Cell-extracellular Matrix Interactions Research Articles

A potential role for the CDH13/CDH15 gene in repeat revascularization after first percutaneous coronary intervention.

Aim: Major drawbacks of percutaneous coronary intervention are the high occurrence of repeat revascularization due to restenosis and disease progression. The aim of this study was to find genetic indicators to predict the risk of repeat revascularization. Materials & methods: From April 2015 to June 2016, 143 patients with percutaneous coronary intervention with genetic test results were enrolled. SNPs were measured by OmniZhongHua-8, and the SNPs in pathways genes related to known stenosis-related processes from the KEGG, BioCarta and Gene Cards databases were selected for analysis. Results: Cell-extracellular matrix interactions were the pathways with the most significant SNP (CDH15 rs72819363) association with repeat revascularization. Compared with CDH13 rs11859453G carriers, the adjusted odds ratio for A carriers was 0.25 and 0.33 at 18 and 30months. Conclusion: We demonstrated a potential role of the cell-extracellular matrix interactions pathway and the possible biomarker CDH13/CDH15 in the development of coronary repeat revascularization.

Biomimetic Materials and Their Utility in Modeling the 3-Dimensional Neural Environment.

The brain is a complex 3-dimensional structure, the organization of which provides a local environment that directly influences the survival, proliferation, differentiation, migration, and plasticity of neurons. To probe the effects of damage and disease on these cells, a synthetic environment is needed. Three-dimensional culturing of stem cells, neural progenitors, and neurons within fabricated biomaterials has demonstrated superior biomimetic properties over conventional 2-dimensional cultureware, offering direct recapitulation of both cell-cell and cell-extracellular matrix interactions. Within this review we address the benefits of deploying biomaterials as advanced cell culture tools capable of influencing neuronal fate and as in vitro models of the native in vivo microenvironment. We highlight recent and promising biomaterials approaches toward understanding neural network and their function relevant to neurodevelopment and provide our perspective on how these materials can be engineered and programmed to study both the healthy and diseased nervous system.

A Novel 3D Scaffold for Cell Growth to Asses Electroporation Efficacy.

Tumor electroporation (EP) refers to the permeabilization of the cell membrane by means of short electric pulses thus allowing the potentiation of chemotherapeutic drugs. Standard plate adhesion 2D cell cultures can simulate the in vivo environment only partially due to lack of cell–cell interaction and extracellular matrix (ECM). In this study, we assessed a novel 3D scaffold for cell cultures based on hyaluronic acid and ionic-complementary self-assembling peptides (SAPs), by studying the growth patterns of two different breast carcinoma cell lines (HCC1569 and MDA-MB231). This 3D scaffold modulates cell shape and induces extracellular matrix deposit around cells. In the MDA-MB 231 cell line, it allows three-dimensional growth of structures known as spheroids, while in HCC1569 it achieves a cell organization similar to that observed in vivo. Interestingly, we were able to visualize the electroporation effect on the cells seeded in the new scaffold by means of standard propidium iodide assay and fluorescence microscopy. Thanks to the presence of cell–cell and cell–ECM interactions, the new 3D scaffold may represent a more reliable support for EP studies than 2D cancer cell cultures and may be used to test new EP-delivered drugs and novel EP protocols.

Expression profiling in exercised mdx suggests a role for extracellular proteins in the dystrophic muscle immune response.

Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disorder caused by mutations in the DMD gene that leads to the absence or severe reduction of dystrophin protein in muscle. The mdx mouse, also dystrophin deficient, is the model most widely used to study the pathology and test potential therapies, but the phenotype is milder than human DMD. This limits the magnitude and range of histological damage parameters and molecular changes that can be measured in pre-clinical drug testing. We used 3 weeks of voluntary wheel running to exacerbate the mdx phenotype. In mdx mice, voluntary exercise increased the amount of damaged necrotic tissue and macrophage infiltration. Global gene expression profiling revealed that exercise induced additional and larger gene expression changes in mdx mice and the pathways most impacted by exercise were all related to immune function or cell-extracellular matrix (ECM) interactions. When we compared the matrisome and inflammation genes that were dysregulated in mdx with those commonly differentially expressed in DMD, we found the exercised mdx molecular signature more closely resembled that of DMD. These gene expression changes in the exercised mdx model thus provide more scope to assess the effects of pre-clinical treatments. Our gene profiling comparisons also highlighted upregulation of ECM proteins involved in innate immunity pathways, proteases that can release them, downstream receptors and signaling molecules in exercised mdx and DMD, suggesting that the ECM could be a major source of pro-inflammatory molecules that trigger and maintain the immune response in dystrophic muscle.

