Control Cell Adhesion Research Articles

Abstract 3160: The actin-binding protein Girdin/GIV regulates collective cancer cell migration by controlling cell adhesion and cytoskeletal organization

Abstract Pathological observations show that cancer cells frequently invade the surrounding stroma in collective groups rather than through single cell migration. The identification of genes and proteins that are specifically involved in the collective behavior of cancer cells has thus far been limited. Here, we studied the role of the actin-binding protein Girdin in collective cancer cell migration. This protein is a specific regulator of collective migration of neuroblasts born in the subventricular zone of the postnatal and adult brains and participates in the progression of cancer. We found that Girdin was essential for the collective migration of the skin cancer cell line A431 on collagen gels as well as their fibroblast-led collective invasion in an organotypic culture model. We provide evidence that Girdin binds to β-catenin that plays important roles in the Wnt signaling pathway and in E-cadherin-mediated cell-cell adhesion. Girdin-depleted cells displayed scattering and impaired E-cadherin-specific cell-cell adhesion. Importantly, Girdin depletion led to impaired cytoskeletal association of the β-catenin complex, which was accompanied by changes in the supracellular actin cytoskeletal organization of cancer cell cohorts on collagen gels. Although the underlying mechanism is unclear, this observation is consistent with the established role of the actin cytoskeletal system and cell-cell adhesion in the collective behavior of cells. Citation Format: Xiaoze Wang, Atsushi Enomoto, Liang Weng, Hisashi Haga, Sumire Ishida, Masahide Takahashi. The actin-binding protein Girdin/GIV regulates collective cancer cell migration by controlling cell adhesion and cytoskeletal organization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3160.

Engineering biointerface with controlled cell adhesion towards cancer diagnostics

Engineering biointerface with controlled cell adhesion towards cancer diagnostics

Mechano-redox control of integrin de-adhesion.

How proteins harness mechanical force to control function is a significant biological question. Here we describe a human cell surface receptor that couples ligand binding and force to trigger a chemical event which controls the adhesive properties of the receptor. Our studies of the secreted platelet oxidoreductase, ERp5, have revealed that it mediates release of fibrinogen from activated platelet αIIbβ3 integrin. Protein chemical studies show that ligand binding to extended αIIbβ3 integrin renders the βI-domain Cys177-Cys184 disulfide bond cleavable by ERp5. Fluid shear and force spectroscopy assays indicate that disulfide cleavage is enhanced by mechanical force. Cell adhesion assays and molecular dynamics simulations demonstrate that cleavage of the disulfide induces long-range allosteric effects within the βI-domain, mainly affecting the metal-binding sites, that results in release of fibrinogen. This coupling of ligand binding, force and redox events to control cell adhesion may be employed to regulate other protein-protein interactions.

Protein Nanosheet Mechanics Controls Cell Adhesion and Expansion on Low-Viscosity Liquids

Adherent cell culture typically requires cell spreading at the surface of solid substrates to sustain the formation of stable focal adhesions and assembly of a contractile cytoskeleton. However, a few reports have demonstrated that cell culture is possible on liquid substrates such as silicone and fluorinated oils, even displaying very low viscosities (0.77 cSt). Such behavior is surprising as low viscosity liquids are thought to relax much too fast (<ms) to enable the stabilization of focal adhesions (with lifetimes on the order of minutes to hours). Here we show that cell spreading and proliferation at the surface of low viscosity liquids are enabled by the self-assembly of mechanically strong protein nanosheets at these interfaces. We propose that this phenomenon results from the denaturation of globular proteins, such as albumin, in combination with the coupling of surfactant molecules to the resulting protein nanosheets. We use interfacial rheology and atomic force microscopy indentation to characterize the mechanical properties of protein nanosheets and associated liquid-liquid interfaces. We identify a direct relationship between interfacial mechanics and the association of surfactant molecules with proteins and polymers assembled at liquid-liquid interfaces. In addition, our data indicate that cells primarily sense in-plane mechanical properties of interfaces, rather than relying on surface tension to sustain spreading, as in the spreading of water striders. These findings demonstrate that bulk and nanoscale mechanical properties may be designed independently, to provide structure and regulate cell phenotype, therefore calling for a paradigm shift for the design of biomaterials in regenerative medicine.

