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. 2018 Aug;24(8):1204-1215.
doi: 10.1038/s41591-018-0086-7. Epub 2018 Jul 2.

Functional Diversity and Cooperativity Between Subclonal Populations of Pediatric Glioblastoma and Diffuse Intrinsic Pontine Glioma Cells

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Free PMC article

Functional Diversity and Cooperativity Between Subclonal Populations of Pediatric Glioblastoma and Diffuse Intrinsic Pontine Glioma Cells

Mara Vinci et al. Nat Med. .
Free PMC article

Abstract

The failure to develop effective therapies for pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively assess the extent to which this was present in these tumors through subclonal genomic analyses and to determine whether distinct tumor subpopulations may interact to promote tumorigenesis by generating subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced tumors revealed multiple tumor subclones, spatially and temporally coexisting in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations that we propose cooperate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, abrogate DNA repair and confer increased invasion and migration on neighboring cells, in vitro and in vivo, through chemokine signaling and modulation of integrins. These data indicate that even rare tumor subpopulations may exert profound effects on tumorigenesis as a whole and may represent a new avenue for therapeutic development. Unraveling the mechanisms of subclonal diversity and communication in pGBM and DIPG will be an important step toward overcoming barriers to effective treatments.

Conflict of interest statement

Competing Financial Interests Statement

The authors have no conflicts of interest pertaining to this manuscript.

Figures

Figure 1
Figure 1. Paediatric GBM and DIPG harbour a complex subclonal architecture.
(A) Inferred heterogeneity. Representative images (from n=142) of six cases of pGBM and DIPG from different anatomical locations and with different histone H3 mutation status. For each case, a CIRCOS plot is given highlighting somatic SNVs and InDels (outer ring), DNA copy number changes (red=gain, blue=loss) and loss of heterozygosity (yellow) on the inner rings, and intra-/inter-chromosomal translocations inside the circle (orange). The CCF for each somatic coding mutation is plotted as a histogram with a kernel density overplotted. In all cases, in addition to a peak of mutations present in 100% cells (clonal) there is a complex pattern of subclonal mutations (<95% CCF) forming multiple peaks at low frequencies within a given tumour. (B) Gene-level clonality. Violin plot of CCFs for a given series of gene mutations across all 142 independent cases of pGBM and DIPG (H3.3 G34R/V, n=10; H3.3 K27M, n=61; H3.1 K27M, n=23; ATRX, n=22; NF1, n=4; ACVR1, n=27; TP53, n=; ATM, n=5; PIK3R1, n=8; PPM1D, n=11; PDGFRA, n=7; BRAF, n=5; PIK3CA, n=15). The shaded area represents a CCF of 95-100% to indicate a clonal mutation. Purported drivers such as histone H3 mutations, ATRX and NF1 are almost wholly found to be clonal (though there are single outliers in some instances). Other genes such as PIK3CA, BRAF and PDGFRA are frequently found to be mutated in smaller subclonal compartments of the tumours. Kernel densities of CCFs are plotted for all samples harbouring a given mutation (number of independent cases listed on figure). (C) Subclonal architecture. The number of subclones present in 142 pGBM and DIPG is calculated from somatic mutation data using the EXPANDS package, and ordered first by the number of subclones (coloured using a rainbow palette) and then by the proportion of the tumour defined by the major clone in each tumour. A single case was found to be clonal, with more than 85% cases harbouring between 3-10 subclones. (D) Mutational burden. Dotplot of the number of somatic coding SNVs (y axis) against the number of subclones (x axis), demonstrating a significant positive relationship (Pearson r2=0.2188, p=4.36x10-9, n=142 independent samples). The horizontal bar represents the median value. Individual tumours are coloured by their histone H3 mutation status, with outliers often seen to harbour H3.3 G34R (blue). (E) Clinical and molecular correlates of subclonal numbers. Boxplots highlighting no differences in the number of subclones on the basis of anatomical location, but an increased number in H3.3 G34R tumours (p=0.044, t-test), and a reduced number in infant cases (<3 years, p=0.0108, t-test) across all n=142 independent samples. The thick line within the box is the median, the lower and upper limits of the boxes represent the first and third quartiles, the whiskers 1.5x the interquartile range, and individual points outliers. (F) Prognostic implications. Kaplan-Meier curves demonstrating H3.3 G34R tumours have a longer overall survival than other pGBM and DIPG (p=3.94x10-6, log-rank test), however despite the association of this subgroup with an increased number of tumour subclones, an elevated subclonal diversity shows a clear trend towards shorter survival across all pGBM and DIPG (p=0.068, log-rank test). Comparisons we made including all n=142 independent samples. * p<0.05. **p<0.01.
Figure 2
Figure 2. DIPGs infiltrate the brain through branching evolution and genotypic convergence.
(A) Multi-region sampling. Thirteen different tumour-harbouring regions of HSJD-DIPG-010 were sampled post-mortem, from within and outside the pons. Scale bar = 100μm. (B) Exome sequencing was carried out for all regions, with CCFs plotted as a heatmap for all variants found in at least one specimen, with anatomical location highlighted and colour-coded. (C) Phylogenetic trees were reconstructed using neighbour-joining algorithms based upon the nested subpopulation phylogenies calculated as part of EXPANDS, with clearly evident laterally-directed evolution and early escape from the pons of tumour cells found in distinct anatomical sites. (D-F) Eight different tumour-harbouring regions of HSJD-DIPG-014 subjected to the same analysis. (G-I) Eight different tumour-harbouring regions of HSJD-DIPG-015 subjected to the same analysis. Scale bar = 100μm.m
Figure 3
Figure 3. Isolation of genotypically and phenotypically diverse single stem-like cell-derived subclones of paediatric GBM and DIPG.
(A) Isolation of subclonal populations. Experimental schema for disaggregation of heterogeneous mixtures of patient-derived tumour cells, flow sorting into single cells in 96-well plates, and allowing colonies to form as either 2D cultures adherent on laminin, or 3D neurospheres, all under stem cell conditions. Individual subclonal colonies are subjected to high-throughput phenotypic analysis and targeted resequencing, and further cultured for detailed in vitro and in vivo mechanistic comparison with heterogeneous bulk populations. (B) Clonogenicity. Percentage of single cells which formed colonies under 2D laminin and 3D neurosphere stem cell conditions are given for six pGBM and DIPG primary patient-derived cell cultures, labelled by anatomical location and histone H3 mutation subgroup. (C) 3D neurosphere culture from single cell-derived colonies from SU-DIPG-VI assessed by Celigo S imaging cytometer. (D) Growth of single cell-derived colonies over time, assessed as diammeter of neurosphere, labelled and colour-coded. (E) Targeted sequencing contingency plot of somatic mutations identified as common to all subclones (blue), shared amongst certain subclones (yellow) and private to individuals (red). (F) 2D laminin culture from single cell-derived colonies from SU-DIPG-VI assessed by Celigo S imaging cytometer. (G) Growth of single cell-derived colonies over time, assessed as diammeter of neurosphere, with subclones taken for later analysis highlighted: A-D10 (fast, purple), A-B8 (intermediate, pink) and A-E6 (slow, violet). (H) Targeted sequencing contingency plot of somatic mutations identified as common to all subclones (blue), shared amongst certain subclones (yellow) and private to individuals (red). Gene names are coloured to highlight private mutations in selected subclones, or common to A-D10 and A-B8 (brown). (I) Growth. Time-course for growth of selected subclones re-plated and grown over 160 hours, highlighting statistically significant differences among subclones and heterogeneous bulk cell populations of SU-DIPG-VI (blue). Representative images at 72 hours are provided from the Celigo S cytometer, with tumour cells marked in green. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. (J) Invasion. Time-course of invasion of cells into a matrigel matrix over 72 hours, either as percentage of the total area in the field of view covered by invading cells, or as a percentage of time zero. Representative images given at 72 hours, with extent of tumour cell invasion marked in green. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. (K) Migration. Time-course of tumour cell migration onto matrigel over 72 hours, either as percentage of the total area of the well covered by migrating cells, or as a percentage of time zero. Representative images given at 72 hours, with extent of tumour cell migration marked in green. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. * p<0.05. **p<0.01. ***p<0.001. All graphs represent mean +/- standard deviation.
Figure 4
Figure 4. Rare DIPG subclones with pathogenic somatic variants driving the cellular phenotype.
(A) Targeted sequencing. Contingency plot of common (blue), shared (yellow) and private (red) somatic mutations in single cell-derived neurospheres from primary patient-derived cell culture HSJD-DIPG-007. NS-F10 is the only subclone to harbour a mutation in KMT5B. (B) Pile-up representation of sequencing reads aligning to the KMT5B locus at 11q13.2. The R187* (c.559G>A) variant is highlighted in red (boxed for clarity) and is present in 2/678 reads of original heterogeneous sample. Cartoon representation of mutations identified in HSJD-DIPG-007 (c.559G>A, R187*, present in 0.47% total reactions by ddPCR) and MCGL-PGBM18 (c.2095G>A, R699*, present in 12.2% total reads by exome sequencing). Amino acid position labelled, and SET domain coloured blue. (C) Digital droplet PCR. Plot of assay for KMT5B wild-type (x axes) and R187* mutation (y axes) for normal human astrocytes, heterogeneous bulk cells, and subclones NS-F10 and NS-F8. Mutant reads are present in 49.77% droplets from NS-F10, equating to 99.64% cells harbouring a heterozygous mutation. They are absent from astrocytes and NS-F8, though are found in 0.48% droplets from the original bulk preparation. Taken from n=3 independent experiments. (D) Immunofluorescence. Heterogeneous bulk HSJD-DIPG-007 cells and subclones were stained using an antibody directed against H4K20me2 (green), or total H4 (red), with nuclei stained with DAPI (blue). Reduced expression of H4K20me2 is observed in KMT5B mutant NS-F10 cells. Representative images taken from n=3 independent experiments. Scale bar = 50μm. (E) PARP inhibition. Effect on cell viability (surviving fraction on y axes) of treatment of heterogeneous bulk cells and subclones with increasing concentrations of two different PARP inhibitors (x axes, log10 scale). ANOVA was used to test for significance of NS-F10 versus NS-F8 and HSJD-DIPG-007 bulk culture for talazoparib and olaparib. *** all p values <0.001. Data derived from n=3 independent experiments. (F) RNAseq. Heatmap of gene expression analysis from RNA sequencing data highlighting differential expression in KMT5B mutant NS-F10 subclones compared to wild-type NS-F8. The most highly elevated genes included a range of extracellular matrix remodellers (represented in gene set enrichment analysis by the gene set “BOWIE RESPONSE TO EXTRACELLULAR MATRIX”) and numerous secreted chemokines (gene set “REACTOME CHEMOKINE RECEPTORS BIND CHEMOKINES”). KMT5B itself is also differentially expressed. All cell preparations were sequenced n=1, and statistical comparisons made by Gene Set Enrichment Analysis using the Kolmogorov-Smirnov test (p) with multiple correction testing using the False Discovery Rate (q). (G) Immunofluorescence of bulk HSJD-DIPG-007 cells and subclones stained using an antibody directed against alpha-5 integrin (red). Nuclei are stained with DAPI (blue). Immunohistochemistry of embedded bulk HSJD-DIPG-007 cells and subclones stained using an antibody directed against alpha-5 integrin, and counterstained with haematoxylin. Representative images taken from n=3 independent experiments. Scale bar = 50μm. (H) Growth. Neurosphere growth of HSJD-DIPG-007 and derived subclones seeded with different cell densities showing significantly elevated growth in the heterogeneous bulk cells, but not among subclones. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. (I) Invasion. Time-course of tumour cell invasion into matrigel over 72 hours, as a percentage of time zero using the Celigo S cytometer. Representative images given at 72 hours, with extent of tumour cell invasion marked in green. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. (J) Migration. Time-course of tumour cell migration onto a fibronectin matrix over 72 hours, as a percentage of time zero using the Celigo S cytometer. Representative images given at 72 hours, with extent of tumour cell migration marked in green. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. (K) Migration in response to stimulation with either conditioned media from HSJD-DIPG-007 heterogeneous bulk cells, or the chemokines CCL2 and CXCL2. Values are given as a percentage of unstimulated cells at 24 hours using the Celigo S cytometer. Representative images are given, with extent of tumour cell migration marked in green. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. All comparisons carried out by ANOVA, * p<0.05. **p<0.01. ***p<0.001. All graphs represent mean +/- standard deviation.
