562 results on '"Xandra O. Breakefield"'
Search Results
2. Heparin interferes with the uptake of liposomes in glioma
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Thomas S. van Solinge, Kristina Pagh Friis, Killian O'Brien, Romy L. Verschoor, Jeroen van Aarle, Arnold Koekman, Xandra O. Breakefield, Pieter Vader, Raymond Schiffelers, and Marike Broekman
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Liposomes ,Heparin ,Glioma ,Glioblastoma ,Uptake ,Delivery ,Pharmacy and materia medica ,RS1-441 - Abstract
In glioblastoma, a malignant primary brain tumor, liposomes have shown promise in pre-clinical and early phase clinical trials as delivery vehicles for therapeutics. However, external factors influencing cellular uptake of liposomes in glioma cells are poorly understood. Heparin and heparin analogues are commonly used in glioma patients to decrease the risk of thrombo-embolic events. Our results show that heparin inhibits pegylated liposome uptake by U87 glioma and GL261 cells in a dose dependent manner in vitro, and that heparin-mediated inhibition of uptake required presence of fetal bovine serum in the media. In a subcutaneous model of glioma, Cy5.5 labeled liposomes could be detected with in vivo imaging after direct intra-tumoral injection. Ex-vivo analysis with flow cytometry showed a decreased uptake of liposomes into tumor cells in mice treated systemically with heparin compared to those treated with vehicle only.
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- 2023
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3. Roles of extracellular vesicles in glioblastoma: foes, friends and informers
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Taral R. Lunavat, Lisa Nieland, Anne B. Vrijmoet, Ayrton Zargani-Piccardi, Youssef Samaha, Koen Breyne, and Xandra O. Breakefield
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glioblastoma ,tumor microenvironement ,extracellular vesicles ,pro-tumorigenic ,immunosuppressive biomarkers ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Glioblastoma (GB) tumors are one of the most insidious cancers which take over the brain and defy therapy. Over time and in response to treatment the tumor and the brain cells in the tumor microenvironment (TME) undergo many genetic/epigenetic driven changes in their phenotypes and this is reflected in the cellular contents within the extracellular vesicles (EVs) they produce. With the result that some EVs try to subdue the tumor (friends of the brain), while others participate in the glioblastoma takeover (foes of the brain) in a dynamic and ever changing process. Monitoring the contents of these EVs in biofluids can inform decisions based on GB status to guide therapeutic intervention. This review covers primarily recent research describing the different cell types in the brain, as well as the tumor cells, which participate in this EV deluge. This includes EVs produced by the tumor which manipulate the transcriptome of normal cells in their environment in support of tumor growth (foes), as well as responses of normal cells which try to restrict tumor growth and invasion, including traveling to cervical lymph nodes to present tumor neo-antigens to dendritic cells (DCs). In addition EVs released by tumors into biofluids can report on the status of living tumor cells via their cargo and thus serving as biomarkers. However, EVs released by tumor cells and their influence on normal cells in the tumor microenvironment is a major factor in immune suppression and coercion of normal brain cells to join the GB “band wagon”. Efforts are being made to deploy EVs as therapeutic vehicles for drugs and small inhibitory RNAs. Increasing knowledge about EVs in the TME is being utilized to track tumor progression and response to therapy and even to weaponize EVs to fight the tumor.
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- 2023
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4. Gene replacement therapy in a schwannoma mouse model of neurofibromatosis type 2
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Shilpa Prabhakar, Roberta L. Beauchamp, Pike See Cheah, Akiko Yoshinaga, Edwina Abou Haidar, Sevda Lule, Gayathri Mani, Katia Maalouf, Anat Stemmer-Rachamimov, David H. Jung, D. Bradley Welling, Marco Giovannini, Scott R. Plotkin, Casey A. Maguire, Vijaya Ramesh, and Xandra O. Breakefield
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neurofibromatosis type 2 ,gene therapy ,adeno-associated viral vector ,schwannoma ,Schwann cells ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Loss of function of the neurofibromatosis type 2 (NF2) tumor suppressor gene leads to the formation of schwannomas, meningiomas, and ependymomas, comprising ∼50% of all sporadic cases of primary nervous system tumors. NF2 syndrome is an autosomal dominant condition, with bi-allelic inactivation of germline and somatic alleles resulting in loss of function of the encoded protein merlin and activation of mammalian target of rapamycin (mTOR) pathway signaling in NF2-deficient cells. Here we describe a gene replacement approach through direct intratumoral injection of an adeno-associated virus vector expressing merlin in a novel human schwannoma model in nude mice. In culture, the introduction of an AAV1 vector encoding merlin into CRISPR-modified human NF2-null arachnoidal cells (ACs) or Schwann cells (SCs) was associated with decreased size and mTORC1 pathway activation consistent with restored merlin activity. In vivo, a single injection of AAV1-merlin directly into human NF2-null SC-derived tumors growing in the sciatic nerve of nude mice led to regression of tumors over a 10-week period, associated with a decrease in dividing cells and an increase in apoptosis, in comparison with vehicle. These studies establish that merlin re-expression via gene replacement in NF2-null schwannomas is sufficient to cause tumor regression, thereby potentially providing an effective treatment for NF2.
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- 2022
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5. CRISPR-Cas knockout of miR21 reduces glioma growth
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Lisa Nieland, Thomas S. van Solinge, Pike See Cheah, Liza M. Morsett, Joseph El Khoury, Joseph I. Rissman, Benjamin P. Kleinstiver, Marike L.D. Broekman, Xandra O. Breakefield, and Erik R. Abels
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glioblastoma ,CRISPR ,gene editing ,microRNA ,miR-21 ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Non-coding RNAs, including microRNAs (miRNAs), support the progression of glioma. miR-21 is a small, non-coding transcript involved in regulating gene expression in multiple cellular pathways, including the regulation of proliferation. High expression of miR-21 has been shown to be a major driver of glioma growth. Manipulating the expression of miRNAs is a novel strategy in the development of therapeutics in cancer. In this study we aimed to target miR-21. Using CRISPR genome-editing technology, we disrupted the miR-21 coding sequences in glioma cells. Depletion of this miRNA resulted in the upregulation of many downstream miR-21 target mRNAs involved in proliferation. Phenotypically, CRISPR-edited glioma cells showed reduced migration, invasion, and proliferation in vitro. In immunocompetent mouse models, miR-21 knockout tumors showed reduced growth resulting in an increased overall survival. In summary, we show that by knocking out a key miRNA in glioma, these cells have decreased proliferation capacity both in vitro and in vivo. Overall, we identified miR-21 as a potential target for CRISPR-based therapeutics in glioma.
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- 2022
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6. Exogenous loading of extracellular vesicles, virus-like particles, and lentiviral vectors with supercharged proteins
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Koen Breyne, Stefano Ughetto, David Rufino-Ramos, Shadi Mahjoum, Emily A. Grandell, Luís P. de Almeida, and Xandra O. Breakefield
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Biology (General) ,QH301-705.5 - Abstract
The development of positive supercharged protein loading of cell membrane-based biovesicles, including extracellular vesicles, virus-like particles, and lentiviral vectors, as a strategy to deliver exogenous plasmid DNA to target cells in vitro and in vivo is presented.
