Actin Fiber Organization Research Articles

Nanosecond pulsed electric field suppresses growth and reduces multi-drug resistance effect in pancreatic cancer

Nanosecond pulsed electric fields (nsPEF) have been shown to exert anticancer effects; however, little is known about the mechanisms triggered in cancer cells by nanosecond-length pulses, especially when low, sub-permeabilization voltage is used. In this study, three human pancreatic cancer cell lines were treated with nsPEF and molecular changes at the cellular level were analyzed. Further, we assessed the efficacy of paclitaxel chemotherapy following nsPEF treatment and correlated that with the changes in the expression of multi-drug resistance (MDR) proteins. Finally, we examined the influence of nsPEF on the adhesive properties of cancer cells as well as the formation and growth of pancreatic cancer spheroids. Cell line response differed with the application of a 200 ns, 100 pulses, 8 kV/cm, 10 kHz PEF treatment. PEF treatment led to (1) the release of microvesicles (MV) in EPP85-181RDB cells, (2) electropermeabilization in EPP85-181RNOV cells and (3) cell shrinkage in EPP85-181P cells. The release of MV’s in EPP85-181RDB cells reduced the membrane content of P-gp and LRP, leading to a transient increase in vulnerability of the cells towards paclitaxel. In all cell lines we observed an initial reduction in size of the cancer spheroids after the nsPEF treatment. Cell line EPP85-181RNOV exhibited a permanent reduction in the spheroid size after nsPEF. We propose a mechanism in which the surface tension of the membrane, regulated by the organization of actin fibers, modulates the response of cancer cells towards nsPEF. When a membrane’s surface tension remains low, we observed some cells form protrusions and release MVs containing MDR proteins. In contrast, when cell surface tension remains high, the cell membrane is being electroporated. The latter effect may be responsible for the reduced tumor growth following nsPEF treatment.

Large-conductance Ca2 +-activated K+ channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) phenotype switching is very important during the pathogenesis and progression of vascular diseases. However, it is not well understood how normal VSMCs maintain the differentiated state. The large-conductance Ca2+-activated K+ (BKCa) channels are widely expressed in VSMCs and regulate vascular tone. Nevertheless, there is limited understanding of the role of the BKCa channel in modulation of the VSMC phenotype. We assessed BKCa channel expression levels in normal and injured carotid arteries from rats of the balloon-injury model. A strong decrease of BKCa-β1 was seen in the injured carotid arteries, accompanied by a parallel decrease of the VSMC contractile markers. BKCa-β1 in primary rat aortic VSMCs was decreased with the increase of passage numbers and the stimulation of platelet-derived growth factor (PDGF)-BB. Conversely, transforming growth factor β upregulated BKCa-β1. Meanwhile, the BKCa-β1 level was positively associated with the levels of VSMC contractile proteins. Intravenous injection of PDGF-BB induced downregulation of BKCa-β1 expression in the carotid arteries. Knockdown of BKCa-β1 favored VSMC dedifferentiation, characterized by altered morphology, abnormal actin fiber organization, decreased contractile proteins expression and reduced contractile ability. Furthermore, the resultant VSMC dedifferentiated phenotype rendered increased proliferation, migration, enhanced inflammatory factors levels, and matrix metalloproteinases activity. Studies using primary cultured aortic VSMCs from human recapitulated key findings. Finally, protein level of BKCa-β1 was reduced in human atherosclerotic arteries. BKCa-β1 is important in the maintenance of the contractile phenotype of VSMCs. As a novel endogenous defender that prevents pathological VSMC phenotype switching, BKCa-β1 may serve as a potential therapeutic target for treating vascular diseases including post-injury restenosis and atherosclerosis.

