Application of advanced surface patterning techniques to study cellular behavior

Surface manipulation for the fabrication of chemical or topographic micro- and nanopatterns, has been central to the evolution of in vitro biology research. A high variety of surface patterning methods have been implemented in a wide spectrum of applications, including fundamental cell biology studies, development of diagnostic tools, biosensors and drug delivery systems, as well as implant design. Surface engineering has increased our understanding of cell functions such as cell adhesion and cell-cell interaction mechanics, cell proliferation, cell spreading and migration. From a plethora of existing surface engineering techniques, we use standard microcontact printing methods followed by click chemistry to study the role of intercellular contacts in collective cancer cell migration. Cell dispersion from a confined area is fundamental in a number of biological processes, including cancer metastasis. To date, a quantitative understanding of the interplay of single cell motility, cell proliferation, and intercellular contacts remains elusive. In particular, the role of E- and N-Cadherin junctions, central components of intercellular contacts, is still controversial. Combining theoretical modeling with in vitro observations, we investigate the collective spreading behavior of colonies of human cancer cells (T24). The spreading of these colonies is driven by stochastic single-cell migration with frequent transient cell-cell contacts. We find that inhibition of E- and N-Cadherin junctions decreases colony spreading and average spreading velocities, without affecting the strength of correlations in spreading velocities of neighboring cells. Based on a biophysical simulation model for cell migration, we show that the behavioral changes upon disruption of these junctions can be explained by reduced repulsive excluded volume interactions between cells. This suggests that in cancer cell migration, cadherin-based intercellular contacts sharpen cell boundaries leading to repulsive rather than cohesive interactions between cells, thereby promoting efficient cell spreading during collective migration. Despite the remarkable progress in surface engineering technology and its applications, a combination of pattern properties such as stability, precision, specificity, high-throughput outcome and spatiotemporal control is highly desirable but challenging to achieve. Here, we introduce a versatile and high-throughput covalent photo-immobilization technique, comprising a light-dose dependent patterning step and a subsequent functionalization of the pattern via click chemistry. This two-step process is feasible on arbitrary surfaces and allows for generation of sustainable patterns and gradients. The method is validated in different biological systems by patterning adhesive ligands on cell repellent surfaces, thereby constraining the growth and migration of cells to the designated areas. We then implement a sequential photopatterning approach by adding a second switchable pattering step, allowing for spatiotemporal control over two distinct surface patterns. As a proof of concept, we reconstruct the dynamics of the tip/stalk cell switch during angiogenesis. Our results show that the spatiotemporal control provided by our “sequential photopatterning” system is essential for mimicking dynamic biological processes, and that our innovative approach has a great potential for further applications in cell science. In summary, this work introduces two novel and versatile paradigms of surface patterning for studying different aspects of cell behaviour in different cell types. The reliability of both setups is experimentally confirmed, providing new insight into the role of cell-cell contacts during collective cancer cell migration as well as the tip/stalk switch behaviour during angiogenesis

Sequential and switchable patterning for studying cellular processes under spatiotemporal control

Attachment of adhesive molecules on cell culture surfaces to restrict cell adhesion to defined areas and shapes has been vital for the progress of in vitro research. In currently existing patterning methods, a combination of pattern properties such as stability, precision, specificity, high-throughput outcome, and spatiotemporal control is highly desirable but challenging to achieve. Here, we introduce a versatile and high-throughput covalent photoimmobilization technique, comprising a light-dose-dependent patterning step and a subsequent functionalization of the pattern via click chemistry. This two-step process is feasible on arbitrary surfaces and allows for generation of sustainable patterns and gradients. The method is validated in different biological systems by patterning adhesive ligands on cell-repellent surfaces, thereby constraining the growth and migration of cells to the designated areas. We then implement a sequential photopatterning approach by adding a second switchable patterning step, allowing for spatiotemporal control over two distinct surface patterns. As a proof of concept, we reconstruct the dynamics of the tip/stalk cell switch during angiogenesis. Our results show that the spatiotemporal control provided by our “sequential photopatterning” system is essential for mimicking dynamic biological processes and that our innovative approach has great potential for further applications in cell science

Interfacial activation of Candida antarctica lipase B: combined evidence from experiment and simulation

Lipase immobilization is frequently used for altering the catalytic properties of these industrially used enzymes. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. Candida antarctica lipase B (CalB), one of the most commonly used biocatalysts, is frequently discussed as an atypical lipase lacking interfacial activation. Here we show that CalB displays an enhanced catalytic rate for large, bulky substrates when adsorbed to a hydrophobic interface composed of densely packed alkyl chains. We attribute this increased activity of more than 7-fold to a conformational change that yields a more open active site. This hypothesis is supported by molecular dynamics simulations that show a high mobility for a small "lid" (helix α5) close to the active site. Molecular docking calculations confirm that a highly open conformation of this helix is required for binding large, bulky substrates and that this conformation is favored in a hydrophobic environment. Taken together, our combined approach provides clear evidence for the interfacial activation of CalB on highly hydrophobic surfaces. In contrast to other lipases, however, the conformational change only affects large, bulky substrates, leading to the conclusion that CalB acts like an esterase for small substrates and as a lipase for substrates with large alcohol substituents

Disentangling cadherin-mediated cell-cell interactions in collective cancer cell migration

Cell dispersion from a confined area is fundamental in a number of biological processes, including cancer metastasis. To date, a quantitative understanding of the interplay of single-cell motility, cell proliferation, and intercellular contacts remains elusive. In particular, the role of E- and N-cadherin junctions, central components of intercellular contacts, is still controversial. Combining theoretical modeling with in vitro observations, we investigate the collective spreading behavior of colonies of human cancer cells (T24). The spreading of these colonies is driven by stochastic single-cell migration with frequent transient cell-cell contacts. We find that inhibition of E- and N-cadherin junctions decreases colony spreading and average spreading velocities, without affecting the strength of correlations in spreading velocities of neighboring cells. Based on a biophysical simulation model for cell migration, we show that the behavioral changes upon disruption of these junctions can be explained by reduced repulsive excluded volume interactions between cells. This suggests that in cancer cell migration, cadherin-based intercellular contacts sharpen cell boundaries leading to repulsive rather than cohesive interactions between cells, thereby promoting efficient cell spreading during collective migration

Interfacial Activation of <i>Candida antarctica</i> Lipase B: Combined Evidence from Experiment and Simulation

Lipase immobilization is frequently used for altering the catalytic properties of these industrially used enzymes. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. <i>Candida antarctica</i> lipase B (CalB), one of the most commonly used biocatalysts, is frequently discussed as an atypical lipase lacking interfacial activation. Here we show that CalB displays an enhanced catalytic rate for large, bulky substrates when adsorbed to a hydrophobic interface composed of densely packed alkyl chains. We attribute this increased activity of more than 7-fold to a conformational change that yields a more open active site. This hypothesis is supported by molecular dynamics simulations that show a high mobility for a small “lid” (helix α5) close to the active site. Molecular docking calculations confirm that a highly open conformation of this helix is required for binding large, bulky substrates and that this conformation is favored in a hydrophobic environment. Taken together, our combined approach provides clear evidence for the interfacial activation of CalB on highly hydrophobic surfaces. In contrast to other lipases, however, the conformational change only affects large, bulky substrates, leading to the conclusion that CalB acts like an esterase for small substrates and as a lipase for substrates with large alcohol substituents

Targeting actin inhibits repair of doxorubicin-induced DNA damage: a novel therapeutic approach for combination therapy

Abstract Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin-binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin