Glioblastomas are composed of genetically divergent clones with distinct tumourigenic potential and variable stem cell-associated phenotypes

Abstract

Glioblastoma (GBM) is known to be a heterogeneous disease; however, the genetic composition of the cells within a given tumour is only poorly explored. In the advent of personalised medicine the understanding of intra-tumoural heterogeneity at the cellular and the genetic level is mandatory to improve treatment and clinical outcome. By combining ploidy-based flow sorting with array-comparative genomic hybridization we show that primary GBMs present as either mono- or polygenomic tumours (64 versus 36%, respectively). Monogenomic tumours were limited to a pseudodiploid tumour clone admixed with normal stromal cells, whereas polygenomic tumours contained multiple tumour clones, yet always including a pseudodiploid population. Interestingly, pseudodiploid and aneuploid fractions carried the same aberrations as defined by identical chromosomal breakpoints, suggesting that evolution towards aneuploidy is a late event in GBM development. Interestingly, while clonal heterogeneity could be recapitulated in spheroid-based xenografts, we find that genetically distinct clones displayed different tumourigenic potential. Moreover, we show that putative cancer stem cell markers including CD133, CD15, A2B5 and CD44 were present on genetically distinct tumour cell populations. These data reveal the clonal heterogeneity of GBMs at the level of DNA content, tumourigenic potential and stem cell marker expression, which is likely to impact glioma progression and treatment response. The combined knowledge of intra-tumour heterogeneity at the genetic, cellular and functional level is crucial to assess treatment responses and to design personalized treatment strategies for primary GBM.

Fig. 1

Mono- and polygenomic GBMs revealed by FS-array CGH analysis of GBM biopsies. a DAPI-based ploidy detection in isolated nuclei of GBM patient biopsies revealed inter-tumoural heterogeneity at the ploidy level. Examples of a monogenomic (T159, one G₁/G₀ DNA peak detectable and one G2/M peak) and a polygenomic tumour (T304, two G₁/G₀ DNA peaks, small G2/M peak) are shown. See supplementary Fig. 1a for flow cytometry gating strategies. Assuming that stromal cells were present in each patient biopsy the first peak recognized was considered as the diploid (2N) fraction (blue). Additional aneuploid fractions are shown in red. b Corresponding array CGH profiles of sorted nuclei from distinct DAPI peaks (shown in a) demonstrating the presence of tumour cells with typical GBM aberrations in all fractions (blue diploid, red aneuploid). Distinct populations from polygenomic tumours (T304) carried similar aberrations. Arrows indicate from left to right, +Chr1, ++EGFR, −Chr10, +Chr19, +Chr20. [++, amplification (log² ratio >2); +, gain (log² ratio >0.35); −, loss (log² ratio < −0.35); −, deletion (log² ratio < −3)]. See supplementary Fig. 2 for additional examples. c Summary of GBM biopsies analysed by FS-array CGH and major chromosomal aberrations identified. Monogenomic tumours in ‘blue’, polygenomic tumours in ‘red’. Of 36 GBMs analysed 23 (64 %) were monogenomic and 13 (36 %) were polygenomic. See supplementary Table 1 for additional samples

Fig. 2

Monogenomic tumours always contain an aberrant pseudodiploid population admixed with normal stromal cells, whereas polygenomic tumours additionally contain one or more aneuploid fractions. a Hoechst-based profiling on viable cells from a monogenomic GBM (T251) showing a wide DNA peak (left). Tumour cells were recognized as EGFR⁺ (‘black’) whereas hematopoietic stromal cells were identified as CD45⁺ (‘grey’) (middle panel). Ploidy analysis of EGFR⁺ tumour cells showed a pseudodiploid peak with a significant shift in the DNA content compared with the CD45⁺ diploid control (2.3N versus 2N peak in right panel). See more examples in supplementary Fig. 4a. b Array CGH profile of T251 confirms typical GBM rearrangements, contributing to the pseudodiploid DNA profile. Arrows indicate (from left to right) EGFR amplification on trisomy 7, deletion of CDKN2A/B, monosomy 10, trisomy 19 and 20. Inset shows a detailed view of the EGFR amplicon on chromosome 7. c Array CGH on single nuclei. Single nuclei of a monogenomic GBM (T16) were individually collected from the same DAPI peak (left panel), amplified and probed by array CGH (right panel). 14/16 sorted nuclei were detected as tumour cells (red), 2/16 were stromal cells with no aberrations detected (green). Blue line corresponds to ‘bulk’ tumour nuclei (non amplified DNA from millions of unsorted nuclei). Numbers indicate large chromosomal losses and gains detected only in ‘bulk’ and isolated tumour nuclei. d Hoechst-based profiling on viable cells from a polygenomic GBM (T238) which contained two ploidy peaks (2N and 3.7N, left panel). Ploidy analysis of EGFR⁺ tumour cells (middle panel; ‘black’) versus haematopoietic CD45⁺ stromal cells (‘grey’) confirmed the presence of two distinct tumour clones (2.1N and 3.7N, right panel). e In-depth comparison of two tumour clones within a polygenomic GBM (T176) showing identical chromosomal breakpoints in the two fractions. The MET amplicon and the PTEN homozygous deletion are shown for the 2N (blue) and 3.8N fraction (red). Arrows indicate the borders of the deletion/amplicon, respectively. See supplementary Fig. 3 for more examples. See supplementary Fig. 1 for flow cytometry gating strategies