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- 2022
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7. Illuminating cellular and extracellular vesicle-mediated communication via a split-Nanoluc reporter in vitro and in vivo
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Thomas S. van Solinge, Shadi Mahjoum, Stefano Ughetto, Alessandro Sammarco, Marike L.D. Broekman, Xandra O. Breakefield, and Killian P. O’Brien
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CP: Cell biology ,Biotechnology ,TP248.13-248.65 ,Biochemistry ,QD415-436 ,Science - Abstract
Summary: Tools to effectively demonstrate and quantify functional delivery in cellular communication have been lacking. This study reports the use of a fluorescently labeled split Nanoluc reporter system to demonstrate and quantify functional transfer between cells in vitro and in a subcutaneous tumor mouse model. Our construct allows monitoring of direct, indirect, and specifically extracellular vesicle-mediated functional communication. Motivation: In tracking protein-mediated cellular communication, few tools distinguish between proteins that escape the endosome and are functional within the cytosol and proteins that are degraded by the lysosome or released back into the extracellular space. We designed a tool that signals functional protein delivery and can be used to study cell-to-cell and extracellular vesicle-mediated communication in vitro and in vivo.
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- 2023
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8. Mutant Allele-Specific CRISPR Disruption in DYT1 Dystonia Fibroblasts Restores Cell Function
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Lilian Cruz, Bence György, Pike See Cheah, Benjamin P. Kleinstiver, William A. Eimer, Sara P. Garcia, Nutan Sharma, Laurie J. Ozelius, D. Cristopher Bragg, J. Keith Joung, Osmar Norberto de Souza, Luis Fernando Saraiva Macedo Timmers, and Xandra O. Breakefield
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CRISPR ,dystonia ,DYT1 ,torsinA ,herpes simplex virus type 1 ,TOR1A ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302–332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function.
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- 2020
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9. Glioblastoma hijacks microglial gene expression to support tumor growth
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Sybren L. N. Maas, Erik R. Abels, Lieke L. Van De Haar, Xuan Zhang, Liza Morsett, Srinjoy Sil, Joana Guedes, Pritha Sen, Shilpa Prabhakar, Suzanne E. Hickman, Charles P. Lai, David T. Ting, Xandra O. Breakefield, Marike L. D. Broekman, and Joseph El Khoury
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Glioblastoma ,Glioma ,Microglia ,Extracellular vesicles ,Exosomes ,Microvesicles ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Abstract Background Glioblastomas are the most common and lethal primary brain tumors. Microglia, the resident immune cells of the brain, survey their environment and respond to pathogens, toxins, and tumors. Glioblastoma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Despite the presence of large numbers of microglia in glioblastoma, the tumors continue to grow, and these neuroimmune cells appear incapable of keeping the tumor in check. To understand this process, we analyzed gene expression in microglia interacting with glioblastoma cells. Methods We used RNASeq of isolated microglia to analyze the expression patterns of genes involved in key microglial functions in mice with glioblastoma. We focused on microglia that had taken up tumor-derived EVs and therefore were within and immediately adjacent to the tumor. Results We show that these microglia have downregulated expression of genes involved in sensing tumor cells and tumor-derived danger signals, as well as genes used for tumor killing. In contrast, expression of genes involved in facilitating tumor spread was upregulated. These changes appear to be in part EV-mediated, since intracranial injection of EVs in normal mice led to similar transcriptional changes in microglia. We observed a similar microglial transcriptomic signature when we analyzed datasets from human patients with glioblastoma. Conclusion Our data define a microgliaGlioblastoma specific phenotype, whereby glioblastomas have hijacked gene expression in the neuroimmune system to favor avoiding tumor sensing, suppressing the immune response, clearing a path for invasion, and enhancing tumor propagation. For further exploration, we developed an interactive online tool at http://www.glioma-microglia.com with all expression data and additional functional and pathway information for each gene.
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- 2020
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10. Long-Term Therapeutic Efficacy of Intravenous AAV-Mediated Hamartin Replacement in Mouse Model of Tuberous Sclerosis Type 1
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Shilpa Prabhakar, Pike See Cheah, Xuan Zhang, Max Zinter, Maria Gianatasio, Eloise Hudry, Roderick T. Bronson, David J. Kwiatkowski, Anat Stemmer-Rachamimov, Casey A. Maguire, Miguel Sena-Esteves, Bakhos A. Tannous, and Xandra O. Breakefield
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Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome caused by mutations in TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins act as a complex that inhibits mammalian target of rapamycin (mTOR)-mediated cell growth and proliferation. Loss of either protein leads to overgrowth in many organs, including subependymal nodules, subependymal giant cell astrocytomas, and cortical tubers in the human brain. Neurological manifestations in TSC include intellectual disability, autism, hydrocephalus, and epilepsy. In a stochastic mouse model of TSC1 brain lesions, complete loss of Tsc1 is achieved in homozygous Tsc1-floxed mice in a subpopulation of neural cells in the brain by intracerebroventricular (i.c.v.) injection at birth of an adeno-associated virus (AAV) vector encoding Cre recombinase. This results in median survival of 38 days and brain pathology, including subependymal lesions and enlargement of neuronal cells. Remarkably, when these mice were injected intravenously on day 21 with an AAV9 vector encoding hamartin, most survived at least up to 429 days in apparently healthy condition with marked reduction in brain pathology. Thus, a single intravenous administration of an AAV vector encoding hamartin restored protein function in enough cells in the brain to extend lifespan in this TSC1 mouse model. Keywords: brain, neurology, tumor suppressor, gene therapy
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- 2019
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11. High levels of AAV vector integration into CRISPR-induced DNA breaks
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Killian S. Hanlon, Benjamin P. Kleinstiver, Sara P. Garcia, Mikołaj P. Zaborowski, Adrienn Volak, Stefan E. Spirig, Alissa Muller, Alexander A. Sousa, Shengdar Q. Tsai, Niclas E. Bengtsson, Camilla Lööv, Martin Ingelsson, Jeffrey S. Chamberlain, David P. Corey, Martin J. Aryee, J. Keith Joung, Xandra O. Breakefield, Casey A. Maguire, and Bence György
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Science - Abstract
In-depth characterization of adeno-associated virus (AAV)-mediated CRISPR delivery is still lacking. Here, the authors show high levels of integration into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in vivo.
