New Salicylanilide Derivatives and Their Peptide Conjugates as Anticancer Compounds: Synthesis, Characterization, and In Vitro Effect on Glioblastoma.

Pharmacologically active salicylanilides (2-hydroxy-N-phenylbenzamides) have been a promising area of interest in medicinal chemistry-related research for quite some time. This group of compounds has shown a wide spectrum of biological activities, including but not limited to anticancer effects. In this study, substituted salicylanilides were chosen to evaluate the in vitro activity on U87 human glioblastoma (GBM) cells. The parent salicylanilide, salicylanilide 5-chloropyrazinoates, a 4-aminosalicylic acid derivative, and the new salicylanilide 4-formylbenzoates were chemically and in vitro characterized. To enhance the internalization of the compounds, they were conjugated to delivery peptides with the formation of oxime bonds. Oligotuftsins ([TKPKG]n, n = 1-4), the ligands of neuropilin receptors, were used as GBM-targeting carrier peptides. The in vitro cellular uptake, intracellular localization, and penetration ability on tissue-mimicking models of the fluorescent peptide derivatives were determined. The compounds and their peptide conjugates significantly decreased the viability of U87 glioma cells. Salicylanilide compound-induced GBM cell death was associated with activation of autophagy, as characterized by immunodetection of autophagy-related processing of light chain 3 protein.

3D cell segregation geometry and dynamics are governed by tissue surface tension regulation.

Tissue morphogenesis and patterning during development involve the segregation of cell types. Segregation is driven by differential tissue surface tensions generated by cell types through controlling cell-cell contact formation by regulating adhesion and actomyosin contractility-based cellular cortical tensions. We use vertebrate tissue cell types and zebrafish germ layer progenitors as in vitro models of 3-dimensional heterotypic segregation and developed a quantitative analysis of their dynamics based on 3D time-lapse microscopy. We show that general inhibition of actomyosin contractility by the Rho kinase inhibitor Y27632 delays segregation. Cell type-specific inhibition of non-muscle myosin2 activity by overexpression of myosin assembly inhibitor S100A4 reduces tissue surface tension, manifested in decreased compaction during aggregation and inverted geometry observed during segregation. The same is observed when we express a constitutively active Rho kinase isoform to ubiquitously keep actomyosin contractility high at cell-cell and cell-medium interfaces and thus overriding the interface-specific regulation of cortical tensions. Tissue surface tension regulation can become an effective tool in tissue engineering.

Malignant pleural mesothelioma nodules remodel their surroundings to vascularize and grow.

Background: The microanatomical steps of malignant pleural mesothelioma (MPM) vascularization and the resistance mechanisms to anti-angiogenic drugs in MPM are unclear. Methods: We investigated the vascularization of intrapleurally implanted human P31 and SPC111 MPM cells. We also assessed MPM cell's motility, invasion and interaction with endothelial cells in vitro. Results: P31 cells exhibited significantly higher two-dimensional (2D) motility and three-dimensional (3D) invasion than SPC111 cells in vitro. In co-cultures of MPM and endothelial cells, P31 spheroids permitted endothelial sprouting (ES) with minimal spatial distortion, whereas SPC111 spheroids repealed endothelial sprouts. Both MPM lines induced the early onset of submesothelial microvascular plexuses covering large pleural areas including regions distant from tumor colonies. The development of these microvascular networks occurred due to both intussusceptive angiogenesis (IA) and ES and was accelerated by vascular endothelial growth factor A (VEGF-A)-overexpression. Notably, SPC111 colonies showed different behavior to P31 cells. P31 nodules incorporated tumor-induced capillary plexuses from the earliest stages of tumor formation. P31 cells deposited a collagenous matrix of human origin which provided "space" for further intratumoral angiogenesis. In contrast, SPC111 colonies pushed the capillary plexuses away and thus remained avascular for weeks. The key event in SPC111 vascularization was the development of a desmoplastic matrix of mouse origin. Continuously invaded by SPC111 cells, this matrix transformed into intratumoral connective tissue trunks, providing a route for ES from the diaphragm. Conclusions: Here, we report two distinct growth patterns of orthotopically implanted human MPM xenografts. In the invasive pattern, MPM cells invade and thus co-opt peritumoral capillary plexuses. In the pushing/desmoplastic pattern, MPM cells induce a desmoplastic response within the underlying tissue which allows the ingrowth of a nutritive vasculature from the pleura.