Plasticity of body axis polarity in Hydra regeneration under constraints

One of the major events in animal morphogenesis is the emergence of a polar body axis. Here, we combine classic grafting techniques with live imaging to explore the plasticity of polarity determination during whole body regeneration in Hydra. Composite tissues are made by fusing two rings, excised from separate animals, in different configurations that vary in the polarity and original positions of the rings along the body axes of the parent animals. Under frustrating initial configurations, body axis polarity that is otherwise stably inherited from the parent animal, can become labile and even be reversed. Importantly, the site of head regeneration exhibits a strong bias toward the edges of the tissue, even when this involves polarity reversal. In particular, we observe head formation at an originally aboral tissue edge, which is not compatible with models of Hydra regeneration based only on preexisting morphogen gradients or an injury response. The site of the new head invariably contains an aster-like defect in the organization of the supra-cellular ectodermal actin fibers. While a defect is neither required nor sufficient for head formation, we show that the defect at the new head site can arise via different routes, either appearing directly following excision as the tissue seals at its edge or through de novo defect formation at the fusion site. Altogether, our results show that the emergence of a polar body axis depends on the original polarity and position of the excised tissues as well as structural factors, suggesting that axis determination is an integrated process that arises from the dynamic interplay of multiple biochemical and mechanical processes.

Rapid Prototypable Biomimetic Peristalsis Bioreactor Capable of Concurrent Shear and Multi-Axial Strain

Peristalsis is a nuanced mechanical stimulus comprised of multi-axial strain (radial and axial strain) and shear stress. Forces associated with peristalsis regulate diverse biological functions including digestion, reproductive function, and urine dynamics. Given the central role peristalsis plays in physiology and pathophysiology, we were motivated to design a bioreactor capable of holistically mimicking peristalsis. We engineered a novel rotating screw-drive based design combined with a peristaltic pump, in order to deliver multi-axial strain and concurrent shear stress to a biocompatible polydimethylsiloxane (PDMS) membrane “wall.” Radial indentation and rotation of the screw drive against the wall demonstrated multi-axial strain evaluated via finite element modeling. Experimental measurements of strain using piezoelectric strain resistors were in close alignment with model-predicted values (15.9 ± 4.2% vs. 15.2% predicted). Modeling of shear stress on the “wall” indicated a uniform velocity profile and a moderate shear stress of 0.4 Pa. Human mesenchymal stem cells (hMSCs) seeded on the PDMS “wall” and stimulated with peristalsis demonstrated dramatic changes in actin filament alignment, proliferation, and nuclear morphology compared to static controls, perfusion, or strain, indicating that hMSCs sensed and responded to peristalsis uniquely. Lastly, significant differences were observed in gene expression patterns of calponin, caldesmon, smooth muscle actin, and transgelin, corroborating the propensity of hMSCs toward myogenic differentiation in response to peristalsis. Collectively, our data suggest that the peristalsis bioreactor is capable of generating concurrent multi-axial strain and shear stress on a “wall.” hMSCs experience peristalsis differently than perfusion or strain, resulting in changes in proliferation, actin fiber organization, smooth muscle actin expression, and genetic markers of differentiation. The peristalsis bioreactor device has broad utility in the study of development and disease in several organ systems.

Morphological signatures of actin organization in single cells accurately classify genetic perturbations using CNNs with transfer learning.

The actin cytoskeleton plays essential roles in countless cell processes, from cell division to migration to signaling. In cancer cells, cytoskeletal dynamics, cytoskeletal filament organization, and overall cell morphology are known to be altered substantially. We hypothesize that actin fiber organization and cell shape may carry specific signatures of genetic or signaling perturbations. We used convolutional neural networks (CNNs) on a small fluorescence microscopy image dataset of retinal pigment epithelial (RPE) cells and triple-negative breast cancer (TNBC) cells for identifying morphological signatures in cancer cells. Using a transfer learning approach, CNNs could be trained to accurately distinguish between normal and oncogenically transformed RPE cells with an accuracy of about 95% or better at the single cell level. Furthermore, CNNs could distinguish transformed cell lines differing by an oncogenic mutation from each other and could also detect knockdown of cofilin in TNBC cells, indicating that each single oncogenic mutation or cytoskeletal perturbation produces a unique signature in actin morphology. Application of the Local Interpretable Model-Agnostic Explanations (LIME) method for visually interpreting the CNN results revealed features of the global actin structure relevant for some cells and classification tasks. Interestingly, many of these features were supported by previous biological observation. Actin fiber organization is thus a sensitive marker for cell identity, and identification of its perturbations could be very useful for assaying cell phenotypes, including disease states.

