MyoScreen, a High-Throughput Phenotypic Screening Platform Enabling Muscle Drug Discovery.

Despite the need for more effective drug treatments to address muscle atrophy and disease, physiologically accurate in vitro screening models and higher information content preclinical assays that aid in the discovery and development of novel therapies are lacking. To this end, MyoScreen was developed: a robust and versatile high-throughput high-content screening (HT/HCS) platform that integrates a physiologically and pharmacologically relevant micropatterned human primary skeletal muscle model with a panel of pertinent phenotypic and functional assays. MyoScreen myotubes form aligned, striated myofibers, and they show nerve-independent accumulation of acetylcholine receptors (AChRs), excitation-contraction coupling (ECC) properties characteristic of adult skeletal muscle and contraction in response to chemical stimulation. Reproducibility and sensitivity of the fully automated MyoScreen platform are highlighted in assays that quantitatively measure myogenesis, hypertrophy and atrophy, AChR clusterization, and intracellular calcium release dynamics, as well as integrating contractility data. A primary screen of 2560 compounds to identify stimulators of myofiber regeneration and repair, followed by further biological characterization of two hits, validates MyoScreen for the discovery and testing of novel therapeutics. MyoScreen is an improvement of current in vitro muscle models, enabling a more predictive screening strategy for preclinical selection of the most efficacious new chemical entities earlier in the discovery pipeline process.

Stem Cell-Like Properties of CK2ß-down Regulated Mammary Cells.

The ubiquitous protein kinase CK2 has been demonstrated to be overexpressed in a number of human tumours. This enzyme is composed of two catalytic α or α' subunits and a dimer of ß regulatory subunits whose expression levels are probably implicated in CK2 regulation. Several recent papers reported that unbalanced expression of CK2 subunits is sufficient to drive epithelial to mesenchymal transition, a process involved in cancer invasion and metastasis. Herein, through transcriptomic and miRNA analysis together with comparison of cellular properties between wild type and CK2ß-knock-down MCF10A cells, we show that down-regulation of CK2ß subunit in mammary epithelial cells induces the acquisition of stem cell-like properties associated with perturbed polarity, CD44high/CD24low antigenic phenotype and the ability to grow under anchorage-independent conditions. These data demonstrate that a CK2ß level establishes a critical cell fate threshold in the control of epithelial cell plasticity. Thus, this regulatory subunit functions as a nodal protein to maintain an epithelial phenotype and its depletion drives breast cell stemness.

Fully Automated One-Step Production of Functional 3D Tumor Spheroids for High-Content Screening.

Adoption of spheroids within high-content screening (HCS) has lagged behind high-throughput screening (HTS) due to issues with running complex assays on large three-dimensional (3D) structures.To enable multiplexed imaging and analysis of spheroids, different cancer cell lines were grown in 3D on micropatterned 96-well plates with automated production of nine uniform spheroids per well. Spheroids achieve diameters of up to 600 µm, and reproducibility was experimentally validated (interwell and interplate CV(diameter) <5%). Biphoton imaging confirmed that micropatterned spheroids exhibit characteristic cell heterogeneity with distinct microregions. Furthermore, central necrosis appears at a consistent spheroid size, suggesting standardized growth.Using three reference compounds (fluorouracil, irinotecan, and staurosporine), we validated HT-29 micropatterned spheroids on an HCS platform, benchmarking against hanging-drop spheroids. Spheroid formation and imaging in a single plate accelerate assay workflow, and fixed positioning prevents structures from overlapping or sticking to the well wall, augmenting image processing reliability. Furthermore, multiple spheroids per well increase the statistical confidence sufficiently to discriminate compound mechanisms of action and generate EC50 values for endpoints of cell death, architectural change, and size within a single-pass read. Higher quality data and a more efficient HCS work chain should encourage integration of micropatterned spheroid models within fundamental research and drug discovery applications.

Spatial organization of the extracellular matrix regulates cell-cell junction positioning.

The organization of cells into epithelium depends on cell interaction with both the extracellular matrix (ECM) and adjacent cells. The role of cell-cell adhesion in the regulation of epithelial topology is well-described. ECM is better known to promote cell migration and provide a structural scaffold for cell anchoring, but its contribution to multicellular morphogenesis is less well-understood. We developed a minimal model system to investigate how ECM affects the spatial organization of intercellular junctions. Fibronectin micropatterns were used to constrain the location of cell-ECM adhesion. We found that ECM affects the degree of stability of intercellular junction positioning and the magnitude of intra- and intercellular forces. Intercellular junctions were permanently displaced, and experienced large perpendicular tensional forces as long as they were positioned close to ECM. They remained stable solely in regions deprived of ECM, where they were submitted to lower tensional forces. The heterogeneity of the spatial organization of ECM induced anisotropic distribution of mechanical constraints in cells, which seemed to adapt their position to minimize both intra- and intercellular forces. These results uncover a morphogenetic role for ECM in the mechanical regulation of cells and intercellular junction positioning.

