Pediatric low-grade gliomas: molecular characterization of patient-derived cellular models

Chiacchiarini, Martina; Besharat, Zein Mersini; Carai, Andrea; Miele, Evelina; Baldo, Giada Del; Mastronuzzi, Angela; Catanzaro, Giuseppina; Ferretti, Elisabetta

doi:10.1007/s00381-020-04559-w

Cited by 4 publications

(5 citation statements)

References 20 publications

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“…Unlike any other PA model to date, inducible SV40-TAg models allow to also study the senescent state of the cells after re-induction of OIS. Long-term expandability overcomes the limitation of low proliferation rates and short lifespans observed in other primary modeling approaches [ 21 ]. SV40-TAg PA models are less cost effective compared to e.g.…”

Section: Discussionmentioning

confidence: 99%

Generation of patient-derived pediatric pilocytic astrocytoma in-vitro models using SV40 large T: evaluation of a modeling workflow

Selt,

El Damaty,

Schuhmann

et al. 2023

J Neurooncol

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Purpose 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. Methods 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. Results 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. Conclusion 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.

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Section: Discussionmentioning

confidence: 99%

Generation of patient-derived pediatric pilocytic astrocytoma in-vitro models using SV40 large T: evaluation of a modeling workflow

Selt,

El Damaty,

Schuhmann

et al. 2023

J Neurooncol

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“…Attempts to generate pLGG patient-derived cell lines yielded varying success rates. Most of them could only be maintained as short-term cultures [ ( 52 , 54 , 60 ), Table 2 ] and/or they acquired additional alterations in culture that made them unrepresentative of the original tumors ( 47 , 50 ). Bax et al.…”

Section: In Vitro Models Of Plgg: the Path To Establishing P...mentioning

confidence: 99%

“…As part of the Pediatric Preclinical Testing Program (PPTP), 2 astrocytoma cell lines were generated, BT-35 cells which had 2 to 5 copies of wild-type (WT) BRAF and BT-40 cells which had 5 copies of activated BRAF V600E [ ( 50 , 51 ), Table 2 )]. BT-40 was further characterized as grade 2-3 astrocytoma as these cells lost CDKN2A and wild-type P53 ( 50 , 54 , 61 ).…”

Section: In Vitro Models Of Plgg: the Path To Establishing P...mentioning

confidence: 99%

“…(2021) reported additional pLGG cellular models which included primary cells derived from Grade I pLGG tumors. 3 of 9 tumors were infratentorial PAs, 1 of 9 tumors was infratentorial ganglioglioma, 3 of 9 tumors were supratentorial DNETs, and 1 of 9 was supratentorial angiocentric gliomas, and 1 of 9 was supratentorial ganglioglioma ( 54 ). Although these patient-derived cells also underwent senescence after about 30 days of culture, they were not genetically modified and thus were more representative of the tumors they were derived from [ ( 54 ), Table 2 )].…”

Section: In Vitro Models Of Plgg: the Path To Establishing P...mentioning

confidence: 99%

“…Second, pLGG samples obtained from young children as patients are usually small, hence only small number of cells can be obtained to culture. Third, the intrinsic slow growth and benign behavior of these tumors coupled with OIS made it difficult to grow these tumor cells in vitro ( 48 , 49 , 52 , 54 , 60 ). Several methods were developed to bypass OIS through genetic modifications of the pLGG cells so that they can be propagated long enough in culture ( 53 , 55 ), but these methods might generate in vitro model systems that incompletely reflect the genetic/epigenetic background of the primary tumors.…”

Section: Leveraging the Strengths And Overcoming The Challenges Of Ge...mentioning

confidence: 99%

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Pediatric low-grade glioma models: advances and ongoing challenges

Yvone,

Breunig

2024

Front. Oncol.

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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.

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Evaluating cell culture reliability in pediatric brain tumor primary cells through DNA methylation profiling

Pedace,

Pizzi,

Abballe

et al. 2024

npj Precis. Onc.

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In vitro models of pediatric brain tumors (pBT) are instrumental for better understanding the mechanisms contributing to oncogenesis and testing new therapies; thus, ideally, they should recapitulate the original tumor. We applied DNA methylation (DNAm) and copy number variation (CNV) profiling to characterize 241 pBT samples, including 155 tumors and 86 pBT-derived cell cultures, considering serum vs serum-free conditions, late vs early passages, and dimensionality (2D vs 3D cultures). We performed a t-SNE classification and identified differentially methylated regions in tumors compared to cell models. Early cell cultures recapitulate the original tumor, but serum media and 2D culturing were demonstrated to significantly contribute to the divergence of DNAm profiles from the parental ones. All divergent cells clustered together acquiring a common deregulated epigenetic signature suggesting a shared selective pressure. We identified a set of hypomethylated genes shared among unfaithful cells converging on response to growth factors and migration pathways, such as signaling cascade activation, tissue organization, and cellular migration. In conclusion, DNAm and CNV are informative tools that should be used to assess the recapitulation of pBT-cells from parental tumors.

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Pediatric low-grade gliomas: molecular characterization of patient-derived cellular models

Cited by 4 publications

References 20 publications

Generation of patient-derived pediatric pilocytic astrocytoma in-vitro models using SV40 large T: evaluation of a modeling workflow

Generation of patient-derived pediatric pilocytic astrocytoma in-vitro models using SV40 large T: evaluation of a modeling workflow

Pediatric low-grade glioma models: advances and ongoing challenges

Evaluating cell culture reliability in pediatric brain tumor primary cells through DNA methylation profiling

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