MicroRNAs Contribute to Breast Cancer Invasiveness.

Cancer statistics in 2018 highlight an 8.6 million incidence in female cancers, and 4.2 million cancer deaths globally. Moreover, breast cancer is the most frequent malignancy in females and twenty percent of these develop metastasis. This provides only a small chance for successful therapy, and identification of new molecular markers for the diagnosis and prognostic prediction of metastatic disease and development of innovative therapeutic molecules are therefore urgently required. Differentially expressed microRNAs (miRNAs) in cancers cause multiple changes in the expression of the tumorigenesis-promoting genes which have mostly been investigated in breast cancers. Herein, we summarize recent data on breast cancer-specific miRNA expression profiles and their participation in regulating invasive processes, in association with changes in cytoskeletal structure, cell-cell adhesion junctions, cancer cell-extracellular matrix interactions, tumor microenvironments, epithelial-to-mesenchymal transitions and cancer cell stem abilities. We then focused on the epigenetic regulation of individual miRNAs and their modified interactions with other regulatory genes, and reviewed the function of miRNA isoforms and exosome-mediated miRNA transfer in cancer invasiveness. Although research into miRNA’s function in cancer is still ongoing, results herein contribute to improved metastatic cancer management.

Identification of matrix physicochemical properties required for renal epithelial cell tubulogenesis by using synthetic hydrogels.

Synthetic hydrogels with controlled physicochemical matrix properties serve as powerful in vitro tools to dissect cell-extracellular matrix (ECM) interactions that regulate epithelial morphogenesis in 3D microenvironments. In addition, these fully defined matrices overcome the lot-to-lot variability of naturally derived materials and have provided insights into the formation of rudimentary epithelial organs. Therefore, we engineered a fully defined synthetic hydrogel with independent control over proteolytic degradation, mechanical properties, and adhesive ligand type and density to study the impact of ECM properties on epithelial tubulogenesis for inner medullary collecting duct (IMCD) cells. Protease sensitivity of the synthetic material for membrane-type matrix metalloproteinase-1 (MT1-MMP, also known as MMP14) was required for tubulogenesis. Additionally, a defined range of matrix elasticity and presentation of RGD adhesive peptide at a threshold level of 2 mM ligand density were required for epithelial tubulogenesis. Finally, we demonstrated that the engineered hydrogel supported organization of epithelial tubules with a lumen and secreted laminin. This synthetic hydrogel serves as a platform that supports epithelial tubular morphogenetic programs and can be tuned to identify ECM biophysical and biochemical properties required for epithelial tubulogenesis.

A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy

BackgroundHuman corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth.MethodsThree placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry.ResultsAME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures.ConclusionAME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.

Understanding the Cellular and Molecular Mechanisms That Control Early Cell Fate Decisions During Appendicular Skeletogenesis.

The formation of the vertebrate skeleton is orchestrated in time and space by a number of gene regulatory networks that specify and position all skeletal tissues. During embryonic development, bones have two distinct origins: bone tissue differentiates directly from mesenchymal progenitors, whereas most long bones arise from cartilaginous templates through a process known as endochondral ossification. Before endochondral bone development takes place, chondrocytes form a cartilage analgen that will be sequentially segmented to form joints; thus, in the cartilage template, either the cartilage maturation programme or the joint formation programme is activated. Once the cartilage differentiation programme starts, the growth plate begins to form. In contrast, when the joint formation programme is activated, a capsule begins to form that contains special articular cartilage and synovium to generate a functional joint. In this review, we will discuss the mechanisms controlling the earliest molecular events that regulate cell fate during skeletogenesis in long bones. We will explore the initial processes that lead to the recruitment of mesenchymal stem/progenitor cells, the commitment of chondrocyte lineages, and the formation of skeletal elements during morphogenesis. Thereafter, we will review the process of joint specification and joint morphogenesis. We will discuss the links between transcription factor activity, cell–cell interactions, cell–extracellular matrix interactions, growth factor signalling, and other molecular interactions that control mesenchymal stem/progenitor cell fate during embryonic skeletogenesis.

