Search Results (31)

Chordomas are rare malignant tumors of the bone, originating from remnants of notochordal cells. The transcription factor brachyury, encoded by TBXT, serves as a critical diagnostic marker and is essential for tumor growth. While brachyury’s role in regulating the cytoskeleton during embryogenesis and tumorigenesis is well understood, the reverse—whether cytoskeletal alterations can influence brachyury levels—remains unclear. Despite advances in understanding chordoma biology, there are currently no approved targeted therapies, underscoring the need for novel therapeutic approaches. Three chordoma cell lines were treated with cytoskeletal inhibitors, including the actin-targeting compounds Cucurbitacin B (CuB) and Latrunculin B (LatB). Morphological changes, TBXT expression, and cell viability were analyzed. The effects of CuB were examined over time and across concentrations, with cell viability assessed via apoptosis and cytotoxicity assays. Microarray gene expression profiling of ten chordoma cell lines was performed to explore CuB-mediated transcriptional changes. Rescue experiments using a TBXT open reading frame vector and co-treatments with autophagy and proteasome inhibitors were conducted to elucidate the mechanisms of brachyury depletion. Both CuB and LatB induced significant morphological changes, but only CuB caused near-complete depletion of brachyury. This effect was time- and concentration-dependent, correlating with reduced cell viability driven primarily by apoptosis. Microarray analysis revealed that CuB treatment upregulated protein refolding pathways and downregulated protein glycosylation. Notably, TBXT transcription was only slightly suppressed, indicating that brachyury depletion was largely post-transcriptional. Rescue experiments and co-treatments implicated dysregulated protein refolding and endoplasmic reticulum (ER) stress as key mechanisms underlying CuB-mediated brachyury loss. This study demonstrates that actin cytoskeleton disruption by CuB depletes brachyury in chordoma cells, primarily through dysregulated protein refolding and ER stress rather than transcriptional repression. These findings suggest that targeting actin cytoskeleton dynamics or protein unfolding pathways may provide novel therapeutic approaches for chordoma treatment. Full article

X-adrenoleukodystrophy (X-ALD) is a peroxisomal metabolic disorder caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). Similar mutations in ABCD1 may result in a spectrum of phenotypes in males with slow progressing adrenomyeloneuropathy (AMN) and fatal cerebral adrenoleukodystrophy (cALD) dominating most cases. Mouse models of X-ALD do not capture the phenotype differences and an appropriate model to investigate the mechanism of disease onset and progress remains a critical need. Here, we generated induced pluripotent stem cell (iPSC) lines from skin fibroblasts of two each of apparently healthy control, AMN, and cALD patients with non-integrating mRNA-based reprogramming. iPSC lines expanded normally and expressed pluripotency markers Oct4, SOX2, NANOG, SSEA, and TRA-1–60. Expression of markers SOX17, Brachyury, Desmin, OXT2, and beta tubulin III demonstrated the ability of the iPSCs to differentiate into all three germ layers. iPSC-derived lines from CTL, AMN, and cALD male patients were differentiated into astrocytes. Differentiated AMN and cALD astrocytes lacked ABCD1 expression and accumulated saturated very long chain fatty acids (VLCFAs), a hallmark of X-ALD, and demonstrated differential mitochondrial bioenergetics, cytokine gene expression, and differences in STAT3 and AMPK signaling between AMN and cALD astrocytes. These patient astrocytes provide disease-relevant tools to investigate the mechanism of differential neuroinflammatory response in X-ALD and will be valuable cell models for testing new therapeutics. Full article

Chordomas are a low-to-intermediate-grade slow-growing subtype of sarcoma, but show propensity to grow and invade locally with recurrence and metastasis in 10–40% of cases. We describe the first case of spontaneous regression of a solid tumor (histologically and immunohistochemically proven chordoma) after COVID-19. A female patient with clival chordoma underwent occipitocervical fixation prior to tumor resection. In the early post-operative stage following the arthrodesis procedure, she was diagnosed with COVID-19. Six months after COVID-19, she finally came back for endoscopic endonasal resection of the tumor and pre-operative MRI surprisingly showed 98.9% regression of the tumor volume. Tumor resection was performed, and both histopathological and immunohistochemistry confirmed diagnosis of chordoma with positive brachyury levels. She showed improvement of right hemiparesis and left-sided tongue palsy. The tumor was comprised of tumor-infiltrating inflammatory cells. CD3 and CD68 were positive, suggesting the presence of T-lymphocytes and macrophages. CD20 and CD56 were negative, suggesting the absence of B-lymphocytes and NK-cells. The authors believe that the onset of COVID-19 exacerbated the patient’s immune response and improved anti-tumor immunity. It was concluded that T-cells, which are involved in the COVID-19 immune response and were found infiltrating the tumor, acted as a critical pathway to this event. Further studies are encouraged in order to gain a better understanding of the SARS-CoV-2–chordoma interaction. Full article

