Brachyury Expression Research Articles

Effects of different concentrations of reversine to enhance conversion of dedifferentiated fat cells into mature cardiomyocytes

Background: There is an essential need for cardiomyocyte regeneration among patients with heart failure. Transplantation of dedifferentiated fat (DFAT) cells may lead to an improvement of cardiomyocyte regeneration among heart failure patients. We believe that DFAT cells are promising candidate cell sources for cardiac regeneration. However, the pathway underlying how DFAT cells of the adipose lineage differentiate into mature cardiomyocytes isn’t fully understood. Methods: We conducted an experimental laboratory study on isolated DFAT cells from adipose tissue of healthy adults. Then, we treated cells with different concentrations of reversine (10, 20 and 40 nM), and performed RNA extraction and cDNA synthesis. Next, we used a ceiling culture method based on the buoyancy properties of mature lipid-filled adipocytes. Stemness expression (Octamer-binding transcription factor 4 [Oct4], brachyury, Fetal liver kinase 1 [Flk-1]) was quantified by reverse transcription-quantitative (RT-q)PCR, while cardiomyocyte expression (Transcription factor GATA-4 [GATA4] and cardiac troponin T [cTnT]) was quantified by immunocytochemistry. Results: ANOVA with Tukey’s post-hoc found that 10 nM reversine increased greater Flk-1 expression compared to the control group (MD: 5.037 + 0.998; p < 0.001), but there were no significant changes among Oct4 (MD: 0.013 + 1.244; p = 0.99) and brachyury expression (MD: 0.157 + 0.084; p = 0.252). Kruskal-Wallis revealed that the expression of GATA4 (1.65 [0.41-1.98] to 0.015 [0.007-0.034]; p = 0.017) reduced significantly from day 7 until day 21 and cTnT (5.07 [6.62-8.91] to 8.22 [6.81-9.40]; p = 0.001) increased significantly from day 7 until day 21. Conclusions: Reversine could increase the expression of Flk-1, but it was unable to stimulate the expression of Oct4 and brachyury related to stem cell-ness. An optimal concentration of 10 nM reversine may have the greatest effect on enhancing the differentiation of DFAT cells into mature cardiomyocytes, as indicated by higher cTnT expression between cells.

Rare germline variants in PALB2 and BRCA2 in familial and sporadic chordoma.

Chordoma is a rare bone tumor with genetic risk factors largely unknown. We conducted a whole-exome sequencing (WES) analysis of germline DNA from 19 familial chordoma cases in five pedigrees and 137 sporadic chordoma patients and identified 17 rare germline variants in PALB2 and BRCA2, whose products play essential roles in homologous recombination (HR) and tumor suppression. One PALB2 variant showed disease cosegregation in a family with four affected people or obligate gene carrier. Chordoma cases had a significantly increased burden of rare variants in both genes when compared to population-based controls. Four of the six PALB2 variants identified from chordoma patients modestly affected HR function and three of the 11 BRCA2 variants caused loss of function in experimental assays. These results, together with previous reports of abnormal morphology and Brachyury expression of the notochord in Palb2 knockout mouse embryos and genomic signatures associated with HR defect and HR gene mutations in advanced chordomas, suggest that germline mutations in PALB2 and BRCA2 may increase chordoma susceptibility. Our data shed light on the etiology of chordoma and support the previous finding that PARP-1 inhibitors may be a potential therapy for some chordoma patients.

Epigenetic regulation mechanism of folic acid deficiency inhibiting Brachyury expression and its effect on differentiation of embryoid bodies

Epigenetic regulation mechanism of folic acid deficiency inhibiting Brachyury expression and its effect on differentiation of embryoid bodies

Epithelial-Mesenchymal Transition Marker Brachyury Is Not Expressed in Spindle Cell Carcinoma of the Head & Neck