Nanoimprinting for high-throughput replication of geometrically precise pillars in fused silica to regulate cell behavior

Developing high-throughput nanopatterning techniques that also allow for precise control over the dimensions of the fabricated features is essential for the study of cell-nanopattern interactions. Here, we developed a process that fulfills both of these criteria. Firstly, we used electron-beam lithography (EBL) to fabricate precisely controlled arrays of submicron pillars with varying values of interspacing on a large area of fused silica. Two types of etching procedures with two different systems were developed to etch the fused silica and create the final desired height. We then studied the interactions of preosteoblasts (MC3T3-E1) with these pillars. Varying interspacing was observed to significantly affect the morphological characteristics of the cell, the organization of actin fibers, and the formation of focal adhesions. The expression of osteopontin (OPN) significantly increased on the patterns, indicating the potential of the pillars for inducing osteogenic differentiation. The EBL pillars were thereafter used as master molds in two subsequent processing steps, namely soft lithography and thermal nanoimprint lithography for high-fidelity replication of the pillars on the substrates of interest. The molding parameters were optimized to maximize the fidelity of the generated patterns and minimize the wear and tear of the master mold. Comparing the replicated feature with those present on the original mold confirmed that the geometry and dimensions of the replicated pillars closely resemble those of the original ones. The method proposed in this study, therefore, enables the precise fabrication of submicron- and nanopatterns on a wide variety of materials that are relevant for systematic cell studies. Statement of significanceSubmicron pillars with specific dimensions on the bone implants have been proven to be effective in controlling cell behaviors. Nowadays, numerous methods have been proposed to produce bio-instructive submicron-topographies. However, most of these techniques are suffering from being low-throughput, low-precision, and expensive. Here, we developed a high-throughput nanopatterning technique that allows for control over the dimensions of the features for the study of cell-nanotopography interactions. Assessing the adaptation of preosteoblast cells showed the potential of the pillars for inducing osteogenic differentiation. Afterward, the pillars were used for high-fidelity replication of the bio-instructive features on the substrates of interest. The results show the advantages of nanoimprint lithography as a unique technique for the patterning of large areas of bio-instructive surfaces.

Atorvastatin Modulates the Efficacy of Electroporation and Calcium Electrochemotherapy

Electroporation is influenced by the features of the targeted cell membranes, e.g., the cholesterol content and the surface tension of the membrane. The latter is eventually affected by the organization of actin fibers. Atorvastatin is a statin known to influence both the cholesterol content and the organization of actin. This work analyzes the effects of the latter on the efficacy of electroporation of cancer cells. In addition, herein, electroporation was combined with calcium chloride (CaEP) to assess as well the effects of the statin on the efficacy of electrochemotherapy. Cholesterol-rich cell lines MDA-MB231, DU 145, and A375 underwent (1) 48 h preincubation or (2) direct treatment with 50 nM atorvastatin. We studied the impact of the statin on cholesterol and actin fiber organization and analyzed the cells’ membrane permeability. The viability of cells subjected to PEF (pulsed electric field) treatments and CaEP with 5 mM CaCl2 was examined. Finally, to assess the safety of the therapy, we analyzed the N-and E-cadherin localization using confocal laser microscopy. The results of our investigation revealed that depending on the cell line, atorvastatin preincubation decreases the total cholesterol in the steroidogenic cells and induces reorganization of actin nearby the cell membrane. Under low voltage PEFs, actin reorganization is responsible for the increase in the electroporation threshold. However, when subject to high voltage PEF, the lipid composition of the cell membrane becomes the regulatory factor. Namely, preincubation with atorvastatin reduces the cytotoxic effect of low voltage pulses and enhances the cytotoxicity and cellular changes induced by high voltage pulses. The study confirms that the surface tension regulates of membrane permeability under low voltage PEF treatment. Accordingly, to reduce the unfavorable effects of preincubation with atorvastatin, electroporation of steroidogenic cells should be performed at high voltage and combined with a calcium supply.

