrusions, actomyosin contractility, variable actin-plasma membrane linkage leading to membrane blebbing, cell-extracellular-matrix adhesion and varying extracellular matrix geometries. This is used to explore the theoretical requirements for rapid migration in different matrix geometries. Confined matrix geometries cause profound shifts in the relationship of adhesion and contractility to cell velocity; indeed, cell-matrix adhesion is dispensable for migration in discontinuous confined environments. The model is challenged to predict the effect of different combinations of kinase inhibitors and integrin depletion in vivo, and in confined matrices based on in vitro two-dimensional measurements. Intravital imaging is used to verify bleb-driven migration at tumour margins, and the predicted response to single and combinatorial manipulations.

Fibroblasts from women with pelvic organ prolapse show differential mechanoresponses depending on surface substrates.

INTRODUCTION AND HYPOTHESIS: Little is known about dynamic cell-matrix interactions in the context of pathophysiology and treatments for pelvic organ prolapse (POP). This study sought to identify differences between fibroblasts from women with varying degrees of prolapse in reaction to mechanical stimuli and matrix substrates in vitro. METHODS: Fibroblasts from the vaginal wall of three patients with POP Quantification (POP-Q) system stages 0, II, and IV were stretched on artificial polymer substrates either coated or not coated with collagen I. Changes in morphology and anabolic/catabolic compounds that affect matrix remodelling were evaluated at protein- and gene-expression levels. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey-Kramer's post hoc test. RESULTS: POP fibroblasts show delayed cell alignment and lower responses to extracellular matrix remodelling factors at both enzymatic- and gene-expression levels compared with healthy fibroblasts. CONCLUSION: POP fibroblasts, when compared with healthy cells, show differential mechanoresponses on two artificial polymer substrates. This should be taken into account when designing or improving implants for treating POP.

Extracellular matrix and fibroblast communication following myocardial infarction.

The extracellular matrix (ECM) provides structural support by serving as a scaffold for cells, and as such the ECM maintains normal tissue homeostasis and mediates the repair response following injury. In response to myocardial infarction (MI), ECM expression is generally upregulated in the left ventricle (LV), which regulates LV remodeling by modulating scar formation. The ECM directly affects scar formation by regulating growth factor release and cell adhesion and indirectly affects scar formation by regulating the inflammatory, angiogenic, and fibroblast responses. This review summarizes the current literature on ECM expression patterns and fibroblast mechanisms in the myocardium, focusing on the ECM response to MI. In addition, we discuss future research areas that are needed to better understand the molecular mechanisms of ECM action, both in general and as a means to optimize infarct healing.

EMT as the ultimate survival mechanism of cancer cells.

Epithelial cancers make up the vast majority of cancer types and, during the transition from benign adenoma to malignant carcinoma and metastasis, epithelial tumor cells acquire a de-differentiated, migratory and invasive behavior. This process of epithelial-mesenchymal transition (EMT) goes along with dramatic changes in cellular morphology, the loss and remodeling of cell-cell and cell-matrix adhesions, and the gain of migratory and invasive capabilities. EMT itself is a multistage process, involving a high degree of cellular plasticity and a large number of distinct genetic and epigenetic alterations, as fully differentiated epithelial cells convert into poorly differentiated, migratory and invasive mesenchymal cells. In the past years, a plethora of genes have been identified that are critical for EMT and metastasis formation. Notably, the EMT process not only induces increased cancer cell motility and invasiveness but also allows cancer cells to avoid apoptosis, anoikis, oncogene addiction, cellular, senescence and general immune defense. Notably, EMT seems to play a critical role in the generation and maintenance of cancer stem cells, highly consistent with the notion that metastatic cells carry the ability to initiate new tumors.

Vitreopapillary traction diagnosed by spectral domain optical coherence tomography.

