Eph-ephrin Signaling Research Articles

TMIC-16. SPECIFIC TARGETING OF THE MRNA EXPRESSION OF TGFB2 LIGAND TO POTENTIALLY ENHANCE IMMUNE THERAPIES IN LOW-GRADE GLIOMAS

Abstract Low-grade glioma (LGG) tumors are characterized by a low infiltration of immune cells, requiring therapies augmenting the immune response in the tumor microenvironment (TME). The Transforming Growth Factor beta (TGFB) pathway contributes to an immunosuppressive environment. We propose targeting the specific inhibition of Transforming Growth Factor Beta 2, TGFB2 mRNA, as an attractive therapeutic strategy using antisense oligonucleotides. We conducted a bioinformatic-led investigation highlighting the ratio of TGFB2:TGFB1 mRNA expression levels in the cohort of 513 TCGA patients, revealing the TGFB2 ratio as a negative prognostic indicator through utilizing a multivariate Cox proportional hazards model that included the TGFB2 ratio and age at diagnosis as linear covariates to predict overall survival (OS). The model showed a 1.8-fold higher TGFB2 ratio relative to TGFB1 mRNA expression correlated to worse OS. Furthermore, the impact of the TGFB2 ratio was significantly affected by the age at diagnosis, in which older patients exhibited worse OS at increased TGFB2 ratios. Increasing the ratio of TGFB2 relative to TGFB1, TGFB3, TGFBR1, or TGFBR2 in LGG patients led to significant increases in multivariate Cox model determined hazards ratios, HR (HR (95% CI range)) of 1.372 (1.224-1.539), 1.434 (1.288-1.560), 1.327 (1.195-1.473), and 1.381 (1.233-1.547) respectively (P<0.0001 for all comparisons). Correlation of TGFB2:TGFB1/TGFB3/TGFBR1/TGFBR2 ratio across 1330 Reactome pathways using Gene Set Enrichment Analysis showed the most positively correlated genes were members of TME pathways including PD-1 signaling, Syndecan interactions, Non-integrin membrane-ECM interactions, EPH-Ephrin signaling, and Collagen biosynthesis and modifying enzymes. These results suggest that the specific targeting TGFB2 in LGG can remodel the TME for immune therapies to improve OS outcomes.

Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses of small extracellular vesicles from C2C12 myoblasts identify specific PTM patterns in ligand-receptor interactions

Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available.In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC–MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided.In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.

Eph-ephrin signaling couples endothelial cell sorting and arterial specification

Cell segregation allows the compartmentalization of cells with similar fates during morphogenesis, which can be enhanced by cell fate plasticity in response to local molecular and biomechanical cues. Endothelial tip cells in the growing retina, which lead vessel sprouts, give rise to arterial endothelial cells and thereby mediate arterial growth. Here, we have combined cell type-specific and inducible mouse genetics, flow experiments in vitro, single-cell RNA sequencing and biochemistry to show that the balance between ephrin-B2 and its receptor EphB4 is critical for arterial specification, cell sorting and arteriovenous patterning. At the molecular level, elevated ephrin-B2 function after loss of EphB4 enhances signaling responses by the Notch pathway, VEGF and the transcription factor Dach1, which is influenced by endothelial shear stress. Our findings reveal how Eph-ephrin interactions integrate cell segregation and arteriovenous specification in the vasculature, which has potential relevance for human vascular malformations caused by EPHB4 mutations.

Exploring the potential of EphA2 receptor signaling pathway: a comprehensive review in cancer treatment.