Nanoimprinting for High-Throughput Fabrication of Geometrically Precise Pillars to Regulate Cell Behavior

Developing high-throughput nanopatterning techniques that also allow for precise control over the dimensions of the fabricated features is essential for the study of cell-nanopattern interactions. Here, we developed a process that fulfills both of these criteria. Firstly, we used electron-beam lithography (EBL) to fabricate precisely controlled arrays of high aspect ratio submicron pillars with varying values of interspacing on a large area (3 × 5 mm2) of fused silica substrates. Two types of etching procedures with two different systems were developed to etch the fused silica and create the final desired height. We then studied the interactions of preosteoblasts (MC3T3-E1) with these pillars. Varying interspacing was observed to significantly affect the morphological characteristics of the cell, the organization of actin fibers, and the formation of focal adhesions. The expression of osteopontin (OPN) significantly increased on the patterns, indicating the potential of the pillars for inducing osteogenic differentiation. The EBL arrays of pillars were thereafter used as master molds in two subsequent processing steps, namely soft lithography (using hybrid PDMS) and thermal nanoimprint lithography for high-fidelity replication of the pillars on the substrates of interest. The molding parameters were optimized to maximize the fidelity of the generated patterns and minimize the wear and tear of the master mold. Comparing the replicated feature with those present on the original mold confirmed that the geometry and dimensions of the replicated pillars closely resemble those of the original ones (i.e., 1% deviation in pillar interspacing, < 20% deviation in pillar diameter). The method proposed in this study, therefore, enables the precise fabrication of submicron- and nanopatterns on a wide variety of substrate materials that are relevant for systematic cell studies.

Cells may feel a hard substrate even on a grafted layer of soft hydrogel.

Introducing or grafting molecules onto biomaterial surfaces to regulate cell destination via biophysical cues is one of the important steps for biomaterial design in tissue engineering. Understanding how cells feel the substrate makes it easier to learn the mechanism behind cell-material interaction. In this study, on a glass substrate, we constructed poly-phenoxyethyl methacrylate (PHEMA) brushes having different lengths via a surface-induced atom transfer radical polymerization (SI-ATRP) method. FTIR-ATR and XPS tests of the formed polymer brushes indicate that these brushes have characteristic chemical structures of PHEMA; the polymer brush length revealed by the AFM tests increases linearly with reaction time. Cell lines of BMSCs, ATDC5, and human chondrocytes (HC) were cultured on these substrates to evaluate proliferation, adhesion, and differentiation. Our results demonstrated that the cells cultured on the substrates with short PHEMA brushes developed a spread morphology and organized actin fibers as compared to the cells cultured on those with long brushes. Different cell lines showed different responses depending on the PHEMA brush length. Cells cultured on long PHEMA brushes displayed a more rounded shape, higher gene expression of FAK and integrin, and lower gene expression of NCAM and N-cadherin as compared to those, especially ATDC5 cells, cultured on short PHEMA brushes. On PHEMA brushes with a long length, the cell lines express higher cartilage-specific genes including Sox9 and Col2 and GAG in ECM. The results suggest that polymer brushes having different lengths may interfere with the behavior of the cells cultured on them.

Tuning the response of osteoblast-like cells to the porous-alumina-assisted mixed-oxide nano-mound arrays.

