Primary glioblastoma cells for precision medicine: a quantitative portrait of genomic (in)stability during the first 30 passages

Abstract

BackgroundPrimary glioblastoma cell (GC) cultures have emerged as a key model in brain tumor research, with the potential to uncover patient-specific differences in therapy response. However, there is limited quantitative information about the stability of such cells during the initial 20–30 passages of culture.MethodsWe interrogated 3 patient-derived GC cultures at dense time intervals during the first 30 passages of culture. Combining state-of-the-art signal processing methods with a mathematical model of growth, we estimated clonal composition, rates of change, affected pathways, and correlations between altered gene dosage and transcription.ResultsWe demonstrate that GC cultures undergo sequential clonal takeovers, observed through variable proportions of specific subchromosomal lesions, variations in aneuploid cell content, and variations in subpopulation cell cycling times. The GC cultures also show significant transcriptional drift in several metabolic and signaling pathways, including ribosomal synthesis, telomere packaging and signaling via the mammalian target of rapamycin, Wnt, and interferon pathways, to a high degree explained by changes in gene dosage. In addition to these adaptations, the cultured GCs showed signs of shifting transcriptional subtype. Compared with chromosomal aberrations and gene expression, DNA methylations remained comparatively stable during passaging, and may be favorable as a biomarker.ConclusionTaken together, GC cultures undergo significant genomic and transcriptional changes that need to be considered in functional experiments and biomarker studies that involve primary glioblastoma cells.

Primary and recurrent glioma patient-derived orthotopic xenografts (PDOX) represent relevant patient avatars for precision medicine

“…These changes are expected in bulk tissue analyses where methylation and transcriptome profiles are biased by TME-derived signals 5,53 . In line with previous reports 26,27 in vitro cell lines showed increased global DNA methylation levels and more profound changes in transcriptomic profiles.…”

“…Some of these shortcomings can be avoided by growing cells in defined serum-free conditions, adapted from neural stem/progenitor cultures 24,25 . These, however, still suffer from a loss of clonal heterogeneity and molecular adaptations to culture conditions 26,27 , in particular loss of focal amplifications 28 . Although patient-derived 3D tumor organoids appear as a robust in vitro alternative, lack of the adequate microenvironment and restricted biological material limit their use 29 .…”

Methods for in vitro modeling of glioma invasion: Choosing tools to meet the need

“…Primary cells can be grown as either nonadherent neurospheres in uncoated plates (Figure 4b) or as adherent cells in laminin or poly-lysine coated plates (Lee et al, 2006). However, with increased passage number (20-30 passages) cultured patient-derived glioma tumor cells exhibit significant genomic and transcriptional changes (Baskaran et al, 2018).…”

Mapping and Quantification of Non-Coding RNA Originating from the rDNA in Human Glioma Cells

“…Sequence alterations in rDNA repeats and their copy number variations have been shown to correlate with high proliferation rates in tumors of different origins as well as high rRNA production and ribosome biosynthesis in cancer cells [24,25]. These observations can also be explained by the increased activity of RNA polymerase I [26], and the fact that rRNA processing and splicing pathways are altered in malignant cells [27]. In addition, human rDNA is known to be regulated epigenetically, and alterations in CpG methylation and histone modification have been shown to upregulate rRNA synthesis in glioblastoma [28,29].…”