Host cell targeting of novel antimycobacterial 4-aminosalicylic acid derivatives with tuftsin carrier peptides.

Mycobacterium tuberculosis is an intracellular pathogen and the uptake of the antimycobacterial compounds by host cells is limited. Novel antimycobacterials effective against intracellular bacteria are needed. New N-substituted derivatives of 4-aminosalicylic acid have been designed and evaluated. To achieve intracellular efficacy and selectivity, these compounds were conjugated to tuftsin peptides via oxime or amide bonds. These delivery peptides can target tuftsin- and neuropilin receptor-bearing cells, such as macrophages and various other cells of lung origin. We have demonstrated that the in vitro antimycobacterial activity of the 4-aminosalicylic derivatives against M. tuberculosis H37Rv was preserved in the peptide conjugates. The free drugs were ineffective on infected cells, but the conjugates were active against the intracellular bacteria and have the selectivity on various types of host cells. The intracellular distribution of the carrier peptides was assessed, and the peptides internalize and display mainly in the cytosol in a concentration-dependent manner. The penetration ability of the most promising carrier peptide OT5 was evaluated using Transwell-inserts and spheroids. The pentapeptide exhibited time- and concentration-dependent penetration across the non-contact monolayers. Also, the pentapeptide has a fair penetration rate towards the center of spheroids formed of EBC-1 cells.

Development and Evaluation of a Human Skin Equivalent in a Semiautomatic Microfluidic Diffusion Chamber.

There is an increasing demand for transdermal transport measurements to optimize topical drug formulations and to achieve proper penetration profile of cosmetic ingredients. Reflecting ethical concerns the use of both human and animal tissues is becoming more restricted. Therefore, the focus of dermal research is shifting towards in vitro assays. In the current proof-of-concept study a three-layer skin equivalent using human HaCaT keratinocytes, an electrospun polycaprolactone mesh and a collagen-I gel was compared to human excised skin samples. We measured the permeability of the samples for 2% caffeine cream using a miniaturized dynamic diffusion cell ("skin-on-a-chip" microfluidic device). Caffeine delivery exhibits similar transport kinetics through the artificial skin and the human tissue: after a rapid rise, a long-lasting high concentration steady state develops. This is markedly distinct from the kinetics measured when using cell-free constructs, where a shorter release was observable. These results imply that both the established skin equivalent and the microfluidic diffusion chamber can serve as a suitable base for further development of more complex tissue substitutes.

Cellular Internalization and Inhibition Capacity of New Anti-Glioma Peptide Conjugates: Physicochemical Characterization and Evaluation on Various Monolayer- and 3D-Spheroid-Based in Vitro Platforms.

Most therapeutic agents used for treating brain malignancies face hindered transport through the blood-brain barrier (BBB) and poor tissue penetration. To overcome these problems, we developed peptide conjugates of conventional and experimental anticancer agents. SynB3 cell-penetrating peptide derivatives were applied that can cross the BBB. Tuftsin derivatives were used to target the neuropilin-1 transport system for selectivity and better tumor penetration. Moreover, SynB3-tuftsin tandem compounds were synthesized to combine the beneficial properties of these peptides. Most of the conjugates showed high and selective efficacy against glioblastoma cells. SynB3 and tandem derivatives demonstrated superior cellular internalization. The penetration profile of the conjugates was determined on a lipid monolayer and Transwell co-culture system with noncontact HUVEC-U87 monolayers as simple ex vivo and in vitro BBB models. Importantly, in 3D spheroids, daunomycin-peptide conjugates possessed a better tumor penetration ability than daunomycin. These conjugates are promising tools for the delivery systems with tunable features.

Multicellular contractility contributes to the emergence of mesothelioma nodules.