Nanostructuring of biomaterials is used to create an appropriate interfacial layer that promotes stable cellular adhesion, proliferation, and differentiation on orthopedic and dental implants. Here, we synthesize self-organized arrays of composite-oxide nano-mounds through anodizing Al/Ta bilayers sputtered on substrates to cover the "missing" smallest size range of 10-40 nm for structuring an advanced inorganic biomaterial-Al2 O3 -doped Ta2 O5 films. The osteoblast-like cells appear to be able to recognize the finest differences in the film nano-morphologies. In the absence of serum proteins, the adhesion and cell growth are substantially enhanced on the 20 and 40 nm nanoarrays while in complete medium the cells show better initial adhesion on the 10 nm nanoarrays. The proliferation assay reveals a significant rise in cell number on the 20 and 40 nm nanoarrays during the first 7 days. A remarkable increase in the alkaline phosphatase activity is noticed on the 40 nm nanoarray. Immunostaining of cells adhered to the nano-mound surfaces shows that the cells are well spread over all the nanostructured films with organized actin fibers. The larger surface areas and improved focal contacts are again associated with the 20 and 40 nm nanoarrays. The findings help improve compatibility of living cells with the metal-oxide nanostructured surfaces developed for tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1645-1654, 2018.

Effect of Sorbus commixta on the invasion and migration of human hepatocellularcarcinoma Hep3Bcells.

Tumor metastasis is a main cause of cancer-related morbidity and mortality. Thus, a number of medicinal herbs and phytochemicals have been investigated as possible candidates for the inhibition of cancer metastasis. Sorbus commixtaHedl.(SC) is a traditional medicinal plant used in the treatment of inflammatory diseases, as it has antioxidant, anti-inflammatory, anti-atherosclerotic and anti-hepatotoxic activities. In this study, we demonstrate that the water extract of SC exerts inhibitory effect on the invasion and migration of hepatocellular carcinoma Hep3Bcells. The activity and expression of matrix metalloproteinase(MMP)-9, which is responsible for the invasion of cancercells, was decreased by SC treatment. The invasive and migratory potentials of the Hep3Bcells were also decreased, as evidence by invitro assay using the Boyden chamber system. In addition, the expression of the chemokine receptors, C-X-C chemokine receptor type4(CXCR)4 and C-X-C chemokine receptor type6(CXCR6), were inhibited by SC in Hep3Bcells. Furthermore, actin fiber organization was markedly suppressed by SC treatment. Taken together, the findings of this study suggest for the first time, to the best of our knowledge, that SC suppresses the invasion and migration of highly metastatic Hep3Bcells.

Structural Inheritance of the Actin Cytoskeletal Organization Determines the Body Axis in Regenerating Hydra

Understanding how mechanics complement bio-signaling in defining patterns during morphogenesis is an outstanding challenge. Here, we utilize the multicellular polyp Hydra to investigate the role of the actomyosin cytoskeleton in morphogenesis. We find that the supra-cellular actin fiber organization is inherited from the parent Hydra and determines the body axis in regenerating tissue segments. This form of structural inheritance is non-trivial because of the tissue folding and dynamic actin reorganization involved. We further show that the emergence of multiple body axes can be traced to discrepancies in actin fiber alignment at early stages of the regeneration process. Mechanical constraints induced by anchoring regenerating Hydra on stiff wires suppressed the emergence of multiple body axes, highlighting the importance of mechanical feedbacks in defining and stabilizing the body axis. Together, these results constitute an important step toward the development of an integrated view of morphogenesis that incorporates mechanics.

Increased expression of integrin-linked kinase during decidualization regulates the morphological transformation of endometrial stromal cells

