NONO enhances mRNA processing of super-enhancer-associated GATA2 and HAND2 genes in neuroblastoma.

High-risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA-binding protein, NONO, in neuroblastoma is associated with poor patient outcome; however, there is little understanding of the mechanism of NONO-dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biochemical and genome-wide molecular analysis to reveal complex NONO-dependent regulation of gene expression. NONO forms RNA- and DNA-tethered condensates throughout the nucleus and undergoes phase separation in vitro, modulated by nucleic acid binding. CLIP analyses show that NONO mainly binds to the 5' end of pre-mRNAs and modulates pre-mRNA processing, dependent on its RNA-binding activity. NONO regulates super-enhancer-associated genes, including HAND2 and GATA2. Abrogating NONO RNA binding, or phase separation activity, results in decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA-binding activity of NONO may have therapeutic potential in this cancer context.

Antenatal creatine supplementation reduces persistent fetal lung inflammation and oxidative stress in an ovine model of chorioamnionitis.

Chorioamnionitis is a common antecedent of preterm birth and induces inflammation and oxidative stress in the fetal lungs. Reducing inflammation and oxidative stress in the fetal lungs may improve respiratory outcomes in preterm infants. Creatine is an organic acid with known anti-inflammatory and antioxidant properties. The objective of the study was to evaluate the efficacy of direct fetal creatine supplementation to reduce inflammation and oxidative stress in fetal lungs arising from an in utero proinflammatory stimulus. Fetal lambs (n = 51) were instrumented at 90 days gestation to receive a continuous infusion of creatine monohydrate (6 mg·kg-1·h-1) or saline for 17 days. Maternal chorioamnionitis was induced with intra-amniotic lipopolysaccharide (LPS; 1 mg, O55:H6) or saline 7 days before delivery at 110 days gestation. Tissue creatine content was assessed with capillary electrophoresis, and inflammatory markers were analyzed with Luminex Magpix and immunohistochemistry. Oxidative stress was measured as the level of protein thiol oxidation. The effects of LPS and creatine were analyzed using a two-way ANOVA. Fetal creatine supplementation increased lung creatine content by 149% (PCr < 0.0001) and had no adverse effects on lung morphology. LPS-exposed groups showed increased levels of interleukin-8 in the bronchoalveolar lavage (PLPS < 0.0001) and increased levels of CD45+ leukocytes (PLPS < 0.0001) and MPO+ (PLPS < 0.0001) cells in the lung parenchyma. Creatine supplementation significantly reduced the levels of CD45+ (PCr = 0.045) and MPO+ cells (PCr = 0.012) in the lungs and reduced thiol oxidation in plasma (PCr < 0.01) and lung tissue (PCr = 0.02). In conclusion, fetal creatine supplementation reduced markers of inflammation and oxidative stress in the fetal lungs arising from chorioamnionitis.NEW & NOTEWORTHY We evaluated the effect of antenatal creatine supplementation to reduce pulmonary inflammation and oxidative stress in the fetal lamb lungs arising from lipopolysaccharide (LPS)-induced chorioamnionitis. Fetal creatine supplementation increased lung creatine content and had no adverse effects on systemic fetal physiology and overall lung architecture. Importantly, fetuses that received creatine had significantly lower levels of inflammation and oxidative stress in the lungs, suggesting an anti-inflammatory and antioxidant benefit of creatine.

CellTrackVis: interactive browser-based visualization for analyzing cell trajectories and lineages.

BACKGROUND: Automatic cell tracking methods enable practitioners to analyze cell behaviors efficiently. Notwithstanding the continuous development of relevant software, user-friendly visualization tools have room for further improvements. Typical visualization mostly comes with main cell tracking tools as a simple plug-in, or relies on specific software/platforms. Although some tools are standalone, limited visual interactivity is provided, or otherwise cell tracking outputs are partially visualized. RESULTS: This paper proposes a self-reliant visualization system, CellTrackVis, to support quick and easy analysis of cell behaviors. Interconnected views help users discover meaningful patterns of cell motions and divisions in common web browsers. Specifically, cell trajectory, lineage, and quantified information are respectively visualized in a coordinated interface. In particular, immediate interactions among modules enable the study of cell tracking outputs to be more effective, and also each component is highly customizable for various biological tasks. CONCLUSIONS: CellTrackVis is a standalone browser-based visualization tool. Source codes and data sets are freely available at http://github.com/scbeom/celltrackvis with the tutorial at http://scbeom.github.io/ctv_tutorial .