Interconnected Microchannels in Hydrogels to Control Cell Adhesion and Mechanotransduction

Cells respond to external mechanical stimuli through the activation of mechanotransduction, which influences cell proliferation, migration and differentiation, and adhesion. Likewise, diseases such as cancer and cardiac dysfunctions are strongly related to cellular mechanotransduction. Here we show an innovative 3D material to serve as a platform for controlling mechanotransduction by mimicking natural 3D cellular environments. Our material contains a novel form of microporous structures represented by micron-sized channels embedded in a hydrogel matrix of a well-defined stiffness and conductivity. The material guarantees pore interconnectivity independently of pore density and size, and different types of surface functionalization are possible. Furthermore, the material also provides a large and spatially controlled cell-surface contact area through the specific architecture of its pores, such that the mechanical properties of the environment have large impact on the cells. We here show data on how different cell types grow in the microporous materials and future applications where cellular mechanotransduction can be exploited.

Facile Strategy to Generate Aligned Polymer Nanofibers: Effects on Cell Adhesion.

Structure of polymer fiber membranes plays a vital role in controlling cell responses as applied to immobilize targets for specific cell interactions. Electrospinning is a simple and powerful method to prepare polymer fiber membranes with scales from nano- to micrometers. In this report, a facile yet versatile strategy has been developed for fabricating polymer nanofiber membranes with well-aligned structures using a glass sheet between the needle and a static drum as the collector. Effects of solution concentration, polymer molecular weight, applied voltage, and collection distance on the morphologies of the formed fibers were systematically studied. Adhesion of cells (e.g., mouse melanoma cells B16-F10 and fibroblast cells NIH-3T3) on the fiber membrane has been further investigated. Our results show that cell morphologies varied from elongated to spherical on the random fiber membrane when the pore area of membrane decreased. In contrast, on the membrane with aligned morphology, when decreasing the gap width of fiber membrane, cell is found to keep elongated state and spread along the alignment direction. This work provides a facile yet effective strategy to engineer surface structures of the fiber membranes for controlling cell adhesion.

Facile Fabrication of Hierarchically Thermoresponsive Binary Polymer Pattern for Controlled Cell Adhesion.

A versatile platform allowing capture and detection of normal and dysfunctional cells on the same patterned surface is important for accessing the cellular mechanism, developing diagnostic assays, and implementing therapy. Here, an original and effective method for fabricating binary polymer brushes pattern is developed for controlled cell adhesion. The binary polymer brushes pattern, composed of poly(N-isopropylacrylamide) (PNIPAAm) and poly[poly(ethylene glycol) methyl ether methacrylate] (POEGMA) chains, is simply obtained via a combination of surface-initiated photopolymerization and surface-activated free radical polymerization. This method is unique in that it does not utilize any protecting groups or procedures of backfilling with immobilized initiator. It is demonstrated that the precise and well-defined binary polymer patterns with high resolution are fabricated using this facile method. PNIPAAm chains capture and release cells by thermoresponsiveness, while POEGMA chains possess high capability to capture dysfunctional cells specifically, inducing a switch of normal red blood cells (RBCs) arrays to hemolytic RBCs arrays on the pattern with temperature. This novel platform composed of binary polymer brush pattern is smart and versatile, which opens up pathways to potential applications as microsensors, biochips, and bioassays.

Optogenetic control of focal adhesion kinase signaling

Focal adhesion kinase (FAK) integrates signaling from integrins, growth factor receptors and mechanical stress to control cell adhesion, motility, survival and proliferation. Here, we developed a single-component, photo-activatable FAK, termed optoFAK, by using blue light-induced oligomerization of cryptochrome 2 (CRY2) to activate FAK-CRY2 fusion proteins. OptoFAK functions uncoupled from physiological stimuli and activates downstream signaling rapidly and reversibly upon blue light exposure. OptoFAK stimulates SRC creating a positive feedback loop on FAK activation, facilitating phosphorylation of paxillin and p130Cas in adherent cells. In detached cells or in mechanically stressed adherent cells, optoFAK is autophosphorylated upon exposure to blue light, however, downstream signaling is hampered indicating that the accessibility to these substrates is disturbed. OptoFAK may prove to be a useful tool to study the biological function of FAK in growth factor and integrin signaling, tension-mediated focal adhesion maturation or anoikis and could additionally serve as test system for kinase inhibitors.

