Single-cell profiling and zebrafish avatars reveal LGALS1 as immunomodulating target in glioblastoma.

Glioblastoma (GBM) remains the most malignant primary brain tumor, with a median survival rarely exceeding 2 years. Tumor heterogeneity and an immunosuppressive microenvironment are key factors contributing to the poor response rates of current therapeutic approaches. GBM-associated macrophages (GAMs) often exhibit immunosuppressive features that promote tumor progression. However, their dynamic interactions with GBM tumor cells remain poorly understood. Here, we used patient-derived GBM stem cell cultures and combined single-cell RNA sequencing of GAM-GBM co-cultures and real-time in vivo monitoring of GAM-GBM interactions in orthotopic zebrafish xenograft models to provide insight into the cellular, molecular, and spatial heterogeneity. Our analyses revealed substantial heterogeneity across GBM patients in GBM-induced GAM polarization and the ability to attract and activate GAMs-features that correlated with patient survival. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock-out experiments in zebrafish subsequently identified LGALS1 as a primary regulator of immunosuppression. Overall, our work highlights that GAM-GBM interactions can be studied in a clinically relevant way using co-cultures and avatar models, while offering new opportunities to identify promising immune-modulating targets.

A 96-wells fluidic system for high-throughput screenings under laminar high wall shear stress conditions.

The ability of endothelial cells to respond to blood flow is fundamental for the correct formation and maintenance of a functional and hierarchically organized vascular network. Defective flow responses, in particular related to high flow conditions, have been associated with atherosclerosis, stroke, arteriovenous malformations, and neurodegenerative diseases. Yet, the molecular mechanisms involved in high flow response are still poorly understood. Here, we described the development and validation of a 96-wells fluidic system, with interchangeable cell culture and fluidics, to perform high-throughput screenings under laminar high-flow conditions. We demonstrated that endothelial cells in our newly developed 96-wells fluidic system respond to fluid flow-induced shear stress by aligning along the flow direction and increasing the levels of KLF2 and KLF4. We further demonstrate that our 96-wells fluidic system allows for efficient gene knock-down compatible with automated liquid handling for high-throughput screening platforms. Overall, we propose that this modular 96-well fluidic system is an excellent platform to perform genome-wide and/or drug screenings to identify the molecular mechanisms involved in the responses of endothelial cells to high wall shear stress.

Tyrosine-protein kinase Yes controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosis.

Vascular endothelial (VE)-cadherin in endothelial adherens junctions is an essential component of the vascular barrier, critical for tissue homeostasis and implicated in diseases such as cancer and retinopathies. Inhibitors of Src cytoplasmic tyrosine kinase have been applied to suppress VE-cadherin tyrosine phosphorylation and prevent excessive leakage, edema and high interstitial pressure. Here we show that the Src-related Yes tyrosine kinase, rather than Src, is localized at endothelial cell (EC) junctions where it becomes activated in a flow-dependent manner. EC-specific Yes1 deletion suppresses VE-cadherin phosphorylation and arrests VE-cadherin at EC junctions. This is accompanied by loss of EC collective migration and exaggerated agonist-induced macromolecular leakage. Overexpression of Yes1 causes ectopic VE-cadherin phosphorylation, while vascular leakage is unaffected. In contrast, in EC-specific Src-deficiency, VE-cadherin internalization is maintained, and leakage is suppressed. In conclusion, Yes-mediated phosphorylation regulates constitutive VE-cadherin turnover, thereby maintaining endothelial junction plasticity and vascular integrity.

WASp controls oriented migration of endothelial cells to achieve functional vascular patterning.