Three-dimensional (3D) tissue culture models provide in vivo-like conditions for studying cell physiology. This study aimed to examine the efficiency of pyramidal microwell geometries in microfluidic devices on spheroid formation, cell growth, viability, and differentiation in mouse embryonic stem cells (mESCs). The static culture using the hanging drop (HD) method served as a control. The microfluidic chips were fabricated to have varying pyramidal tip angles, including 66°, 90°, and 106°. From flow simulations, when the tip angle increased, streamline distortion decreased, resulting in more uniform flow and a lower velocity gradient near the spheroids. These findings demonstrate the significant influence of microwell geometry on fluid dynamics. The 90° microwells provide optimal conditions, including uniform flow and reduced shear stress, while maintaining the ability for waste removal, resulting in superior spheroid growth compared to the HD method and other microwell designs. From the experiments, by Day 3, spheroids in the 90° microwells reached approximately 400 µm in diameter which was significantly larger than those in the 66° microwells, 106° microwells, and HD cultures. Brachyury gene expression in the 90° microwells was four times higher than the HD method, indicating enhanced mesodermal differentiation essential for cardiac differentiation. Immunofluorescence staining confirmed cardiomyocyte differentiation. In conclusion, microwell geometry significantly influences 3D cell culture outcomes. The pyramidal microwells with a 90° tip angle proved most effective in promoting spheroid growth and cardiac differentiation of mESC differentiation, providing insights for optimizing microfluidic systems in tissue engineering and regenerative medicine. Full article

The notochord is an axial structure required for the development of all chordate embryos, from sea squirts to humans. Over the course of more than half a billion years of chordate evolution, in addition to its structural function, the notochord has acquired increasingly relevant patterning roles for its surrounding tissues. This process has involved the co-option of signaling pathways and the acquisition of novel molecular mechanisms responsible for the precise timing and modalities of their deployment. To reconstruct this evolutionary route, we surveyed the expression of signaling molecules in the notochord of the tunicate Ciona, an experimentally amenable and informative chordate. We found that several genes encoding for candidate components of diverse signaling pathways are expressed during notochord development, and in some instances, display distinctive regionalized and/or lineage-specific patterns. We identified and deconstructed notochord enhancers associated with TGF-β and Ctgf, two evolutionarily conserved signaling genes that are expressed dishomogeneously in the Ciona notochord, and shed light on the cis-regulatory origins of their peculiar expression patterns. Full article

The significant deposition of tail fat in sheep has a profound impact on the economic benefits of animal husbandry. Furthermore, increasing the litter size is a crucial means of enhancing economic benefits. The BMPR1B and T/Brachyury genes are considered major functional genes that could affect sheep litter size and tail bone number, respectively. In this study, we employed direct sequencing to identify specific mutations of the BMPR1B gene in Gobi short tail sheep and carried out genotyping using MassARRAY technology for each variant of both the BMPR1B and T genes. Significant associations were demonstrated between the c.687G>A mutation of BMPR1B and the litter size in both the Gobi short tail sheep and Ujimqin sheep breeds. Meanwhile, the g.30058882_30058873GCAGATTAAAIndel mutation was significantly associated with the litter size in Gobi short tail sheep. These findings may provide valuable genetic markers for expanding sheep litter size. In addition, we also confirmed that the frequency of tail-bone-number-related T alleles was significantly higher in Gobi short tail sheep than in longer-tailed Ujimqin sheep. Full article

Chordomas, arising from notochord remnants, are rare neoplasms with aggressive growth patterns despite their histologically low-grade nature. This review explores their embryological origins, molecular markers like brachyury, and genetic alterations driving pathogenesis. Diagnosis relies on advanced imaging and biopsy confirmation due to overlapping features with chondrosarcoma. The WHO classification distinguishes conventional, dedifferentiated, and poorly differentiated chordomas, each with distinct prognostic implications. Recent genomic analyses uncovered recurrent mutations in PI3K signaling pathways and chromatin remodeling genes, informing prognostic models. Surgery remains the cornerstone of treatment, though adjuvant radiation complements surgical resection. Although chordomas are generally considered refractory to medical therapy, emerging targeted molecular strategies show potential promise in ongoing trials. This review aims to provide a concise yet comprehensive overview of chordomas, guiding clinicians in diagnosis, treatment, and prognostication for improved patient outcomes. Full article