Galectin-3 orchestrates the histology of mesentery and protects liver during lupus-like syndrome induced by pristane

Galectin-3 (Gal-3) controls intercellular and cell-extracellular matrix interactions during immunological responses. In chronic inflammation, Gal-3 is associated with fibrotic events, regulates B cell differentiation and delays lupus progression. Gal-3 deficient mice (Lgals3−/−) have intense germinal center formation and atypical plasma cell generation correlated to high levels IgG, IgE, and IgA. Here, we used pristane (2,6,10,14-tetramethylpentadecane) to induce lupus-like syndrome in Lgals3−/− and Lgals3+/+ BALB/c mice. Mesentery and peritoneal cells were monitored because promptly react to pristane injected in the peritoneal cavity. For the first time, mesenteric tissues have been associated to the pathogenesis of experimental lupus-like syndrome. In Lgals3+/+ pristane-induced mice, mesentery was hallmarked by intense fibrogranulomatous reaction restricted to submesothelial regions and organized niches containing macrophages and B lymphocytes and plasma cells. In contrast, Lgals3−/− pristane-treated mice had diffuse mesenteric fibrosis affecting submesothelium and peripheral tissues, atypical M1/M2 macrophage polarization and significant DLL1+ cells expansion, suggesting possible involvement of Notch/Delta pathways in the disease. Early inflammatory reaction to pristane was characterized by significant disturbances on monocyte recruitment, macrophage differentiation and dendritic cell (DC) responses in the peritoneal cavity of pristane-induced Lgals3−/− mice. A correlative analysis showed that mesenteric damages in the absence of Gal-3 were directly associated with severe portal inflammation and hepatitis. In conclusion, it has suggested that Gal-3 orchestrates histological organization in the mesentery and prevents lupoid hepatitis in experimental lupus-like syndrome by controlling macrophage polarization, Notch signaling pathways and DC differentiation in mesenteric structures.

Laminin-driven Epac/Rap1 regulation of epithelial barriers on decellularized matrix

Decellularized tissues offer a unique tool for developing regenerative biomaterials or in vitro platforms for the study of cell-extracellular matrix (ECM) interactions. One main challenge associated with decellularized lung tissue is that ECM components can be stripped away or altered by the detergents used to remove cellular debris. Without characterizing the composition of lung decellularized ECM (dECM) and the cellular response caused by the altered composition, it is difficult to utilize dECM for regeneration and specifically, engineering the complexities of the alveolar-capillary barrier. This study takes steps towards uncovering if dECM must be enhanced with lost ECM proteins to achieve proper epithelial barrier formation. To achieve this, the epithelial barrier function was assessed on dECM coatings with and without the systematic addition of several key basement membrane proteins. After comparing barrier function on collagen I, fibronectin, laminin, and dECM in varying combinations as an in vitro coating, the alveolar epithelium exhibited superior barrier function when dECM was supplemented with laminin as evidenced by trans-epithelial electrical resistance (TEER) and permeability assays. Increased barrier resistance with laminin addition was associated with upregulation of Claudin-18, E-cadherin, and junction adhesion molecule (JAM)-A, and stabilization of zonula occludens (ZO)-1 at junction complexes. The Epac/Rap1 pathway was observed to play a role in the ECM-mediated barrier function determined by protein expression and Epac inhibition. These findings revealed potential ECM coatings and molecular therapeutic targets for improved regeneration with decellularized scaffolds. Statement of significanceEfforts to produce a transplantable organ-scale biomaterial for lung regeneration has not been entirely successful to date, due to incomplete cell-cell junction formation, ultimately leading to severe edema in vivo. To fully understand the process of alveolar junction formation on ECM-derived biomaterials, this research has characterized and tailored decellularized ECM (dECM) to mitigate reductions in barrier strength or cell attachment caused by abnormal ECM compositions or detergent damage to dECM.These results indicate that laminin-driven Epac signaling plays a vital role in the stabilization of the alveolar barrier. Addition of laminin or Epac agonists during alveolar regeneration can reduce epithelial permeability within bioengineered lungs.

GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.