The protein encoded by the ephrin type-A receptor 2 (EphA2) gene is a member of the ephrin receptor subfamily of the receptor tyrosine kinase family (RTKs). Eph receptors play a significant role in various biological processes, particularly cancer progression, development, and pathogenesis. They have been observed to regulate cancer cell growth, migration, invasion, tumor development, invasiveness, angiogenesis, and metastasis. To target EphA2 activity, various molecular, genetic, biochemical, and pharmacological strategies have been extensively tested in laboratory cultures and animal models. Notably, drugs, such as dasatinib, initially designed to target the kinase family, have demonstrated an additional capability to target EphA2 activity. Additionally, a novel monoclonal antibody named EA5 has emerged as a promising option to counteract the effects of EphA2 overexpression and restore tamoxifen sensitivity in EphA2-transfected MCF-7 cells during in vitro experiments. This antibody mimicked the binding of Ephrin A to EphA2. These methods offer potential avenues for inhibiting EphA2 activity, which could significantly decelerate breast cancer progression and restore sensitivity to certain drugs. This review article comprehensively covers EphA2's involvement in multiple malignancies, including ovarian, colorectal, breast, lung, glioma, and melanoma. Furthermore, we discuss the structure of EphA2, the Eph-Ephrin signaling pathway, various EphA2 inhibitors, and the mechanisms of EphA2 degradation. This article provides an extensive overview of EphA2's vital role in different types of cancers and outlines potential therapeutic approaches to target EphA2, shedding light on the underlying molecular mechanisms that make it an attractive target for cancer treatment.

The long non-coding RNA TAZ-AS202 promotes lung cancer progression via regulation of the E2F1 transcription factor and activation of Ephrin signaling

Long non-coding RNAs (lncRNAs) are transcripts without coding potential that are pervasively expressed from the genome and have been increasingly reported to play crucial roles in all aspects of cell biology. They have been also heavily implicated in cancer development and progression, with both oncogenic and tumor suppressor functions. In this work, we identified and characterized a novel lncRNA, TAZ-AS202, expressed from the TAZ genomic locus and exerting pro-oncogenic functions in non-small cell lung cancer. TAZ-AS202 expression is under the control of YAP/TAZ-containing transcriptional complexes. We demonstrated that TAZ-AS202 is overexpressed in lung cancer tissue, compared with surrounding lung epithelium. In lung cancer cell lines TAZ-AS202 promotes cell migration and cell invasion. TAZ-AS202 regulates the expression of a set of genes belonging to cancer-associated pathways, including WNT and EPH-Ephrin signaling. The molecular mechanism underlying TAZ-AS202 function does not involve change of TAZ expression or activity, but increases the protein level of the transcription factor E2F1, which in turn regulates the expression of a large set of target genes, including the EPHB2 receptor. Notably, the silencing of both E2F1 and EPHB2 recapitulates TAZ-AS202 silencing cellular phenotype, indicating that they are essential mediators of its activity. Overall, this work unveiled a new regulatory mechanism that, by increasing E2F1 protein, modifies the non-small cell lung cancer cells transcriptional program, leading to enhanced aggressiveness features. The TAZ-AS202/E2F1/EPHB2 axis may be the target for new therapeutic strategies.

Cannabigerol Activates Cytoskeletal Remodeling via Wnt/PCP in NSC-34: An In Vitro Transcriptional Study

Cannabigerol (CBG) is a non-psychoactive phytocannabinoid present in the Cannabis sativa L. plant. In our study, CBG at the concentration of 10 µM was used to treat NSC-34 motor neuron-like cells. The aim of the study was to evaluate the effects of CBG on NSC-34 cells, using next-generation sequencing (NGS) technology. Analysis showed the activation of the WNT/planar cell polarity (PCP) pathway and Ephrin-Eph signaling. The results revealed that CBG increases the expression of genes associated with the onset process of cytoskeletal remodeling and axon guidance.