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- 2019
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12. Versatile Role of Rab27a in Glioma: Effects on Release of Extracellular Vesicles, Cell Viability, and Tumor Progression
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Thomas S. van Solinge, Erik R. Abels, Lieke L. van de Haar, Killian S. Hanlon, Sybren L. N. Maas, Rosalie Schnoor, Jeroen de Vrij, Xandra O. Breakefield, and Marike L. D. Broekman
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glioma ,glioblastoma ,extracellular vesicles ,exosomes ,tumor microenvironment ,Rab27a ,Biology (General) ,QH301-705.5 - Abstract
Introduction: Glioma cells exert influence over the tumor-microenvironment in part through the release of extracellular vesicles (EVs), membrane-enclosed structures containing proteins, lipids, and RNAs. In this study, we evaluated the function of Ras-associated protein 27a (Rab27a) in glioma and evaluated the feasibility of assessing its role in EV release in glioma cells in vitro and in vivo.Methods: Rab27a was knocked down via a short hairpin RNA (shRNA) stably expressed in mouse glioma cell line GL261, with a scrambled shRNA as control. EVs were isolated by ultracentrifugation and quantified with Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS). CellTiter-Glo viability assays and cytokine arrays were used to evaluate the impact of Rab27a knockdown. GL261.shRab27a cells and GL261.shControl were implanted into the left striatum of eight mice to assess tumor growth and changes in the tumor microenvironment.Results: Knockdown of Rab27a in GL261 glioma cells decreased the release of small EVs isolated at 100,000 × g in vitro (p = 0.005), but not the release of larger EVs, isolated at 10,000 × g. GL261.shRab27a cells were less viable compared to the scramble control in vitro (p < 0.005). A significant increase in CCL2 expression in shRab27a GL261 cells was also observed (p < 0.001). However, in vivo there was no difference in tumor growth or overall survival between the two groups, while shRab27a tumors showed lower proliferation at the tumor borders. Decreased infiltration of IBA1 positive macrophages and microglia, but not FoxP3 positive regulatory T cells was observed.Conclusion: Rab27a plays an important role in the release of small EVs from glioma cells, and also in their viability and expression of CCL2 in vitro. As interference in Rab27a expression influences glioma cell viability and expression profiles, future studies should be cautious in using the knockdown of Rab27a as a means of studying the role of small EVs in glioma growth.
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- 2020
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13. Discovery and Verification of Extracellular miRNA Biomarkers for Non-invasive Prediction of Pre-eclampsia in Asymptomatic Women
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Srimeenakshi Srinivasan, Ryan Treacy, Tiffany Herrero, Richelle Olsen, Trevor R. Leonardo, Xuan Zhang, Peter DeHoff, Cuong To, Lara G. Poling, Aileen Fernando, Sandra Leon-Garcia, Katharine Knepper, Vy Tran, Morgan Meads, Jennifer Tasarz, Aishwarya Vuppala, Soojin Park, Clara D. Laurent, Tony Bui, Pike See Cheah, Rachael Tabitha Overcash, Gladys A. Ramos, Hilary Roeder, Ionita Ghiran, Mana Parast, Xandra O. Breakefield, Amir J. Lueth, Sharon R. Rust, Max T. Dufford, Angela C. Fox, Durlin E. Hickok, Julja Burchard, J. Jay Boniface, Louise C. Laurent, Kim A. Boggess, George R. Saade, Scott A. Sullivan, Glenn R. Markenson, Jay D. Iams, Dean V. Coonrod, Leonardo M. Pereira, M. Sean Esplin, Larry M. Cousins, Garrett K. Lam, and Matthew K. Hoffman
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pre-eclampsia ,biomarkers ,bivariate ,miRNA ,extracellular RNA ,small RNA-seq ,Medicine (General) ,R5-920 - Abstract
Summary: Development of effective prevention and treatment strategies for pre-eclampsia is limited by the lack of accurate methods for identification of at-risk pregnancies. We performed small RNA sequencing (RNA-seq) of maternal serum extracellular RNAs (exRNAs) to discover and verify microRNAs (miRNAs) differentially expressed in patients who later developed pre-eclampsia. Sera collected from 73 pre-eclampsia cases and 139 controls between 17 and 28 weeks gestational age (GA), divided into separate discovery and verification cohorts, are analyzed by small RNA-seq. Discovery and verification of univariate and bivariate miRNA biomarkers reveal that bivariate biomarkers verify at a markedly higher rate than univariate biomarkers. The majority of verified biomarkers contain miR-155-5p, which has been reported to mediate the pre-eclampsia-associated repression of endothelial nitric oxide synthase (eNOS) by tumor necrosis factor alpha (TNF-α). Deconvolution analysis reveals that several verified miRNA biomarkers come from the placenta and are likely carried by placenta-specific extracellular vesicles.
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- 2020
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14. Glioma-Derived miRNA-Containing Extracellular Vesicles Induce Angiogenesis by Reprogramming Brain Endothelial Cells
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Rocco Lucero, Valentina Zappulli, Alessandro Sammarco, Oscar D. Murillo, Pike See Cheah, Srimeenakshi Srinivasan, Eric Tai, David T. Ting, Zhiyun Wei, Matthew E. Roth, Louise C. Laurent, Anna M. Krichevsky, Xandra O. Breakefield, and Aleksandar Milosavljevic
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Biology (General) ,QH301-705.5 - Abstract
Summary: Glioblastoma (GBM) is characterized by aberrant vascularization and a complex tumor microenvironment. The failure of anti-angiogenic therapies suggests pathways of GBM neovascularization, possibly attributable to glioblastoma stem cells (GSCs) and their interplay with the tumor microenvironment. It has been established that GSC-derived extracellular vesicles (GSC-EVs) and their cargoes are proangiogenic in vitro. To further elucidate EV-mediated mechanisms of neovascularization in vitro, we perform RNA-seq and DNA methylation profiling of human brain endothelial cells exposed to GSC-EVs. To correlate these results to tumors in vivo, we perform histoepigenetic analysis of GBM molecular profiles in the TCGA collection. Remarkably, GSC-EVs and normal vascular growth factors stimulate highly distinct gene regulatory responses that converge on angiogenesis. The response to GSC-EVs shows a footprint of post-transcriptional gene silencing by EV-derived miRNAs. Our results provide insights into targetable angiogenesis pathways in GBM and miRNA candidates for liquid biopsy biomarkers. : Extensive intercellular interactions occur within the notoriously heterogeneous tumor microenvironment of GBM. Lucero et al. identify distinct angiogenic gene regulatory responses of brain endothelial cells to growth factors and to extracellular vesicles (EVs) secreted by GBM stem-like cells. The response to EVs shows a footprint of EV-derived miRNAs. Keywords: angiogenesis, biomarker, cancer stem cell, deconvolution, glioblastoma, exRNA, extracellular vesicle, miRNA, reprogramming, tumor microenvironment
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- 2020
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15. Large and small extracellular vesicles released by glioma cells in vitro and in vivo
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Anudeep Yekula, Valentina R. Minciacchi, Matteo Morello, Huilin Shao, Yongil Park, Xuan Zhang, Koushik Muralidharan, Michael R. Freeman, Ralph Weissleder, Hakho Lee, Bob Carter, Xandra O. Breakefield, Dolores Di Vizio, and Leonora Balaj
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extracellular vesicles ,large extracellular vesicles ,small extracellular vesicles ,biomarkers ,glioma ,Cytology ,QH573-671 - Abstract
Tumour cells release diverse populations of extracellular vesicles (EVs) ranging in size, molecular cargo, and function. We sought to characterize mRNA and protein content of EV subpopulations released by human glioblastoma (GBM) cells expressing a mutant form of epidermal growth factor receptor (U87EGFRvIII) in vitro and in vivo with respect to size, morphology and the presence of tumour cargo. The two EV subpopulations purified from GBM U87EGFRvIII cancer cells, non-cancer human umbilical vein endothelial cells (HUVEC; control) and serum of U87EGFRvIII glioma-bearing mice using differential centrifugation (EVs that sediment at 10,000 × g or 100,000 × g are termed large EVs and small EVs, respectively) were characterized using transmission electron microscopy (TEM), confocal microscopy, nanoparticle tracking analysis (NTA), flow cytometry, immunofluorescence (IF), quantitative-polymerase chain reaction (qPCR), droplet digital polymerase chain reaction (ddPCR) and micro-nuclear magnetic resonance (μNMR). We report that both U87EGFRvIII and HUVEC release a similar number of small EVs, but U87EGFRvIII glioma cells alone release a higher number of large EVs compared to non-cancer HUVEC. The EGFRvIII mRNA from the two EV subpopulations from U87EGFRvIII glioma cells was comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Similarly, EGFRvIII mRNA in large and small EVs isolated from the serum of U87EGFRvIII glioma-bearing mice is comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Here we report for the first time a direct comparison of large and small EVs released by glioma U87EGFRvIII cells and from serum of U87EGFRvIII glioma-bearing mice. Both large and small EVs contain tumour-specific EGFRvIII mRNA and proteins and combining these platforms may be beneficial in detecting rare mutant events in circulating biofluids.