Introduction. Epithelial-mesenchymal transition (EMT) is a complex biologic process by which epithelial cells lose their cytomorphologic characteristics and assume the phenotype of mesenchymal cells. EMT is essential to normal human embryogenesis, and also occurs in wound healing, tissue repair and cancer progression. Acquisition of a mesenchymal phenotype via EMT enhances the migratory capacity of carcinoma cells, increasing their metastatic capability and worsening prognosis for patients. One emerging marker of EMT is brachyury, a T-box transcription factor protein. Recent studies have demonstrated upregulation of brachyury in several carcinoma types, includ- ing squamous cell carcinoma (SCC). Spindle cell carcinoma (SpCC) is a variant of SCC in which cancer cells have a predominantly spindled phenotype. Whether brachyury is expressed in SpCC and contributes to this pheno- typic change is not clear. Objective.To examine brachyury expression by immunohistochemistry in SpCC of the head & neck, hypothesizing that brachyury expression is increased in SpCC, as compared with other forms of SCC. Methods.Immunohistochemical staining for brachyury was performed on 20 head and neck carcinoma cases per group in each of four groups: SpCC; moderately- to well-differentiated SCC; moderately- to poorly-differentiated SCC; and verrucous carcinoma (VC). Uninflamed fibroma was the negative control; and human chordoma, along with a 14.5-day-old mouse embryo, were positive controls. Evaluation of brachyury reactivity was completed by light microscopy. Results.Brachyury expression was not seen in any of the experimental groups. Nuclear reactiv- ity was present in both positive controls, including within chordoma cells, and within the nucleus pulposus of the mouse embryo. Conclusions.Brachyury does not appear to be involved in the cytomorphologic changes present in SpCC, nor is it expressed in any head & neck SCC variants. Our results are contrary to those found in previous reports. This result supports the hypothesis that an alternate mechanism contributes to the phenotype of SpCC.

Effect of hyperthermia and proton beam radiation as a novel approach in chordoma cells death and its clinical implication to treat chordoma

Purpose Chordoma is a locally aggressive tumor that most commonly affects the base of the skull/clivus, cervical, and sacral spine. Conventional radiotherapy (RT), cannot be safely increased further to improve disease control due to the risk of toxicity to the surrounding critical structures. Tumor-targeted hyperthermia (HT) combined with Proton Beam Radiation Therapy (PBRT) is known to act as a potent radiosensitizer in cancer control. In this study, we investigated whether PBRT efficacy for chordoma can be enhanced in combination with HT as a radiosensitizer. Material and methods Human chordoma cell lines, U-CH2 and Mug-chor1 were treated in vitro with HT followed by PBRT with variable doses. The colony-forming assay was performed, and dose-response was characterized by linear-quadratic model fits. HSP-70 and Brachyury (TBXT) biomarkers for chordoma aggression levels were quantified by western blot analysis. Gene microarray analysis was performed by U133 Arrays. Pathway Analysis was also performed using IPA bioinformatic software. Results Our findings in both U-CH2 and Mug–Chor1 cell lines demonstrate that hyperthermia followed by PBRT has an enhanced cell killing effect when compared with PBRT-alone (p < .01). Western blot analysis showed HT decreased the expression of Brachyury protein (p < .05), which is considered a biomarker for chordoma tumor aggression. HT with PBRT also exhibited an RT-dose-dependent decrease of Brachyury expression (p < .05). We also observed enhanced HSP-70 expression due to HT, RT, and HT + RT combined in both cell lines. Interestingly, genomic data showed 344 genes expressed by the treatment of HT + RT compared to HT (68 genes) or RT (112 genes) as individual treatment. We also identified activation of death receptor and apoptotic pathway in HT + RT treated cells. Conclusion We found that Hyperthermia (HT) combined with Proton Beam Radiation (PBRT) could significantly increase chordoma cell death by activating the death receptor pathway and apoptosis which has the promise to treat metastatic chordoma.