Endothelial cell migration and proliferation are essential for the establishment of a hierarchical organization of blood vessels and optimal distribution of blood. However, how these cellular processes are quantitatively coordinated to drive vascular network morphogenesis remains unknown. Here, using the zebrafish vasculature as a model system, we demonstrate that the balanced distribution of endothelial cells, as well as the resulting regularity of vessel calibre, is a result of cell migration from veins towards arteries and cell proliferation in veins. We identify the Wiskott-Aldrich Syndrome protein (WASp) as an important molecular regulator of this process and show that loss of coordinated migration from veins to arteries upon wasb depletion results in aberrant vessel morphology and the formation of persistent arteriovenous shunts. We demonstrate that WASp achieves its function through the coordination of junctional actin assembly and PECAM1 recruitment and provide evidence that this is conserved in humans. Overall, we demonstrate that functional vascular patterning in the zebrafish trunk is established through differential cell migration regulated by junctional actin, and that interruption of differential migration may represent a pathomechanism in vascular malformations.

Long-lived tumor-associated macrophages in glioma.

BACKGROUND: The tumor microenvironment plays a major tumor-supportive role in glioma. In particular, tumor-associated macrophages (TAMs), which can make up to one-third of the tumor mass, actively support tumor growth, invasion, and angiogenesis. Predominantly alternatively activated (M2-polarized) TAMs are found in late-stage glioma in both human and mouse tumors, as well as in relapse samples from patients. However, whether tumor-educated M2 TAMs can actively contribute to the emergence and growth of relapse is currently debated. METHODS: To investigate whether tumor-educated stromal cells remaining in the brain after surgical removal of the primary tumor can be long-lived and retain their tumor-supporting function, we developed a transplantation mouse model and performed lineage-tracing. RESULTS: We discovered that macrophages can survive transplantation and stay present in the tumor much longer than previously suggested, while sustaining an M2-polarized protumorigenic phenotype. Transplanted tumors showed a more aggressive growth and faster polarization of the TAMs toward an M2 phenotype compared with primary tumors, a process dependent on the presence of few cotransplanted macrophages. CONCLUSIONS: Overall, we propose a new way for tumor-educated TAMs to contribute to glioma aggressiveness by long survival and stable protumorigenic features. These properties could have a relapse-supporting effect.

Multiscale Modeling of Dyadic Structure-Function Relation in Ventricular Cardiac Myocytes.

Cardiovascular disease is often related to defects of subcellular components in cardiac myocytes, specifically in the dyadic cleft, which include changes in cleft geometry and channel placement. Modeling of these pathological changes requires both spatially resolved cleft as well as whole cell level descriptions. We use a multiscale model to create dyadic structure-function relationships to explore the impact of molecular changes on whole cell electrophysiology and calcium cycling. This multiscale model incorporates stochastic simulation of individual L-type calcium channels and ryanodine receptor channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell. We found action potential duration, systolic, and diastolic [Ca2+] to respond most sensitively to changes in L-type calcium channel current. The ryanodine receptor channel cluster structure inside dyadic clefts was found to affect all biomarkers investigated. The shape of clusters observed in experiments by Jayasinghe et al. and channel density within the cluster (characterized by mean occupancy) showed the strongest correlation to the effects on biomarkers.

Spatial modeling of the membrane-cytosolic interface in protein kinase signal transduction.

The spatial architecture of signaling pathways and the interaction with cell size and morphology are complex, but little understood. With the advances of single cell imaging and single cell biology, it becomes crucial to understand intracellular processes in time and space. Activation of cell surface receptors often triggers a signaling cascade including the activation of membrane-attached and cytosolic signaling components, which eventually transmit the signal to the cell nucleus. Signaling proteins can form steep gradients in the cytosol, which cause strong cell size dependence. We show that the kinetics at the membrane-cytosolic interface and the ratio of cell membrane area to the enclosed cytosolic volume change the behavior of signaling cascades significantly. We suggest an estimate of average concentration for arbitrary cell shapes depending on the cell volume and cell surface area. The normalized variance, known from image analysis, is suggested as an alternative measure to quantify the deviation from the average concentration. A mathematical analysis of signal transduction in time and space is presented, providing analytical solutions for different spatial arrangements of linear signaling cascades. Quantification of signaling time scales reveals that signal propagation is faster at the membrane than at the nucleus, while this time difference decreases with the number of signaling components in the cytosol. Our investigations are complemented by numerical simulations of non-linear cascades with feedback and asymmetric cell shapes. We conclude that intracellular signal propagation is highly dependent on cell geometry and, thereby, conveys information on cell size and shape to the nucleus.