Gliomas are the most common primary brain tumors. Their highly invasive character and the heterogeneity of active oncogenic pathways within single tumors complicate the development of curative therapies and cause poor patient prognosis. Glioma cells express the intermediate filament protein glial fibrillary acidic protein (GFAP), and the level of its alternative splice variant GFAP-δ, relative to its canonical splice variant GFAP-α, is higher in grade IV compared with lower-grade and lower malignant glioma. In this study we show that a high GFAP-δ/α ratio induces the expression of the dual-specificity phosphatase 4 (DUSP4) in focal adhesions. By focusing on pathways up- and downstream of DUSP4 that are involved in the cell-extracellular matrix interaction, we show that a high GFAP-δ/α ratio equips glioma cells to better invade the brain. This study supports the hypothesis that glioma cells with a high GFAP-δ/α ratio are highly invasive and more malignant cells, thus making GFAP alternative splicing a potential therapeutic target.-Van Bodegraven, E. J., van Asperen, J. V., Sluijs, J. A., van Deursen, C. B. J., van Strien, M. E., Stassen, O. M. J. A., Robe, P. A. J., Hol, E. M. GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.

Isolation and culture of primary embryonic zebrafish neural tissue

BackgroundPrimary cell culture is a valuable tool to utilize in parallel with in vivo studies in order to maximize our understanding of the mechanisms surrounding neurogenesis and central nervous system (CNS) regeneration and plasticity. The zebrafish is an important model for biomedical research and primary neural cells are readily obtainable from their embryonic stages viatissue dissociation. Further, transgenic reporter lines with cell type-specific expression allows for observation of distinct cell populations within the dissociated tissue. New methodHere, we define an efficient method for ex vivo quantification and characterization of neuronal and glial tissue dissociated from embryonic zebrafish. ResultsZebrafish brain dissociated cells have been documented to survive in culture for at least 9 days in vitro (div). Anti-HuC/D and anti-Acetylated Tubulin antibodies were used to identify neurons in culture; at 3 div approximately 48% of cells were HuC/D positive and 85% expressed serotonin, suggesting our protocol can efficiently isolate neurons from whole embryonic zebrafish brains. Live time-lapse imaging was also carried out to analyze cell migration in vitro. Comparison with existing methodsPrimary cultures of zebrafish neural cells typically have low rates of survivability in vitro. We have developed a culture system that has long term cell viability, enabling direct analysis of cell-cell and cell-extracellular matrix interactions. ConclusionsThese results demonstrate a practical method for isolating, dissociating and culturing of embryonic zebrafish neural tissue. This approach could further be utilized to better understand zebrafish regeneration in vitro.

Spatiotemporal pattern of glucose in a microfluidic device depend on the porosity and permeability of the medium: A finite element study

BackgroundGlucose plays an important role as a source of nutrients and influence cellular processes such as differentiation, proliferation and migration. In vitro models based on microfluidic devices represent an alternative to study several biological processes in a more reproducible and controllable method compared to in vivo models. Glucose concentration across a microfluidic chip and its behavior in experimental conditions is not completely understood. ObjectiveThis paper investigated the spatiotemporal distribution of glucose across the hydrogel inside a microfluidic chip. The influence of different parameters, boundary and initial conditions of experiments on glucose concentration was studied. MethodsA finite element model using a two dimensional geometry was developed. With this model, patterns of glucose concentration were investigated for different combinations of flow rate of culture medium, permeability and porosity of the medium. Patterns were also studied for two hydrogels made of collagen type I and fibrin with different initial and boundary conditions for pressure and glucose concentration. ResultsPorosity influenced significantly on the chemical gradients generated when interstitial fluid flow was null or neglectable. A difference in concentration lower than 15% was obtained at the input of microchamber and after 90 min, when porosity changed from 0.5 to 0.99. In addition, no significant effects of modifications in permeability were observed. Regarding the collagen and fibrin matrices, in the presence of a pressure gradient of 40 Pa, the permeability significantly influenced on the concentration gradients generated. ConclusionsPorosity influences importantly on patterns when diffusion is the main transport mechanism. Permeability is the most influencing parameter when a fluid flow is present. Common insertion rates of culture medium does not significantly modify the patterns of glucose inside the chips. Thus, new experiments must consider the impact of such parameters on the distribution and the time span that nutrients occupy the medium. To better contribute with experimental trials, other studies involving cell-cell and cell-extracellular matrix interactions, and different chip geometries should be developed. The results of the present work could assist to develop specific systems for experimentation, to design new experiments and to improve the analysis of the obtained results.