A comparative study of mesenchymal stem cell transplantation and NTG-101 molecular therapy to treat degenerative disc disease

Cellular replacement therapy using mesenchymal stem cells (MSCs) and/or the delivery of growth factors are at the forefront of minimally invasive biological treatment options for Degenerative Disc Disease (DDD). In this study, we compared the therapeutic potential of a novel drug candidate, NTG-101 to MSCs, including rat cartilage derived stem cells (rCDSCs), bone marrow stem cells (rBMSCs) and human Umbilical Cord Derived Mesenchymal Stem Cells (hUCMSCs) for the treatment of DDD. We induced DDD using a validated image-guided needle puncture injury in rat-tail IVDs. Ten weeks post-injury, animals were randomized and injected with MSCs, NTG-101 or vehicle. At the end of the study, histological analysis of the IVD-Nucleus Pulposus (NPs) injected with NTG-101 or rCDSCs showed a healthy or mild degenerative phenotype in comparison to vehicle controls. Immunohistochemical analysis revealed strong expression of aggrecan, collagen 2, brachyury and Oct4 in IVD-NPs injected with NTG-101. Our results also demonstrated suppression of inflammation induced p38 and NFκB resulting in inhibition of catabolic genes, but activation of Smad-2/3, Erk-1/2 and Akt-dependent signaling inducing anabolic genes in IVD-NP on treatment with NTG-101. In conclusion, a single injection of NTG-101 into the degenerative disc demonstrated superior benefits compared to stem cell transplantation.

Clinical impact of brachyury expression in Ewing sarcoma patients

PurposeThe T-box transcription factor brachyury has been demonstrated as a prognostic factor in a variety of cancer types and considered a novel oncotarget in solid tumors. Brachyury acts as a regulator of the epithelial-mesenchymal transition (EMT) process, leading to more aggressive behavior and poorer prognosis. However, recent literature evidence suggests a tumor suppressor role in other neoplasms. In the present study, we aimed to study brachyury expression and its prognostic impact in Ewing sarcoma, an aggressive neoplasm of young individuals. MethodsWe analyzed the expression of brachyury by immunohistochemistry in a series of 96 Ewing sarcomas in a tissue microarray and investigated the association of the protein expression with the clinical parameters and overall survival. ResultsMore than half of the cases (51%, n ​= ​49) depicted positive nuclear brachyury expression, while a lack of expression was observed in 49% (n ​= ​47) of cases. Nuclear brachyury staining was significantly associated with non-white ethnicity (p ​= ​0.04) and axial localization (p ​= ​0.025). Importantly, lack of brachyury expression was significantly associated with lower overall survival in multivariate analyses (hazard ratio – HR: 2.227, p ​= ​0.008). ConclusionsOur findings indicate, that brachyury is an independent prognostic biomarker in Ewing sarcoma, which might suggest a tumor suppressor role and which yet to be fully elucidated.

Abstract LB216: Targeted brachyury degradation disrupts a highly specific autoregulatory program controlling chordoma cell identity

Abstract Chordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape, autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Both transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition which globally downregulates transcription leading to cell death, brachyury degradation is much more selective, inducing senescence and sensitizing cells to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop. Citation Format: Hadley E. Sheppard, Alessandra Dall'Agnese, Woojun D. Park, Hamza Shamim, Julien Dubrulle, Hannah L. Johnson, Fabio Stossi, Patricia Cogswell, Josh Sommer, Joan Levy, Tanaz Sharifnia, Mathias J. Wawer, Paul A. Clemons, Behnam Nabet, Nathanael S. Gray, Stuart L. Schreiber, Paul Workman, Richard A. Young, Charles Y. Lin. Targeted brachyury degradation disrupts a highly specific autoregulatory program controlling chordoma cell identity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB216.

Abstract 630: Response to nivolumab in a pediatric chordoma with overexpression of brachyury

Abstract Chordomas are neoplasms derived from notochord cells that typically form in the spine or skull base, and affect both adults and children. Poorly differentiated chordomas, associated with particularly poor outcomes, occur predominantly in children and are characterized by expression of the transcription factor brachyury and loss of INI1/SMARCB1. The use of immune checkpoint inhibitors in pediatric cancer is of growing interest, with reported anti-tumour activity in pediatric Hodgkin lymphoma and gliomas with mismatch repair deficiency. However, responses to immune checkpoint inhibitors are infrequent and unpredictable in pediatric solid tumours, and the molecular markers that can distinguish the seemingly indiscriminate response remain to be defined. Understanding which pediatric patient populations may benefit from immune checkpoint inhibitor therapies is essential. Here we describe the molecular profiles of two pediatric poorly differentiated chordomas using whole-genome, transcriptome and whole-genome bisulfite sequencing as part of the Personalized Oncogenomics program at BC Cancer (NCT02155621) that studies the impact of embedding comprehensive genomic profiling into the clinical care of advanced cancer patients. Genomic profiling and multiplex IHC revealed a high level of CD8+ T cells in both chordoma samples, despite low mutation burden. DNA methylation-based clustering revealed similarities with SMARCB1-deficient rhabdoid tumour subtypes that are have been characterized by increased levels of immune cell infiltration. A dominant tumour-associated T cell receptor clone was identified in one patient sample, which was demonstrated to have affinity for reconstituted brachyury-MHC complexes, indicating a potential anti-tumour immune response directed against brachyury-derived antigens. As these collective observations pointed to a potential benefit of immune targeted therapy, the patient was treated with nivolumab and subsequently achieved a partial radiographic response by RANO criteria. These observations provide support for the benefit of immune checkpoint inhibitors in poorly differentiated chordomas with high levels of infiltrating CD8+ T cells. Citation Format: Laura Williamson, Craig Rive, Daniela Di Francesco, Emma Titmuss, Elizabeth Chun, Scott Brown, Katy Milne, Erin Pleasance, Anna F. Lee, Stephen Yip, David Dix, Daniel Renouf, Robert Holt, Brad H. Nelson, Martin Hirst, Steven Jones, Shahrad Rassekh, Rebecca Deyell, Janessa Laskin, Macro A. Marra. Response to nivolumab in a pediatric chordoma with overexpression of brachyury [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 630.

Effect of BMP-Wnt-Nodal signal on stem cell differentiation.

The generation of germ cells from embryonic stem cells in vitro has current historical significance. Western blot, qPCR, immunofluorescence and flow cytometry assays were used to investigate the differences in expression levels of totipotency and specific markers for Wnt regulation and the related signalling pathways during primordial germ cell-like cell (PGCLC) induction and differentiation. During PGCLC induction, activation of WNT3a increased the expression of NANOG, SOX2 and OCT4, but Mvh, DAZL, Blimp1, TFAP2C, Gata4, SOX17, EOMES, Brachyury and PRDM1 expression levels were significantly reduced. Inhibition of the WNT signal demonstrated the opposite effect. Similarly, inhibitors of BMP and the Nodal/Activin signal were used to determine the effect of signal pathways on differentiation. CER1 affected the Wnt signal and differentiation, but the inhibitor SB only regulated differentiation. BMP-WNT-NODAL were mainly responsible for regulating differentiation. Our results provide a reliable theoretical basis and feasibility for further clinical medical research.

A Benchmark Side-by-Side Comparison of Two Well-Established Protocols for in vitro Hematopoietic Differentiation From Human Pluripotent Stem Cells.

The generation of transplantable hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) remains challenging. Current differentiation protocols from hPSCs generate mostly hematopoietic progenitors of the primitive HSC-independent program, and it remains unclear what is the best combination of cytokines and hematopoietic growth factors (HGFs) for obtaining functional hematopoietic cells in vitro. Here, we have used the AND1 and H9 hESC lines and the H9:dual-reporter RUNX1C-GFP-SOX17-Cherry to compare the hematopoietic differentiation in vitro based on the treatment of embryoid bodies (EBs) with the ventral mesoderm inducer BMP4 plus HGFs in the absence (protocol 1) or presence (protocol 2) of stage-specific activation of Wnt/β-catenin and inhibition of Activin/Nodal. Despite a slight trend in favor of protocol 1, no statistically significant differences were observed between protocols at any time point analyzed throughout EB development regarding the frequency of hemogenic endothelial (HE) precursors; CD43+ CD45−, CD45+, and CD45 + CD34 + hematopoietic derivatives; or the output of clonogenic progenitors. Similarly, the kinetics of emergence throughout EB development of both SOX17 + HE and RUNX1C + definitive hematopoiesis was very similar for both protocols. The expression of the early master mesendodermal transcription factors Brachyury, MIXL1, and KDR revealed similar gene expression kinetics prior to the emergence of RUNX1C + definitive hematopoiesis for both protocols. Collectively, the simpler protocol 1 is, at least, as efficient as protocol 2, suggesting that supplementation with additional morphogens/HGFs and modulation of Activin/Nodal and Wnt/β-catenin pathways seem dispensable for in vitro hematopoietic differentiation of hPSCs.