Multimodal Bioactivation of Hydrophilic Electrospun Nanofibers Enables Simultaneous Tuning of Cell Adhesivity and Immunomodulatory Effects

AbstractBiomaterials research usually focuses on functional and structural mimicry of the extracellular matrix or tissue hierarchy and morphology. Most recently, material‐induced modulatory effects on the immune system to arouse a healing response is another upcoming strategy. Approaches, however, that integrate both aspects to induce healing and facilitate specific cell adhesion are so far little explored. This study exploits manifold but chemical crosslinker free functionalization of hydrophilic and nonadhesive electrospun fiber surfaces with peptides for controlled cell adhesion, and with neutra­lizing antibodies targeting the master cytokine tumor necrosis factor (TNF) to dampen proinflammatory reactions by the fiber adherent cells. It is demonstrated that cell attachment and immunomodulatory properties of a textile can be tailored at the same time to generate meshes that combine immunosuppressive activity with specific cell adhesion properties.

Photo-responsive smart surfaces with controllable cell adhesion

In the past few years, photo-responsive smart surfaces have emerged as the promising candidates to investigate cell adhesion due to the noninvasive property and high spatiotemporal precision of photo stimuli. Under external optical stimulation, photo-responsive molecules can cleave their chemical bond or switch their configuration, thereby dynamically changing surface physicochemical properties such as wettability, polarity, and charge. Furthermore, the switchable physicochemical properties can directly or indirectly affect the interaction between cells and the smart surfaces, resulting in the controllable cell adhesion. This review briefly discusses the recent development of photo-responsive smart surfaces in the field of controllable cell adhesion taking advantage of photo cleavage, photo isomerization or photothermal effect. In doing so, we summarize the photo-responsive smart surfaces, including construction, modification and physicochemical properties switch, and their bioapplications in cell diagnostic, tissue engineering and cell medicine.

Keratins Regulate the Adhesive Properties of Desmosomal Cadherins through Signaling

Tightly controlled intercellular adhesion is crucial for the integrity and function of the epidermis. The keratin filament cytoskeleton anchors desmosomes, supramolecular complexes required for strong intercellular adhesion. We tested whether keratin filaments control cell adhesion by regulating the adhesive properties of desmosomal cadherins such as desmoglein (Dsg) 3. Atomic force microscopy and fluorescence recovery after photobleaching experiments showed reduced Dsg3 adhesive forces and membrane stability in murine keratinocytes lacking all keratin filaments. Impairment of the actin cytoskeleton also resulted in decreased Dsg3 immobilization but did not affect Dsg3 binding properties, indicating that the latter are exclusively controlled by keratins. Reduced binding forces were dependent on p38 mitogen-activated protein kinase activity, which was deregulated in keratin-deficient cells. In contrast, inhibition of protein kinase C signaling, which is known to be controlled by keratins, promoted and spatially stabilized Dsg3-mediated interactions in the membrane. These results show a previously unreported mechanism for how keratins stabilize intercellular adhesion on the level of single desmosomal adhesion molecules.

Beyond adhesion:emerging roles for integrins in control of the tumor microenvironment.

While integrins were originally discovered as cell adhesion receptors, recent studies have reinforced the concept that integrins have central roles in cancer that extend far beyond controlling cell adhesion and migration. Indeed, as transmembrane cell surface receptors that occupy a critical position at the interface of cellular and extracellular interactions and are capable of both “inside-out” and “outside-in” signaling, integrins are uniquely poised to regulate the cell’s ability to promote, sense, and react to changes in the tumor microenvironment. Moreover, integrins are present on all cell types in the tumor microenvironment, and they have important roles in regulating intercellular communication. Decades of promising pre-clinical studies have implicated certain integrins as attractive therapeutic targets in the cancer clinic. Nevertheless, results of the few clinical trials that target integrins in cancer have thus far been disappointing. Importantly, these clinical failures likely reflect the emerging complexity of individual and combinatorial integrin function within both tumor cells and other cell types of the tumor microenvironment, together with a need to explore integrin-targeting agents not just as monotherapies but also as adjuvants to more conventional radiotherapies or chemotherapies. In this review, we will examine recent advances toward understanding how integrins regulate cancer progression, including their roles in intercellular communication and modulation of the tumor microenvironment. Additionally, we will discuss factors that underlie the limited efficacy of current efforts to target integrins in the cancer clinic as well as potential strategies to overcome these challenges.

