Cell Extrusion Research Articles

Epithelial Cell Extrusion Zones Observed on Confocal Laser Endomicroscopy Correlates with Immunohistochemical Staining of Mucosal Biopsy Samples.

The density of epithelial cell extrusion zones in the intestinal lining, also known as gap density (number of gaps/1000 epithelial cells counted), can be quantitated using probe-based confocal laser endomicroscopy (pCLE). Gap density has been reported to be higher than normal in both inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) patients. Epithelial cells destined for extrusion from the intestinal surface would stain positive for either activated caspase-1 or caspase-3 on mucosal biopsy samples. The aim of this study was to determine whether epithelial gap density on pCLE correlates with quantitative analysis of activated caspase staining of mucosal biopsy samples from patients. We obtained pCLE images and biopsy samples of the terminal ileum during colonoscopies of healthy controls and patients with either IBD or IBS. The pCLE images and biopsy samples were blindly analyzed for gap density and for cells staining positive for activated caspases, respectively. The degree of correlation was determined using nonparametric statistical tests. The median results were 10gaps/1000 cells counted for controls versus 33 gaps/1000 cells counted for chronic intestinal disorder patients (p=0.02). Activated caspase staining showed 13positive cells/1000 epithelial cells counted versus 26positive cells/1000 epithelial cells counted, respectively (p=0.02), thus showing a strong correlation with a Spearman's coefficient ρ of 0.61 (strong correlation for ρ=0.4-0.75, p=0.01). Intestinal epithelial gap density via pCLE correlated strongly with quantitative analysis of immunohistochemical staining of mucosal biopsy samples.

Twinstar/cofilin is required for regulation of epithelial integrity and tissue growth in Drosophila.

Regulation of actin assembly and depolymerization is important for the organization of epithelia. Recent studies have shown that the actin-capping proteins are required to prevent cell extrusion and inappropriate activation of Yorkie (Yki) activity in Drosophila, implicating the importance of actin regulation for epithelial integrity and Yki-dependent tissue growth. However, the role of Twinstar (Tsr), the Drosophila homolog for cofilin/actin depolymerization factor (ADF), in epithelial integrity and Hippo signaling is unknown. Here we demonstrate that reduction of Tsr by RNA interference (RNAi) or mutant clones in wing imaginal disc induces not only F-actin accumulation but also ectopic expression of Wingless (Wg) and Yki target gene expanded (ex). Knockdown of Yki in Tsr-depleted cells reduced the level of ectopic Wg expression. Reduced Tsr also led to downregulation of cell junction proteins and extrusion of affected cells from the basal part of the epithelium. Rho GTPase 1 was upregulated in Tsr-depleted tissue, supporting the Tsr function in the inhibition of cell extrusion from the epithelium. Tsr is also required for blocking cell death and c-Jun N-terminal kinase (JNK) signaling. Ectopic JNK activation induced caspase activation but did not cause cell extrusion. Further, the invasiveness of Tsr-depleted cells was not suppressed by inhibition of cell death or JNK signaling. In contrast, Yki upregulation was significantly suppressed by cell death inhibition. Taken together, our data suggest that Tsr is required for cell survival and tissue growth by regulating JNK and Yki signaling while maintaining the epithelial integrity by controlling cell junctions. This study provides an insight into potential roles of ADF/cofilin in invasive cell migration and tumor suppression in higher animals.

Epithelial Contractility: A Crowning Achievement

Epithelial cells transmit contractile force with adherens junctions to mediate morphological changes like the extrusion of apoptotic cells. In this issue of Developmental Cell, Michael and colleagues (2016) show that the actin binding protein Coronin plays a critical role in actin cytoskeleton reorganization and association with junctions to promote contractility.