Calcium in Vascular Smooth Muscle Cell Elasticity and Adhesion: Novel Insights Into the Mechanism of Action.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the arterial wall. These cells play a critical role in maintaining vascular homeostasis including vasoconstriction and vasodilatation through active contraction and relaxation. Dysregulation of VSMC function alters the response of blood vessels to mechanical stress, contributing to the pathogenesis of vascular diseases, particularly atherosclerosis and hypertension. The stiffness of VSMCs is a major regulator of vascular function. Previous studies suggest that intracellular Ca2+ controls the stiffness of VSMCs by a mechanism involving myosin contractile apparatus. More recent studies highlight important functions of cytoskeletal α-smooth muscle actin (α-SMA), α5β1 integrin, and integrin-mediated cell-extracellular matrix (ECM) interactions in Ca2+-dependent regulation of VSMC stiffness and adhesion to the ECM, providing novel insights into the mechanism of calcium action.

Laminin-Driven Epac/Rap1 Regulation of Epithelial Barriers on Decellularized Matrix

Decellularized tissues offer a unique tool for developing regenerative biomaterials or in vitro platforms for the study of cell-extracellular matrix (ECM) interactions. One main challenge associated with decellularized lung tissue is that ECM components can be stripped away or altered by the detergents used to remove cellular debris. Without characterizing the composition of lung decellularized ECM (dECM) and the cellular response caused by the altered composition, it is difficult to utilize dECM for regeneration and specifically, engineering the complexities of the alveolar-capillary barrier. This study takes steps towards uncovering if dECM must be enhanced with lost ECM proteins to achieve proper epithelial barrier formation. To achieve this, the epithelial barrier function was assessed on dECM coatings with and without the systematic addition of several key basement membrane proteins. After comparing barrier function on collagen, fibronectin, laminin, and dECM in varying combinations as an in vitro coating, the alveolar epithelium exhibited superior barrier function when dECM was supplemented with laminin as evidenced by trans-epithelial electrical resistance (TEER) and permeability assays. Increased barrier resistance with laminin addition was associated with upregulation of Claudin-18, E-cadherin, and junction adhesion molecule (JAM)-A, and stabilization of zonula occludens (ZO)-1 at junction complexes. The Epac/Rap1 pathway was observed to play a role in the ECM-mediated barrier function determined by protein expression and Epac inhibition. These findings reveal potential ECM coatings and molecular therapeutic targets for improved regeneration with decellularized scaffolds or edema related pathologies.

Abstract 1299: Potential antitumor effects of a Golgi disrupting agent, M-COPA, via targeting cell-extracellular matrix interaction under the spheroid culture conditions

Abstract The Golgi apparatus plays an essential role in the transport, processing, and sorting of cell surface proteins. We previously demonstrated that a Golgi disrupting agent, 2-methylcoprophilinamide (M-COPA), exhibited antitumor effect in human cancer cells in vitro and in vivo; however, in vivo antitumor efficacy of M-COPA did not simply reflect its in vitro efficacy, probably due to the lack of microenvironmental features of tumor tissues in the monolayer culture conditions. To bridge the gap between in vitro and in vivo efficacies, we exploited 3-dimensional (3D) spheroid culture models, which can recapitulate physiological characteristics of tumor tissues; i.e., nutrient/oxygen gradient, cell-cell interaction and cell-extracellular matrix (ECM) interaction, if cancer cells synthesize their own ECM molecules. Interestingly, human gastric cancer MKN1 cells displayed hypersensitivity to M-COPA under the 3D culture conditions compared to those in the monolayer culture conditions. Especially, we found that M-COPA caused loss of spheroid integrity. We previously demonstrated that M-COPA exerted antitumor effects via inhibiting intracellular transport of receptor tyrosine kinases onto the cell surface. Therefore, we examined the involvement of cell adhesion molecules in the antitumor effect of M-COPA. As a result, we found that specific integrins, which serve as cell surface receptors responsible for cell-ECM adhesion, were overexpressed and their downstream signaling molecules such as Src, Akt and MEK/ERK were highly phosphorylated in the cells under the 3D culture conditions compared to those under the monolayer culture conditions. Upon exposure to M-COPA, cell surface expression of these integrins was efficiently decreased and the downstream signaling molecules were dephosphorylated along with loss of spheroid integrity. Moreover, expression knockdown or neutralizing antibody of the integrins clearly suppressed sphere formation of MKN1 cells. These results suggest that M-COPA exerted in vivo antitumor effects via targeting specific integrins that mediate cell-matrix adhesion and its downstream intracellular signaling pathway observed in 3D spheroid culture models. Citation Format: Yoshimi Ohashi, Kazuma Takeuchi, Mutsumi Okamura, Akinobu Akatsuka, Isamu Shiina, Kentaro Yoshimatsu, Shingo Dan. Potential antitumor effects of a Golgi disrupting agent, M-COPA, via targeting cell-extracellular matrix interaction under the spheroid culture conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1299.