Distribution, composition, and activity of airway-associated adipose tissue in the porcine lung.

Patients with comorbid asthma-obesity experience greater disease severity and are less responsive to therapy. We have previously reported adipose tissue within the airway wall that positively correlated with body mass index. Accumulation of biologically active adipose tissue may result in the local release of adipokines and disrupt large and small airway function depending on its anatomical distribution. This study therefore characterized airway-associated adipose tissue distribution, lipid composition, and adipokine activity in a porcine model. Airway segments were systematically dissected from different locations of the bronchial tree in inflation-fixed lungs. Cryosections were stained with hematoxylin and eosin (H&E) for airway morphology, oil red O to distinguish adipose tissue, and Nile blue A for lipid subtype delineation. Excised airway-associated adipose tissue was cultured for 72 h to quantify adipokine release using immunoassays. Results showed that airway-associated adipose tissue extended throughout the bronchial tree and occupied an area proportionally similar to airway smooth muscle within the wall area. Lipid composition consisted of pure neutral lipids (61.7 ± 3.5%), a mixture of neutral and acidic lipids (36.3 ± 3.4%), or pure acidic lipids (2.0 ± 0.8%). Following tissue culture, there was rapid release of IFN-γ, IL-1ß, and TNF-α at 12 h. Maximum IL-4 and IL-10 release was at 24 and 48 h, and peak leptin release occurred between 48 and 72 h. These data extend previous findings and demonstrate that airway-associated adipose tissue is prevalent and biologically active within the bronchial tree, providing a local source of adipokines that may be a contributing factor in airway disease.

Reprogramming anchorage dependency by adherent-to-suspension transition promotes metastatic dissemination.

BACKGROUND: Although metastasis is the foremost cause of cancer-related death, a specialized mechanism that reprograms anchorage dependency of solid tumor cells into circulating tumor cells (CTCs) during metastatic dissemination remains a critical area of challenge. METHODS: We analyzed blood cell-specific transcripts and selected key Adherent-to-Suspension Transition (AST) factors that are competent to reprogram anchorage dependency of adherent cells into suspension cells in an inducible and reversible manner. The mechanisms of AST were evaluated by a series of in vitro and in vivo assays. Paired samples of primary tumors, CTCs, and metastatic tumors were collected from breast cancer and melanoma mouse xenograft models and patients with de novo metastasis. Analyses of single-cell RNA sequencing (scRNA-seq) and tissue staining were performed to validate the role of AST factors in CTCs. Loss-of-function experiments were performed by shRNA knockdown, gene editing, and pharmacological inhibition to block metastasis and prolong survival. RESULTS: We discovered a biological phenomenon referred to as AST that reprograms adherent cells into suspension cells via defined hematopoietic transcriptional regulators, which are hijacked by solid tumor cells to disseminate into CTCs. Induction of AST in adherent cells 1) suppress global integrin/ECM gene expression via Hippo-YAP/TEAD inhibition to evoke spontaneous cell-matrix dissociation and 2) upregulate globin genes that prevent oxidative stress to acquire anoikis resistance, in the absence of lineage differentiation. During dissemination, we uncover the critical roles of AST factors in CTCs derived from patients with de novo metastasis and mouse models. Pharmacological blockade of AST factors via thalidomide derivatives in breast cancer and melanoma cells abrogated CTC formation and suppressed lung metastases without affecting the primary tumor growth. CONCLUSION: We demonstrate that suspension cells can directly arise from adherent cells by the addition of defined hematopoietic factors that confer metastatic traits. Furthermore, our findings expand the prevailing cancer treatment paradigm toward direct intervention within the metastatic spread of cancer.

Thyroid-Friendly Soft Materials as 3D Cell Culture Tool for Stimulating Thyroid Cell Function.

The disruption of thyroid hormones because of chemical exposure is a significant societal problem. Chemical evaluations of environmental and human health risks are conventionally based on animal experiments. However, owing to recent breakthroughs in biotechnology, the potential toxicity of chemicals can now be evaluated using 3D cell cultures. In this study, the interactive effects of thyroid-friendly soft (TS) microspheres on thyroid cell aggregates are elucidated and their potential as a reliable toxicity assessment tool is evaluated. Using state-of-the-art characterization methods coupled with cell-based analysis and quadrupole time-of-flight mass spectrometry, it is shown that TS-microsphere-integrated thyroid cell aggregates exhibit improved thyroid function. Specifically, the responses of zebrafish embryos, which are used for thyroid toxicity analysis, and the TS-microsphere-integrated cell aggregates to methimazole (MMI), a known thyroid inhibitor, are compared. The results show that the thyroid hormone disruption response of the TS-microsphere-integrated thyroid cell aggregates to MMI is more sensitive compared with those of the zebrafish embryos and conventionally formed cell aggregates. This proof-of-concept approach can be used to control cellular function in the desired direction and hence evaluate thyroid function. Thus, the proposed TS-microsphere-integrated cell aggregates may yield new fundamental insights for advancing in vitro cell-based research.