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- 2020
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16. Analysis of extracellular mRNA in human urine reveals splice variant biomarkers of muscular dystrophies
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Layal Antoury, Ningyan Hu, Leonora Balaj, Sudeshna Das, Sofia Georghiou, Basil Darras, Tim Clark, Xandra O. Breakefield, and Thurman M. Wheeler
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Science - Abstract
Patients with myotonic dystrophy need to undergo invasive muscle biopsies to monitor disease progression and response to therapy. Here, the authors show that extracellular RNAs in human urine can be used as biomarkers to differentiate patients from unaffected controls, and to monitor exon skipping in patients with Duchenne muscular dystrophy taking the drug eteplirsen.
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- 2018
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17. Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours
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Franz L. Ricklefs, Cecile L. Maire, Rudolph Reimer, Lasse Dührsen, Katharina Kolbe, Mareike Holz, Enja Schneider, Anne Rissiek, Anna Babayan, Claudia Hille, Klaus Pantel, Susanne Krasemann, Markus Glatzel, Dieter Henrik Heiland, Jörg Flitsch, Tobias Martens, Nils Ole Schmidt, Sven Peine, Xandra O. Breakefield, Sean Lawler, E. Antonio. Chiocca, Boris Fehse, Bernd Giebel, André Görgens, Manfred Westphal, and Katrin Lamszus
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imaging flow cytometry ,extracellular vesicle ,biomarker ,glioma ,tetraspanin ,Cytology ,QH573-671 - Abstract
Cells release heterogeneous nano-sized vesicles either as exosomes, being derived from endosomal compartments, or through budding from the plasma membrane as so-called microvesicles, commonly referred to as extracellular vesicles (EVs). EVs are known for their important roles in mammalian physiology and disease pathogenesis and provide a potential biomarker source in cancer patients. EVs are generally often analysed in bulk using Western blotting or by bead-based flow-cytometry or, with limited parameters, through nanoparticle tracking analysis. Due to their small size, single EV analysis is technically highly challenging. Here we demonstrate imaging flow cytometry (IFCM) to be a robust, multiparametric technique that allows analysis of single EVs and the discrimination of distinct EV subpopulations. We used IFCM to analyse the tetraspanin (CD9, CD63, CD81) surface profiles on EVs from human and murine cell cultures as well as plasma samples. The presence of EV subpopulations with specific tetraspanin profiles suggests that EV-mediated cellular responses are tightly regulated and dependent on cell environment. We further demonstrate that EVs with double positive tetraspanin expression (CD63+/CD81+) are enriched in cancer cell lines and patient plasma samples. In addition, we used IFCM to detect tumour-specific GFP-labelled EVs in the blood of mice bearing syngeneic intracerebral gliomas, indicating that this technique allows unprecedented disease modelling. In summary, our study highlights the heterogeneous and adaptable nature of EVs according to their marker profile and demonstrates that IFCM facilitates multiparametric phenotyping of EVs not only in vitro but also in patient plasma at a single EV level, with the potential for future functional studies and clinically relevant applications. Abbreviation: EDTA = ethylenediamine tetraacetic acid
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- 2019
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18. CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease
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Bence György, Camilla Lööv, Mikołaj P. Zaborowski, Shuko Takeda, Benjamin P. Kleinstiver, Caitlin Commins, Ksenia Kastanenka, Dakai Mu, Adrienn Volak, Vilmantas Giedraitis, Lars Lannfelt, Casey A. Maguire, J. Keith Joung, Bradley T. Hyman, Xandra O. Breakefield, and Martin Ingelsson
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Alzheimer's disease ,Swedish mutation ,adeno-associated virus ,genome editing ,CRISPR ,amyloid precursor protein ,amyloid-β ,Therapeutics. Pharmacology ,RM1-950 - Abstract
The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer’s disease (AD) as a result of increased β-secretase cleavage of the amyloid-β (Aβ) precursor protein. This leads to abnormally high Aβ levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aβ. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo—and thereby decrease pathogenic Aβ. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aβ.
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- 2018
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19. Mutant torsinA in the heterozygous DYT1 state compromises HSV propagation in infected neurons and fibroblasts
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Bence György, Lilian Cruz, David Yellen, Massimo Aufiero, Isabel Alland, Xuan Zhang, Maria Ericsson, Cornel Fraefel, Yu-Ching Li, Shuko Takeda, Bradley T. Hyman, and Xandra O. Breakefield
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Medicine ,Science - Abstract
Abstract Most cases of early onset torsion dystonia (DYT1) are caused by a 3-base pair deletion in one allele of the TOR1A gene causing loss of a glutamate in torsinA, a luminal protein in the nuclear envelope. This dominantly inherited neurologic disease has reduced penetrance and no other medical manifestations. It has been challenging to understand the neuronal abnormalities as cells and mouse models which are heterozygous (Het) for the mutant allele are quite similar to wild-type (WT) controls. Here we found that patient fibroblasts and mouse neurons Het for this mutation showed significant differences from WT cells in several parameters revealed by infection with herpes simplex virus type 1 (HSV) which replicates in the nucleus and egresses out through the nuclear envelope. Using a red fluorescent protein capsid to monitor HSV infection, patient fibroblasts showed decreased viral plaque formation as compared to controls. Mouse Het neurons had a decrease in cytoplasmic, but not nuclear HSV fluorescence, and reduced numbers of capsids entering axons as compared to infected WT neurons. These findings point to altered dynamics of the nuclear envelope in cells with the patient genotype, which can provide assays to screen for therapeutic agents that can normalize these cells.
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- 2018
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20. Engineered nanointerfaces for microfluidic isolation and molecular profiling of tumor-specific extracellular vesicles
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Eduardo Reátegui, Kristan E. van der Vos, Charles P. Lai, Mahnaz Zeinali, Nadia A. Atai, Berent Aldikacti, Frederick P. Floyd, Aimal H. Khankhel, Vishal Thapar, Fred H. Hochberg, Lecia V. Sequist, Brian V. Nahed, Bob S. Carter, Mehmet Toner, Leonora Balaj, David T. Ting, Xandra O. Breakefield, and Shannon L. Stott
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Science - Abstract
Extracellular vesicles can carry many different types of biological cargo and have been investigated as a biomarker for cancer diagnosis. Here the authors develop a microfluidic platform for rapid and sensitive isolation of tumor-specific extracellular vesicles.