Malignant pleural mesothelioma (MPM) has an overall poor prognosis and unsatisfactory treatment options. MPM nodules, protruding into the pleural cavity may have growth and spreading dynamics distinct that of other solid tumors. We demonstrate that multicellular aggregates can develop spontaneously in the majority of tested MPM cell lines when cultured at high cell density. Surprisingly, the nodule-like aggregates do not arise by excessive local cell proliferation, but by myosin II-driven cell contractility. Prominent actin cables, spanning several cells, are abundant both in cultured aggregates and in MPM surgical specimens. We propose a computational model for in vitro MPM nodule development. Such a self-tensioned Maxwell fluid exhibits a pattern-forming instability that was studied by analytical tools and computer simulations. Altogether, our findings may underline a rational for targeting the actomyosin system in MPM.

Author Correction: Enhanced endothelial motility and multicellular sprouting is mediated by the scaffold protein TKS4.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Enhanced endothelial motility and multicellular sprouting is mediated by the scaffold protein TKS4.

Endothelial cell motility has fundamental role in vasculogenesis and angiogenesis during developmental or pathological processes. Tks4 is a scaffold protein known to organize the cytoskeleton of lamellipodia and podosomes, and thus modulating cell motility and invasion. In particular, Tks4 is required for the localization and activity of membrane type 1-matrix metalloproteinase, a key factor for extracellular matrix (ECM) cleavage during cell migration. While its role in transformed cells is well established, little is known about the function of Tks4 under physiological conditions. In this study we examined the impact of Tks4 gene silencing on the functional activity of primary human umbilical vein endothelial cells (HUVEC) and used time-lapse videomicrosopy and quantitative image analysis to characterize cell motility phenotypes in culture. We demonstrate that the absence of Tks4 in endothelial cells leads to impaired ECM cleavage and decreased motility within a 3-dimensional ECM environment. Furthermore, absence of Tks4 also decreases the ability of HUVEC cells to form multicellular sprouts, a key requirement for angiogenesis. To establish the involvement of Tks4 in vascular development in vivo, we show that loss of Tks4 leads sparser vasculature in the fetal chorion in the Tks4-deficient 'nee' mouse strain.

Matrigel patterning reflects multicellular contractility.

Non-muscle myosin II (NMII)-induced multicellular contractility is essential for development, maintenance and remodeling of tissue morphologies. Dysregulation of the cytoskeleton can lead to birth defects or enable cancer progression. We demonstrate that the Matrigel patterning assay, widely used to characterize endothelial cells, is a highly sensitive tool to evaluate cell contractility within a soft extracellular matrix (ECM) environment. We propose a computational model to explore how cell-exerted contractile forces can tear up the cell-Matrigel composite material and gradually remodel it into a network structure. We identify measures that are characteristic for cellular contractility and can be obtained from image analysis of the recorded patterning process. The assay was calibrated by inhibition of NMII activity in A431 epithelial carcinoma cells either directly with blebbistatin or indirectly with Y27632 Rho kinase inhibitor. Using Matrigel patterning as a bioassay, we provide the first functional demonstration that overexpression of S100A4, a calcium-binding protein that is frequently overexpressed in metastatic tumors and inhibits NMIIA activity by inducing filament disassembly, effectively reduces cell contractility.

Software tools for cell culture-related 3D printed structures.

Three-dimensional (3D) printing technology allowed fast and cheap prototype fabrication in numerous segments of industry and it also became an increasingly versatile experimental platform in life sciences. Yet, general purpose software tools to control printer hardware are often suboptimal for bioprinting applications. Here we report a package of open source software tools that we developed specifically to meet bioprinting requirements: Machine movements can be (i) precisely specified using high level programming languages, and (ii) easily distributed across a batch of tissue culture dishes. To demonstrate the utility of the reported technique, we present custom fabricated, biocompatible 3D-printed plastic structures that can control cell spreading area or medium volume, and exhibit excellent optical properties even at 50 ul sample volumes. We expect our software tools to be helpful not only to manufacture customized in vitro experimental chambers, but for applications involving printing cells and extracellular matrices as well.

Soluble VEGFR1 signaling guides vascular patterns into dense branching morphologies.