Chondrocyte-specific response to stiffness-mediated primary cilia formation and centriole positioning.

Mechanical stress and the stiffness of the extracellular matrix are key drivers of tissue development and homeostasis. Aberrant mechanosensation is associated with a wide range of pathologies, including osteoarthritis. Matrix (or substrate) stiffness plays a major role in cell spreading, adhesion, proliferation, and differentiation. However, how specific cells sense substrate stiffness still remains unclear. The primary cilium is an essential cellular organelle that senses and integrates mechanical and chemical signals from the extracellular environment. We hypothesized that the primary cilium dynamically alters its length and position to fine-tune cell mechanosignaling based on substrate stiffness alone. We used a hydrogel system of varying substrate stiffness to examine the role of stiffness on cilia frequency, length, and centriole position as well as cell and nuclei area over time. Contrary to other cell types, we show that chondrocyte primary cilia shorten on softer substrates, demonstrating tissue-specific mechanosensing that is aligned with the tissue stiffness the cells originate from. We further show that stiffness determines centriole positioning to either the basal or apical membrane during attachment and spreading, with centrioles positioned toward the basal membrane on stiffer substrates. These phenomena are mediated by force generation actin-myosin stress fibers in a time-dependent manner. Finally, we show on stiff substrates that primary cilia are involved in tension-mediated cell spreading. We propose that substrate stiffness plays a role in cilia positioning, regulating cellular responses to external forces, and maybe a key driver of mechanosignaling-associated diseases.

Subcellular mechano-microscopy: high resolution three-dimensional elasticity mapping using optical coherence microscopy.

The importance of cellular-scale mechanical properties is well-established, yet it is challenging to map subcellular elasticity in three dimensions. We present subcellular mechano-microscopy, an optical coherence microscopy (OCM)-based variant of three-dimensional (3-D) compression optical coherence elastography (OCE) that provides an elasticity system resolution of 5 × 5 × 5 µm: a 7-fold improvement in system resolution over previous OCE studies of cells. The improved resolution is achieved through a ∼5-fold improvement in optical resolution, refinement of the strain estimation algorithm, and demonstration that mechanical deformation of subcellular features provides feature resolution far greater than that demonstrated previously on larger features with diameter >250 µm. We use mechano-microscopy to image adipose-derived stem cells encapsulated in gelatin methacryloyl. We compare our results with compression OCE and demonstrate that mechano-microscopy can provide contrast from subcellular features not visible using OCE.

Role of Graphene Family Nanomaterials in Skin Wound Healing and Regeneration.

Owing to astonishing properties such as the large surface area to volume ratio, mechanical stability, antimicrobial property, and collagen crosslinking, graphene family nanomaterials (GFNs) have been widely used in various biomedical applications including tissue regeneration. Many review literatures are available to compile the role of GFNs in cardiac, bone, and neuronal tissue regeneration. However, the contribution of GFNs in skin wound healing and tissue regeneration was not yet discussed. In the present review, we have highlighted the properties of GFNs and their application in skin wound healing. In addition, we have included challenges and future directions of GFNs in skin tissue regeneration in the portion of conclusion and perspectives.

Stem cell migration and mechanotransduction on linear stiffness gradient hydrogels.

The spatial presentation of mechanical information is a key parameter for cell behavior. We have developed a method of polymerization control in which the differential diffusion distance of unreacted cross-linker and monomer into a prepolymerized hydrogel sink results in a tunable stiffness gradient at the cell-matrix interface. This simple, low-cost, robust method was used to produce polyacrylamide hydrogels with stiffness gradients of 0.5, 1.7, 2.9, 4.5, 6.8, and 8.2 kPa/mm, spanning the in vivo physiological and pathological mechanical landscape. Importantly, three of these gradients were found to be nondurotactic for human adipose-derived stem cells (hASCs), allowing the presentation of a continuous range of stiffnesses in a single well without the confounding effect of differential cell migration. Using these nondurotactic gradient gels, stiffness-dependent hASC morphology, migration, and differentiation were studied. Finally, the mechanosensitive proteins YAP, Lamin A/C, Lamin B, MRTF-A, and MRTF-B were analyzed on these gradients, providing higher-resolution data on stiffness-dependent expression and localization.