Coronin 1B Reorganizes the Architecture of F-Actin Networks for Contractility at Steady-State and Apoptotic Adherens Junctions

In this study we sought to identify how contractility at adherens junctions influences apoptotic cell extrusion. We first found that the generation of effective contractility at steady-state junctions entails a process of architectural reorganization whereby filaments that are initially generated as poorly organized networks of short bundles are then converted into co-aligned perijunctional bundles. Reorganization requires coronin 1B, which is recruited to junctions by E-cadherin adhesion and is necessary to establish contractile tension at the zonula adherens. When cells undergo apoptosis within an epithelial monolayer, coronin 1B is also recruited to the junctional cortex at the apoptotic/neighbor cell interface in an E-cadherin-dependent fashion to support actin architectural reorganization, contractility, and extrusion. We propose that contractile stress transmitted from the apoptotic cell through E-cadherin adhesions elicits a mechanosensitive response in neighbor cells that is necessary for the morphogenetic event of apoptotic extrusion to occur.

Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium.

Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring non-dying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, contraction of actomyosin cables formed in apoptotic and neighboring cells drives cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs) between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. We propose a model in which modulation of tissue tension represents a mechanism of apoptotic cell extrusion.

BMP signaling and cellular dynamics during regeneration of airway epithelium from basal progenitors.

The pseudostratified epithelium of the lung contains ciliated and secretory luminal cells and basal stem/progenitor cells. To identify signals controlling basal cell behavior we screened factors that alter their self-renewal and differentiation in a clonal organoid (tracheosphere) assay. This revealed that inhibitors of the canonical BMP signaling pathway promote proliferation but do not affect lineage choice, whereas exogenous Bmp4 inhibits proliferation and differentiation. We therefore followed changes in BMP pathway components in vivo in the mouse trachea during epithelial regeneration from basal cells after injury. The findings suggest that BMP signaling normally constrains proliferation at steady state and this brake is released transiently during repair by the upregulation of endogenous BMP antagonists. Early in repair, the packing of epithelial cells along the basal lamina increases, but density is later restored by active extrusion of apoptotic cells. Systemic administration of the BMP antagonist LDN-193189 during repair initially increases epithelial cell number but, following the shedding phase, normal density is restored. Taken together, these results reveal crucial roles for both BMP signaling and cell shedding in homeostasis of the respiratory epithelium.

Emergence of an Apical Epithelial Cell Surface In Vivo

Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, wecombine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium invivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging celland mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis.

Direct printing of living cell composed 3D objects with complex and large shape: a scaffold-free approach

Event Abstract Back to Event Direct printing of living cell composed 3D objects with complex and large shape: a scaffold-free approach Léa Pourchet1, Christophe Marquette1 and Amélie Thépot2 1 université Lyon 1, ICBMS, France 2 LabSkin Creations, France In the past few years, the advances in 3D printing and especially in bioprinting, gave an excellent overview of what can be the future of the regenerative medicine. Scaffolds for tissues growing and other biomaterial applications were then largely covered by the developed technologies. Nevertheless, instruments, biomaterial formulation and technologies being able to print directly living cells in real 3D format with hollow structures and multi-component composition are still very few. In our present approach, we had created an open source bioprinter on the basis of a 3D extrusion printer (TOBECA®, France), by substituting the extruders’ head with a rack of syringe pumps, enabling the bio-extrusion process. All parts of the new bioprinter were then printed using the initial PLA 3D printer, following the self-replication concept. All source code and design files are now open source available. Ink formulation optimization was carried on in order to permit living cell extrusion. An alginate based ink was selected, together with the additives necessary to the stabilization of the ink and the cell proliferation. The developed ink has a perfect biocompatibility, printability, and an ideal polymerization process adapted to the cell culture. We were thus able to print bi-component 3D hydrogels with high potential applications in the field of regenerative medicine and 3D organ model systems. We have demonstrated the feasibility of printing functional tissue. Indeed, once the objet printed and after few weeks of incubation, we observed the development of cells 3D network as well as the formation of neo-synthesized extracellular matrix. The tissue characterization shows the formation of a dermis and epidermis. These amazing results allow seeing how could be the future of the personalized medicine. The latest developments of the technique and the results of the last living printed 3D objects will be presented and discussed in the light of the biomaterial and tissue engineering community requirements. Keywords: Hydrogel, Bioprinting, complex tissue orgnization Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Three-dimensional fabrication Citation: Pourchet L, Marquette C and Thépot A (2016). Direct printing of living cell composed 3D objects with complex and large shape: a scaffold-free approach. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01181 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Léa Pourchet Christophe Marquette Amélie Thépot Google Léa Pourchet Christophe Marquette Amélie Thépot Google Scholar Léa Pourchet Christophe Marquette Amélie Thépot PubMed Léa Pourchet Christophe Marquette Amélie Thépot Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Continuous Exposure to Bendamustine (BDM) Results in Stable Upregulation of CD30 and Increased Sensitivity to Brentuximab Vedotin (BV) in Tumor Cells of Hodgkin Lymphoma (HL)

Background: Given the consistent antitumor activity and favorable toxicity profile in heavily pretreated patients, BDM represents a suitable platform for combination with target-based agents in the setting of recurrent HL. The anti-CD30 antibody-monomethyl auristatin E (MMAE) conjugate brentuximab vedotin (BV) appears a most valuable candidate by coupling an impressive clinical activity with the lack of serious overlapping toxicities with BDM. While the combination of BDM and BV is being evaluated in Phase II studies, no information is available as to possible mechanisms through which BDM may regulate the antitumor efficacy of BV towards HL cells.Methods: We evaluated the effects of acute and extended exposure to BDM on CD30 expression and sensitivity to the cytotoxic activity BV in the HL cell line L1236. Cells were cultured in the presence of BDM at its IC50 for different time points of acute exposure. Through continuous exposure to increasing concentrations (25.0, 50.0, 75.0, 100 micromol/L) of BDM, we then performed a serial in vitro selection for BDM-resistant (R) L1236 cell clones and determined their CD30 expression by flow cytometry, qRT-PCR, and Western analysis.Results: Acute exposure to BDM (48 to 72 hrs) of L1236 cells led to a sizeable upregulation of CD30 as shown by flow cytometry. This effect was unsustained since CD30 intensity returned to baseline levels upon culturing in BDM-free medium for one week. Expression of other surface antigens, i.e. HLA-DR, was unaffected by BDM while acute exposure to doxorubicin and other cytotoxic drugs did not modify the CD30 expression. We established four L1236-derived cell clones (R25, R50, R75 and R100) able to proliferate across the different concentrations of BDM with growth/viability curves superimposable to native cells. Clonal identity among clones and with parental cells was confirmed by sequencing of V3-21 (FR2/FR3) and JH3-JH4 Ig DNA regions. All R-clones displayed an up to 900% increase in CD30 median fluorescence intensity, relative to native L1236 cells (Figure 1A). The sustained CD30 upregulation was confirmed by qRT-PCR and Western blotting since R-clones expressed up to 10-fold higher levels of CD30-specific mRNA and CD30-specific 120 kDa components as compared to parental cells. To rule out a general upregulation of surface molecules as a result of the chronic exposure to BDM we evaluated the expression of other surface antigens. As opposed to CD30, HLA-DR and PDL-1 relative transcript levels and cell surface expression were significantly reduced in R-cells. Intriguingly, BDM-induced CD30 upregulation was specific to L1236 cells since extended exposure to BDM did not modify expression levels of CD30 in other lymphoma cell types (SUDHL1, JEKO). BDM-induced CD30 overexpression significantly enhanced the sensitivity of HL cells to the cytotoxic effects of BV. MTS assay showed the IC50 of R100 cells to BV shifted to 0.21 ± 0.06 mcg/mL (48 hrs) and 0.19 ± 0.05 mcg/mL (72 hrs) vs. 3.16 ± 0.75 mcg/mL (48 hrs) and 3.87 ± 0.68 mcg/mL (72 hrs) of parental cells, a 15- and 20-fold increase, respectively (p<0.001; Figure 1B). Finally, qRT-PCR studies disclosed that R-clones did not show any upregulation of the drug exporter MDR1 gene, involved in MMAE extrusion and BV resistance in HL cells, as compared to parental L1236 cells.Conclusions: Whileacute exposure to BDM induced a transient upregulation of CD30, extended exposure to the agent resulted in the selection of L1236 subclones with a stable overexpression of surface CD30 and reduced levels of HLA-DR and PDL-1. Upregulation of CD30 was associated with a significantly enhanced sensitivity to cytotoxic effects of BV. Translated into a clinical context these findings suggest that HL patients progressing upon BDM treatment may turn exquisitely sensitive to BV and prompt the exploration of a sequential treatment with BDM and BV in late and earlier disease phases. Results also provide a mechanistic insight as to the efficacy of the BDM-BV combination in HL, highlighting non-overlapping resistance pathways between BDM and MMAE. BDM-induced selection of CD30hi, HLA-DRlow/PDL-1low tumor cell clones might be relevant to the design of novel treatment strategies for HL based on the combination of BDM with BV and anti-PD1/PDL-1 antibodies. [Display omitted] [Display omitted] DisclosuresPinto:Takeda: Research Funding; Takeda, Celgene, Roche, TEVA: Honoraria.