To study the impact of integrin-linked kinase (ILK) in endometrial stromal cells (ESCs) during decidualization. Laboratory study with the use of human endometrium. University hospital. Fertile reproductive-age women who had not received hormonal treatment for 3months before tissue collection. Endometrium tissue collection, invitro decidualization of isolated ESCs, and small interfering (si) RNA transfection. Immunohistochemistry, ELISA, Western blot analysis, methylthiazolyl tetrazolium assay, and immunofluorescence staining. Invivo expression of ILK is significantly increased in distended-fusiform stromal cells of late secretory endometrium and in cobblestone-shaped decidual cells of early pregnancy. During invitro decidualization for up to 8days, confluent cultures of isolated ESCs consistently displayed increased ILK expression and morphologic transformation from fibroblast-like to polygonal cells. Subsequent ILK knockdown by siRNA transfection reversed this transformation, accompanied by decreased phosphorylation of glycogen synthase kinase (GSK) 3β and decreased viable cell numbers. Immunofluorescence staining of the decidualized ESCs demonstrated linkage of increased levels of ILK at the tips of the fan-shaped organization of actin stress fibers located in the submembranous area, which expanded the decidual cells into a typical polygonal appearance. Knock-down of ILK abrogated the polymerization and organization of actin fibers, which reverted the cells to their undecidualized morphology. During human endometrial decidualization, ILK is essential for morphologic transformation of ESCs through organization of the actin cytoskeleton; it may also function through subsequent GSK3β signaling, which requires further studies.

Effect of Actin Organization on the Stiffness of Living Breast Cancer Cells Revealed by Peak-Force Modulation Atomic Force Microscopy.

We study the correlation between cytoskeleton organization and stiffness of three epithelial breast cancer cells lines with different degrees of malignancy: MCF-10A (healthy), MCF-7 (tumorigenic/noninvasive), and MDA-MB-231 (tumorigenic/invasive). Peak-force modulation atomic force microscopy is used for high-resolution topography and stiffness imaging of actin filaments within living cells. In healthy cells, local stiffness is maximum where filamentous actin is organized as well-aligned stress fibers, resulting in apparent Young's modulus values up to 1 order of magnitude larger than those in regions where these structures are not observed, but these organized actin fibers are barely observed in tumorigenic cells. We further investigate cytoskeleton conformation in the three cell lines by immunofluorescence confocal microscopy. The combination of both techniques determines that actin stress fibers are present at apical regions of healthy cells, while in tumorigenic cells they appear only at basal regions, where they cannot contribute to stiffness as probed by atomic force microscopy. These results substantiate that actin stress fibers provide a dominant contribution to stiffness in healthy cells, while the elasticity of tumorigenic cells appears not predominantly determined by these structures. We also discuss the effects of the high-frequency indentations inherent to peak-force atomic force microscopy for the identification of mechanical cancer biomarkers. Whereas conventional low loading rate indentations (1 Hz) result in slightly differentiated average stiffness for each cell line, in high-frequency measurements (250 Hz) healthy cells are clearly discernible from both tumorigenic cells with an enhanced stiffness ratio; however, the two cancerous cell lines produced indistinguishable results.

Growth on elastic silicone substrate elicits a partial myogenic response in periodontal ligament derived stem cells

The processes of cellular differentiation and phenotypic maintenance can be influenced by stimuli from a variety of different factors. One commonly overlooked factor is the mechanical properties of the growth substrate in which stem cells are maintained or differentiated down various lineages. Here we explored the effect that growth on an elastic silicone substrate had on the myogenic expression and cytoskeletal morphology of periodontal ligament derived stem cells. Cells were grown on either collagen I coated tissue culture polystyrene plates or collagen I coated elastic silicone membranes for a period of 4 days without further induction from soluble factors in the culture media. Following the 4-day growth, gene expression and immunohistochemical analysis for key cardiomyogenic markers was performed along with a morphological assessment of cytoskeletal organization. Results show that cells grown on the elastic substrate significantly upregulate key markers associated with contractile activity in muscle tissues. Namely, the myosin light chain polypeptides 2 and 7, as well as the myosin heavy chain polypeptide 7 genes underwent a statistically significant upregulation in the cells grown on elastic silicone membranes. Similarly, the cells on the softer elastic substrate stained positive for both sarcomeric actin and cardiac troponin t proteins following just 4 days of growth on the softer material. Cytoskeletal analysis showed that substrate stiffness had a marked effect on the organization and distribution of filamentous actin fibers within the cell body. Growth on silicone membranes produced flatter and shorter cellular morphologies with filamentous actin fibers projecting anisotropically throughout the cell body. These results demonstrate how crucial the mechanical properties of the growth substrate of cells can be on the ultimate cellular phenotype. These observations highlight the need to further optimize differentiation protocols to enhance the application of cellular products in the fields of regenerative medicine and stem cell therapies.