Microcontact printing of polydopamine on thermally expandable hydrogels for controlled cell adhesion and delivery of geometrically defined microtissues.

Harvest of artificial tissue with controlled cellular arrangement independently from external materials has been widely studied in cell-based assay and regenerative medicine. In this study, we developed scaffold-free harvest system of microtissues with anisotropic arrangement and controlled width by exploiting thermally expandable hydrogels with cell-adhesive patterns of polydopamine formed by simple microcontact printing. Cultured strips of human dermal fibroblasts on the hydrogels were rapidly delivered to various targets ranging from flat coverglass to mice subcutaneous tissue by thermal expansion of the hydrogel at 4°C for 10min. These were further utilized as a drug screening model responding to ROCK1 inhibitor, which imply its versatile applicability.

Laser Patterning of Self‐Assembled Monolayers on PEDOT:PSS Films for Controlled Cell Adhesion

Conducting polymers have shown great potential as a means to interface electronics with living tissues, toward a plethora of different biological applications ranging from in vitro to in vivo systems. However, the development of effective functionalization approaches to render this interface biomimetic still remains rather challenging, due to the lack of inherent surface functionalities in such polymers. Here, a straightforward and versatile modification strategy of poly(3,4‐ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) surfaces is demonstrated for preferential and spatially confined cell adhesion and growth. By combining three simple surface modification steps, including chemical modification using self‐assembled monolayers and their selective laser ablation, this study is able to design either cell‐adhesive or cell‐repulsive patterns of various shapes on PEDOT:PSS films. Studies using Madin–Darby canine kidney II epithelial cells reveal preferential cell adhesion and growth with good precision following the preformed patterns. The proposed surface modification approach can be extended to encompass a variety of polymeric biomaterials, without affecting their bulk properties.

Peptide-functionalized poly[oligo(ethylene glycol) methacrylate] brushes on dopamine-coated stainless steel for controlled cell adhesion.

Stainless steel (SS) implants are widely used clinically for orthopaedic, spinal, dental and cardiovascular applications. However, non-specific adsorption of biomolecules onto implant surfaces results in sub-optimal integration with host tissue. To allow controlled cell-SS interactions, we have developed a strategy to grow non-fouling polymer brushes that prevent protein adsorption and cell adhesion and can be subsequently functionalized with adhesive peptides to direct cell adhesion and signaling. This approach has broad application to improve osseointegration onto stainless steel implants in bone repair.

Genetic variants in the genes encoding rho GTPases and related regulators predict cutaneous melanoma-specific survival.

Rho GTPases control cell division, motility, adhesion, vesicular trafficking and phagocytosis, which may affect progression and/or prognosis of cancers. Here, we investigated associations between genetic variants of Rho GTPases-related genes and cutaneous melanoma-specific survival (CMSS) by re-analyzing a published melanoma genome-wide association study (GWAS) and validating the results in another melanoma GWAS. In the single-locus analysis of 36,018 SNPs in 129 Rho-related genes, 427 SNPs were significantly associated with CMSS (p < 0.050 and false-positive report probability <0.2) in the discovery dataset, and five SNPs were replicated in the validation dataset. Among these, four SNPs (i.e., RHOU rs10916352 G > C, ARHGAP22 rs3851552 T > C, ARHGAP44 rs72635537 C > T and ARHGEF10 rs7826362 A > T) were independently predictive of CMSS (a meta-analysis derived p = 9.04 × 10-4 , 9.58 × 10-4 , 1.21 × 10-4 and 8.47 × 10-4 , respectively). Additionally, patients with an increasing number of unfavorable genotypes (NUGs) of these loci had markedly reduced CMSS in both discovery dataset and validation dataset (ptrend =1.47 × 10-7 and 3.12 × 10-5 ). The model including the NUGs and clinical variables demonstrated a significant improvement in predicting the five-year CMSS. Moreover, rs10916352C and rs3851552C alleles were significantly associated with an increased mRNA expression levels of RHOU (p = 1.8 × 10-6 ) and ARHGAP22 (p = 5.0 × 10-6 ), respectively. These results may provide promising prognostic biomarkers for CM personalized management and treatment.