Vascular patterning is a key process during development and disease. The diffusive decoy receptor sVEGFR1 (sFlt1) is a known regulator of endothelial cell behavior, yet the mechanism by which it controls vascular structure is little understood. We propose computational models to shed light on how vascular patterning is guided by self-organized gradients of the VEGF/sVEGFR1 factors. We demonstrate that a diffusive inhibitor can generate structures with a dense branching morphology in models where the activator elicits directed growth. Inadequate presence of the inhibitor leads to compact growth, while excessive production of the inhibitor blocks expansion and stabilizes existing structures. Model predictions were compared with time-resolved experimental data obtained from endothelial sprout kinetics in fibrin gels. In the presence of inhibitory antibodies against VEGFR1 vascular sprout density increases while the speed of sprout expansion remains unchanged. Thus, the rate of secretion and stability of extracellular sVEGFR1 can modulate vascular sprout density.

Microglia control the spread of neurotropic virus infection via P2Y12 signalling and recruit monocytes through P2Y12-independent mechanisms.

Neurotropic herpesviruses can establish lifelong infection in humans and contribute to severe diseases including encephalitis and neurodegeneration. However, the mechanisms through which the brain's immune system recognizes and controls viral infections propagating across synaptically linked neuronal circuits have remained unclear. Using a well-established model of alphaherpesvirus infection that reaches the brain exclusively via retrograde transsynaptic spread from the periphery, and in vivo two-photon imaging combined with high resolution microscopy, we show that microglia are recruited to and isolate infected neurons within hours. Selective elimination of microglia results in a marked increase in the spread of infection and egress of viral particles into the brain parenchyma, which are associated with diverse neurological symptoms. Microglia recruitment and clearance of infected cells require cell-autonomous P2Y12 signalling in microglia, triggered by nucleotides released from affected neurons. In turn, we identify microglia as key contributors to monocyte recruitment into the inflamed brain, which process is largely independent of P2Y12. P2Y12-positive microglia are also recruited to infected neurons in the human brain during viral encephalitis and both microglial responses and leukocyte numbers correlate with the severity of infection. Thus, our data identify a key role for microglial P2Y12 in defence against neurotropic viruses, whilst P2Y12-independent actions of microglia may contribute to neuroinflammation by facilitating monocyte recruitment to the sites of infection.

Cell Dispersal Influences Tumor Heterogeneity and Introduces a Bias in NGS Data Interpretation.

Short and long distance cell dispersal can have a marked effect on tumor structure, high cellular motility could lead to faster cell mixing and lower observable intratumor heterogeneity. Here we evaluated a model for cell mixing that investigates how short-range dispersal and cell turnover will account for mutational proportions. We show that cancer cells can penetrate neighboring and distinct areas in a matter of days. In next generation sequencing runs, higher proportions of a given cell line generated frequencies with higher precision, while mixtures with lower amounts of each cell line had lower precision manifesting in higher standard deviations. When multiple cell lines were co-cultured, cellular movement altered observed mutation frequency by up to 18.5%. We propose that some of the shared mutations detected at low allele frequencies represent highly motile clones that appear in multiple regions of a tumor owing to dispersion throughout the tumor. In brief, cell movement will lead to a significant technical (sampling) bias when using next generation sequencing to determine clonal composition. A possible solution to this drawback would be to radically decrease detection thresholds and increase coverage in NGS analyses.

Collective motion of cells: from experiments to models.

Swarming or collective motion of living entities is one of the most common and spectacular manifestations of living systems that have been extensively studied in recent years. A number of general principles have been established. The interactions at the level of cells are quite different from those among individual animals, therefore the study of collective motion of cells is likely to reveal some specific important features which we plan to overview in this paper. In addition to presenting the most appealing results from the quickly growing related literature we also deliver a critical discussion of the emerging picture and summarize our present understanding of collective motion at the cellular level. Collective motion of cells plays an essential role in a number of experimental and real-life situations. In most cases the coordinated motion is a helpful aspect of the given phenomenon and results in making a related process more efficient (e.g., embryogenesis or wound healing), while in the case of tumor cell invasion it appears to speed up the progression of the disease. In these mechanisms cells both have to be motile and adhere to one another, the adherence feature being the most specific to this sort of collective behavior. One of the central aims of this review is to present the related experimental observations and treat them in light of a few basic computational models so as to make an interpretation of the phenomena at a quantitative level as well.

Collective cell streams in epithelial monolayers depend on cell adhesion.

