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000285655 1001_ $$0P:(DE-He78)a23e88cc676489fe05be8c178ceaf58e$$aSelt, Florian$$b0$$eFirst author$$udkfz
000285655 245__ $$aGeneration of patient-derived pediatric pilocytic astrocytoma in-vitro models using SV40 large T: evaluation of a modeling workflow.
000285655 260__ $$aDordrecht [u.a.]$$bSpringer Science + Business Media B.V$$c2023
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000285655 500__ $$a#EA:B310#LA:B310# / 2023 Dec;165(3):467-478
000285655 520__ $$aAlthough pediatric low-grade gliomas (pLGG) are the most common pediatric brain tumors, patient-derived cell lines reflecting pLGG biology in culture are scarce. This also applies to the most common pLGG subtype pilocytic astrocytoma (PA). Conventional cell culture approaches adapted from higher-grade tumors fail in PA due to oncogene-induced senescence (OIS) driving tumor cells into arrest. Here, we describe a PA modeling workflow using the Simian Virus large T antigen (SV40-TAg) to circumvent OIS.18 pLGG tissue samples (17 (94%) histological and/or molecular diagnosis PA) were mechanically dissociated. Tumor cell positive-selection using A2B5 was perfomed in 8/18 (44%) cases. All primary cell suspensions were seeded in Neural Stem Cell Medium (NSM) and Astrocyte Basal Medium (ABM). Resulting short-term cultures were infected with SV40-TAg lentivirus. Detection of tumor specific alterations (BRAF-duplication and BRAF V600E-mutation) by digital droplet PCR (ddPCR) at defined time points allowed for determination of tumor cell fraction (TCF) and evaluation of the workflow. DNA-methylation profiling and gene-panel sequencing were used for molecular profiling of primary samples.Primary cell suspensions had a mean TCF of 55% (+/- 23% (SD)). No sample in NSM (0/18) and ten samples in ABM (10/18) were successfully transduced. Three of these ten (30%) converted into long-term pLGG cell lines (TCF 100%), while TCF declined to 0% (outgrowth of microenvironmental cells) in 7/10 (70%) cultures. Young patient age was associated with successful model establishment.A subset of primary PA cultures can be converted into long-term cell lines using SV40-TAg depending on sample intrinsic (patient age) and extrinsic workflow-related (e.g. type of medium, successful transduction) parameters. Careful monitoring of sample-intrinsic and extrinsic factors optimizes the process.
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000285655 650_7 $$2Other$$aCircumvention of OIS
000285655 650_7 $$2Other$$aIn-vitro models
000285655 650_7 $$2Other$$aInducible SV40 large T
000285655 650_7 $$2Other$$aPediatric low-grade glioma cell lines
000285655 650_7 $$2Other$$aPilocytic astrocytoma
000285655 7001_ $$aEl Damaty, Ahmed$$b1
000285655 7001_ $$aSchuhmann, Martin U$$b2
000285655 7001_ $$0P:(DE-He78)a5e60710c7515b3e1de74ced6928a9dd$$aSigaud, Romain$$b3$$udkfz
000285655 7001_ $$0P:(DE-He78)3de637452ba900e2bdd359b8f41953bf$$aEcker, Jonas$$b4$$udkfz
000285655 7001_ $$0P:(DE-He78)8aad075b17d93a5636a34942bdbd7ee6$$aSievers, Philipp$$b5$$udkfz
000285655 7001_ $$0P:(DE-He78)4679059a19b1f3cd341bd27d0ddb7c45$$aKocher, Daniela$$b6$$udkfz
000285655 7001_ $$aHerold-Mende, Christel$$b7
000285655 7001_ $$0P:(DE-He78)908367a659dea9e28dac34592b3c46e5$$aOehme, Ina$$b8$$udkfz
000285655 7001_ $$0P:(DE-He78)a8a10626a848d31e70cfd96a133cc144$$avon Deimling, Andreas$$b9$$udkfz
000285655 7001_ $$0P:(DE-He78)f746aa965c4e1af518b016de3aaff5d9$$aPfister, Stefan$$b10$$udkfz
000285655 7001_ $$0P:(DE-He78)a1f4b408b9155beb2a8f7cba4d04fe88$$aSahm, Felix$$b11$$udkfz
000285655 7001_ $$0P:(DE-He78)551bb92841f634070997aa168d818492$$aJones, David$$b12$$udkfz
000285655 7001_ $$0P:(DE-He78)143af26de9d57bf624771616318aaf7c$$aWitt, Olaf$$b13$$udkfz
000285655 7001_ $$0P:(DE-He78)0be2f86573954f87e97f8a4dbb05cb0f$$aMilde, Till$$b14$$eLast author$$udkfz
000285655 773__ $$0PERI:(DE-600)2007293-4$$a10.1007/s11060-023-04500-6$$n3$$p467-478$$tJournal of neuro-oncology$$v165$$x0167-594X$$y2023
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