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Article: Pediatric low-grade glioma models: advances and ongoing challenges.

Yvone, Griselda Metta / Breunig, Joshua J

2024 Volume 13, Page(s) 1346949

Abstract: Pediatric low-grade gliomas represent the most common childhood brain tumor class. While often curable, some tumors fail to respond and even successful treatments can have life-long side effects. Many clinical trials are underway for pediatric low-grade ... ...

Abstract	Pediatric low-grade gliomas represent the most common childhood brain tumor class. While often curable, some tumors fail to respond and even successful treatments can have life-long side effects. Many clinical trials are underway for pediatric low-grade gliomas. However, these trials are expensive and challenging to organize due to the heterogeneity of patients and subtypes. Advances in sequencing technologies are helping to mitigate this by revealing the molecular landscapes of mutations in pediatric low-grade glioma. Functionalizing these mutations in the form of preclinical models is the next step in both understanding the disease mechanisms as well as for testing therapeutics. However, such models are often more difficult to generate due to their less proliferative nature, and the heterogeneity of tumor microenvironments, cell(s)-of-origin, and genetic alterations. In this review, we discuss the molecular and genetic alterations and the various preclinical models generated for the different types of pediatric low-grade gliomas. We examined the different preclinical models for pediatric low-grade gliomas, summarizing the scientific advances made to the field and therapeutic implications. We also discuss the advantages and limitations of the various models. This review highlights the importance of preclinical models for pediatric low-grade gliomas while noting the challenges and future directions of these models to improve therapeutic outcomes of pediatric low-grade gliomas.
Language	English
Publishing date	2024-01-22
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2649216-7
ISSN	2234-943X
ISSN	2234-943X
DOI	10.3389/fonc.2023.1346949
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: In vitro

Hatanaka, Emily A / Breunig, Joshua J

Frontiers in oncology

2024 Volume 14, Page(s) 1360358

Abstract: Ependymomas are rare brain tumors that can occur in both children and adults. Subdivided by the tumors' initial location, ependymomas develop in the central nervous system in the supratentorial or infratentorial/posterior fossa region, or the spinal cord. ...

Abstract	Ependymomas are rare brain tumors that can occur in both children and adults. Subdivided by the tumors' initial location, ependymomas develop in the central nervous system in the supratentorial or infratentorial/posterior fossa region, or the spinal cord. Supratentorial ependymomas (ST-EPNs) are predominantly characterized by common driver gene fusions such as
Language	English
Publishing date	2024-02-26
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2649216-7
ISSN	2234-943X
ISSN	2234-943X
DOI	10.3389/fonc.2024.1360358
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures.

Grausam, Katie B / Breunig, Joshua J

Journal of visualized experiments : JoVE

2023 , Issue 196

Abstract: Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent ...

Abstract	Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.
MeSH term(s)	Mice ; Animals ; Neural Stem Cells/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Electroporation/methods ; Plasmids/genetics ; Brain Neoplasms/genetics ; Brain Neoplasms/therapy ; Brain Neoplasms/metabolism ; DNA/genetics ; Mutation
Chemical Substances	RNA, Guide, CRISPR-Cas Systems ; DNA (9007-49-2)
Language	English
Publishing date	2023-06-23
Publishing country	United States
Document type	Journal Article ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/65286
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Article ; Online: Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain.

Linesch, Paul W / Akhtar, Aslam Abbasi / Breunig, Joshua J

Current protocols

2023 Volume 3, Issue 6, Page(s) e792

Abstract: Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline-inducible genetic element of interest (pB-tet-GOI) plasmid system ... ...

Abstract	Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline-inducible genetic element of interest (pB-tet-GOI) plasmid system allows for stable piggyBac transposition-mediated integration into cells, identification of cells that have been transfected using a fluorescent nuclear reporter, and robust transgene activation or suppression upon the addition of doxycycline (dox) to the cell culture or the diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. More recently, we have developed a transgenic system as an alternative to piggyBac called mosaic analysis by dual recombinase-mediated cassette exchange (MADR), as well as additional in vitro transfection techniques and in vivo dox chow applications. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning of respective genetic element of interest (GOI) into response plasmid Basic Protocol 2: In vitro nucleofection of iPSC-derived human/mouse neural progenitor cells and subsequent derivation of stable inducible cell lines Alternate Protocol: In vitro electroporation of iPSC-derived human/mouse neural progenitor cells Support Protocol: Recovery stage after in vitro transfection Basic Protocol 3: Adding doxycycline to cells to induce/reverse GOI Basic Protocol 4: Assessing gene expression in vitro by non-invasive bioluminescence imaging of luciferase activity.
MeSH term(s)	Humans ; Animals ; Mice ; Doxycycline/pharmacology ; Doxycycline/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Genes, Reporter ; Genetic Vectors ; DNA Transposable Elements ; Anti-Bacterial Agents/metabolism ; Tetracycline/pharmacology ; Tetracycline/metabolism ; Luciferases/genetics ; Luciferases/metabolism ; Gene Expression ; Brain ; Mammals/genetics ; Mammals/metabolism
Chemical Substances	Doxycycline (N12000U13O) ; DNA Transposable Elements ; Anti-Bacterial Agents ; Tetracycline (F8VB5M810T) ; Luciferases (EC 1.13.12.-)
Language	English
Publishing date	2023-06-07
Publishing country	United States
Document type	Journal Article
ISSN	2691-1299
ISSN (online)	2691-1299
DOI	10.1002/cpz1.792
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Article ; Online: Murine-Derived Glioma Organoids and Cell Line Culture Systems.