A Novel Approach to Assess Podocyte Damage at Single Cell Level

Diseases affecting podocytes in kidney glomeruli are heterogeneous with respect to triggers, molecular mechanisms, and speed of progression. Frequently, healthy and diseased podocytes are present in parallel. Single cell technology is ideally suited to capture such heterogeneity including temporal resolution, which can be achieved by trajectory analysis. However, trajectories require large numbers of cells, which are difficult to obtain for rare cell populations such as podocytes. Here, we propose a novel analysis approach for single‐cell data in rare cell types, which relies on a podocyte disease score (PDS) to monitor the health status of individual cells. PDS is based on integrative meta‐analysis of published and our own bulk and single‐cell transcriptomic data of glomeruli and primary podocytes obtained from a multitude (n=37) of mouse models featuring podocyte damage. Among others, podocyte damage triggers integrated in the analysis include diabetes, ageing, mutations in slit diaphragm proteins, alterations of transcription factors, and adriamycin‐induced podocytopathy. In a rank‐based approach, a geneset was derived, whose expression quantitatively predicts podocyte damage irrespective of the trigger and at single‐cell resolution. Our PDS is robust, correlates with albuminuria in mice and humans, and allows for aligning disease states across independent and novel datasets. Applying PDS to published and novel glomerular single‐nucleus RNAseq data from mouse models recapitulated established molecular pathways relevant to the models, and identified common and model‐specific pathways related to podocyte damage. As such, Eph‐ephrin signaling was identified as a pathway that is universally altered upon podocyte damage at transcriptional level, and was confirmed by STED microscopy on the protein level in two mouse models. In conclusion, PDS is a valid and versatile method to assess podocyte damage at single‐cell level and allows to characterize cellular processes in podocytes in mice and humans.

Ephrin Ligands are Upregulated in the Saliva of SARS‐CoV‐2 Infected Patients

BackgroundPatients infected with SARS‐CoV‐2 may develop severe lung disease characterized by respiratory distress, systemic thrombosis, or death. Molecular mechanisms responsible for severe cases of COVID‐19 remain unclear. We investigated the role of the Eph family receptor‐interacting ligands (ephrin‐A1 and ephrin‐B2) in COVID‐19 patients. Ephrin binding to its tyrosine kinase Eph receptor plays important roles in injury and inflammation. Serum levels of ephrin‐A1 ligand correlates positively with COVID‐19 severity (Mendoza et al, CellPress 2021). Based on this recent report, and the emerging role of Ephrin‐Eph signaling in lung injury, we hypothesize that ephrin‐mediated cytokine signaling is upregulated in epithelial and immune cells assayed from saliva samples obtained from seriously ill COVID‐19 patients seeking emergency evaluation and treatment.MethodsSaliva samples were collected from patients (± SARS‐CoV‐2) in the University of Utah Emergency Department (ED) with IRB‐approved written consent. Saliva samples were incubated 1:1 in Streck Preservative (prior to EpCAM and CD45 labeling) or with 0.008% glutaraldehyde (following ELISA and multiplex immunoassays) in order to inactivate virus.ResultsFlow cytometric evaluation of saliva for EpCAM+ and CD45+ cells indicated that SARS‐CoV‐2 positive samples contain both epithelial and immune cell types ( see Fig 1 ). Uninfected saliva samples (control) primarily contained EpCAM+ cells. COVID‐19 samples (n=67 patients) expressed higher levels of type 1 interferon [IFNγ1 and IFNα2 (p< 0.001)] vs control (n=64 patients). Interferon induced protein [IP‐10] was also upregulated in COVID‐19 patients (p<0.001), as well as several cytokines in the interleukin family [IL‐1β, IL‐6, IL‐8 (p<0.001) and IL‐12p70 (p<0.05)]. TNF‐α did not differ significantly between the ± SARS‐CoV‐2 samples evaluated. Using a subset of the +SARS‐CoV‐2 samples (n=20) and uninfected controls obtained from the ED (n=20), we show that both ephrin‐A1 and ephrin‐B2 ligands are significantly elevated in +SARS‐CoV‐2 samples vs. ED control samples (p<0.001). Ephrin‐B2 levels correlated negatively with IL‐12p70 (Pearson Correlation =‐0.53, p=.01).ConclusionSaliva is low risk to obtain and is a biologically relevant sample that can be used to reliably measure changes in epithelial and immune cell responses of COVID‐19 patients. Specifically, our study showed that ephrin‐A1 and ephrin‐B2 ligands are upregulated in the saliva of COVID‐19 patients in a TNF‐α independent manner and that ephrin‐B2 correlates negatively with IL‐12p70 (a potent pro‐inflammatory cytokine). Future studies are aimed at establishing ephrin ligands as a biomarker for severity of lung injury and improving understanding the patho‐physiology of SARS‐CoV‐2 infection.

