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  • br Results and Discussion Using the

    2018-11-02


    Results and Discussion Using the neurosphere protocol (Piccirillo et al., 2009), we established stable patient-derived cell cultures from dissociated tumor tissue from 12 cases of primary GBM (Table S1 available online). In order to exclude the possibility of in vitro aberrations and culture selection, as soon as the primary include formed neurospheres, they were dissociated into single cells and used for intracerebral transplantation (and retransplantation) into NOD-SCID mice. We used high-resolution SNP arrays performed on DNA extracted from the primary GBM tumor cells to identify “driver” copy number alterations (CNAs), defined as recurrent regions of amplification or deletion (Table S2). FISH probes were designed for selected (preferably focal) CNAs, and three-color FISH was applied to neurosphere cells as well as cells isolated from tumors after secondary transplantation in NOD-SCID mice. We then compared the subclonal genetic architecture and clonal phylogenies of the neurospheres and the tumors generated in the mice. In all cases, the driver CNAs chosen from analysis of tumor DNA were present in the neurospheres and subclones that were present in the mouse xenografts could be backtracked to the original tumor, confirming the validity of this approach for investigating clonal progression. A schematic overview of the workflow is given in Figure S1. Ten of the original 12 GBM neurosphere cultures resulted in tumors in the mice (Table S3). Four of these (GBM 2, GBM 5, GBM 8, and GBM 11) had at least three “driver” lesions that could be tracked by FISH in both the neurospheres and secondary xenografts in the mice. The remaining cases were not included because they had fewer than three “trackable” lesions by SNP array (GBM 3 and GBM 6), because various aneuploid conditions were observed in the derived neurosphere cell line (GBM 1) (Table S2), or because there were too few cells for FISH at secondary transplantation (GBM 4, GBM 7, and GBM 9). In all four cases studied by multicolor FISH, there was genetic heterogeneity in the neurosphere cells, and each case showed a unique, branched phylogenetic architecture. In each case, more than one subclone was capable of propagating tumors in secondary transplanted mice (Figures 1, 2, 3, and 4). Analyses of clonal architecture by multicolor FISH for CNAs inevitably underestimate the extent of clonal diversity (Anderson et al., 2011). In GBM 5, we had sufficient material for a more detailed genetic analysis. The SNP arrays of primary tumor GBM 5 revealed high-level, focal amplification of EGFR; homozygous loss of CDKN2A (one large deletion and one small focal deletion); and loss of TP53 due to a deletion of 17p (Figure S2). We observed seven subclones in the neurosphere cells and a branching subclonal structure. The major clone in the neurospheres had high-level focal amplification of EGFR, heterozygous TP53, and homozygous CDKN2A loss. Only two subclones read out in the secondary transplant tumor cells, and include both had high-level EGFR amplification and homozygous CDKN2A loss. In contrast to the neurospheres, the major clone in the secondary mouse xenograft had two copies of TP53. We performed mutation screening of the TP53 gene by capillary electrophoresis single-strand conformation analysis in primary patient tumor DNA, followed by Sanger sequencing to characterize any mobility shifts thus identified. This revealed a mutation in exon 5: c.454C > T: p.152S. The same TP53 mutation was also found in xenograft cells after secondary transplantation (mouse 1 and mouse 3). Both wild-type and mutated TP53 sequences were present in the tumor DNA, but only the TP53 mutated sequence was present in the xenograft DNA, indicating that the mutation was present in all subclones of the mouse xenografts. In order to investigate this further, we carried out whole-exome sequencing and single-cell analysis for the simultaneous occurrence of CNAs and selected single-nucleotide variants (SNVs) in this case. For the latter, we used multiplex-targeted DNA amplification of flow-sorted single cells followed by high-throughput quantitative PCR (qPCR) using the BioMark HD microfluidic platform (Potter et al., 2013).