Regulation of epithelial to mesenchymal transition: CK2ß on stage.

Protein kinase CK2 participates in the regulation of fundamental cellular processes. Among these processes, cell polarity and cell morphology are controlled by this enzyme probably through the phosphorylation of key proteins. To further study the involvement of CK2 in these processes, we showed that in epithelial cells, the regulatory CK2ß subunit was required for LKB1-dependent polarization and cell adhesion. Moreover, CK2ß silencing in MCF10A mammary epithelial cells triggered changes in their morphology correlated with the acquisition of mesenchymal phenotype, which were reminiscent to TGFß-induced epithelial-to-mesenchymal-transition (EMT). TGFß has emerged as a major inducer of EMT both in vitro and in vivo. We found that among the TGFß isoforms, TGFß2 expression was strongly induced in CK2ß-knockdown cells. However, the EMT phenotype induced in response to CK2ß silencing was not abolished by blocking the TGFß signaling pathway at TGFß receptor level, suggesting that alternative pathways might be involved. Given the importance of CK2 in tumorigenesis, a dysregulation of CK2ß expression might contribute to EMT induction during cancer progression.

A new micropatterning method of soft substrates reveals that different tumorigenic signals can promote or reduce cell contraction levels.

In tissues, cell microenvironment geometry and mechanics strongly impact on cell physiology. Surface micropatterning allows the control of geometry while deformable substrates of tunable stiffness are well suited for the control of the mechanics. We developed a new method to micropattern extracellular matrix proteins on poly-acrylamide gels in order to simultaneously control cell geometry and mechanics. Microenvironment geometry and mechanics impinge on cell functions by regulating the development of intra-cellular forces. We measured these forces in micropatterned cells. Micropattern geometry was streamlined to orient forces and place cells in comparable conditions. Thereby force measurement method could be simplified and applied to large-scale experiment on chip. We applied this method to mammary epithelial cells with traction force measurements in various conditions to mimic tumoral transformation. We found that, contrary to the current view, all transformation phenotypes were not always associated to an increased level of cell contractility.

Protein kinase CK2α subunit over-expression correlates with metastatic risk in breast carcinomas: quantitative immunohistochemistry in tissue microarrays.

BACKGROUND: CK2α is a signalling molecule that participates in major events in solid tumour progression. The aim of this study was to evaluate the prognostic significance of the immunohistochemical expression of CK2α in breast carcinomas. METHODS: Quantitative measurements of immunohistochemical expression of 33 biomarkers using high-throughput densitometry, assessed on digitised microscopic tissue micro-array images were correlated with clinical outcome in 1000 breast carcinomas using univariate and multivariate analyses. RESULTS: In univariate analysis, CK2α was a significant prognostic indicator (p<0.001). Moreover, a multivariable model allowed the selection of the best combination of the 33 biomarkers to predict patients' outcome through logistic regression. A nine-marker signature highly predictive of metastatic risk, associating SHARP-2, STAT1, eIF4E, pmapKAPk-2, pAKT, caveolin, VEGF, FGF-1 and CK2α permitted to classify well 82.32% of patients (specificity 81.59%, sensitivity 92.55%, area under ROC curve 0.939). Importantly, in a node negative subset of patients an even more (86%) clinically relevant association of eleven markers was found predictive of poor outcome. CONCLUSION: A strong quantitative CK2α immunohistochemical expression in breast carcinomas is individually a significant indicator of poor prognosis. Moreover, an immunohistochemical signature of 11 markers including CK2α accurately (86%) well classifies node negative patients in good and poor outcome subsets. Our results suggest that CK2α evaluation together with key downstream CK2 targets might be a useful tool to identify patients at high risk of distant metastases and that CK2 can be considered as a relevant target for potential specific therapy.

Antitumor activity of pyridocarbazole and benzopyridoindole derivatives that inhibit protein kinase CK2.

The alkyloid compound ellipticine derived from the berrywood tree is a topoisomerase II poison that is used in ovarian and breast cancer treatment. In this study, we report the identification of ellipticine derivatives and their tetracyclic angular benzopyridoindole analogues as novel ATP-competitive inhibitors of the protein kinase CK2. In vitro and in vivo assays showed that these compounds have a good pharmacologic profile, causing a marked inhibition of CK2 activity associated with cell cycle arrest and apoptosis in human cancer cells. Further, in vivo assays demonstrate antitumor activity in a mouse xenograft model of human glioblastoma. Finally, crystal structures of CK2-inhibitor complex provide structural insights on the molecular basis of CK2 inhibition. Our work lays the foundation for development of clinically useful CK2 inhibitors derived from a well-studied scaffold with suitable pharmacokinetics parameters.