Patterns of brachyury expression in chordomas

IntroductionChordomas are rare malignant midline tumors, presumed to arise from notochordal remnants. This was further suggested by the discovery of the brachyury in chordomas pathogenesis. Its immunohistochemical expression has become the principal adjunct in the diagnosis of chordomas. However, studies about brachyury expression in chordomas are not fully comparable, mainly because they use different primary antibodies. Thus, the aim of this study is to investigate the expression of brachyury expression in a series of chordomas in conjunction to clinicopathological characteristics and to review the relevant literature providing all the details needed in the immunohistochemical study of brachyury. Materials and methodsThis is a retrospective study of 62 chordomas, diagnosed over a 22-year period. No dedifferentiated or poorly differentiated cases were included. A monoclonal primary antibody (clone A-4) was used and brachyury expression was evaluated by the H-score. Clinicopathological parameters studied were age, sex, tumor localization, decalcification status and tissue age. Fetal notochords were used for comparison. ResultsMean H-score of nuclear brachyury expression was 129.8. The tissue age significantly influenced brachyury expression, the older samples expressing less brachyury. Decalcification demonstrated a trend to weaken brachyury expression. Clinical characteristics were not correlated with the patterns of brachyury expression. Notochords were negative. Literature review reveals several polyclonal antibodies used and a positivity of 75%–100% in chordomas with even more variable results in notochords. ConclusionIn chordomas, as in other tumor types, an uniformization of studies about brachyury expression is needed, by considering the clone used, and the decalcification and the age of the sample, given the growing importance of brachyury in diagnosis and therapeutic steps.

Machine learning based tissue analysis reveals Brachyury has a diagnosis value in breast cancer.

Background: The aim of the present study was to confirm the role of Brachyury in breast cancer and to verify whether four types of machine learning models can use Brachyury expression to predict the survival of patients.Methods: We conducted a retrospective review of the medical records to obtain patient information, and made the patient’s paraffin tissue into tissue chips for staining analysis. We selected 303 patients for research and implemented four machine learning algorithms, including multivariate logistic regression model, decision tree, artificial neural network and random forest, and compared the results of these models with each other. Area under the receiver operating characteristic (ROC) curve (AUC) was used to compare the results.Results: The chi-square test results of relevant data suggested that the expression of Brachyury protein in cancer tissues was significantly higher than that in paracancerous tissues (P=0.0335); patients with breast cancer with high Brachyury expression had a worse overall survival (OS) compared with patients with low Brachyury expression. We also found that Brachyury expression was associated with ER expression (P=0.0489). Subsequently, we used four machine learning models to verify the relationship between Brachyury expression and the survival of patients with breast cancer. The results showed that the decision tree model had the best performance (AUC = 0.781).Conclusions: Brachyury is highly expressed in breast cancer and indicates that patients had a poor prognosis. Compared with conventional statistical methods, decision tree model shows superior performance in predicting the survival status of patients with breast cancer.