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- 2018
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21. Coding and noncoding landscape of extracellular RNA released by human glioma stem cells
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Zhiyun Wei, Arsen O. Batagov, Sergio Schinelli, Jintu Wang, Yang Wang, Rachid El Fatimy, Rosalia Rabinovsky, Leonora Balaj, Clark C. Chen, Fred Hochberg, Bob Carter, Xandra O. Breakefield, and Anna M. Krichevsky
- Subjects
Science - Abstract
While circulating DNA has been extensively explored as a potential cancer biomarker, RNA potential has been overlooked so far. Here the authors present a comprehensive analysis of extracellular RNA secreted by glioblastoma cells that could prove a valuable resource for biomarker discovery and a means of intercellular communication.
- Published
- 2017
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22. Living Proof of Activity of Extracellular Vesicles in the Central Nervous System
- Author
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Shadi Mahjoum, David Rufino-Ramos, Luís Pereira de Almeida, Marike L. D. Broekman, Xandra O. Breakefield, and Thomas S. van Solinge
- Subjects
extracellular vesicle ,central nervous system ,in vivo experiments ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The central nervous system (CNS) consists of a heterogeneous population of cells with highly specialized functions. For optimal functioning of the CNS, in disease and in health, intricate communication between these cells is vital. One important mechanism of cellular communication is the release and uptake of extracellular vesicles (EVs). EVs are membrane enclosed particles actively released by cells, containing a wide array of proteins, lipids, RNA, and DNA. These EVs can be taken up by neighboring or distant cells, and influence a wide range of processes. Due to the complexity and relative inaccessibility of the CNS, our current understanding of the role of EVs is mainly derived in vitro work. However, recently new methods and techniques have opened the ability to study the role of EVs in the CNS in vivo. In this review, we discuss the current developments in our understanding of the role of EVs in the CNS in vivo.
- Published
- 2021
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23. Glioblastoma-Associated Microglia Reprogramming Is Mediated by Functional Transfer of Extracellular miR-21
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Erik R. Abels, Sybren L.N. Maas, Lisa Nieland, Zhiyun Wei, Pike See Cheah, Eric Tai, Christy-Joy Kolsteeg, Sophie A. Dusoswa, David T. Ting, Suzanne Hickman, Joseph El Khoury, Anna M. Krichevsky, Marike L.D. Broekman, and Xandra O. Breakefield
- Subjects
Biology (General) ,QH301-705.5 - Abstract
Summary: Gliomas are primary, diffusely infiltrating brain tumors. Microglia are innate immune cells in the CNS and make up a substantial portion of the tumor mass. Glioma cells shape their microenvironment, communicating with and reprogramming surrounding cells, resulting in enhanced angiogenesis, immune suppression, and remodeling of the extracellular matrix. Glioma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Mouse glioma cells stably expressing a palmitoylated GFP to label EVs were implanted intracranially into syngeneic miR-21-null mice. Here, we demonstrate functional delivery of miR-21, regulating specific downstream mRNA targets in microglia after uptake of tumor-derived EVs. These findings attest to EV-dependent microRNA delivery as studied in an in vivo-based model and provide insight into the reprograming of microglial cells by tumor cells to create a favorable microenvironment for cancer progression. : Abels et al. show miR-21 transfer from glioma to microglia by palmitoylated GFP-labeled extracellular vesicles in vivo. This transfer results in miR-21 target-specific mRNA downregulation. Following downregulation of Btg2, proliferation in microglia is increased, suggesting reprogramming of microglia in the tumor microenvironment through extracellular vesicles shed by glioma cells.
- Published
- 2019
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24. Methods for Systematic Identification of Membrane Proteins for Specific Capture of Cancer-Derived Extracellular Vesicles
- Author
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Mikołaj Piotr Zaborowski, Kyungheon Lee, Young Jeong Na, Alessandro Sammarco, Xuan Zhang, Marcin Iwanicki, Pike See Cheah, Hsing-Ying Lin, Max Zinter, Chung-Yu Chou, Giulia Fulci, Bakhos A. Tannous, Charles Pin-Kuang Lai, Michael J. Birrer, Ralph Weissleder, Hakho Lee, and Xandra O. Breakefield
- Subjects
Biology (General) ,QH301-705.5 - Abstract
Summary: Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type. : Cancer cell-derived extracellular vesicles (EVs) can be used in diagnostics, but their enrichment remains challenging. Zaborowski et al. identify membrane proteins enriched on the surface of cancer cells compared with normal tissues using TCGA, the Human Protein Atlas, and GTEx and present methods to measure immunocapture of cancer EVs in vitro and in animal models. Keywords: extracellular vesicles, membrane proteins, biomarker, The Cancer Genome Atlas, Genotype-Tissue Expression Project, Human Protein Atlas, palmitoylated fluorescent protein, membrane-bound Gaussia luciferase
- Published
- 2019
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25. Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells
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Naoto Ito, William T. Hendriks, Jyotsna Dhakal, Christine A. Vaine, Christina Liu, David Shin, Kyle Shin, Noriko Wakabayashi-Ito, Marisela Dy, Trisha Multhaupt-Buell, Nutan Sharma, Xandra O. Breakefield, and D. Cristopher Bragg
- Subjects
X-linked dystonia-parkinsonism ,Induced pluripotent stem cells ,TAF1 ,Medicine ,Pathology ,RB1-214 - Abstract
X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known, in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containing TAF1, a large gene with at least 38 exons, and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 of TAF1, as well as a neural-specific TAF1 isoform, N-TAF1, which showed decreased expression in post-mortem XDP brain compared with control tissue. Here, we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model, we compared expression of TAF1 and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells, XDP fibroblasts exhibited decreased expression of TAF1 transcript fragments derived from exons 32-36, a region spanning the SVA insertion site. N-TAF1, which incorporates an alternative exon (exon 34′), was not expressed in fibroblasts, but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP, but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration.
- Published
- 2016
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26. Survival benefit and phenotypic improvement by hamartin gene therapy in a tuberous sclerosis mouse brain model
- Author
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Shilpa Prabhakar, Xuan Zhang, June Goto, Sangyeul Han, Charles Lai, Roderick Bronson, Miguel Sena-Esteves, Vijaya Ramesh, Anat Stemmer-Rachamimov, David J. Kwiatkowski, and Xandra O. Breakefield
- Subjects
Tuberous sclerosis complex ,TSC ,TSC1 ,TSC2 ,Gene therapy ,AAV ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
We examined the potential benefit of gene therapy in a mouse model of tuberous sclerosis complex (TSC) in which there is embryonic loss of Tsc1 (hamartin) in brain neurons. An adeno-associated virus (AAV) vector (serotype rh8) expressing a tagged form of hamartin was injected into the cerebral ventricles of newborn pups with the genotype Tsc1cc (homozygous for a conditional floxed Tsc1 allele) SynI-cre+, in which Tsc1 is lost selectively in neurons starting at embryonic day 12. Vector-treated Tsc1ccSynIcre+ mice showed a marked improvement in survival from a mean of 22 days in non-injected mice to 52 days in AAV hamartin vector-injected mice, with improved weight gain and motor behavior in the latter. Pathologic studies showed normalization of neuron size and a decrease in markers of mTOR activation in treated as compared to untreated mutant littermates. Hence, we show that gene replacement in the brain is an effective therapeutic approach in this mouse model of TSC1. Our strategy for gene therapy has the advantages that therapy can be achieved from a single application, as compared to repeated treatment with drugs, and that AAV vectors have been found to have minimal to no toxicity in clinical trials for other neurologic conditions. Although there are many additional issues to be addressed, our studies support gene therapy as a useful approach in TSC patients.