Dynamics of cell wall elasticity pattern shapes the cell during yeast mating morphogenesis.

The cell wall defines cell shape and maintains integrity of fungi and plants. When exposed to mating pheromone, Saccharomyces cerevisiae grows a mating projection and alters in morphology from spherical to shmoo form. Although structural and compositional alterations of the cell wall accompany shape transitions, their impact on cell wall elasticity is unknown. In a combined theoretical and experimental approach using finite-element modelling and atomic force microscopy (AFM), we investigated the influence of spatially and temporally varying material properties on mating morphogenesis. Time-resolved elasticity maps of shmooing yeast acquired with AFM in vivo revealed distinct patterns, with soft material at the emerging mating projection and stiff material at the tip. The observed cell wall softening in the protrusion region is necessary for the formation of the characteristic shmoo shape, and results in wider and longer mating projections. The approach is generally applicable to tip-growing fungi and plants cells.

Influence of cell shape, inhomogeneities and diffusion barriers in cell polarization models.

In silico experiments bear the potential for further understanding of biological transport processes by allowing a systematic modification of any spatial property and providing immediate simulation results. Cell polarization and spatial reorganization of membrane proteins are fundamental for cell division, chemotaxis and morphogenesis. We chose the yeast Saccharomyces cerevisiae as an exemplary model system which entails the shuttling of small Rho GTPases such as Cdc42 and Rho, between an active membrane-bound form and an inactive cytosolic form. We used partial differential equations to describe the membrane-cytosol shuttling of proteins. In this study, a consistent extension of a class of 1D reaction-diffusion systems into higher space dimensions is suggested. The membrane is modeled as a thin layer to allow for lateral diffusion and the cytosol is modeled as an enclosed volume. Two well-known polarization mechanisms were considered. One shows the classical Turing-instability patterns, the other exhibits wave-pinning dynamics. For both models, we investigated how cell shape and diffusion barriers like septin structures or bud scars influence the formation of signaling molecule clusters and subsequent polarization. An extensive set of in silico experiments with different modeling hypotheses illustrated the dependence of cell polarization models on local membrane curvature, cell size and inhomogeneities on the membrane and in the cytosol. In particular, the results of our computer simulations suggested that for both mechanisms, local diffusion barriers on the membrane facilitate Rho GTPase aggregation, while diffusion barriers in the cytosol and cell protrusions limit spontaneous molecule aggregations of active Rho GTPase locally.

Yeast mating and image-based quantification of spatial pattern formation.

Communication between cells is a ubiquitous feature of cell populations and is frequently realized by secretion and detection of signaling molecules. Direct visualization of the resulting complex gradients between secreting and receiving cells is often impossible due to the small size of diffusing molecules and because such visualization requires experimental perturbations such as attachment of fluorescent markers, which can change diffusion properties. We designed a method to estimate such extracellular concentration profiles in vivo by using spatiotemporal mathematical models derived from microscopic analysis. This method is applied to populations of thousands of haploid yeast cells during mating in order to quantify the extracellular distributions of the pheromone α-factor and the activity of the aspartyl protease Bar1. We demonstrate that Bar1 limits the range of the extracellular pheromone signal and is critical in establishing α-factor concentration gradients, which is crucial for effective mating. Moreover, haploid populations of wild type yeast cells, but not BAR1 deletion strains, create a pheromone pattern in which cells differentially grow and mate, with low pheromone regions where cells continue to bud and regions with higher pheromone levels and gradients where cells conjugate to form diploids. However, this effect seems to be exclusive to high-density cultures. Our results show a new role of Bar1 protease regulating the pheromone distribution within larger populations and not only locally inside an ascus or among few cells. As a consequence, wild type populations have not only higher mating efficiency, but also higher growth rates than mixed MATa bar1Δ/MATα cultures. We provide an explanation of how a rapidly diffusing molecule can be exploited by cells to provide spatial information that divides the population into different transcriptional programs and phenotypes.