Roles of Eph-Ephrin Signaling in the Eye Lens Cataractogenesis, Biomechanics, and Homeostasis.

The eye lens is responsible for fine focusing of light onto the retina, and its function relies on tissue transparency and biomechanical properties. Recent studies have demonstrated the importance of Eph-ephrin signaling for the maintenance of life-long lens homeostasis. The binding of Eph receptor tyrosine kinases to ephrin ligands leads to a bidirectional signaling pathway that controls many cellular processes. In particular, dysfunction of the receptor EphA2 or the ligand ephrin-A5 lead to a variety of congenital and age-related cataracts, defined as any opacity in the lens, in human patients. In addition, a wealth of animal studies reveal the unique and overlapping functions of EphA2 and ephrin-A5 in lens cell shape, cell organization and patterning, and overall tissue optical and biomechanical properties. Significant differences in lens phenotypes of mouse models with disrupted EphA2 or ephrin-A5 signaling indicate that genetic modifiers likely affect cataract phenotypes and progression, suggesting a possible reason for the variability of human cataracts due to Eph-ephrin dysfunction. This review summarizes the roles of EphA2 and ephrin-A5 in the lens and suggests future avenues of study.

DNA Methylation Markers and Prediction Model for Depression and Their Contribution for Breast Cancer Risk.

BackgroundMajor depressive disorder (MDD) has become a leading cause of disability worldwide. However, the diagnosis of the disorder is dependent on clinical experience and inventory. At present, there are no reliable biomarkers to help with diagnosis and treatment. DNA methylation patterns may be a promising approach for elucidating the etiology of MDD and predicting patient susceptibility. Our overarching aim was to identify biomarkers based on DNA methylation, and then use it to propose a methylation prediction score for MDD, which we hope will help us evaluate the risk of breast cancer.MethodsMethylation data from 533 samples were extracted from the Gene Expression Omnibus (GEO) database, of which, 324 individuals were diagnosed with MDD. Statistical difference of DNA Methylation between Promoter and Other body region (SIMPO) score for each gene was calculated based on the DNA methylation data. Based on SIMPO scores, we selected the top genes that showed a correlation with MDD in random resampling, then proposed a methylation-derived Depression Index (mDI) by combining the SIMPO of the selected genes to predict MDD. A validation analysis was then performed using additional DNA methylation data from 194 samples extracted from the GEO database. Furthermore, we applied the mDI to construct a prediction model for the risk of breast cancer using stepwise regression and random forest methods.ResultsThe optimal mDI was derived from 426 genes, which included 245 positive and 181 negative correlations. It was constructed to predict MDD with high predictive power (AUC of 0.88) in the discovery dataset. In addition, we observed moderate power for mDI in the validation dataset with an OR of 1.79. Biological function assessment of the 426 genes showed that they were functionally enriched in Eph Ephrin signaling and beta-catenin Wnt signaling pathways. The mDI was then used to construct a predictive model for breast cancer that had an AUC ranging from 0.70 to 0.67.ConclusionOur results indicated that DNA methylation could help to explain the pathogenesis of MDD and assist with its diagnosis.

EphA2 Affects Development of the Eye Lens Nucleus and the Gradient of Refractive Index.