A role of the sphingosine-1-phosphate (S1P)-S1P receptor 2 pathway in epithelial defense against cancer (EDAC).

At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)-S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells.

Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle: A STUDY USING GENETICALLY ENCODED CALCIUM INDICATORS

Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca(2+) oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca(2+) enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca(2+) changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca(2+) oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca(2+) influx. This is the first study showing, in real time, Ca(2+) signals preceding egress and their direct link with motility, an essential virulence trait.

Photosynthetic pigments, cell extrusion and relative leaf water content of the castor bean under silicon and salinity

ABSTRACTThe castor bean crop plays an important social, environmental and economic role, especially due to possibility of its use as feedstock for the generation of clean energy. Thus, this study aimed to evaluate the contents of photosynthetic pigments, cell membrane extrusion and the relative water content in the leaves of the castor bean cultivar ‘BRS Energia’ under silicon and salinity levels. The experiment was carried out at Embrapa Cotton, in a completely randomized design, in a 4 x 4 factorial scheme, and the treatments consisted of four silicon levels (0, 100, 200 and 300 mg L-1) and four water salinity levels of (ECw 0, 2, 4 and 6 dS m-1), with three replicates. The gradual increase in salinity levels reduced the concentration of photosynthetic pigments, relative water content in the leaves and increased cell membrane extrusion. The increase in the concentration of silicon reduced the harmful effects of salts on the analysed variables in the castor bean ‘BRS Energia’.

The elimination of an adult segment by the Hox gene Abdominal-B

Hox gene activity leads to morphological diversity of organs or structures in different species. One special case of Hox function is the elimination of a particular structure. The Abdominal-B Hox gene of Drosophila melanogaster provides an example of such activity, as this gene suppresses the formation of the seventh abdominal segment in the adult. This elimination occurs only in males, and is characteristic of more advanced Diptera. The elimination requires the differential expression or activity of genes that are downstream Abdominal-B, or that work together with it, and which regulate cell proliferation or cell extrusion. Here, we review the mechanisms responsible for such elimination and provide some new data on processes taking place within this segment.

Toxicity mechanisms of ionic silver and polymer-coated silver nanoparticles with interactions of functionalized carbon nanotubes on early development stages of sea urchin

Exposures of aquatic organisms to multiple contaminants are likely to take place in estuarine and coastal areas and combined effects on early life stages have to be examined. Among emerging contaminants, ionic silver (Ag+) and silver nanoparticles (AgNps) have demonstrated contrasting effects on marine invertebrates, but their interactions with functionalized carbon nanotubes (f-SWCNTs) have not yet been investigated in details. In order to observe the impacts and understand the toxicity mechanism of Ag+ and polymer-coated AgNps, and their combined effects with f-SWCNTs, successive development stages of embryos of sea urchin, Strongylocentrotus droebachiensis, were exposed to Ag+, AgNps and f-SWCNTs, separately and in mixtures using moderate environmental concentrations. We also assessed long-term effects of treatments under recovery conditions. Morphological endpoints such as archenteron elongation, primary and secondary mesenchyme cells fate, pigment cells migration, spiculogenic cells and gut development indicated different effects of silver and nanosilver forms during successive development stages. Whereas Ag+ induced vegetalization and extrusion of mesenchyme cells on early embryos; f-SWCNTs+Ag+ strongly interfered with gut regionalization in late larvae. Sensitive blastocoelar cells got vacuolized and shapeless with AgNps, but not with mixtures with f-SWCNTs. Increased concentrations of Ag+ and f-SWCNTs+Ag+ led to the most disruptive effects during development, but f-SWCNTs+Ag+ caused the highest mortality rate during the recovery period, which indicated far-reaching effects driven by f-SWCNTs and their ability to keep silver more available during exposure period.