Surface Patterning of Gold Nanoparticles on PEG-Based Hydrogels to Control Cell Adhesion.

We report on a versatile and easy approach to micro-pattern gold nanoparticles (Au NPs) on 8-arm poly(ethylene glycol)-vinyl sulfone thiol (8PEG-VS-SH) hydrogels, and the application of these patterned Au NPs stripes in controlling cell adhesion. Firstly, the Au NPs were patterned on silicon wafers, and then they were transferred onto reactive, multifunctional 8PEG-VS-SH hydrogels. The patterned, micrometer-sized Au NPs stripes with variable spacings ranging from 20 μm to 50 μm were created by our recently developed micro-contact deprinting method. For this micro-contact deprinting approach, four different PEG-based stamp materials have been tested and it was found that the triblock copolymer PEG-PPG-PEG-(3BC) stamp established the best transfer efficiency and has been used in the ongoing work. After the successful creation of micro-patterns of Au NPs stripes on silicon, the patterns can be transferred conveniently and accurately to 8PEG-VS-SH hydrogel films. Subsequently these Au NPs patterns on 8PEG-VS-SH hydrogels have been investigated in cell culture with murine fibroblasts (L-929). The cells have been observed to adhere to and spread on those nano-patterned micro-lines in a remarkably selective and ordered manner.

Cell adhesive spectra along surface wettability gradient from superhydrophilicity to superhydrophobicity

Surface wettability is important to design biointerfaces and functional biomaterials in various biological applications. However, to date, it remains some confusions about how cells would response to the surfaces with different wettabilities. Herein, we systematically explore the adhesive spectra of cells to the surface with wettability gradient from superhydrophilicity to superhydrophobicity, clarifying the effect of wettability on cell adhesion. We envision that this study may provide valuable information for the design of biomedical implants with controllable cell adhesion, such as neural interface devices and flexible implant.

Fabrication of biocomposites composed of natural rubber latex and bone tissue derived from MC3T3-E1 mouse preosteoblastic cells

Natural rubber latex (NRL) is mainly used around traditional industrial products, but currently their target application is continuously expanding into tissue engineering. To broaden our knowledge of application in tissue engineering of NRL, we have focused on the surface modification of NRL nanoparticles through the biomineralization of hydroxyapatite (HA) using simulated body fluid in order to create a better cytocompatibility with controlled cell adhesion and mineralization properties in osteogenic culture to determine the effect on bone outcomes. Using MC3T3-E1 mouse osteoblastic-like cells incubated with NRL nanoparticles coated with HA layer, we have examined the osteogenic differentiation and expressions of multiple proteins and characteristic genes of mature osteoblast. We have successfully prepared the biocomposites composed of NRL and bone tissue.

Controlling cell adhesion in antibody purification by expanded bed adsorption chromatography

Abstract This study introduces an improved approach for determining the most suitable mobile phase maximizing repulsion between biomass and adsorbent to reduce biomass fouling in expanded bed adsorption (EBA) chromatography. Extended DLVO theory (xDLVO) has been utilized to comprehensively calculate optimal mobile phase compositions, thus increasing efficiency, improving hydrodynamics as well as product recovery. Biomass-adsorbent interaction energy surfaces were generated as a function of pH, ionic strength, and salt type, investigating the interactions of CHO cells with MabDirect® Protein-A beads. We have shown that mobile phases consisting of Na2SO4 were best suited at low ionic strength, whereas NaCl was more favorable at higher ionic strength. This was found to increase product recovery while maintaining a constant level of purity of monoclonal antibodies. Our method provides a tool for rapid method development of EBA purifications that can be dynamically adapted to operational requirements. In our case, the resulting optimized process was able to outperform the current protocol by up to 10% in terms of product recovery.