Fig. 3

Genetic heterogeneity is retained in spheroid-based xenografts. a Monogenomic (T331) and polygenomic (T341) GBMs were used for derivation of spheroid-based xenografts in NOD/Scid mice. In xenografts, tumour nuclei (‘black’) were recognized by human-specific laminA/C positivity (middle panel). DAPI staining of human nuclei in xenografts showed that tumours retained the ploidy detected in the parental biopsy (right). See supplementary Fig. 4c, d and supplementary Table 2 for more examples. b The polygenomic T101 GBM was serially transplanted in NOD/Scid mice (G1 generation 1, G6 generation 6). Diploid (2N, blue) and aneuploid (AN, red) tumour clones detected by nuclear DAPI staining were sorted from the parental biopsy and corresponding xenografts for array CGH analysis. Clones retained their genetic profile upon serial transplantation (see highlighted aberrations). Arrows indicate (from left to right), amplification of EGFR, deletion of CDKN2A/B and amplification of MDM2

Fig. 4

Distinct ploidy-based tumour clones show differential growth characteristics in vivo. a DsRed⁺ polygenomic GBM (T16) established in eGFP⁺ mice was used for Hoechst-based ploidy detection in viable cells and for sorting of distinct tumour clones. See supplementary Fig. 1b for the gating strategy. Tumour cells were recognized as DsRed⁺ eGFP⁻ cells (‘orange’) and gated out from DsRed⁻ eGFP⁺ mouse stromal cells (green) (left panel). Two distinct clones were detected within the tumour population (red peaks in middle panel) in addition to the diploid stromal cells (green). The diploid (2N, blue) and aneuploid (AN, red) tumour cells were sorted (right panel) and used for subsequent analysis. b Both diploid and aneuploid tumour clones formed spheroids in vitro within 10 days of culture. c Representative image of intracranial tumour developed from DsRed⁺ spheroids in eGFP⁺ mice. d Mice implanted with the aneuploid clone (red) died significantly earlier compared with those carrying the diploid clone (blue) or the bulk tumour (orange) (p = 0.006). No significant difference was detected between sorted diploid and unsorted bulk tumour (p = 0.173) (Kaplan–Meier plot, n = 5 per group). e Hoechst-based ploidy analysis of xenografts derived from sorted clones indicating that the implanted clones retained their initial pre-sort ploidy. f Serial transplantation of T16 spheroids of bulk tumour showing an overgrowth of aneuploid cells in late generations. The ratio between diploid and aneuploid cells in the tumour cell compartment is indicated for successive generations (g)

Fig. 5

Genetically divergent clones display variable cancer stem cell-associated markers. a CSC-associated marker expression (CD133, CD15 and A2B5) as determined by flow cytometry in monogenomic GBM xenografts, showing a high variability between different GBMs. Expression (black) is displayed compared with negative controls (grey line). Intra-tumoural heterogeneity in some GBMs is presented as percentage of cells expressing high levels of CSC-associated markers. b Two polygenomic tumours (T16, T238) chosen for Hoechst-based ploidy profiling combined with cell membrane marker expression. c CD56 expression is similar in diploid and aneuploid clones of T16 and T238 GBMs. d Differential expression of CD133, CD15, A2B5 and CD44 in diploid and aneuploidy clones of T16 and T238 GBMs. Intra-tumoural heterogeneity is detected for all markers in the bulk tumour cells (‘black’). The discrimination between diploid (‘blue’) and aneuploid (‘red’) cells showed that marker expression varied between the two clones. The expression profiles and the percentage of positive/strongly positive cells for a given marker are displayed for each clone compared with negative controls (grey line). e The ratio of diploid versus aneuploid cells within a given marker-positive population is indicated, in comparison with the ratio detected in the bulk tumour cells (dotted line), indicating a high variability in marker expression between the genetically distinct clones. See supplementary Fig. 1b for the gating strategy