Generation, characterization and drug sensitivities of twelve patient-derived IDH1 mutant glioma cell cultures

Abstract

Background: Mutations of the isocitrate dehydrogenase (IDH) gene occur in over 80% of low-grade gliomas and secondary glioblastomas. Despite considerable efforts, endogenous in vitro IDH-mutated glioma models remain scarce. Availability of these models is key for the development of new therapeutic interventions. Methods: Cell cultures were established from fresh tumor material and expanded in serum-free culture media. D-2-Hydroxyglutarate levels were determined by mass-spectrometry. Genomic and transcriptomic profiling were carried out on the Illumina Novaseq platform, methylation profiling was performed with the Infinium MethylationEpic BeadChip array. Mitochondrial respiration was measured with the Seahorse XF24 Analyzer. Drug screens were performed with an NIH FDA-approved anti-cancer drug set and two IDH-mutant specific inhibitors. Results: A set of twelve patient-derived IDHmt cell cultures were established. We confirmed high concordance in driver mutations, copy number and methylation profiles between the tumors and derived cultures. Homozygous deletion of CDKN2A/B was observed in all cultures. IDH-mutant cultures had lower mitochondrial reserve capacity. IDH-mutant specific inhibitors did not affect cell viability or global gene expression. Screening of 107 FDA-approved anti-cancer drugs identified nine compounds with potent activity against IDHmt gliomas, including three compounds with favorable pharmacokinetic characteristics for CNS penetration: teniposide, omacetaxine mepesuccinate, and marizomib. Conclusions: Our twelve IDH-mutant cell cultures show high similarity to the parental tissues and offer a unique tool to study the biology and drug sensitivities of high-grade IDHmt gliomas in vitro. Our drug screening studies reveal lack of sensitivity to IDHmt inhibitors, but sensitivity to a set of nine available anti-cancer agents.

3D‐Engineered Scaffolds to Study Microtubes and Localization of Epidermal Growth Factor Receptor in Patient‐Derived Glioma Cells

“…GS-830 and GS-921 are isocitrate dehydrogenase (IDH)wild-type glioblastoma cells, and GS-580 and GS-827 are IDH-mutant astrocytomas. [5,78] EGFR is expressed in both (IDH)-wild-type and (IDH)mutant cell cultures used in the experiments, but EGFR amplification is lost in culture. [78] The use of patient tissue was approved by the Medical Ethical Review Committee Erasmus MC, code MEC-2013-090, and all patients signed informed consent forms according to the guidelines of the Institutional review board.…”

“…The cell culture steps were adopted from a published protocol. [5,78] Unless otherwise specified, 20 ng mL −1 basic fibroblast growth factor, 20 ng mL −1 EGF, and 5 µg mL −1 heparin was used in Dulbecco's modified Eagle medium/Nutrient Mixture F-12 (DMEM/F-12, 11320-033, Gibco, USA) as the cell culture media. Each sample set, containing three scaffolds and four pedestals fabricated on one ITO substrate, was placed in one well of a 6-well plate and washed with ethanol 70% followed by two washes with phosphate buffered saline (PBS).…”

“…[ 1 ] One contributor to the absence of novel effective therapies is the lack of cell culture models that can faithfully capture the native glioma phenotype [ 2,3 ] even despite increasing use of patient‐derived cell cultures (that more accurately capture the cancer cell genotypes compared to standard cell lines). [ 4,5 ] Current in vitro models for example, rapidly lose high copy epidermal growth factor receptor (EGFR) amplification and have limited ability to monitor cells and (microtube‐based) networks. [ 6,7 ] High copy EGFR amplification and microtube‐based networks are two important features that are known to promote glioma progression.…”

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cell genotypes compared to standard cell lines). [4,5] Current in vitro models for example, rapidly lose high copy epidermal growth factor receptor (EGFR) amplification and have limited ability to monitor cells and (microtube-based) networks. [6,7] High copy EGFR amplification and microtube-based networks are two important features that are known to promote glioma progression.

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Successes and challenges in modeling heterogeneous BRAFV600E mutated central nervous system neoplasms

“…Indeed, certain copy number alterations have been associated with robust in vitro growth, including gain of chromosome 7p, and loss of chromosome 10q. Loss of PTEN (126) and CDKN2A loci (127) have been positively associated with the establishment of PDCLs. While high-grade tumors tend to have a higher frequency of establishing cell lines, it is challenging to maintain the phenotypic intra-tumoral heterogeneity that is so characteristic of high-grade CNS tumors; in addition, certain genetic alterations (ATRX, IDH1, and hTert) tend to get lost in cultured cells presumably due to negative selective pressure from culture conditions (128).…”