Brachyury expression in intracranial SMARCB1-deficient tumors: important points for distinguishing poorly differentiated chordoma from atypical teratoid/rhabdoid tumor

Loss of SMARCB1 protein expression has recently been identified in a variety of tumor types such as poorly differentiated chordoma (PCh) and malignant rhabdoid tumor (MRT) including atypical teratoid/rhabdoid tumor (AT/RT). PCh is characterized by poorly differentiated epithelioid tumor cells, sheet arrangement, and coexpression of nonepithelial and epithelial markers. Rhabdoid cells are sometimes present. Therefore, the differentiation of these tumors is often difficult. Brachyury is a transcription factor within the T-box family typically expressed in notochord tissue and chordomas. Some studies have reported high specificity and sensitivity of brachyury expression in chordomas. In the present study, we analyzed immunohistochemical brachyury expression in SMARCB1-deficient tumors and discuss important clinicopathological and diagnostic points, especially in cases of intracranial SMARCB1-deficient tumors with brachyury expression. Brachyury and cytokeratin immunoexpression status was examined in 42 formalin-fixed paraffin-embedded SMARCB1-deficient tumor specimens (PCh, 6 cases; extra-central nervous system [CNS] MRT, 26 cases; AT/RT, 10 cases) and 25 cases of conventional chordoma(CCh). All cases of PCh and CCh showed diffuse immunopositivities for cytokeratin 8, pan-cytokeratin, and brachyury. Brachyury immunoexpression was present in 2 extra-CNS MRT (8%) and 5 AT/RT (50%) cases, but immunopositivity was focal not diffuse. Indeed, in almost all cases of AT/RT (cytokeratin 8, 7/10 cases; pan-cytokeratin, 7/10 cases) and extra-CNS MRT (cytokeratin 8, 23/26 cases; pan-cytokeratin, 25/26 cases), fewer than 50% of cells showed immunoreactivity. Although the histological and clinical features of PCh resemble those of AT/RT, semiquantitative evaluations of the degree of brachyury and cytokeratin immunoexpressivity may help to distinguish PCh from AT/RT.

Clinicopathological features of notochordal tumors: a study of 48 cases

Objective: To investigate the clinicopathological features, diagnosis and differential diagnosis of notochordal tumors. Methods: The clinical, radiologic and pathologic data of 48 notochordal tumors were collected from 2008 to 2019 at Shanghai Jiaotong University Sixth People's Hospital. Expression of cytokertin, S-100 protein, vimentin, brachyury and INI1 was detected by immunohistochemistry. The pathologic differential diagnoses and biologic behavior of various types of notochordal tumors were analyzed using the new standard in the 5th edition of WHO tumor classification. Results: Four cases of benign notochordal cell tumor were confined to vertebral body. Histopathologically, they lacked lobular architecture and extracellular myxoid matrix. The tumor cells were vacuolated and had centrally or peripherally located round to oval nuclei, with small nucleoli, without atypia, mimicking mature adipocytes. No mitotic figures were seen. Two cases of poorly differentiated chordoma, from patients aged 12 years and 21 years respectively, were located in cervical vertebra, and were composed of cohesive sheets or nests of epithelioid cells, with focal rhabdoid morphology. There was relatively abundant eosinophilic cytoplasm and scattered cytoplasmic vacuoles. The moderately pleomorphic nuclei were round to ovoid with vesicular chromatin and mitotic figures could be seen. Extracellular myxoid stroma was observed focally. Forty cases of conventional chordoma and two cases of extra-axis chordoma had similar histologic features. All 48 cases expressed cytokeretin, 45 cases expressed brachyury, and two poorly differentiated tumors showed loss of INI1/SMARCB1. Conclusions: There are four subtypes of chordomas: conventional, dedifferentiated, poorly differentiated and extra-axis. Chondroid chordoma is no longer thought to be a distinct entity. Each type has its unique clinicopathological characteristics. Brachyury is highly specific and sensitive for the diagnosis of various notochordal tumors. Poorly differentiated chordoma shows distinct clinicopathological features, including young age and loss of immunohistochemical expression of INI1/SMARCB1, and its diagnosis requires the combined detection of brachyury and INI1/SMARCB1.

