Generation of patient-derived pediatric pilocytic astrocytoma in-vitro models using SV40 large T: evaluation of a modeling workflow.
Circumvention of OIS
In-vitro models
Inducible SV40 large T
Pediatric low-grade glioma cell lines
Pilocytic astrocytoma
Journal
Journal of neuro-oncology
ISSN: 1573-7373
Titre abrégé: J Neurooncol
Pays: United States
ID NLM: 8309335
Informations de publication
Date de publication:
24 Nov 2023
24 Nov 2023
Historique:
received:
27
09
2023
accepted:
02
11
2023
medline:
24
11
2023
pubmed:
24
11
2023
entrez:
24
11
2023
Statut:
aheadofprint
Résumé
Although pediatric low-grade gliomas (pLGG) are the most common pediatric brain tumors, patient-derived cell lines reflecting pLGG biology in culture are scarce. This also applies to the most common pLGG subtype pilocytic astrocytoma (PA). Conventional cell culture approaches adapted from higher-grade tumors fail in PA due to oncogene-induced senescence (OIS) driving tumor cells into arrest. Here, we describe a PA modeling workflow using the Simian Virus large T antigen (SV40-TAg) to circumvent OIS. 18 pLGG tissue samples (17 (94%) histological and/or molecular diagnosis PA) were mechanically dissociated. Tumor cell positive-selection using A2B5 was perfomed in 8/18 (44%) cases. All primary cell suspensions were seeded in Neural Stem Cell Medium (NSM) and Astrocyte Basal Medium (ABM). Resulting short-term cultures were infected with SV40-TAg lentivirus. Detection of tumor specific alterations (BRAF-duplication and BRAF V600E-mutation) by digital droplet PCR (ddPCR) at defined time points allowed for determination of tumor cell fraction (TCF) and evaluation of the workflow. DNA-methylation profiling and gene-panel sequencing were used for molecular profiling of primary samples. Primary cell suspensions had a mean TCF of 55% (+/- 23% (SD)). No sample in NSM (0/18) and ten samples in ABM (10/18) were successfully transduced. Three of these ten (30%) converted into long-term pLGG cell lines (TCF 100%), while TCF declined to 0% (outgrowth of microenvironmental cells) in 7/10 (70%) cultures. Young patient age was associated with successful model establishment. A subset of primary PA cultures can be converted into long-term cell lines using SV40-TAg depending on sample intrinsic (patient age) and extrinsic workflow-related (e.g. type of medium, successful transduction) parameters. Careful monitoring of sample-intrinsic and extrinsic factors optimizes the process.
Identifiants
pubmed: 37999877
doi: 10.1007/s11060-023-04500-6
pii: 10.1007/s11060-023-04500-6
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : DKTK German Cancer Consortium (DKTK JF Upgrade Next Gen LOGGIC)
ID : B310-JF-LOGGIC-MDE
Organisme : The Brain Tumour Charity, UK, The Everest Centre for Low-Grade Paediatric Brain Tumours
ID : GN- 370 000707
Informations de copyright
© 2023. The Author(s).
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