This report describes a case of vitreopapillary traction with right inferior altitudinal defect similar to anterior ischemic optic neuropathy. A 35-year-old man with inferior altitudinal defect in his right eye was referred. The right optic disc was elevated and the superior disc margin was blurred. Spectral domain optical coherence tomography (SD-OCT) of the right optic disc showed the vitreous cortex was highly reflective and pulling the optic disc margin superiorly. The posterior hyaloid membrane on the nasal side of the optic disc was less reflective than that superior to the disc, suggesting that vitreopapillary traction contributed to this condition. During a careful 12-month observation, no change was noted in the optic disc appearance or visual field. Thus, SD-OCT was useful in visualizing the posterior hyaloid membrane and was helpful in the diagnosis of vitreopapillary traction syndrome.

Genetics of nephrotic syndrome: connecting molecular genetics to podocyte physiology.

Urinary losses of macromolecules in nephrotic syndrome (NS) reflect a dysfunction of the highly permselective glomerular filtration barrier. Genetic studies of hereditary forms of NS have led to the identification of proteins playing a crucial role in slit-diaphragm signalling, regulation of actin cytoskeleton dynamics, maintenance of podocyte integrity and cell-matrix interactions. This review will focus on recent molecular and clinical findings in the field of genetics of NS, thereby providing a better understanding of the complex glomerular filtration barrier physiology.

Molecular mechanisms of epithelial-barrier disruption by Helicobacter pylori.

Intact intercellular junctions and cell-matrix contacts are important structures in the formation and maintenance of epithelial-barrier functions against microbes. The human gastric pathogen Helicobacter pylori developed a remarkable network of strategies to alter these epithelial cell-cell and cell-matrix adhesions, which are implicated in inflammation, proliferation, cell migration and invasive growth. This review focuses on recent findings on H. pylori-induced host-cell signaling. We propose a stepwise model for how H. pylori interacts with components of focal adhesions and intercellular tight and adherens junctions to disrupt the epithelial layer, providing novel insights into the pathogenesis of H. pylori.

Fluorescence- and spin-labeled carbonic anhydrase inhibitors.

Carbonic anhydrase IX (hCA IX) is a membrane-associated glycoprotein that is observed in many tumor tissues and is strongly overexpressed by hypoxia conditions. Hypoxia is a clinically important tumor parameter and this enzyme can play an important role as a potential marker of hypoxic tumor and as a therapeutic target too. In the last years, Carbonic Anhydrase IX Inhibitors which possess fluorescent probe were largely used for visualize hypoxic tumor cell lines and for understanding the biological roles of hCA IX in acidification of the external matrix. Here we resume the development pathways of such compounds from the design to the final biological evaluation. Furthermore, spin-labeled CAIs were included to have a complete overview of the potentiality of this enzyme as marker of hypoxic tumors.

The role of actin bundling protein fascin in the progression of ovarian neoplasms.

PURPOSE OF INVESTIGATION: The aim of the study was to investigate the role of fascin in tumor progression and to investigate the role of fascin on endothelial cell migration and angiogenesis in ovarian neoplasms. METHODS: In the study, 94 malign epithelial ovarian neoplasms, 13 borderline epithelial ovarian neoplasms, 25 serous and mucinous cystadenomas and four normal ovarian tissues were examined by means of immunohistochemistry, using monoclonal antihuman fascin antibody, clone IM20. RESULTS: Total stromal fascin score in cases of borderline and malign epithelial ovarian tumors was significantly higher compared to normal ovaries and benign epithelial ovarian tumors (.000, p < 0.001). There was no statistically significant difference in terms of total epithelial fascin scores of samples between groups (.080, p > 0.05). Presence of vascular invasion (.000, p < 0.001), psammomatous calcifications (.001, p = 0.001), and lymphocytic infiltration (.000, p < 0.001) were significantly higher in malign neoplasms. There was no significant difference in terms of mean microvessel count and homogeneous or heterogeneous fascin expression of microvessels between the benign and malign groups (respectively p = .228 and p = .143). CONCLUSIONS: This study suggests that up-regulation of fascin in tumoral tissue may promote invasion of ovarian carcinoma by cell-matrix adhesion.