Chordomas are very rare malignant neoplasms of the bone occurring almost exclusively along the spine. As the tumours are thought to arise from notochordal remnants, the vast majority of chordomas express the TBXT gene, resulting in detectable nuclear amounts of its gene product brachyury. This T-Box transcription factor is commonly recognised as being essential in chordoma cells, and limiting TBXT expression is thought to be the key factor in controlling this tumour. Although the tumour is rare, distinct molecular differences and vulnerabilities have been described with regard to its location and the progression status of the disease, rendering it mandatory for novel cell lines to reflect all relevant chordoma subtypes. Here, we describe a novel chordoma cell line arising from the pleural effusion of a disseminated, poorly differentiated chordoma. This cell line, U-CH22, represents a highly aggressive terminal chordoma and, therefore, fills a relevant gap within the panel of available cell culture models for this orphan disease. CDK7 and CDK9 inhibition was lately identified as being effective in reducing viability in four chordoma cell lines, most likely due to a reduction in brachyury levels. In this study, we determined the capability of the CDK7 inhibitor THZ1 and the CDK1/2/5/9 inhibitor dinaciclib to reduce TBXT expression at mRNA and protein levels in a broad range of nine cell lines that are models of primary, recurrent, and metastasised chordoma of the clivus and the sacrum. Full article

Pluripotent stem cells can be differentiated into all three germ-layers including ecto-, endo-, and mesoderm in vitro. However, the early identification and rapid characterization of each germ-layer in response to chemical and physical induction of differentiation is limited. This is a long-standing issue for rapid and high-throughput screening to determine lineage specification efficiency. Here, we present deep learning (DL) methodologies for predicting and classifying early mesoderm cells differentiated from embryoid bodies (EBs) based on cellular and nuclear morphologies. Using a transgenic murine embryonic stem cell (mESC) line, namely OGTR1, we validated the upregulation of mesodermal genes (Brachyury (T): DsRed) in cells derived from EBs for the deep learning model training. Cells were classified into mesodermal and non-mesodermal (representing endo- and ectoderm) classes using a convolutional neural network (CNN) model called InceptionV3 which achieved a very high classification accuracy of 97% for phase images and 90% for nuclei images. In addition, we also performed image segmentation using an Attention U-Net CNN and obtained a mean intersection over union of 61% and 69% for phase-contrast and nuclear images, respectively. This work highlights the potential of integrating cell culture, imaging technologies, and deep learning methodologies in identifying lineage specification, thus contributing to the advancements in regenerative medicine. Collectively, our trained deep learning models can predict the mesoderm cells with high accuracy based on cellular and nuclear morphologies. Full article

Mesenchymal-epithelial transition (MET) is a widely spread and evolutionarily conserved process across species during development. In Ciona embryogenesis, the notochord cells undergo the transition from the non-polarized mesenchymal state into the polarized endothelial-like state to initiate the lumen formation between adjacent cells. Based on previously screened MET-related transcription factors by ATAC-seq and Smart-Seq of notochord cells, Ciona robusta Snail (Ci-Snail) was selected for its high-level expression during this period. Our current knockout results demonstrated that Ci-Snail was required for notochord cell MET. Importantly, overexpression of the transcription factor Brachyury in notochord cells resulted in a similar phenotype with failure of lumen formation and MET. More interestingly, expression of Ci-Snail in the notochord cells at the late tailbud stage could partially rescue the MET defect caused by Brachyury-overexpression. These results indicated an inverse relationship between Ci-Snail and Brachyury during notochord cell MET, which was verified by RT-qPCR analysis. Moreover, the overexpression of Ci-Snail could significantly inhibit the transcription of Brachyury, and the CUT&Tag-qPCR analysis demonstrated that Ci-Snail is directly bound to the upstream region of Brachyury. In summary, we revealed that Ci-Snail promoted the notochord cell MET and was essential for lumen formation via transcriptionally repressing Brachyury. Full article

During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of Xenopus midgastrula embryos and full-transcriptome sequencing, we identified genes with altered expression in response to external mechanical stretching. Importantly, mechanically activated genes appeared to be expressed during normal development in the trunk, i.e., in the stretched region only. By contrast, genes inhibited by mechanical stretching were normally expressed in the anterior neuroectoderm, where mechanical stress is low. These results indicate that mechanical tensions may play the role of a long-range signaling factor that regulates patterning of the embryo, serving as a link coupling morphogenesis and cell differentiation. Full article

The vertebrate body comprises four distinct cell populations: cells derived from (1) ectoderm, (2) mesoderm, (3) endoderm, and (4) neural crest cells, often referred to as the fourth germ layer. Neural crest cells arise when the neural plate edges fuse to form a neural tube, which eventually develops into the brain and spinal cord. To date, the embryonic origin of exocrine glands located in the head and neck remains under debate. In this study, transgenic TRiCK mice were used to investigate the germinal origin of the salivary and lacrimal glands. TRiCK mice express fluorescent proteins under the regulatory control of Sox1, T/Brachyury, and Sox17 gene expressions. These genes are representative marker genes for neuroectoderm (Sox1), mesoderm (T), and endoderm (Sox17). Using this approach, the cellular lineages of the salivary and lacrimal glands were examined. We demonstrate that the salivary and lacrimal glands contain cells derived from all three germ layers. Notably, a subset of Sox1-driven fluorescent cells differentiated into epithelial cells, implying their neural crest origin. Also, these Sox1-driven fluorescent cells expressed high levels of stem cell markers. These cells were particularly pronounced in duct ligation and wound damage models, suggesting the involvement of neural crest-derived epithelial cells in regenerative processes following tissue injury. This study provides compelling evidence clarifying the germinal origin of exocrine glands and the contribution of neural crest-derived cells within the glandular epithelium to the regenerative response following tissue damage. Full article

