The microenvironment dictates glycocalyx construction and immune surveillance.

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium.

Tissue tension permits ß-catenin phosphorylation to drive mesoderm specification in human embryonic stem cells.

The role of morphogenetic forces in cell fate specification is an area of intense interest. Our prior studies suggested that the development of high cell-cell tension in human embryonic stem cells (hESC) colonies permits the Src-mediated phosphorylation of junctional ß-catenin that accelerates its release to potentiate Wnt-dependent signaling critical for initiating mesoderm specification. Using an ectopically expressed nonphosphorylatable mutant of ß-catenin (Y654F), we now provide direct evidence that impeding tension-dependent Src-mediated ß-catenin phosphorylation impedes the expression of Brachyury (T) and the epithelial-to-mesenchymal transition (EMT) necessary for mesoderm specification. Addition of exogenous Wnt3a or inhibiting GSK3ß activity rescued mesoderm expression, emphasizing the importance of force dependent Wnt signaling in regulating mechanomorphogenesis. Our work provides a framework for understanding tension-dependent ß-catenin/Wnt signaling in the self-organization of tissues during developmental processes including gastrulation.

Mechanical Tension Promotes Formation of Gastrulation-like Nodes and Patterns Mesoderm Specification in Human Embryonic Stem Cells.

Embryogenesis is directed by morphogens that induce differentiation within a defined tissue geometry. Tissue organization is mediated by cell-cell and cell-extracellular matrix (ECM) adhesions and is modulated by cell tension and tissue-level forces. Whether cell tension regulates development by modifying morphogen signaling is less clear. Human embryonic stem cells (hESCs) exhibit an intrinsic capacity for self-organization, which motivates their use as a tractable model of early human embryogenesis. We engineered patterned substrates that recapitulate the biophysical properties of the early embryo and mediate the self-organization of "gastrulation-like" nodes in cultured hESCs. Tissue geometries that generated local nodes of high cell-adhesion tension directed the spatial patterning of the BMP4-dependent "gastrulation-like" phenotype by enhancing phosphorylation and junctional release of ß-catenin to promote Wnt signaling and mesoderm specification. Furthermore, direct force application via mechanical stretching promoted BMP-dependent mesoderm specification, confirming that tissue-level forces can directly regulate cell fate specification in early human development.

Targeting acid ceramidase inhibits YAP/TAZ signaling to reduce fibrosis in mice.

Hepatic stellate cells (HSCs) drive hepatic fibrosis. Therapies that inactivate HSCs have clinical potential as antifibrotic agents. We previously identified acid ceramidase (aCDase) as an antifibrotic target. We showed that tricyclic antidepressants (TCAs) reduce hepatic fibrosis by inhibiting aCDase and increasing the bioactive sphingolipid ceramide. We now demonstrate that targeting aCDase inhibits YAP/TAZ activity by potentiating its phosphorylation-mediated proteasomal degradation via the ubiquitin ligase adaptor protein ß-TrCP. In mouse models of fibrosis, pharmacologic inhibition of aCDase or genetic knockout of aCDase in HSCs reduces fibrosis, stromal stiffness, and YAP/TAZ activity. In patients with advanced fibrosis, aCDase expression in HSCs is increased. Consistently, a signature of the genes most down-regulated by ceramide identifies patients with advanced fibrosis who could benefit from aCDase targeting. The findings implicate ceramide as a critical regulator of YAP/TAZ signaling and HSC activation and highlight aCDase as a therapeutic target for the treatment of fibrosis.

Challenges in the Regulation of High-Cost Treatments: An Overview From Brazil.

Regulation of drug prices that ensures adequate access to effective treatments and promotes innovation is a global challenge. In the United States, the government does not regulate drug prices when they come onto market. On the other hand, in countries such as France and Brazil, government agencies are responsible for setting up price limits by leveraging the interests of the companies and the countries' population. In Brazil, safety and efficacy of drugs are regulated by the Brazilian Health Regulatory Agency, and drug prices are regulated by the Pharmaceutical Market Regulation Chamber with a participation of Brazilian Health Regulatory Agency. Here, we introduce the current challenges faced by the Brazilian government in the drug price regulation and present proposed initiatives aiming to streamline access to innovative treatments for its citizens.

Tissue mechanics, an important regulator of development and disease.

A growing body of work describes how physical forces in and around cells affect their growth, proliferation, migration, function and differentiation into specialized types. How cells receive and respond biochemically to mechanical signals is a process termed mechanotransduction. Disease may arise if a disruption occurs within this mechanism of sensing and interpreting mechanics. Cancer, cardiovascular diseases and developmental defects, such as during the process of neural tube formation, are linked to changes in cell and tissue mechanics. A breakdown in normal tissue and cellular forces activates mechanosignalling pathways that affect their function and can promote disease progression. The recent advent of high-resolution techniques enables quantitative measurements of mechanical properties of the cell and its extracellular matrix, providing insight into how mechanotransduction is regulated. In this review, we will address the standard methods and new technologies available to properly measure mechanical properties, highlighting the challenges and limitations of probing different length-scales. We will focus on the unique environment present throughout the development and maintenance of the central nervous system and discuss cases where disease, such as brain cancer, arises in response to changes in the mechanical properties of the microenvironment that disrupt homeostasis. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

Computational fluid dynamic analysis of physical forces playing a role in brain organoid cultures in two different multiplex platforms.

BACKGROUND: Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Multiplex systems for organoid cultivation have been proposed, but whether they meet similar shear stress parameters as the regularly used spinner flask and its correlation with the successful generation of brain organoids has not been determined. RESULTS: Here we used computational fluid dynamics (CFD) to simulate two multiplex culture conditions: steering plates on an orbital shaker and the use of a previously described bioreactor. The bioreactor had low speed and high shear stress regions that may affect cell aggregate growth, depending on volume, whereas the computed variables of the steering plates were closer to those of the spinning flask. CONCLUSION: Our protocol improves the initial steps of the standard brain organoid formation, and the produced organoids displayed regionalized brain structures, including retinal pigmented cells. Overall, we conclude that suspension culture on orbital steering plates is a cost-effective practical alternative to previously described platforms for the cultivation of brain organoids for research and multiplex testing.