Abstract 5184: N-CoR2 epigenetically regulates adhesion-dependent branching morphogenesis and its loss correlates with malignant progression of the mammary gland

Abstract Branching morphogenesis depends upon extracellular matrix remodeling and dynamic cell-cell and cell-extracellular matrix interactions, although how this developmental program is coordinated remains unclear. Expression profiling of mammary tissue at different stages of branching morphogenesis combined with histological and functional studies identified the histone deacetylase (HDAC) regulator, nuclear receptor co-repressor 2 (N-CoR2), as a key orchestrator of adhesion-dependent branching morphogenesis in the mammary gland. Molecular studies revealed that N-CoR2 actively represses fibronectin (FN) and thrombospondin-1 (THBS-1) promoter accessibility in an HDAC-dependent manner. Consistently, histological analysis revealed that N-CoR2 is abundantly expressed in the luminal epithelial cells localized along the outer rim of invading terminal end buds (TEB) and that its expression is lowest at sites of TEB branching bifurcation. In addition, reducing N-CoR2 expression drove the scattering of human mammary acini in vitro and promoted promiscuous branching of humanized mouse mammary tissue in vivo, suggesting that downregulation of N-CoR2 is required for branching morphogenesis in mammary gland. Furthermore, downregulation of N-CoR2 in breast cancer cells increased the expression of both FN and THBS-1 and reduced the expression of E-cadherin and enhanced the migratory capability of breast cancer cells. Intriguingly, immunohistochemical and clinical correlative analysis indicated that loss of N-CoR2 correlates with perturbed cell-cell and enhanced cell-ECM adhesion and acquisition of an invasive phenotype and local lymph node metastasis in a subset of breast carcinomas. These findings suggest N-CoR2 may epigenetically regulate adhesion-dependent branching morphogenesis in the mammary gland and that its loss could promote malignant progression of some breast cancers. Citation Format: Shenq-Shyang Huang, Valerie M. Weaver, Kelvin K.C. Tsai. N-CoR2 epigenetically regulates adhesion-dependent branching morphogenesis and its loss correlates with malignant progression of the mammary gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5184.

Abstract LB-127: Loss of function of GAS7 promotes neuroblastoma metastasis in the context of MYCN overexpression

Abstract Neuroblastoma is an embryonal tumor arising in the peripheral sympathetic nervous system (PSNS), and often presents with widespread metastasis at diagnosis. We recently demonstrated that overexpression of LMO1 can synergize with MYCN to promote tumor metastasis in our zebrafish model system. Here, we identify a “metastasis suppressor” gene – growth arrest specific 7 (GAS7) – whose loss of function or reduced expression can promote dissemination and migration of neuroblastoma cells with MYCN overexpression, both in vitro and in vivo. GAS7 resides on chromosome 17p13.1, a region that is deleted in high-risk neuroblastomas with higher frequency in MYCN-amplified cases. Expression levels of GAS7 are also significantly downregulated in MYCN-amplified or otherwise MYCN-overexpressing human neuroblastoma cells, as well as MYCN-overexpressing zebrafish PSNS cells, suggesting an inverse relationship between the oncogenic activities of MYCN and GAS7downregulation. To determine whether loss of function or reduced expression of GAS7 could contribute to neuroblastoma pathogenesis in the context of MYCN overexpression, we developed a gas7 knockout zebrafish line using a transcription activator-like effector nucleases (TALEN) method. Loss of function of gas7 promoted hematogenous metastasis of MYCN-overexpressing tumor cells to tissues and organs that paralleled the common sites of metastasis in neuroblastoma patients. Further RNA-sequencing and qRT-PCR analyses showed that representative signature genes encoding proteins involved in tumor cell-cell or cell-extracellular matrix interactions were significantly downregulated in fish tumors with loss of function of gas7, consistent with our electron microscopic finding in the fish model showing a loss of contact among tumor cells in fish lacking functional gas7, suggesting that GAS7 may physically act as a metastasis suppressor. Thus, in high-risk neuroblastoma with MYCNamplification or overexpression, we propose that deletion or reduced expression of GAS7 cooperates with MYCN to promote neuroblastoma cell dissemination. Although the precise mechanism of this interaction remains unclear, we suggest that it relies, in part, on the downregulation of pivotal genes involved in tumor cell-cell or cell-extracellular matrix contacts. Citation Format: Zhiwei Dong, Gonzalo Lepez Garcia, Cheng Zhang, Erin N. Dankert, Jo Lynne Harenza, Xiaonan Hou, Hu Li, John M. Maris, Shizhen (Jane) Zhu. Loss of function of GAS7 promotes neuroblastoma metastasis in the context of MYCN overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-127.