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome that targets a highly selective subset of organs causing specific types of tumors. The biological basis for this principle of organ selectivity and tumor specificity is not well understood. VHL-associated hemangioblastomas share similar molecular and morphological features with embryonic blood and vascular precursor cells. Therefore, we suggest that VHL hemangioblastomas are derived from developmentally arrested hemangioblastic lineage keeping their potential of further differentiation. Due to these common features, it is of major interest to investigate whether VHL-associated tumors other than hemangioblastoma also share these pathways and molecular features. The expression of hemangioblast proteins has not yet been assessed in other VHL-related tumors. To gain a better understanding of VHL tumorigenesis, the expression of hemangioblastic proteins in different VHL-associated tumors was investigated. The expression of embryonic hemangioblast proteins Brachyury and TAL1 (T-cell acute lymphocytic leukemia protein 1) was assessed by immunohistochemistry staining on 75 VHL-related tumors of 51 patients: 47 hemangioblastomas, 13 clear cell renal cell carcinomas, 8 pheochromocytomas, 5 pancreatic neuroendocrine tumors, and 2 extra-adrenal paragangliomas. Brachyury and TAL1 expression was, respectively, observed in 26% and 93% of cerebellar hemangioblastomas, 55% and 95% of spinal hemangioblastomas, 23% and 92% of clear cell renal cell carcinomas, 38% and 88% of pheochromocytomas, 60% and 100% of pancreatic neuroendocrine tumors, and 50% and 100% of paragangliomas. We concluded that the expression of hemangioblast proteins in different VHL-associated tumors indicates a common embryological origin of these lesions. This may also explain the specific topographic distribution of VHL-associated tumors. Full article

Chordomas account for approximately 1–4% of all malignant bone tumors and 20% of primary tumors of the spinal column. It is a rare disease, with an incidence estimated to be approximately 1 per 1,000,000 people. The underlying causative mechanism of chordoma is unknown, which makes it challenging to treat. Chordomas have been linked to the T-box transcription factor T (TBXT) gene located on chromosome 6. The TBXT gene encodes a protein transcription factor TBXT, or brachyury homolog. Currently, there is no approved targeted therapy for chordoma. Here, we performed a small molecule screening to identify small chemical molecules and therapeutic targets for treating chordoma. We screened 3730 unique compounds and selected 50 potential hits. The top three hits were Ribociclib, Ingenol-3-angelate, and Duvelisib. Among the top 10 hits, we found a novel class of small molecules, including proteasomal inhibitors, as promising molecules that reduce the proliferation of human chordoma cells. Furthermore, we discovered that proteasomal subunits PSMB5 and PSMB8 are increased in human chordoma cell lines U-CH1 and U-CH2, confirming that the proteasome may serve as a molecular target whose specific inhibition may lead to better therapeutic strategies for chordoma. Full article

Chordomas are rare primary malignant tumours of notochordal origin usually arising along the axial skeleton with particular predilection of the skull base and sacrococcygeal region. Albeit usually slow-growing, chordomas can be aggressive mostly depending on their invasive behaviour and according to different histotypes and molecular alterations, including TBXT duplication and SMARCB1 homozygous deletion. Partial or complete PTEN deficiency has also been observed. PTEN is a negative regulator of the Akt/mTOR pathway and hyperactivation of Akt/mTOR in cells lacking PTEN expression contributes to cell proliferation and invasiveness. This pathway is targeted by mTOR inhibitors and the availability of in vitro models of chordoma cells will aid in further investigating this issue. However, isolation and maintenance of chordoma cell lines are challenging and PTEN-deleted chordoma cell lines are exceedingly rare. Hereby, we established and characterized a novel human PTEN-deleted chordoma cell line (CH3) from a primary skull base chordoma. Cells exhibited morphological and molecular features of the parent tumour, including PTEN loss and expression of Brachyury and EMA. Moreover, we investigated the activation of the mTOR pathway and cell response to mTOR inhibitors. CH3 cells were sensitive to Rapamycin treatment suggesting that mTOR inhibitors may represent a valuable option for patients suffering from PTEN-deleted chordomas. Full article