Ayala-Sarmiento, Alberto E / Pacheco, David Rincon Fernandez / Breunig, Joshua J

Current protocols

2023 Volume 3, Issue 2, Page(s) e665

Abstract: Research models in cancer have greatly evolved in the last decade, with the advent of several new methods both in vitro and in vivo. While in vivo models remain the gold standard for preclinical studies, these methods present a series of disadvantages ... ...

Abstract	Research models in cancer have greatly evolved in the last decade, with the advent of several new methods both in vitro and in vivo. While in vivo models remain the gold standard for preclinical studies, these methods present a series of disadvantages such as a high cost and long periods of time to produce results compared with in vitro models. We have previously developed a method named Mosaic Analysis by Dual Recombinase-mediated cassette exchange (MADR) that generates autochthonous gliomas in immunocompetent mice through the transgenesis of personalized driver mutations, which highly mimic the spatial and temporal tumor development of their human counterparts. Due to the control of single-copy expression of transgenes, it allows for comparing the visualization of tumor cells and non-tumor cells. Here we describe a method to generate murine-derived glioma organoids (MGOs) and cell line cultures from these murine models by physical and enzymatic methods for in vitro downstream applications. Tumor cells can be readily distinguished from non-tumor cell populations, in both organoids and monolayer cell cultures, and isolated due to the use of personalized fluorescent reporter transgenes. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D murine-derived glioma organoids Basic Protocol 2: Generation of 2D glioma monolayer cell lines.
MeSH term(s)	Mice ; Humans ; Animals ; Glioma/genetics ; Glioma/pathology ; Cell Line ; Cell Culture Techniques/methods ; Transgenes ; Organoids/pathology
Language	English
Publishing date	2023-02-03
Publishing country	United States
Document type	Journal Article
ISSN	2691-1299
ISSN (online)	2691-1299
DOI	10.1002/cpz1.665
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Article ; Online: De Novo

Ayala-Sarmiento, Alberto E / Kobritz, Naomi / Breunig, Joshua J

STAR protocols

2020 Volume 1, Issue 3, Page(s) 100184

Abstract: Mosaic analysis by dual recombinase-mediated cassette exchange (MADR) is a technology that allows stable and locus-specific integration of transgenic elements into recipient cells carrying loxP and FRT sites. Nevertheless, most cell lines lack these ... ...

Abstract	Mosaic analysis by dual recombinase-mediated cassette exchange (MADR) is a technology that allows stable and locus-specific integration of transgenic elements into recipient cells carrying loxP and FRT sites. Nevertheless, most cell lines lack these recombination-specific sites. This protocol describes a method to introduce the minimum requirements into cells, leading to the generation of
MeSH term(s)	Animals ; Cell Culture Techniques/methods ; Cell Line ; Genetic Loci ; HEK293 Cells ; Humans ; Mice ; Recombinases/metabolism ; Reproducibility of Results ; Transgenes
Chemical Substances	Recombinases
Language	English
Publishing date	2020-11-25
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2020.100184
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Article ; Online: Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain.

Akhtar, Aslam Abbasi / Breunig, Joshua J

Current protocols in stem cell biology

2017 Volume 41, Page(s) 5A.9.1–5A.9.12

Abstract: The pB-tet-GOI plasmid system allows for stable piggyBac transposition-mediated integration into cells, a fluorescent nuclear reporter to identify cells that have been transfected, and robust transgene activation or suppression upon the addition of dox ... ...