- Published
- 2015
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27. Mutant human torsinA, responsible for early-onset dystonia, dominantly suppresses GTPCH expression, dopamine levels and locomotion in Drosophila melanogaster
- Author
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Noriko Wakabayashi-Ito, Rami R. Ajjuri, Benjamin W. Henderson, Olugbenga M. Doherty, Xandra O. Breakefield, Janis M. O'Donnell, and Naoto Ito
- Subjects
Dystonia ,Drosophila ,GTP cyclohydrolase ,TorsinA ,Movement disorder ,Science ,Biology (General) ,QH301-705.5 - Abstract
Dystonia represents the third most common movement disorder in humans with over 20 genetic loci identified. TOR1A (DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. Most cases of DYT1 dystonia are caused by a 3 bp (ΔGAG) deletion that results in the loss of a glutamic acid residue (ΔE302/303) in the carboxyl terminal region of torsinA. This torsinAΔE mutant protein has been speculated to act in a dominant-negative manner to decrease activity of wild type torsinA. Drosophila melanogaster has a single torsin-related gene, dtorsin. Null mutants of dtorsin exhibited locomotion defects in third instar larvae. Levels of dopamine and GTP cyclohydrolase (GTPCH) proteins were severely reduced in dtorsin-null brains. Further, the locomotion defect was rescued by the expression of human torsinA or feeding with dopamine. Here, we demonstrate that human torsinAΔE dominantly inhibited locomotion in larvae and adults when expressed in neurons using a pan-neuronal promoter Elav. Dopamine and tetrahydrobiopterin (BH4) levels were significantly reduced in larval brains and the expression level of GTPCH protein was severely impaired in adult and larval brains. When human torsinA and torsinAΔE were co-expressed in neurons in dtorsin-null larvae and adults, the locomotion rates and the expression levels of GTPCH protein were severely reduced. These results support the hypothesis that torsinAΔE inhibits wild type torsinA activity. Similarly, neuronal expression of a Drosophila DtorsinΔE equivalent mutation dominantly inhibited larval locomotion and GTPCH protein expression. These results indicate that both torsinAΔE and DtorsinΔE act in a dominant-negative manner. We also demonstrate that Dtorsin regulates GTPCH expression at the post-transcriptional level. This Drosophila model of DYT1 dystonia provides an important tool for studying the differences in the molecular function between the wild type and the mutant torsin proteins.
- Published
- 2015
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28. Molecular pathways in dystonia
- Author
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D. Cristopher Bragg, Ioanna A. Armata, Flavia C. Nery, Xandra O. Breakefield, and Nutan Sharma
- Subjects
Dystonia ,TorsinA ,THAP1 ,PACT ,PRKRA ,Dopamine ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
The hereditary dystonias comprise a set of diseases defined by a common constellation of motor deficits. These disorders are most likely associated with different molecular etiologies, many of which have yet to be elucidated. Here we discuss recent advances in three forms of hereditary dystonia, DYT1, DYT6 and DYT16, which share a similar clinical picture: onset in childhood or adolescence, progressive spread of symptoms with generalized involvement of body regions and a steady state affliction without treatment. Unlike DYT1, the genes responsible for DYT6 and DYT16 have only recently been identified, with relatively little information about the function of the encoded proteins. Nevertheless, recent data suggest that these proteins may fit together within interacting pathways involved in dopaminergic signaling, transcriptional regulation, and cellular stress responses. This review focuses on these molecular pathways, highlighting potential common themes among these dystonias which may serve as areas for future research. This article is part of a Special Issue entitled “Advances in dystonia”.
- Published
- 2011
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29. Dystonia-causing mutant torsinA inhibits cell adhesion and neurite extension through interference with cytoskeletal dynamics
- Author
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Jeffrey W. Hewett, Juan Zeng, Brian P. Niland, D. Cristopher Bragg, and Xandra O. Breakefield
- Subjects
Intermediate filaments ,Dystonia ,DYT1 ,Vimentin ,Fibroblasts ,Cell adhesion ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Early onset torsion dystonia is a movement disorder inherited as an autosomal dominant syndrome with reduced penetrance. Symptoms appear to result from altered neuronal circuitry within the brain with no evidence of neuronal loss. Most cases are caused by loss of a glutamic acid residue in the AAA+ chaperone protein, torsinA, encoded in the DYT1 gene. In this study, torsinA was found to move in conjunction with vimentin in three cell culture paradigms—recovery from microtubule depolymerization, expression of a dominant-negative form of kinesin light chain and respreading after trypsinization. Co-immune precipitation studies revealed association between vimentin and torsinA in a complex including other cytoskeletal elements, actin and tubulin, as well as two proteins previously shown to interact with torsinA—the motor protein, kinesin light chain 1, and the nuclear envelope protein, LAP1. Morphologic and functional differences related to vimentin were noted in primary fibroblasts from patients carrying this DYT1 mutation as compared with controls, including an increased perinuclear concentration of vimentin and a delayed rate of adhesion to the substratum. Overexpression of mutant torsinA inhibited neurite extension in human neuroblastoma cells, with torsinA and vimentin immunoreactivity enriched in the perinuclear region and in cytoplasmic inclusions. Collectively, these studies suggest that mutant torsinA interferes with cytoskeletal events involving vimentin, possibly by restricting movement of these particles/filaments, and hence may affect development of neuronal pathways in the brain.
- Published
- 2006
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30. A Novel Method for Imaging Apoptosis Using a Caspase-1 Near-Infrared Fluorescent Probe
- Author
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Shanta M. Messerli, Shilpa Prabhakar, Yi Tang, Khalid Shah, Maria L. Cortes, Vidya Murthy, Ralph Weissleder, Xandra O. Breakefield, and Ching-Hsuan Tung
- Subjects
Caspase-1 ,near-infrared fluorescence (NIRF) ,apoptosis ,brain tumors ,HSV ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Here we describe a novel method for imaging apoptosis in cells using a near-infrared fluorescent (NIRF) probe selective for caspase-1 (interleukin β-converting enzyme, ICE). This biocompatible, optically quenched ICE-NIRF probe incorporates a peptide substrate, which can be selectively cleaved by caspase-1, resulting in the release of fluorescence signal. The specificity of this probe for caspase-1 is supported by various lines of evidence: 1) activation by purified caspase-1, but not another caspase in vitro; 2) activation of the probe by infection of cells with a herpes simplex virus amplicon vector (HGC-ICE-IacZ) expressing a catalytically active caspase-1-IacZ fusion protein; 3) inhibition of HGC-ICE-IacZ vector-induced activation of the probe by coincubation with the caspase-1 inhibitor YVAD-cmk, but not with a caspase-3 inhibitor; and 4) activation of the probe following standard methods of inducing apoptosis with staurosporine, ganciclovir, or ionizing radiation in culture. These results indicate that this novel ICE-NIRF probe can be used in monitoring endogenous and vector-expressed caspase-1 activity in cells. Furthermore, tumor implant experiments indicate that this ICE-NIRF probe can be used to detect caspase-1 activity in living animals. This novel ICE-NIRF probe should prove useful in monitoring endogenous and vector-expressed caspase-1 activity, and potentially apoptosis in cell culture and in vivo.