PurposeOur studies in mouse eye lenses demonstrate that ephrin-A5 and EphA2 are needed for normal epithelial cells and lens transparency. We sought to determine whether EphA2 and ephrin-A5 are important for lens morphometrics, nucleus formation, and refractive index.MethodsWe performed tissue morphometric measurements, electron microscopy, Western blots, and interferometric measurements using an X-ray synchrotron beam source to measure the gradient of refractive index (GRIN) to compare mouse lenses with genetic disruption of EphA2 or ephrin-A5.ResultsMorphometric analysis revealed that although there is no change in the overall lens volume, there is a change in lens shape in both EphA2−/− lenses and ephrin-A5−/− lenses. Surprisingly, EphA2−/− lenses had small and soft lens nuclei different from hard lens nuclei of control lenses. SEM images revealed changes in cell morphology of EphA2−/− fiber cells close to the center of the lens. Inner EphA2−/− lens fibers had more pronounced tongue-and-groove interdigitations and formed globular membrane morphology only in the deepest layers of the lens nucleus. We did not observe nuclear defects in ephrin-A5−/− lenses. There was an overall decrease in magnitude of refractive index across EphA2−/− lenses, which is most pronounced in the nucleus.ConclusionsThis work reveals that Eph-ephrin signaling plays a role in fiber cell maturation, nuclear compaction, and lens shape. Loss of EphA2 disrupts the nuclear compaction resulting in a small lens nucleus. Our data suggest that Eph-ephrin signaling may be required for fiber cell membrane reorganization and compaction and for establishing a normal GRIN.

Single-cell RNA landscape of the osteoimmunology microenvironment in periodontitis

Single-cell RNA sequencing (scRNA-seq) enables specific profiling of cell populations at single-cell resolution. The osteoimmunology microenvironment in the occurrence and development of periodontitis remains poorly understood at the single-cell level. In this study, we used single-cell transcriptomics to comprehensively reveal the complexities of the molecular components and differences with counterparts residing in periodontal tissues.Methods: We performed scRNA-seq to identify 51248 single cells from healthy controls (n=4), patients with severe chronic periodontitis (n=5), and patients with severe chronic periodontitis after initial periodontal therapy within 1 month (n=3). Uniform manifold approximation and projection (UMAP) were further conducted to explore the cellular composition of periodontal tissues. Pseudotime cell trajectory and RNA velocity analysis, combined with gene enrichment analysis were used to reveal the molecular pathways underlying cell fate decisions. CellPhoneDB were performed to identify ligand-receptor pairs among the major cell types in the osteoimmunology microenvironment of periodontal tissues.Results: A cell atlas of the osteoimmunology microenvironment in periodontal tissues was characterized and included ten major cell types, such as fibroblasts, monocytic cells, endothelial cells, and T and B cells. The enrichment of TNFRSF21+ fibroblasts with high expression of CXCL1, CXCL2, CXCL5, CXCL6, CXCL13, and IL24 was detected in patients with periodontitis compared to healthy individuals. The fractions of CD55+ mesenchymal stem cells (MSCs), APOE+ pre-osteoblasts (pre-OBs), and IBSP+ osteoblasts decreased significantly in response to initial periodontal therapy. In addition, CXCL12+ MSC-like pericytes could convert their identity into a pre-OB state during inflammatory responses even after initial periodontal therapy confirmed by single-cell trajectory. Moreover, we portrayed the distinct subtypes of monocytic cells and abundant endothelial cells significantly involved in the immune response. The heterogeneity of T and B cells in periodontal tissues was characterized. Finally, we mapped osteoblast/osteoclast differentiation mediators to their source cell populations by identifying ligand-receptor pairs and highlighted the effects of Ephrin-Eph signaling on bone regeneration after initial periodontal therapy.Conclusions: Our analyses uncovered striking spatiotemporal dynamics in gene expression, population composition, and cell-cell interactions during periodontitis progression. These findings provide insights into the cellular and molecular underpinning of periodontal bone regeneration.