Global Deletion of Ankrd1 Results in a Wound-Healing Phenotype Associated with Dermal Fibroblast Dysfunction

The expression of ankyrin repeat domain protein 1 (Ankrd1), a transcriptional cofactor and sarcomeric component, is strongly elevated by wounding and tissue injury. We developed a conditional Ankrd1(fl/fl) mouse, performed global deletion with Sox2-cre, and assessed the role of this protein in cutaneous wound healing. Although global deletion of Ankrd1 did not affect mouse viability or development, Ankrd1(-/-) mice had at least two significant wound-healing phenotypes: extensive necrosis of ischemic skin flaps, which was reversed by adenoviral expression of ANKRD1, and delayed excisional wound closure, which was characterized by decreased contraction and reduced granulation tissue thickness. Skin fibroblasts isolated from Ankrd1(-/-) mice did not spread or migrate on collagen- or fibronectin-coated surfaces as efficiently as fibroblasts isolated from Ankrd1(fl/fl) mice. More important, Ankrd1(-/-) fibroblasts failed to contract three-dimensional floating collagen gels. Reconstitution of ANKRD1 by adenoviral infection stimulated both collagen gel contraction and actin fiber organization. These invitro data were consistent with invivo wound closure studies, and suggest that ANKRD1 is important for the proper interaction of fibroblasts with a compliant collagenous matrix both invitro and invivo.

Marine algal fucoxanthin inhibits the metastatic potential of cancer cells

Metastasis is major cause of malignant cancer-associated mortality. Fucoxanthin has effect on various pharmacological activities including anti-cancer activity. However, the inhibitory effect of fucoxanthin on cancer metastasis remains unclear. Here, we show that fucoxanthin isolated from brown alga Saccharina japonica has anti-metastatic activity. To check anti-metastatic properties of fucoxanthin, in vitro models including assays for invasion, migration, actin fiber organization and cancer cell–endothelial cell interaction were used. Fucoxanthin inhibited the expression and secretion of MMP-9 which plays a critical role in tumor invasion and migration, and also suppressed invasion of highly metastatic B16-F10 melanoma cells as evidenced by transwell invasion assay. In addition, fucoxanthin diminished the expressions of the cell surface glycoprotein CD44 and CXC chemokine receptor-4 (CXCR4) which play roles in migration, invasion and cancer–endothelial cell adhesion. Fucoxanthin markedly suppressed cell migration in wound healing assay and inhibited actin fiber formation. The adhesion of B16-F10 melanoma cells to the endothelial cells was significantly inhibited by fucoxanthin. Moreover, in experimental lung metastasis in vivo assay, fucoxanthin resulted in significant reduction of tumor nodules. Taken together, we demonstrate, for the first time, that fucoxanthin suppresses metastasis of highly metastatic B16-F10 melanoma cells in vitro and in vivo.

The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH

The extracellular-matrix protein laminin forms polymers both in vivo and in vitro. Acidification of pH leads to the formation of an artificial polymer with biomimetic properties, named polylaminin (polyLM). Follicle cells in the thyroid are in close contact with laminin, but their response to this important extracellular signal is still poorly understood. PCCL3 thyroid follicular cells cultured on glass, on regular laminin (LM) or on laminin previously polymerized in acidic pH (polyLM) showed different cell morphologies and propensities to proliferate, as well as differences in the organization of their actin cytoskeleton. On polyLM, cells displayed a typical epithelial morphology and radially organized actin fibers; whereas on LM, they spread irregularly on the substrate, lost cell contacts, and developed thick actin fibers extending through the entire cytoplasm. Iodide uptake decreased similarly in response to both laminin substrates, in comparison to glass. On both the LM and polyLM substrates, the expression of the sodium iodide symporter (NIS) decreased slightly but not significantly. NIS showed dotted immunostaining at the plasma membrane in the cells cultured on glass; on polyLM, NIS was observed mainly in the perinuclear region, and more diffusely throughout the cytoplasm on the LM substrate. Additionally, polyLM specifically favored the maintenance of cell polarity in culture. These findings indicate that PCCL3 cells can discriminate between LM and polyLM and that they respond to the latter by better preserving the phenotype observed in the thyroid tissue.