The art of magnesium transport.

Patients with hypomagnesemia suffer from a wide range of symptoms including muscle cramps, cardiac arrhythmias and epilepsy. Disturbances in body Mg(2+) homeostasis can often be attributed to increased Mg(2+) excretion by the kidney. Within the kidney, the distal convoluted tubule (DCT) segment determines the final Mg(2+) excretion, since no reabsorption takes place beyond this segment of the nephron. On 21(st) of January 2015, Jeroen de Baaij defended his thesis "The Distal Convoluted Tubule: the Art of Magnesium Transport", in which he aimed to identify new genes involved in Mg(2+) reabsorption in the DCT. This review summarizes the main findings of his graduate research. TRPM6 mediates apical Mg(2+) entry into the DCT cell and is highly regulated by EGF, insulin and pH. ATP and flavagline compounds have been characterized as new regulators of TRPM6 activity, providing novel pathways to target Mg(2+) disturbances. Using isolated primary DCT cells from mice, PCBD1 was identified as a new transcriptional regulator of Mg(2+) transport in the DCT. Indeed, patients with PCBD1 mutations were shown to suffer hypomagnesemia and MODY5-like diabetes. Subsequently, the work presented in the thesis focused on the elucidation of the basolateral Mg(2+) extrusion of the DCT cell. In vivo studies using SLC41A3-knockout mice suggest that SLC41A3 may act as Mg(2+) extrusion mechanism. CNNM2 has long been hypothesized to transport Mg(2+) at the basolateral membrane of the DCT. However, by determining the protein topology and homology modeling of the CBS domains, it was argued that CNNM2 is rather an Mg(2+)-sensing mechanism. Follow-up studies using (25)Mg(2+) isotopes showed that CNNM2 increases Mg(2+) uptake when overexpressed in HEK293 cells. Additionally, by knocking down cnnm2 in zebrafish, CNNM2 was demonstrated to be essential for brain development and Mg(2+) homeostasis. Mutations in CNNM2 were shown to cause hypomagnesemia, seizures and intellectual disability. Altogether, this thesis established the importance of Mg(2+) reabsorption in the DCT to health and disease. Combined, continued efforts of clinicians, geneticists, and researchers are necessary to improve the care of hypomagnesemic patients and increase our understanding of Mg(2+) reabsorption in the DCT.

The exocyst gene Sec10 regulates renal epithelial monolayer homeostasis and apoptotic sensitivity.

The highly conserved exocyst protein complex regulates polarized exocytosis of subsets of secretory vesicles. A previous study reported that shRNA knockdown of an exocyst central subunit, Sec10 (Sec10-KD) in Madin-Darby canine kidney (MDCK) cells disrupted primary cilia assembly and 3D cyst formation. We used three-dimensional collagen cultures of MDCK cells to further investigate the mechanisms by which Sec10 and the exocyst regulate epithelial polarity, morphogenesis, and homeostasis. Sec10-KD cysts initially demonstrated undisturbed lumen formation although later displayed significantly fewer and shorter primary cilia than controls. Later in cystogenesis, control cells maintained normal homeostasis, while Sec10-KD cysts displayed numerous apoptotic cells extruded basally into the collagen matrix. Sec10-KD MDCK cells were also more sensitive to apoptotic triggers than controls. These phenotypes were reversed by restoring Sec10 expression with shRNA-resistant human Sec10. Apico-basal polarity appeared normal in Sec10-KD cysts, whereas mitotic spindle angles differed significantly from controls, suggesting a planar cell polarity defect. In addition, analysis of renal tubules in a newly generated kidney-specific Sec10-knockout mouse model revealed significant defects in primary cilia assembly and in the targeted renal tubules; abnormal epithelial cell extrusion was also observed, supporting our in vitro results. We hypothesize that, in Sec10-KD cells, the disrupted exocyst activity results in increased apoptotic sensitivity through defective primary cilia signaling and that, in combination with an increased basal cell extrusion rate, it affects epithelial barrier integrity and homeostasis.