EphA2 and Ephrin-A5 Guide Eye Lens Suture Alignment and Influence Whole Lens Resilience.

PurposeFine focusing of light by the eye lens onto the retina relies on the ability of the lens to change shape during the process of accommodation. Little is known about the cellular structures that regulate elasticity and resilience. We tested whether Eph–ephrin signaling is involved in lens biomechanical properties.MethodsWe used confocal microscopy and tissue mechanical testing to examine mouse lenses with genetic disruption of EphA2 or ephrin-A5.ResultsConfocal imaging revealed misalignment of the suture between each shell of newly added fiber cells in knockout lenses. Despite having disordered sutures, loss of EphA2 or ephrin-A5 did not affect lens stiffness. Surprisingly, knockout lenses were more resilient and recovered almost completely after load removal. Confocal microscopy and quantitative image analysis from live lenses before, during, and after compression revealed that knockout lenses had misaligned Y-sutures, leading to a change in force distribution during compression. Knockout lenses displayed decreased separation of fiber cell tips at the anterior suture at high loads and had more complete recovery after load removal, which leads to improved whole-lens resiliency.ConclusionsEphA2 and ephrin-A5 are needed for normal patterning of fiber cell tips and the formation of a well-aligned Y-suture with fiber tips stacked on top of previous generations of fiber cells. The misalignment of lens sutures leads to increased resilience after compression. The data suggest that alignment of the Y-suture may constrain the overall elasticity and resilience of the lens.

Eph-ephrin Signaling Affects Eye Lens Fiber Cell Intracellular Voltage and Membrane Conductance.

The avascular eye lens generates its own microcirculation that is required for maintaining lifelong lens transparency. The microcirculation relies on sodium ion flux, an extensive network of gap junction (GJ) plaques between lens fiber cells and transmembrane water channels. Disruption of connexin proteins, the building blocks of GJs, or aquaporins, which make up water and adhesion channels, lead to lens opacification or cataracts. Recent studies have revealed that disruption of Eph-ephrin signaling, in particular the receptor EphA2 and the ligand ephrin-A5, in humans and mice lead to congenital and age-related cataracts. We investigated whether changes in lens transparency in EphA2 or ephrin-A5 knockout (–/–) mice is related to changes in GJ coupling and lens fluid and ion homeostasis. Immunostaining revealed changes in connexin 50 (Cx50) subcellular localization in EphA2–/– peripheral lens fibers and alteration in aquaporin 0 (Aqp0) staining patterns in ephrin-A5–/– and EphA2–/– inner mature fiber cells. Surprisingly, there was no obvious change in GJ coupling in knockout lenses. However, there were changes in fiber cell membrane conductance and intracellular voltage in knockout lenses from 3-month-old mice. These knockout lenses displayed decreased conductance of mature fiber membranes and were hyperpolarized compared to control lenses. This is the first demonstration that the membrane conductance of lens fibers can be regulated. Together these data suggest that EphA2 may be needed for normal Cx50 localization to the cell membrane and that conductance of lens fiber cells requires normal Eph-ephrin signaling and water channel localization.

Interplay of Eph-Ephrin Signalling and Cadherin Function in Cell Segregation and Boundary Formation.

The segregation of distinct cell populations to form sharp boundaries is crucial for stabilising tissue organisation, for example during hindbrain segmentation in craniofacial development. Two types of mechanisms have been found to underlie cell segregation: differential adhesion mediated by cadherins, and Eph receptor and ephrin signalling at the heterotypic interface which regulates cell adhesion, cortical tension and repulsion. An interplay occurs between these mechanisms since cadherins have been found to contribute to Eph-ephrin-mediated cell segregation. This may reflect that Eph receptor activation acts through multiple pathways to decrease cadherin-mediated adhesion which can drive cell segregation. However, Eph receptors mainly drive cell segregation through increased heterotypic tension or repulsion. Cadherins contribute to cell segregation by antagonising homotypic tension within each cell population. This suppression of homotypic tension increases the difference with heterotypic tension triggered by Eph receptor activation, and it is this differential tension that drives cell segregation and border sharpening.