The role of spectrin in cell adhesion and cell–cell contact

Spectrins are proteins that are responsible for many aspects of cell function and adaptation to changing environments. Primarily the spectrin-based membrane skeleton maintains cell membrane integrity and its mechanical properties, together with the cytoskeletal network a support cell shape. The occurrence of a variety of spectrin isoforms in diverse cellular environments indicates that it is a multifunctional protein involved in numerous physiological pathways. Participation of spectrin in cell–cell and cell–extracellular matrix adhesion and formation of dynamic plasma membrane protrusions and associated signaling events is a subject of interest for researchers in the fields of cell biology and molecular medicine. In this mini-review, we focus on data concerning the role of spectrins in cell surface activities such as adhesion, cell–cell contact, and invadosome formation. We discuss data on different adhesion proteins that directly or indirectly interact with spectrin repeats. New findings support the involvement of spectrin in cell adhesion and spreading, formation of lamellipodia, and also the participation in morphogenetic processes, such as eye development, oogenesis, and angiogenesis. Here, we review the role of spectrin in cell adhesion and cell–cell contact.Impact statementThis article reviews properties of spectrins as a group of proteins involved in cell surface activities such as, adhesion and cell–cell contact, and their contribution to morphogenesis. We show a new area of research and discuss the involvement of spectrin in regulation of cell–cell contact leading to immunological synapse formation and in shaping synapse architecture during myoblast fusion. Data indicate involvement of spectrins in adhesion and cell–cell or cell–extracellular matrix interactions and therefore in signaling pathways. There is evidence of spectrin’s contribution to the processes of morphogenesis which are connected to its interactions with adhesion molecules, membrane proteins (and perhaps lipids), and actin. Our aim was to highlight the essential role of spectrin in cell–cell contact and cell adhesion.

DOWN-EXPRESSION OF TETRASPANIN CD9 IS A SENSITIVE MARKER FOR IDENTIFYING PRE-MALIGNANT CHANGES IN THE ORAL EPITHELIUM

Objectives Tetraspanins, cell surface proteins that mediate cell-cell and cell-extracellular matrix interactions, are capable to modify cell motility, thus being potential diagnostic markers in pre-malignant conditions. We examined the immunohistochemical expression of tetraspanins CD9, CD81 and CD63 in normal oral mucosa as well as in inflamed, dysplastic and neoplastic epithelial lesions. Findings Included were cases of normal oral mucosa (NOR, N=15), oral lichen planus (OLP, N=51), hyperkeratosismild dysplasia (HK, N=29), moderate-severe dysplasia (DYSP, N=22), oral squamous cell carcinoma (OSCC, N=31), and normal-looking mucosa nearby OSCC (N-OSCC, N=18). Staining, assessed as percent of stained cells multiplied by staining intensity (1=weak, 2=strong), was evaluated per epithelial thirds (basal, middle and upper) and then as total staining score (sum of all thirds). Statistical analysis was performed using One-way ANOVA. Receiver operating characteristic (ROC) curve was used for diagnostic sensitivity. Statistical significance was set at P Conclusions CD9 could accurately discriminate between normal (high expression) and all other types of pathologies (lower expression) with high diagnostic sensitivity. In addition, expression of CD9 in neoplasia and the nearby histologically “normal-looking” epithelium was similar but significantly lower than in dysplasia and OLP. Therefore, the expression of CD9 could aid in defining the nature of equivocal histopathological changes in oral epithelial lesions.