Figure 5
Figure 5. Distinct infiltrative phenotypes of genotypically divergent DIPG subclones in vivo.
(A) Tumour burden and infiltration. Heterogeneous HSJD-DIPG-007 bulk cells and NS-F10 and NS-F8 subclones were implanted directly into the pons of NOD-SCID mice and tumours allowed to form over 8 months. At weeks 23/24, bulk cells and NS-F10 formed diffusely infiltrating tumours throughout the brain, as seen by haematoxylin and eosin staining as well as immunohistochemistry with anti-human nuclei antigen (HNA), whereas NS-F8 had formed considerably less infiltrative lesions even at 30 weeks. Representative images from a total of n=4 mice per group. Scale bar = 1000μm (inset scale bar = 50μm). (B) Survival. Tumour-bearing animals implanted with NS-F8 subclones had significantly longer survival than heterogeneous HSJD-DIPG-007 bulk cells and NS-F10 (p=0.0236, log-rank test, n=4 mice per group). * p<0.05. (C) Digital droplet PCR. Plot of assay for KMT5B wild-type (x axes) and R187* mutation (y axes) for normal human astrocytes and tumours from mice implanted with heterogeneous bulk cells, and subclones NS-F10 and NS-F8. Mutant reads are present in 51.33% droplets from NS-F10 and 0.23% droplets from the original bulk preparation. Taken from n=3 independent experiments. (D) Tumour burden and infiltration. Heterogeneous SU-DIPG-VI bulk cells and A-D10 and A-E6 subclones were implanted directly into the pons of nude mice and tumours allowed to form over 8 months. At week 10, bulk cells and A-D10 formed highly cellular, infiltrating tumours, as seen by haematoxylin and eosin staining as well as immunohistochemistry with anti-HNA, whereas A-E6 had formed considerably less cellular lesions even at 14 weeks. Representative images from a total of n=8 mice per group. Scale bar = 1000μm (inset scale bar = 50μm). (E) Survival. Tumour-bearing animals implanted with A-E6 subclones had significantly longer survival than heterogeneous SU-DIPG-VI bulk cells and A-D10 (p=0.037, log-rank test, n=8 mice per group).
Figure 6
Figure 6. DIPG subclones co-operate to enhance tumorigenic phenotypes
Individual subclones of SU-DIPG-VI (A-D) and HSJD-DIPG-007 (E-H) were differentially labelled and cultured either as pure populations or mixed in equal ratios. (A) Growth of co-cultured (yellow) and mono-cultured E6 (green) and D10 (red) cells plated as single neurospheres after 96 hours, measured as diammeter of the sphere, with representative images provided from the Celigo S cytometer under phase contrast and fluorescence. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. (B) Invasion of co-cultured (yellow) and mono-cultured E6 (green) and D10 (red) into matrigel over 72 hours, with area assessed by ImageJ software from representative images provided from the Celigo S cytometer under phase contrast and fluorescence. Co-cultures and D10 have significantly enhanced invasive capabilities compared to E6. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. (C) Migration of mono- and co-cultured E6 (green) and D10 (red) on matrigel, assessed by the number of differentially labelled distant cells at 24 hours, with representative images provided from the IncuCyte Zoom live-cell analysis system under phase contrast and fluorescence. Cells from individual subclones have enhanced migratory properties when cultured together compared to alone. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. (D) Confocal microscopy analysis of invasion of mono- and co-cultured E6 (green) and D10 (red) into matrigel after 4 days, with nuclei stained with DAPI. Poorly motile E6 cells are found to invade further and in greater numbers alongside D10 cells than when cultured alone. Representative images taken from n=3 independent experiments. Scale bar = 200μm. (E) Growth of co-cultured (yellow) and mono-cultured NS-F8 (green) and NS-F10 (red) cells plated as single neurospheres after 96 hours, measured as diammeter of the sphere, with representative images provided from the Celigo S cytometer under phase contrast and fluorescence. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. (F) Invasion of co-cultured (yellow) and mono-cultured NS-F8 (green) and NS-F10 (red) into matrigel over 72 hours, with area assessed by ImageJ software from representative images provided from the Celigo S cytometer under phase contrast and fluorescence. Co-cultures and NS-F10 have significantly enhanced invasive capabilities compared to NS-F8. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. (G) Migration of mono- and co-cultured NS-F8 (green) and NS-F10 (red) on fibronectin, assessed by the number of differentially labelled distant cells at 48 hours, with representative images provided from the IncuCyte Zoom live-cell analysis system under phase contrast and fluorescence. Cells from NS-F8 have enhanced migratory properties when cultured with NS-F10 compared to alone. Data derived and representative images taken from n=3 independent experiments. Scale bar = 500μm. (H) Confocal microscopy analysis of migration of mono- and co-cultured NS-F8 (green) and NS-F10 (red) on fibronectin after 3 days, with nuclei stained with DAPI. Poorly motile NS-F8 cells are found to migrate further and in greater numbers alongside NS-F10 cells than when cultured alone. Representative images taken from n=3 independent experiments. Scale bar = 200μm. All comparisons carried out by ANOVA, * p<0.05. **p<0.01. ***p<0.001. All graphs represent mean +/- standard deviation.

Comment in

  • Subclones work together.
    Seton-Rogers S. Seton-Rogers S. Nat Rev Cancer. 2018 Sep;18(9):530-531. doi: 10.1038/s41568-018-0047-y. Nat Rev Cancer. 2018. PMID: 30061713 No abstract available.

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