- Published
- 2004
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31. HSV-1-Based Vectors for Gene Therapy of Neurological Diseases and Brain Tumors: Part II. Vector Systems and Applications
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Andreas Jacobs, Xandra O. Breakefield, and Cornel Fraefel
- Subjects
glioma ,gene therapy ,recombinant HSV-1 ,amplicon ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Many properties of HSV-1 are especially suitable for using this virus as a vector to treat diseases affecting the central nervous system (CNS), such as Parkinson's disease or malignant gliomas. These advantageous properties include natural neurotropism, high transduction efficiency, large transgene capacity, and the ability of entering a latent state in neurons. Selective oncolysis in combination with modulation of the immune response mediated by replication-conditional HSV-1 vectors appears to be a highly promising approach in the battle against malignant glioma. Helper virus-free HSV/AAV hybrid amplicon vectors have great promise in mediating long-term gene expression in the PNS and CNS for the treatment of various neurodegenerative disorders or chronic pain. Current research focuses on the design of HSV-1-derived vectors which are targeted to certain cell types and support transcriptionally regulatable transgene expression. Here, we review the recent developments on HSV-1-based vector systems and their applications in experimental and clinical gene therapy protocols.
- Published
- 1999
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32. HSV-1-Based Vectors for Gene Therapy of Neurological Diseases and Brain Tumors: Part I. HSV-1 Structure, Replication and Pathogenesis
- Author
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Andreas Jacobs, Xandra O. Breakefield, and Cornel Fraefel
- Subjects
herpes simplex virus ,gene therapy ,recombinant HSV-1 ,amplicon ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
The design of effective gene therapy strategies for brain tumors and other neurological disorders relies on the understanding of genetic and pathophysiological alterations associated with the disease, on the biological characteristics of the target tissue, and on the development of safe vectors and expression systems to achieve efficient, targeted and regulated, therapeutic gene expression. The herpes simplex virus type 1 (HSV-1) virion is one of the most efficient of all current gene transfer vehicles with regard to nuclear gene delivery in central nervous system-derived cells including brain tumors. HSV-1-related research over the past decades has provided excellent insight into the structure and function of this virus, which, in turn, facilitated the design of innovative vector systems. Here, we review aspects of HSV-1 structure, replication and pathogenesis, which are relevant for the engineering of HSV-1-based vectors.
- Published
- 1999
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33. Functional Coexpression of HSV-1 Thymidine Kinase and Green Fluorescent Protein: Implications for Noninvasive Imaging of Transgene Expression
- Author
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Andreas Jacobs, Michael Dubrovin, Jeff Hewett, Miguel Sena-Esteves, Cui-Wen Tan, Mark Slack, Michele Sadelain, Xandra O. Breakefield, and Juri G. Tjuvajev
- Subjects
thymidine kinase ,ganciclovir ,FIAU ,cancer gene therapy ,fusion genes ,imaging ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk) marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp) and HSV-1-tk genes was generated (tkgfp gene) and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS). TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1-tk-specific radiolabeled substrate, 2′-fluoro-2′-deoxy-1β-D-arabino-furanosyl-5-iodo-uracil (FIAU), in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT]), and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.
- Published
- 1999
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34. Correction: Stochastic Model of Lesions in Mouse Brain.
- Author
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Shilpa Prabhakar, June Goto, Xuan Zhang, Miguel Sena-Esteves, Roderick Bronson, Jillian Brockmann, Davide Gianni, Gregory R. Wojtkiewicz, John W. Chen, Anat Stemmer-Rachamimov, David J. Kwiatkowski, and Xandra O. Breakefield
- Subjects
Medicine ,Science - Published
- 2013
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35. Correction: Dopa-Responsive Dystonia: Functional Analysis of Single Nucleotide Substitutions within the 5’ Untranslated Region.
- Author
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Ioanna A. Armata, Leonora Balaj, John K. Kuster, Xuan Zhang, Shelun Tsai, Andreas A. Armatas, Trisha J. Multhaupt-Buell, Roy Soberman, Xandra O. Breakefield, Hiroshi Ichinose, and Nutan Sharma
- Subjects
Medicine ,Science - Published
- 2013
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- View/download PDF
36. Extracellular Vesicles and Their Convergence with Viral Pathways
- Author
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Thomas Wurdinger, NaTosha N. Gatson, Leonora Balaj, Balveen Kaur, Xandra O. Breakefield, and D. Michiel Pegtel
- Subjects
Microbiology ,QR1-502 - Abstract
Extracellular vesicles (microvesicles), such as exosomes and shed microvesicles, contain a variety of molecules including proteins, lipids, and nucleic acids. Microvesicles appear mostly to originate from multivesicular bodies or to bud from the plasma membrane. Here, we review the convergence of microvesicle biogenesis and aspects of viral assembly and release pathways. Herpesviruses and retroviruses, amongst others, recruit several elements from the microvesicle biogenesis pathways for functional virus release. In addition, noninfectious pleiotropic virus-like vesicles can be released, containing viral and cellular components. We highlight the heterogeneity of microvesicle function during viral infection, addressing microvesicles that can either block or enhance infection, or cause immune dysregulation through bystander action in the immune system. Finally, endogenous retrovirus and retrotransposon elements deposited in our genomes millions of years ago can be released from cells within microvesicles, suggestive of a viral origin of the microvesicle system or perhaps of an evolutionary conserved system of virus-vesicle codependence. More research is needed to further elucidate the complex function of the various microvesicles produced during viral infection, possibly revealing new therapeutic intervention strategies.
- Published
- 2012
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37. Untethering the Nuclear Envelope and Cytoskeleton: Biologically Distinct Dystonias Arising from a Common Cellular Dysfunction
- Author
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Nadia A. Atai, Scott D. Ryan, Rashmi Kothary, Xandra O. Breakefield, and Flávia C. Nery
- Subjects
Cytology ,QH573-671 - Abstract
Most cases of early onset DYT1 dystonia in humans are caused by a GAG deletion in the TOR1A gene leading to loss of a glutamic acid (ΔE) in the torsinA protein, which underlies a movement disorder associated with neuronal dysfunction without apparent neurodegeneration. Mutation/deletion of the gene (Dst) encoding dystonin in mice results in a dystonic movement disorder termed dystonia musculorum, which resembles aspects of dystonia in humans. While torsinA and dystonin proteins do not share modular domain architecture, they participate in a similar function by modulating a structural link between the nuclear envelope and the cytoskeleton in neuronal cells. We suggest that through a shared interaction with the nuclear envelope protein nesprin-3α, torsinA and the neuronal dystonin-a2 isoform comprise a bridge complex between the outer nuclear membrane and the cytoskeleton, which is critical for some aspects of neuronal development and function. Elucidation of the overlapping roles of torsinA and dystonin-a2 in nuclear/endoplasmic reticulum dynamics should provide insights into the cellular mechanisms underlying the dystonic phenotype.