The mechanotransduction machinery at work at adherens junctions.

The shaping of a multicellular body, and the maintenance and repair of adult tissues require fine-tuning of cell adhesion responses and the transmission of mechanical load between the cell, its neighbors and the underlying extracellular matrix. A growing field of research is focused on how single cells sense mechanical properties of their micro-environment (extracellular matrix, other cells), and on how mechanotransduction pathways affect cell shape, migration, survival as well as differentiation. Within multicellular assemblies, the mechanical load imposed by the physical properties of the environment is transmitted to neighboring cells. Force imbalance at cell-cell contacts induces essential morphogenetic processes such as cell-cell junction remodeling, cell polarization and migration, cell extrusion and cell intercalation. However, how cells respond and adapt to the mechanical properties of neighboring cells, transmit forces, and transform mechanical signals into chemical signals remain open questions. A defining feature of compact tissues is adhesion between cells at the specialized adherens junction (AJ) involving the cadherin super-family of Ca(2+)-dependent cell-cell adhesion proteins (e.g., E-cadherin in epithelia). Cadherins bind to the cytoplasmic protein β-catenin, which in turn binds to the filamentous (F)-actin binding adaptor protein α-catenin, which can also recruit vinculin, making the mechanical connection between cell-cell adhesion proteins and the contractile actomyosin cytoskeleton. The cadherin-catenin adhesion complex is a key component of the AJ, and contributes to cell assembly stability and dynamic cell movements. It has also emerged as the main route of propagation of forces within epithelial and non-epithelial tissues. Here, we discuss recent molecular studies that point toward force-dependent conformational changes in α-catenin that regulate protein interactions in the cadherin-catenin adhesion complex, and show that α-catenin is the core mechanosensor that allows cells to locally sense, transduce and adapt to environmental mechanical constrains.

Convergence and extrusion are required for normal fusion of the mammalian secondary palate.

The fusion of two distinct prominences into one continuous structure is common during development and typically requires integration of two epithelia and subsequent removal of that intervening epithelium. Using confocal live imaging, we directly observed the cellular processes underlying tissue fusion, using the secondary palatal shelves as a model. We find that convergence of a multi-layered epithelium into a single-layer epithelium is an essential early step, driven by cell intercalation, and is concurrent to orthogonal cell displacement and epithelial cell extrusion. Functional studies in mice indicate that this process requires an actomyosin contractility pathway involving Rho kinase (ROCK) and myosin light chain kinase (MLCK), culminating in the activation of non-muscle myosin IIA (NMIIA). Together, these data indicate that actomyosin contractility drives cell intercalation and cell extrusion during palate fusion and suggest a general mechanism for tissue fusion in development.

Sa1709 Antitumor Effect of the Potent FGFR Inhibitor Ponatinib on an FGFR2-Fusion Driven Cholangiocarcinoma Mouse Model