We report a spontaneously emerging, randomly oriented, collective streaming behavior within a monolayer culture of a human keratinocyte cell line, and explore the effect of modulating cell adhesions by perturbing the function of calcium-dependent cell adhesion molecules. We demonstrate that decreasing cell adhesion induces narrower and more anisotropic cell streams, reminiscent of decreasing the Taylor scale of turbulent liquids. To explain our empirical findings, we propose a cell-based model that represents the dual nature of cell-cell adhesions. Spring-like connections provide mechanical stability, while a cellular Potts model formalism represents surface-tension driven attachment. By changing the relevance and persistence of mechanical links between cells, we are able to explain the experimentally observed changes in emergent flow patterns.

Collective motion of cells mediates segregation and pattern formation in co-cultures.

Pattern formation by segregation of cell types is an important process during embryonic development. We show that an experimentally yet unexplored mechanism based on collective motility of segregating cells enhances the effects of known pattern formation mechanisms such as differential adhesion, mechanochemical interactions or cell migration directed by morphogens. To study in vitro cell segregation we use time-lapse videomicroscopy and quantitative analysis of the main features of the motion of individual cells or groups. Our observations have been extensive, typically involving the investigation of the development of patterns containing up to 200,000 cells. By either comparing keratocyte types with different collective motility characteristics or increasing cells' directional persistence by the inhibition of Rac1 GTP-ase we demonstrate that enhanced collective cell motility results in faster cell segregation leading to the formation of more extensive patterns. The growth of the characteristic scale of patterns generally follows an algebraic scaling law with exponent values up to 0.74 in the presence of collective motion, compared to significantly smaller exponents in case of diffusive motion.

Inhibition of myosin II triggers morphological transition and increased nuclear motility.

We investigate the effect of myosin II inhibition on cell shape and nuclear motility in cultures of mouse radial glia-like neural progenitor and rat glioma C6 cells. Instead of reducing nucleokinesis, the myosin II inhibitor blebbistatin provokes an elongated bipolar morphology and increased nuclear motility in both cell types. When myosin II is active, time-resolved traction force measurements indicate a pulling force between the leading edge and the nucleus of C6 cells. In the absence of myosin II activity, traction forces during nucleokinesis are diminished below the sensitivity threshold of our assay. By visualizing the centrosome position in C6 cells with GFP-centrin, we show that in the presence or absence of myosin II activity, the nucleus tends to overtake or lag behind the centrosome, respectively. We interpret these findings with the help of a simple viscoelastic model of the cytoskeleton consisting active contractile and passive compressed elements.

Multicellular sprouting in vitro.

Cell motility and its guidance through cell-cell contacts is instrumental in vasculogenesis and in other developmental or pathological processes as well. During vasculogenesis, multicellular sprouts invade rapidly into avascular areas, eventually creating a polygonal pattern. Sprout elongation, in turn, depends on a continuous supply of endothelial cells, streaming along the sprout toward its tip. As long-term videomicroscopy of in vitro cell cultures reveal, cell lines such as C6 gliomas or 3T3 fibroblasts form multicellular linear arrangements in vitro, similar to the multicellular vasculogenic sprouts. We show evidence that close contact with elongated cells enhances and guides cell motility. To model the patterning process we augmented the widely used cellular Potts model with an inherently nonequilibrium interaction whereby surfaces of elongated cells become more preferred adhesion substrates than surfaces of well-spread, isotropic cells.

Dystroglycan is involved in laminin-1-stimulated motility of Müller glial cells: combined velocity and directionality analysis.

We investigate the role of dystroglycan, a major laminin-1 receptor and central member of the dystrophin-glycoprotein complex, in the laminin-1 induced motility of cultured Muller glial cells. Binding of laminin-1 to dystroglycan was prevented by IIH6, a function-blocking monoclonal antibody against alpha-dystroglycan. As an alternative means of inhibition, we used heparin to mask the dystroglycan binding site of the laminin-1, known to overlap with heparin binding sites. Cell motility was characterized in a two-dimensional motility assay based on computer-controlled videomicroscopy and statistical analysis of cellular trajectories. We obtained data on both the cell velocity and the diffusion index, a measure of direction-changing frequency. Both means of inhibition of dystroglycan function led to a significant decrease in the ability of laminin-1 to stimulate cell migration. At the same time, dystroglycan function does not appear to be involved in laminin-1-dependent increase in process dynamism and direction-changing activity.