45 Embryonic disc development invitro in ovine embryos

Embryonic mortality during the second week of pregnancy has an important economic impact on farming. At this time, the embryo undergoes critical developmental events that cannot be recapitulated invitro, limiting our understanding of these pregnancy losses. After the blastocyst stage, the hypoblast migrates to cover the inner surface of the embryo and the epiblast forms a flat embryonic disc (ED) that initiates gastrulation. The aim of this study was to develop an invitro culture system to support sheep embryo development after the blastocyst stage. Day 6/7 invitro-produced blastocysts were cultured over agarose gels to prevent attachment and allocated to different media: synthetic oviductal fluid with 10% fetal bovine serum (SOF-FBS, n=52), an invitro culture medium (hIVC, n=35) supporting ED formation in human embryos (Deglincerti et al. 2016 Nature 533, 251-254; https://doi.org/10.1038/nature17948), and chemically defined N2B27 medium (n=38) supporting ED formation in bovine embryos (Ramos-Ibeas et al. 2020 Reproduction 160, 579-589, https://doi.org/10.1530/REP-20-0243). At Day 14, survival and embryo area were recorded, the abundance of transcripts encoding interferon Tau (TP1) and metabolic enzymes was analysed by RT-qPCR, and the development of epiblast and hypoblast was assessed by immunostaining for SOX2 and SOX17. Embryo survival and size and the percentage of embryos achieving complete hypoblast migration were significantly reduced in SOF-FBS (Chi-squared test and one-way ANOVA; P&amp;lt;0.05). Only N2B27 medium supported epiblast survival in 11/28 embryos. TP1 expression increased at Day 14 in all culture conditions and metabolism-related genes revealed a shift from anaerobic glycolysis to oxidative phosphorylation after culture in hIVC and N2B27. Next, to promote epiblast development, we allocated blastocysts to N2B27 medium supplemented with activin A (n=45), rho-associated protein kinase (ROCK) inhibitor (ROCKi, n=42), or insulin growth factor 1 (IGF1, n=29). IGF1 reduced significantly the percentage of embryos showing an ED-like structure, whereas activin A supplementation significantly increased epiblast survival (Chi-squared test; P&amp;lt;0.05) and SOX2+ cell number was higher in embryos cultured with ROCK inhibitor. When we combined activin A and ROCKi supplementation (N2B27+A+R, n=151), SOX2+ cell number and the percentage of embryos showing an ED-like structure increased significantly (165.1±53.1 and 31/35 embryos with SOX2+ epiblast cells; ∼89%) compared with N2B27 alone (49.4±12.7 and 5/11; ∼45%) (one-way ANOVA and Chi-squared test; P&amp;lt;0.05). Moreover, 18/31 (∼58%) ED-like structures developed in N2B27+A+R lost the Rauber’s layer (polar trophoblast), and BRACHYURY expression, denoting the onset of gastrulation, was observed in 3/14. In conclusion, we have developed a culture system that supports complete hypoblast migration and ED development invitro, which represents a valuable tool to explore early embryo mortality in livestock species without the need for experimental animals.

Targeted brachyury degradation disrupts a highly specific autoregulatory program controlling chordoma cell identity

SummaryChordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition, leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape and autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition, which globally downregulates transcription, leading to cell death, brachyury degradation is much more selective, inducing senescence and sensitizing cells to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop.

RARE-58. CONGENITAL METASTATIC CHORDOMA OF THE CLIVUS

Chordomas are rare midline axial skeletal neoplasms that typically present in adults. They are infrequent in childhood with typical localization in the spheno-occipital skull base. They are derived from remnants of the embryonic notochord. We present the case of 4 months old girl, who was born with „blueberry muffin” syndrome and was first negatively diagnosed for neuroblastoma and leukemia (two negative skin biopsies were performed) was admitted with axial laxity. In imaging testes there was a tumor of the scull base, metastases in the lungs and kidneys (that were not seen at previous assessments) and a small lesion in the heart. The third biopsy of skin lesion was performed and pathological examination revealed a neoplasm composed of cords, clusters, and chains of multivacuolated cells embedded within a myxoid matrix and separated by fibrous septa. No atypical and dedifferentiated features were present. Mitotic activity was not observed. Neoplastic cells showed the typical cytoplasmic immunostaining for EMA, S100 and cytokeratin AE1/AE3, strong nuclear brachyury expression, and retention of nuclear INI-1 expression. The diagnosis of chordoma was established. Neoplastic tissue and blood samples were obtained for molecular analysis using next generation sequencing, including germline mutations assessment (are ongoing). Chemotherapy as for soft tissue sarcomas was undertaken. Currently a patient is on treatment with improvement of neurological status.