A three-dimensional spheroid-specific role for Wnt–β-catenin and Eph–ephrin signaling in nasopharyngeal carcinoma cells

One of the greatest unmet needs hindering the successful treatment of nasopharyngeal carcinomas (NPCs) is for representative physiological and cost-effective models. Although Epstein-Barr virus (EBV) infection is consistently present in NPCs, most studies have focused on EBV-negative NPCs. For the first time, we established and analyzed three-dimensional (3D) spheroid models of EBV-positive and EBV-negative NPC cells and compared these to classical two-dimensional (2D) cultures in various aspects of tumor phenotype and drug responses. Compared to 2D monolayers, the 3D spheroids showed significant increases in migration capacity, stemness characteristics, hypoxia and drug resistance. Co-culture with endothelial cells, which mimics essential interactions in the tumor microenvironment, effectively enhanced spheroid dissemination. Furthermore, RNA sequencing revealed significant changes at the transcriptional level in 3D spheroids compared to expression in 2D monolayers. In particular, we identified known (VEGF, AKT and mTOR) and novel (Wnt-β-catenin and Eph-ephrin) cell signaling pathways that are activated in NPC spheroids. Targeting these pathways in 3D spheroids using FDA-approved drugs was effective in monoculture and co-culture. These findings provide the first demonstration of the establishment of EBV-positive and EBV-negative NPC 3D spheroids with features that resemble advanced and metastatic NPCs. Furthermore, we show that NPC spheroids have potential use in identifying new drug targets.

Differential expression of microRNAs in the developing avian auditory hindbrain.

The avian auditory hindbrain is a longstanding model for studying neural circuit development. Information on gene regulatory network (GRN) components underlying this process, however, is scarce. Recently, the spatiotemporal expression of 12 microRNAs (miRNAs) was investigated in the mammalian auditory hindbrain. As a comparative study, we here investigated the spatiotemporal expression of the orthologous miRNAs during development of the chicken auditory hindbrain. All miRNAs were expressed both at E13, an immature stage, and P14, a mature stage of the auditory system. In most auditory nuclei, a homogeneous expression pattern was observed at both stages, like the mammalian system. An exception was the nucleus magnocellularis (NM). There, at E13, nine miRNAs showed a differential expression pattern along the cochleotopic axis with high expression at the rostromedial pole. One of them showed a gradient expression whereas eight showed a spatially selective expression at the rostral pole that reflected the different rhombomeric origins of this composite nucleus. The miRNA differential expression persisted in the NM to the mature stage, with the selective expression changed to linear gradients. Bioinformatics analysis predicted mRNA targets that are associated with neuronal developmental processes such as neurite and synapse organization, calcium and ephrin-Eph signaling, and neurotransmission. Overall, this first analysis of miRNAs in the chicken central auditory system reveals shared and strikingly distinct features between chicken and murine orthologues. The embryonic gradient expression of these GRN elements in the NM adds miRNA patterns to the list of cochleotopic and developmental gradients in the central auditory system.

Single-Cell RNA-Sequencing Reveals Lineage-Specific Regulatory Changes of Fibroblasts and Vascular Endothelial Cells in Keloids