Graphene-Functionalized Biomimetic Scaffolds for Tissue Regeneration.

Graphene is a two-dimensional atomic layer of graphite, where carbon atoms are assembled in a honeycombed lattice structure. Recently, graphene family nanomaterials, including pristine graphene, graphene oxide and reduced graphene oxide, have increasingly attracted a great deal of interest from researchers in a variety of science, engineering and industrial fields because of their unique structural and functional features. In particular, extensive studies have been actively conducted in the biomedical and related fields, including multidisciplinary and emerging areas, as their stimulating effects on cell behaviors have been becoming an increasing concern. Herein, we are attempting to summarize some of recent findings in the fields of tissue regeneration concerning the graphene family nanomaterial-functionalized biomimetic scaffolds, and to provide the promising perspectives for the possible applications of graphene family nanomaterial.

Interplay of matrix stiffness and protein tethering in stem cell differentiation.

Stem cells regulate their fate by binding to, and contracting against, the extracellular matrix. Recently, it has been proposed that in addition to matrix stiffness and ligand type, the degree of coupling of fibrous protein to the surface of the underlying substrate, that is, tethering and matrix porosity, also regulates stem cell differentiation. By modulating substrate porosity without altering stiffness in polyacrylamide gels, we show that varying substrate porosity did not significantly change protein tethering, substrate deformations, or the osteogenic and adipogenic differentiation of human adipose-derived stromal cells and marrow-derived mesenchymal stromal cells. Varying protein-substrate linker density up to 50-fold changed tethering, but did not affect osteogenesis, adipogenesis, surface-protein unfolding or underlying substrate deformations. Differentiation was also unaffected by the absence of protein tethering. Our findings imply that the stiffness of planar matrices regulates stem cell differentiation independently of protein tethering and porosity.

Digital Plasmonic Patterning for Localized Tuning of Hydrogel Stiffness.

The mechanical properties of the extracellular matrix (ECM) can dictate cell fate in biological systems. In tissue engineering, varying the stiffness of hydrogels-water-swollen polymeric networks that act as ECM substrates-has previously been demonstrated to control cell migration, proliferation, and differentiation. Here, "digital plasmonic patterning" (DPP) is developed to mechanically alter a hydrogel encapsulated with gold nanorods using a near-infrared laser, according to a digital (computer-generated) pattern. DPP can provide orders of magnitude changes in stiffness, and can be tuned by laser intensity and speed of writing. In vitro cellular experiments using A7R5 smooth muscle cells confirm cell migration and alignment according to these patterns, making DPP a useful technique for mechanically patterning hydrogels for various biomedical applications.

In situ mechanotransduction via vinculin regulates stem cell differentiation.

Human mesenchymal stem cell (hMSC) proliferation, migration, and differentiation have all been linked to extracellular matrix stiffness, yet the signaling pathway(s) that are necessary for mechanotransduction remain unproven. Vinculin has been implicated as a mechanosensor in vitro, but here we demonstrate its ability to also regulate stem cell behavior, including hMSC differentiation. RNA interference-mediated vinculin knockdown significantly decreased stiffness-induced MyoD, a muscle transcription factor, but not Runx2, an osteoblast transcription factor, and impaired stiffness-mediated migration. A kinase binding accessibility screen predicted a cryptic MAPK1 signaling site in vinculin which could regulate these behaviors. Indeed, reintroduction of vinculin domains into knocked down cells indicated that MAPK1 binding site-containing vinculin constructs were necessary for hMSC expression of MyoD. Vinculin knockdown does not appear to interfere with focal adhesion assembly, significantly alter adhesive properties, or diminish cell traction force generation, indicating that its knockdown only adversely affected MAPK1 signaling. These data provide some of the first evidence that a force-sensitive adhesion protein can regulate stem cell fate.

Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels.

In obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), the extracellular matrix (ECM) protein amount and composition of the airway smooth muscle (ASM) is often remodelled, likely altering tissue stiffness. The underlying mechanism of how human ASM cell (hASMC) mechanosenses the aberrant microenvironment is not well understood. Physiological stiffnesses of the ASM were measured by uniaxial compression tester using porcine ASM layers under 0, 5 and 10% longitudinal stretch above in situ length. Linear stiffness gradient hydrogels (230 kPa range) were fabricated and functionalized with ECM proteins, collagen I (ColI), fibronectin (Fn) and laminin (Ln), to recapitulate the above-measured range of stiffnesses. Overall, hASMC mechanosensation exhibited a clear correlation with the underlying hydrogel stiffness. Cell size, nuclear size and contractile marker alpha-smooth muscle actin (αSMA) expression showed a strong correlation to substrate stiffness. Mechanosensation, assessed by Lamin-A intensity and nuc/cyto YAP, exhibited stiffness-mediated behaviour only on ColI and Fn-coated hydrogels. Inhibition studies using blebbistatin or Y27632 attenuated most mechanotransduction-derived cell morphological responses, αSMA and Lamin-A expression and nuc/cyto YAP (blebbistatin only). This study highlights the interplay and complexities between stiffness and ECM protein type on hASMC mechanosensation, relevant to airway remodelling in obstructive airway diseases.

In situ stress estimation in quantitative micro-elastography.

In quantitative micro-elastography (QME), a pre-characterized compliant layer with a known stress-strain curve is utilized to map stress at the sample surface. However, differences in the boundary conditions of the compliant layer when it is mechanically characterized and when it is used in QME experiments lead to inconsistent stress estimation and consequently, inaccurate elasticity measurements. Here, we propose a novel in situ stress estimation method using an optical coherence tomography (OCT)-based uniaxial compression testing system integrated with the QME experimental setup. By combining OCT-measured axial strain with axial stress determined using a load cell in the QME experiments, we can estimate in situ stress for the compliant layer, more accurately considering its boundary conditions. Our proposed method shows improved accuracy, with an error below 10%, compared to 85% using the existing QME technique with no lubrication. Furthermore, demonstrations on hydrogels and cells indicate the potential of this approach for improving the characterization of the micro-scale mechanical properties of cells and their interactions with the surrounding biomaterial, which has potential for application in cell mechanobiology.

Tumor Biomechanics Alters Metastatic Dissemination of Triple Negative Breast Cancer via Rewiring Fatty Acid Metabolism.

In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin ß1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.

Getting physical: Material mechanics is an intrinsic cell cue.

Advances in biomaterial science have allowed for unprecedented insight into the ability of material cues to influence stem cell function. These material approaches better recapitulate the microenvironment, providing a more realistic ex vivo model of the cell niche. However, recent advances in our ability to measure and manipulate niche properties in vivo have led to novel mechanobiological studies in model organisms. Thus, in this review, we will discuss the importance of material cues within the cell niche, highlight the key mechanotransduction pathways involved, and conclude with recent evidence that material cues regulate tissue function in vivo.

Volume adaptation of neonatal cardiomyocyte spheroids in 3D stiffness gradient GelMA.

Present understandings of cardiomyocyte mechanobiology have primarily been developed using 2-dimensional, monocellular cell cultures, however the emergence of 3-dimensional (3D) multicellular cardiac constructs has enabled us to develop more sophisticated recapitulations of the cardiac microenvironment. Several of these strategies have illustrated that incorporating elements of the extracellular matrix (ECM) can promote greater maturation and enhance desirable cardiac functions, such as contractility, but the responses of these cardiac constructs to biophysically aberrant conditions, such as in the post-infarct heart, has remained relatively unexplored. In our study, we employ a stiffness gradient gelatin methacryloyl (GelMA) hydrogel platform to unpack the mechanobiology of cardiac spheroids. We encapsulated neonatal rat cardiac cell spheroids in a 4.4-18.7 kPa linear stiffness gradient up to 120 h. We found the proportion of viable cells within the spheroids increased over time, but the cell number per spheroid decreased. Spheroids expand more in softer matrices while stiffer matrices promote larger nuclei without changing nuclei shape. Volume expansion came primarily from cells expressing vimentin. We did not observe any correlations between stiffness and mechanomarker expression, however we found that after 120 h post-encapsulation, the localization of YAP, the localization of MRTF-A and the expression of Lamin-A was correlated with spheroid morphology. The same trends were not observed 24 h post-encapsulation, indicating that volume adaptation can take a relatively long time. Our data demonstrates that cardiac spheroids are mechanosensitive and that their capacity to respond to ECM-based cues depends on their capacity to adapt their volume with a 3D microenvironment.