- Published
- 2012
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38. Correction: , the Ortholog of the Early-Onset Dystonia (), Plays a Novel Role in Dopamine Metabolism.
- Author
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Noriko Wakabayashi-Ito, Olugbenga M. Doherty, Hideaki Moriyama, Xandra O. Breakefield, James F. Gusella, Janis M. O'Donnell, and Naoto Ito
- Subjects
Medicine ,Science - Published
- 2011
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39. Engineered EVs designed to target diseases of the CNS
- Author
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Lisa Nieland, Shadi Mahjoum, Emily Grandell, Koen Breyne, and Xandra O. Breakefield
- Subjects
Pharmaceutical Science - Published
- 2023
40. Extracellular communication between brain cells through functional transfer of Cre mRNA mediated by extracellular vesicles
- Author
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David Rufino-Ramos, Kevin Leandro, Pedro R.L. Perdigão, Killian O'Brien, Maria Manuel Pinto, Magda M. Santana, Thomas S. van Solinge, Shadi Mahjoum, Xandra O. Breakefield, Koen Breyne, and Luís Pereira de Almeida
- Subjects
Pharmacology ,Drug Discovery ,Genetics ,Molecular Medicine ,Molecular Biology - Published
- 2023
41. Comparison of Therapeutic Efficacy of Gene Therapy for Tuberous Sclerosis Type 2 with Standard of Care Everolimus in Preclinical Model (S14.002)
- Author
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Edwina Abou Haidar, Shilpa Prabhakar, Pike See Cheah, Sevda Lule, Roberta L. Beauchamp, Akiko Yoshinaga, Alexandra L. Geffrey, Anat Stemmer-Rachamimov, Vijaya Ramesh, Casey A. Maguire, and Xandra O. Breakefield
- Published
- 2023
42. Supplementary Figure Legend from Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity
- Author
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Agnieszka Bronisz, Jakub Godlewski, E. Antonio Chiocca, Xandra O. Breakefield, Ralph Weissleder, Al Charest, Ichiro Nakano, Arun K. Rooj, Marco Mineo, and Franz Ricklefs
- Abstract
Figure Legend to all supplementary data
- Published
- 2023
43. Data from Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity
- Author
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Agnieszka Bronisz, Jakub Godlewski, E. Antonio Chiocca, Xandra O. Breakefield, Ralph Weissleder, Al Charest, Ichiro Nakano, Arun K. Rooj, Marco Mineo, and Franz Ricklefs
- Abstract
A lack of experimental models of tumor heterogeneity limits our knowledge of the complex subpopulation dynamics within the tumor ecosystem. In high-grade gliomas (HGG), distinct hierarchical cell populations arise from different glioma stem-like cell (GSC) subpopulations. Extracellular vesicles (EV) shed by cells may serve as conduits of genetic and signaling communications; however, little is known about how HGG heterogeneity may impact EV content and activity. In this study, we performed a proteomic analysis of EVs isolated from patient-derived GSC of either proneural or mesenchymal subtypes. EV signatures were heterogeneous, but reflected the molecular make-up of the GSC and consistently clustered into the two subtypes. EV-borne protein cargos transferred between proneural and mesenchymal GSC increased protumorigenic behaviors in vitro and in vivo. Clinically, analyses of HGG patient data from the The Cancer Genome Atlas database revealed that proneural tumors with mesenchymal EV signatures or mesenchymal tumors with proneural EV signatures were both associated with worse outcomes, suggesting influences by the proportion of tumor cells of varying subtypes in tumors. Collectively, our findings illuminate the heterogeneity among tumor EVs and the complexity of HGG heterogeneity, which these EVs help to maintain. Cancer Res; 76(10); 2876–81. ©2016 AACR.
- Published
- 2023
44. Supplementary TABLE 2 from Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity
- Author
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Agnieszka Bronisz, Jakub Godlewski, E. Antonio Chiocca, Xandra O. Breakefield, Ralph Weissleder, Al Charest, Ichiro Nakano, Arun K. Rooj, Marco Mineo, and Franz Ricklefs
- Abstract
Genes coding for protein sets that vary coherently between GSC subtypes within 89- protein signature, were retrieved from TCGA GBM dataset and identified by subtype prediction.
- Published
- 2023
45. Supplementary Figure 4 from Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity
- Author
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Agnieszka Bronisz, Jakub Godlewski, E. Antonio Chiocca, Xandra O. Breakefield, Ralph Weissleder, Al Charest, Ichiro Nakano, Arun K. Rooj, Marco Mineo, and Franz Ricklefs
- Abstract
Supplementary Figure 4 related to Figure 4. GSC EV proteome predicts patients' outcome
- Published
- 2023
46. Supplementary TABLE 1 from Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity
- Author
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Agnieszka Bronisz, Jakub Godlewski, E. Antonio Chiocca, Xandra O. Breakefield, Ralph Weissleder, Al Charest, Ichiro Nakano, Arun K. Rooj, Marco Mineo, and Franz Ricklefs
- Abstract
List of the 89 proteins from GSC EV proteome profile distinguishing P from M GSC subtypes; signal intensity
- Published
- 2023
47. Supplementary video from Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity
- Author
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Agnieszka Bronisz, Jakub Godlewski, E. Antonio Chiocca, Xandra O. Breakefield, Ralph Weissleder, Al Charest, Ichiro Nakano, Arun K. Rooj, Marco Mineo, and Franz Ricklefs
- Abstract
M and P GSCs display distinct intra-spherical organization in vitro. Representative time lapse movie
- Published
- 2023
48. Supplementary Figure 1 from Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity
- Author
-
Agnieszka Bronisz, Jakub Godlewski, E. Antonio Chiocca, Xandra O. Breakefield, Ralph Weissleder, Al Charest, Ichiro Nakano, Arun K. Rooj, Marco Mineo, and Franz Ricklefs
- Abstract
Supplementary Figure 1 related to Figure 1. Analysis of GSC EV proteome heterogeneity reveals coherent molecular modules.
- Published
- 2023
49. Supplementary Figure 3 from Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity
- Author
-
Agnieszka Bronisz, Jakub Godlewski, E. Antonio Chiocca, Xandra O. Breakefield, Ralph Weissleder, Al Charest, Ichiro Nakano, Arun K. Rooj, Marco Mineo, and Franz Ricklefs
- Abstract
Supplementary Figure 3 related to Figure 3. Presence of P GSC supports pro-oncogenic traits of M GSCs in vivo
- Published
- 2023
50. Supplementary Figure 2 from Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity
- Author
-
Agnieszka Bronisz, Jakub Godlewski, E. Antonio Chiocca, Xandra O. Breakefield, Ralph Weissleder, Al Charest, Ichiro Nakano, Arun K. Rooj, Marco Mineo, and Franz Ricklefs
- Abstract
Supplementary Figure 2 related to Figure 2. Presence of EVM supports pro-oncogenic traits of P GSCs in vitro.
- Published
- 2023
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