Keloids are a benign dermal fibrotic disorder with features similar to malignant tumors. keloids remain a therapeutic challenge and lack medical therapies, which is partially due to the incomplete understanding of the pathogenesis mechanism. We performed single-cell RNA-sequencing of 28,064 cells from keloid skin tissue and adjacent relatively normal tissue. Unbiased clustering revealed substantial cellular heterogeneity of keloid tissue, which included 21 clusters assigned to 11 cell lineages. We observed significant expansion of fibroblast and vascular endothelial cell subpopulations in keloids, reflecting their strong association with keloid pathogenesis. Comparative analyses were performed to identify the dysregulated pathways, regulators and ligand-receptor interactions in keloid fibroblasts and vascular endothelial cells. Our results highlight the roles of TGFβ and Eph-ephrin signaling pathways in both the aberrant fibrogenesis and angiogenesis of keloids. Critical regulators probably involved in the fibrogenesis of keloid fibroblasts, such as TWIST1, FOXO3 and SMAD3, were identified. TWIST1 inhibitor harmine could significantly suppress the fibrogenesis of keloid fibroblasts. In addition, tumor-related pathways were activated in keloid fibroblasts and vascular endothelial cells, which may be responsible for the malignant features of keloids. Our study put insights into the pathogenesis of keloids and provides potential targets for medical therapies.

Tumor Spheroids of an Aggressive Form of Central Neurocytoma Have Transit-Amplifying Progenitor Characteristics with Enhanced EGFR and Tumor Stem Cell Signaling.

Central neurocytoma (CN) has been known as a benign neuronal tumor. In rare cases, CN undergoes malignant transformation to glioblastomas (GBM). Here we examined its cellular origin by characterizing differentiation potential and gene expression of CN-spheroids. First, we demonstrate that both CN tissue and cultured primary cells recapitulate the hierarchal cellular composition of subventricular zone (SVZ), which is comprised of neural stem cells (NSCs), transit amplifying progenitors (TAPs), and neuroblasts. We then derived spheroids from CN which displayed EGFR+/MASH+ TAP and BLBP+ radial glial cell (RGC) characteristic, and mitotic neurogenesis and gliogenesis by single spheroids were observed with cycling multipotential cells. CN-spheroids expressed increased levels of pluripotency and tumor stem cell genes such as KLF4 and TPD5L1, when compared to their differentiated cells and human NSCs. Importantly, Gene Set Enrichment Analysis showed that gene sets of GBM-Spheroids, EGFR Signaling, and Packaging of Telomere Ends are enriched in CN-spheroids in comparison with their differentiated cells. We speculate that CN tumor stem cells have TAP and RGC characteristics, and upregulation of EGFR signaling as well as downregulation of eph-ephrin signaling have critical roles in tumorigenesis of CN. And their ephemeral nature of TAPs destined to neuroblasts, might reflect benign nature of CN.

Aggressive and recurrent ovarian cancers upregulate ephrinA5, a non-canonical effector of EphA2 signaling duality

Erythropoietin producing hepatocellular (Eph) receptors and their membrane-bound ligands ephrins are variably expressed in epithelial cancers, with context-dependent implications to both tumor-promoting and -suppressive processes in ways that remain incompletely understood. Using ovarian cancer tissue microarrays and longitudinally collected patient cells, we show here that ephrinA5/EFNA5 is specifically overexpressed in the most aggressive high-grade serous carcinoma (HGSC) subtype, and increased in the HGSC cells upon disease progression. Among all the eight ephrin genes, high EFNA5 expression was most strongly associated with poor overall survival in HGSC patients from multiple independent datasets. In contrast, high EFNA3 predicted improved overall and progression-free survival in The Cancer Genome Atlas HGSC dataset, as expected for a canonical inducer of tumor-suppressive Eph receptor tyrosine kinase signaling. While depletion of either EFNA5 or the more extensively studied, canonically acting EFNA1 in HGSC cells increased the oncogenic EphA2-S897 phosphorylation, EFNA5 depletion left unaltered, or even increased the ligand-dependent EphA2-Y588 phosphorylation. Moreover, treatment with recombinant ephrinA5 led to limited EphA2 tyrosine phosphorylation, internalization and degradation compared to ephrinA1. Altogether, our results suggest a unique function for ephrinA5 in Eph-ephrin signaling and highlight the clinical potential of ephrinA5 as a cell surface biomarker in the most aggressive HGSCs.