Poorly Differentiated Chordoma with Rhabdoid Features

Abstract Casestudy: Chordoma is an uncommon primary malignant tumor of the bone showing notochordal differentiation. The current World Health Organization classifies chordoma into three subtypes: conventional, chondroid, and dedifferentiated. Poorly differentiated chordoma (PDC) is a newly described variant of chordoma, with its own distinct features in terms of age distribution (common in pediatric and young adults), histopathology (lack of physaliphorous cells and chondromyxoid matrix), immunohistochemical and molecular characteristics (loss of SMARCB1/INI1 expression) and clinical outcome (poor prognosis with rapid onset of local recurrence and metastasis). Herein, we report the case of a 43 year old man with history of fall and injury of tailbone 2 years prior, who presented with pain and swelling at the sacrcoccygeal region and intermittent numbness in the legs. MRI of the pelvis showed a 9.8 cm lobulated mass centered on the sacrum with osseous destruction of S5 through coccyx, with intralesional hemorrhage, and mild heterogeneous enhancement. Biopsy of the sacrococcygeal mass demonstrated a malignant neoplasm composed of lobules of epithelioid cells with abundant eosinophilic cytoplasm showing focal vacuolization, and mild to moderate nuclear pleomorphism with an average 3 mitoses per high power field (400x). Immunohistochemically, the neoplastic cells were positive for pancytokeratin, CAM 5.2, vimentin and brachyury. They had loss of SMARCB1 (INI1) and were negative for S100, CK7, CK20, desmin and actin. The location, imaging studies, morphologic features and immunohistochemical profile were consistent with poorly differentiated chordoma with rhabdoid features. With its specific clinical, morphologic, immunophenotypical, and molecular features, PDC may be recognized as a chordoma subtype in a future WHO classification. According to current knowledge, the diagnosis of PDC should be restricted to cases with brachyury expression (specific for notochord tumors) and INI1 protein loss, especially in tumors of pediatric and young patients, regardless of cellular characteristics.

Delayed notochordal cell disappearance through integrin α5β1 mechanotransduction during ex-vivo dynamic loading-induced intervertebral disc degeneration.

The loss of nucleus pulposus (NP) notochordal cells is one of the key initial hallmarks of age-related intervertebral disc degeneration. Although the transmembrane mechanoreceptor integrin α5β1 is important in the process of disc degeneration, the relationship between integrin α5β1 and notochordal cell disappearance remains unclear. The purpose of this study was to elucidate the roleof integrin α5β1 in the homeostasis of notochordal cells using an ex-vivo dynamic loading culture system that we developed. Rat tail functional spinal units (n = 80 from 40 rats) were cultured under unloading or 1.3-MPa, 1.0-Hz dynamic compressive loading for 48 or 144 h with or without an integrin α5β1 inhibitor. Disc histomorphology, cell viability, apoptosis, senescence, and phenotypic expression were investigated. Consequently, histological degenerative disc changes with decreased cell viability and increased cell apoptosis and senescence were observed with an extended loading duration. Immunofluorescence revealed that the expression of notochordal cell markers, CD24 and brachyury, and chondrocyte markers, collagen type II and SRY-box 9, declined with loading. In particular, reduction in notochordal cell marker expression was more dramatic than that in chondrocyte marker expression. Apoptotic terminal deoxynucleotidyl transferase dUTP nick-end labelingpositivity was also higher in brachyury-positive notochordal cells. Furthermore, all these changes were delayed by inhibiting integrin α5β1. Findings of our dynamic loading regimen with a relatively high pressure suggest reproducibility of the cellularity and phenotypic disappearance of NP notochordal cells during adolescence, the susceptibility of notochordal cells tomechanical stimuli partially through the integrin α5β1 pathway, and future potential treatment of integrin regulation for intervertebral disc disease.