Abstract	The pB-tet-GOI plasmid system allows for stable piggyBac transposition-mediated integration into cells, a fluorescent nuclear reporter to identify cells that have been transfected, and robust transgene activation or suppression upon the addition of dox to the cell culture or diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. Specifically, a detailed protocol is provided to illustrate: (1) cloning of the respective GOI (genetic element(s) of interest); (2) nucleofection of the plasmid system into human induced pluripotent stem cell (iPSC)-derived neural progenitors; (3) dox-induced activation in vitro or in vivo; and (4) non-invasive assessment of gene activity in vivo by bioluminescence imaging. © 2017 by John Wiley & Sons, Inc.
Language	English
Publishing date	2017-05-16
Publishing country	United States
Document type	Journal Article
ISSN	1938-8969
ISSN (online)	1938-8969
DOI	10.1002/cpsc.28
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Article: In vivo discovery of RNA proximal proteins via proximity-dependent biotinylation

Lin, Xianzhi / Fonseca, Marcos A. S. / Breunig, Joshua J. / Corona, Rosario I. / Lawrenson, Kate

RNA biology. 2021 Dec. 02, v. 18, no. 12

2021

Abstract: RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely ... ...

Abstract	RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins in vivo via proximity-based biotinylation. RPL applied to U1 identified proteins involved in both U1 canonical and noncanonical functions. Profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs and provided additional evidence for 5ʹ-3ʹ proximity and unexplored subcellular localizations of poly(A)⁺ RNA. Our results suggest that RPL allows rapid identification of target RNA binding proteins in native cellular contexts, and is expected to pave the way for discovery of novel RNA–protein interactions important for health and disease.
Keywords	CRISPR-Cas systems ; ascorbate peroxidase ; biotinylation ; gene expression ; genome
Language	English
Dates of publication	2021-1202
Size	p. 2203-2217.
Publishing place	Taylor & Francis
Document type	Article
ISSN	1555-8584
DOI	10.1080/15476286.2021.1917215
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Preparation, Assembly, and Transduction of Transgenic Elements Using Mosaic Analysis with Dual Recombinases (MADR).

Rincon Fernandez Pacheco, David / Sabet, Sara / Breunig, Joshua J

STAR protocols

2020 Volume 1, Issue 3, Page(s) 100199

Abstract: This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES ... ...

Abstract	This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES elements. Additionally, we describe how to generate a neural stem cell culture from Gt(ROSA)26Sort
MeSH term(s)	Animals ; Animals, Genetically Modified ; Electroporation ; Genetic Engineering/methods ; Genetic Vectors/chemistry ; Genetic Vectors/genetics ; Green Fluorescent Proteins/genetics ; Luminescent Proteins/genetics ; Mice ; Plasmids/genetics ; Recombinases/genetics ; Recombinases/metabolism ; Transfection ; Red Fluorescent Protein
Chemical Substances	Luminescent Proteins ; Recombinases ; Green Fluorescent Proteins (147336-22-9)
Language	English
Publishing date	2020-12-09
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2020.100199
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Article ; Online: RELA Fusion-Positive Ependymoma in a Child with Down Syndrome: A Case Report.

Chan, Julie L / Kashanian, Alon / Bannykh, Serguei I / Majlessipour, Fataneh / Breunig, Joshua J / Danielpour, Moise

Pediatric neurosurgery

2021 Volume 56, Issue 2, Page(s) 146–151

Abstract: Introduction: Down syndrome (DS) is the most common multiple malformation syndrome in humans and is associated with an increased risk of childhood malignancy, particularly leukemia. Incidence of brain tumors in patients with DS is limited to sporadic ... ...

Abstract	Introduction: Down syndrome (DS) is the most common multiple malformation syndrome in humans and is associated with an increased risk of childhood malignancy, particularly leukemia. Incidence of brain tumors in patients with DS is limited to sporadic cases. We report the first case of a RELA fusion-positive ependymoma in a 3-year-old boy with DS. Case presentation: Imaging prompted by new left-sided hemiparesis demonstrated an 8-cm hemorrhagic right temporal-parietal mass. Subsequent image-complete resection confirmed a RELA fusion-positive anaplastic ependymoma with 90% OLIG2 staining. Postoperatively, the patient, unfortunately, experienced fatal recurrence and drop metastases with leptomeningeal involvement. Conclusion: To our knowledge, this is the first reported case of a confirmed RELA fusion-positive ependymoma in a child with DS. We discuss this finding in the context of intracranial tumors in children with DS, as well as the finding of 90% positive OLIG2 expression and its potential as a prognostic marker.
MeSH term(s)	Brain Neoplasms/diagnostic imaging ; Brain Neoplasms/genetics ; Brain Neoplasms/surgery ; Child ; Child, Preschool ; Down Syndrome/complications ; Ependymoma/complications ; Ependymoma/diagnostic imaging ; Ependymoma/genetics ; Glioma ; Humans ; Male ; Supratentorial Neoplasms ; Transcription Factor RelA
Chemical Substances	RELA protein, human ; Transcription Factor RelA
Language	English
Publishing date	2021-03-09
Publishing country	Switzerland
Document type	Case Reports
ZDB-ID	1091757-3
ISSN	1423-0305 ; 1016-2291
ISSN (online)	1423-0305
ISSN	1016-2291
DOI	10.1159/000511673
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