Directed endothelial cell morphogenesis in micropatterned gelatin methacrylate hydrogels

Engineering of organized vasculature is a crucial step in the development of functional and clinically relevant tissue constructs. A number of previous techniques have been proposed to spatially regulate the distribution of angiogenic biomolecules and vascular cells within biomaterial matrices to promote vascularization. Most of these approaches have been limited to two-dimensional (2D) micropatterned features or have resulted in formation of random vasculature within three-dimensional (3D) microenvironments. In this study, we investigate 3D endothelial cord formation within micropatterned gelatin methacrylate (GelMA) hydrogels with varying geometrical features (50–150 μm height). We demonstrated the significant dependence of endothelial cells proliferation, alignment and cord formation on geometrical dimensions of the patterned features. The cells were able to align and organize within the micropatterned constructs and assemble to form cord structures with organized actin fibers and circular/elliptical cross-sections. The inner layer of the cord structure was filled with gel showing that the micropatterned hydrogel constructs guided the assembly of endothelial cells into cord structures. Notably, the endothelial cords were retained within the hydrogel microconstructs for all geometries after two weeks of culture; however, only the 100 μm-high constructs provided the optimal microenvironment for the formation of circular and stable cord structures. Our findings suggest that endothelial cord formation is a preceding step to tubulogenesis and the proposed system can be used to develop organized vasculature for engineered tissue constructs.

384 - The Role of Antiestrogens in Breast Canser Cells' Invasiveness

ABSTRACT Aim The current study investigates the effect of two selective antagonists of the estrogen receptor (ER), fulvestrant (FL) and tamoxifen (Tam), on the invasive ability of breast cancer cells. ER blockage using anti-estrogens is associated with a small induction of invasiveness. Moreover, matrix metalloproteinases (MMPs) contribute to this procedure by cleaving components of the extracellular matrix, forming a path for the migrating cells. In addition, the focal adhesion kinase (FAK) plays a central role in invasiveness. Methods We used two ER+ breast cancer cell lines, MCF7 and T47D. Cells were stimulated by estradiol (E2) and then treated with FL, Tam and the metabolites endoxifen (End) and 4OH-tamoxifen (4OHT). The invasiveness was evaluated using the matrigel assay and MMPs expression with gelatin zymography assay. Using immunofluoresence, we study the expression and localization of phospho FAK and its correlation with F-actin. Results We found that E2 exerts a protective role in invasiveness. On the contrary, the anti-estrogens, as well as their metabolites reversed the effect of E2, a finding that was more obvious with Tam. The effect of the agents on MMPs expression was more pronounced at 48 h, when Tam and 4OHT were found to reduce the levels of both MMP-2 and MMP-9 compared to FL. Following E2 exposure, the maximum autophosphorylation of FAK (Y397) was observed at 10 min. At the same time point we assessed the effect of our agents, regarding the spatial organization of actin fibers. The concurrent administration of E2 with FL, Tam or End was associated with rearrangement of cytoskeleton, a finding that was not observed in untreated cells, as well as after the use of 4OHT and E2 or E2 alone. Conclusions Our results indicate that the metabolite 4OHT is superior to the pro-drug Tam as well as the pure anti-estrogen FL with respect to invasiveness, MMPs induction and actin rearrangement. Regarding the comparison of Tam with FL, the effect on MMPs and rearrangement was similar. However, FL was found to be slightly superior to Tam concerning invasiveness. Disclosure All authors have declared no conflicts of interest.