Background: Overcrowding always lead to the shedding of live cells to maintain homeostasis in epithelial cell sheets. Recently, some reports have shown that overcrowded cancer cells can be basally extruded by their neighbors, and then form metastases. However, this interesting phenomenon needs to be further clarified, and the exact mechanisms driving the extrusion of crowded cancer cell sheets remain elusive. We have previously confirmed that BVES, a novel adhesion molecule regulating tight junction formation, could inhibit liver cancer cell extrusion in a simply and skillfully designed 2D cell culture model, as cancer cells are always basally extruded, in this study, we performed a 3D cell culture model to further confirm the inhibition role of BVES in liver cancer cell extrusion. Methods: We designed a 3D cell culture model, after centrifuge, 5×10 6 cells were resuspended by 120μl 10% sucrose solutions, and quickly mixed with equal volume of 0.5% hydrogel solution( BeaverNanoTM ), the cells were then seeded in a Class Bottom Cell Culture Dish(NEST), and allowed to grow for six days. Then, cells were fixed with 4 % paraformaldehyde, incubated with primary antibody and secondary antibody, observed under a confocal microscopy. Extracted cell number were also investigated after enhanced or silenced expression of BVES. RhoA agonist U-46619 and inhibitor C3 enzyme were used to verify the role of RhoA/Rho-kinase pathway in BVES inhibited cancer cell extrusion. Results: Cells successfully grown to form an overcrowded cell mass in the 3D cell culture model, the number of extruded SK-Hep-1 cells, a high metastasis potential cell line, was significantly more than Huh7 cells with low metastasis potential, the normal liver cell line LO2 cells nearly had no cell extruded. Meanwhile, the extruded cells had decreased expression of BVES. Overexpression of BVES inhibited cells extrusion in the 3D cell culture model and increased RhoA activity in SK-Hep-1 cells, while stable knockdown of BVES remarkably promoted cells extrusion and decreased RhoA activity in Huh7 cells. U-46619 increased Huh7 cells extrusion while treated with C3 enzyme significantly diminished cells extrusion. Conclusion: These results suggest that in crowding liver cancer, BVES inactivate RhoA signaling pathway to decrease liver cancer cells extrusion, subsequently may inhibits tumor metastasis. (This study is supported by the National Natural Science Foundation of China NO:81472311 and the Fundamental Research Funds for the Central Universities 2014ZHYX020)

Sa1710 Suppression of Tumor Progression in Patients With Advanced Stage of Hepatocellular Carcinoma by M1 Macrophage Induction

Background: Overcrowding always lead to the shedding of live cells to maintain homeostasis in epithelial cell sheets. Recently, some reports have shown that overcrowded cancer cells can be basally extruded by their neighbors, and then form metastases. However, this interesting phenomenon needs to be further clarified, and the exact mechanisms driving the extrusion of crowded cancer cell sheets remain elusive. We have previously confirmed that BVES, a novel adhesion molecule regulating tight junction formation, could inhibit liver cancer cell extrusion in a simply and skillfully designed 2D cell culture model, as cancer cells are always basally extruded, in this study, we performed a 3D cell culture model to further confirm the inhibition role of BVES in liver cancer cell extrusion. Methods: We designed a 3D cell culture model, after centrifuge, 5×10 6 cells were resuspended by 120μl 10% sucrose solutions, and quickly mixed with equal volume of 0.5% hydrogel solution( BeaverNanoTM ), the cells were then seeded in a Class Bottom Cell Culture Dish(NEST), and allowed to grow for six days. Then, cells were fixed with 4 % paraformaldehyde, incubated with primary antibody and secondary antibody, observed under a confocal microscopy. Extracted cell number were also investigated after enhanced or silenced expression of BVES. RhoA agonist U-46619 and inhibitor C3 enzyme were used to verify the role of RhoA/Rho-kinase pathway in BVES inhibited cancer cell extrusion. Results: Cells successfully grown to form an overcrowded cell mass in the 3D cell culture model, the number of extruded SK-Hep-1 cells, a high metastasis potential cell line, was significantly more than Huh7 cells with low metastasis potential, the normal liver cell line LO2 cells nearly had no cell extruded. Meanwhile, the extruded cells had decreased expression of BVES. Overexpression of BVES inhibited cells extrusion in the 3D cell culture model and increased RhoA activity in SK-Hep-1 cells, while stable knockdown of BVES remarkably promoted cells extrusion and decreased RhoA activity in Huh7 cells. U-46619 increased Huh7 cells extrusion while treated with C3 enzyme significantly diminished cells extrusion. Conclusion: These results suggest that in crowding liver cancer, BVES inactivate RhoA signaling pathway to decrease liver cancer cells extrusion, subsequently may inhibits tumor metastasis. (This study is supported by the National Natural Science Foundation of China NO:81472311 and the Fundamental Research Funds for the Central Universities 2014ZHYX020)