Glioblastoma stem-like cells give rise to tumour endothelium

Journal name:
Nature
Volume:
468,
Pages:
829–833
Date published:
DOI:
doi:10.1038/nature09624
Received
Accepted
Published online
Corrected online

Glioblastoma (GBM) is among the most aggressive of human cancers1. A key feature of GBMs is the extensive network of abnormal vasculature characterized by glomeruloid structures and endothelial hyperplasia2. Yet the mechanisms of angiogenesis and the origin of tumour endothelial cells remain poorly defined3, 4, 5. Here we demonstrate that a subpopulation of endothelial cells within glioblastomas harbour the same somatic mutations identified within tumour cells, such as amplification of EGFR and chromosome 7. We additionally demonstrate that the stem-cell-like CD133+ fraction includes a subset of vascular endothelial-cadherin (CD144)-expressing cells that show characteristics of endothelial progenitors capable of maturation into endothelial cells. Extensive in vitro and in vivo lineage analyses, including single cell clonal studies, further show that a subpopulation of the CD133+ stem-like cell fraction is multipotent and capable of differentiation along tumour and endothelial lineages, possibly via an intermediate CD133+/CD144+ progenitor cell. The findings are supported by genetic studies of specific exons selected from The Cancer Genome Atlas6, quantitative FISH and comparative genomic hybridization data that demonstrate identical genomic profiles in the CD133+ tumour cells, their endothelial progenitor derivatives and mature endothelium. Exposure to the clinical anti-angiogenesis agent bevacizumab7 or to a γ-secretase inhibitor8 as well as knockdown shRNA studies demonstrate that blocking VEGF or silencing VEGFR2 inhibits the maturation of tumour endothelial progenitors into endothelium but not the differentiation of CD133+ cells into endothelial progenitors, whereas γ-secretase inhibition or NOTCH1 silencing blocks the transition into endothelial progenitors. These data may provide new perspectives on the mechanisms of failure of anti-angiogenesis inhibitors currently in use. The lineage plasticity and capacity to generate tumour vasculature of the putative cancer stem cells within glioblastoma are novel findings that provide new insight into the biology of gliomas and the definition of cancer stemness, as well as the mechanisms of tumour neo-angiogenesis.

At a glance

Figures

  1. CD105+ endothelial cells in GBM harbour genomic aberrations.
    Figure 1: CD105+ endothelial cells in GBM harbour genomic aberrations.

    a, FACS analysis and quantification of GBM-derived CD105+ cells shows co-expression of other endothelial cell markers (CD31, VEGFR2) and uptake of DiI-AcLDL(n = 3). FITC, fluorescein isothiocyanate; PE, phycoerythrin b, CD105 immunostaining in GBMs delineates microvessels co-labelling with CD31 and glomeruloid vessels surrounded by caldesmon (CALD)-expressing pericytes. c, Functional neovessel formation by GBM-derived CD105+ cells in the flank of NOD-SCID mice. Confocal immunofluorescence demonstrates co-localization of a human mitochondria marker with CD31 and uptake of lectin by the CD105+ vessels in the implants. d, Immuno-FISH of CD105+ vessels in GBM specimens (case 76, 78) shows multiple copies of the EGFR amplicon (arrows). e, FISH on CD105+ cells sorted from GBMs confirms amplification of EGFR (red) and chromosome 7 centromere (Chr7, green) (arrows). Control nuclei, individually contoured, are from normal human fibroblasts. Scale bars, 50μm. Error bars, s.d.

  2. GBM-derived CD133+ cells include a fraction of endothelial progenitors
    Figure 2: GBM-derived CD133+ cells include a fraction of endothelial progenitors

    a, Representative FACS analysis of a GBM specimen with fractionation into four cell subpopulations based on the expression of CD133 and vascular E-cadherin (CD144). b, Immunofluorescence analysis of DP (CD133+/CD144+) cells upon differentiation demonstrates co-expression of endothelial markers and DiI-AcLDL uptake. c, d, In Matrigel, DP cells will exhibit DiI-AcLDL uptake and form tubular networks comparable to those shown by normal endothelial cells, as well as areas of thickened walls where cells are more proliferative. Scale bars, 100μm in b and d; 300μm in c.

  3. CD133+/CD144- cells are multipotential and give rise to endothelial cells via an endothelial progenitor intermediate.
    Figure 3: CD133+/CD144 cells are multipotential and give rise to endothelial cells via an endothelial progenitor intermediate.

    a, b, Co-cultures of CD133+/CD144 cells with tumour cells give rise to endothelial progenitors that co-express CD133 and CD144 (DP) as shown and quantified by FACS analysis (n = 3). APC, allophycocyanin. c, GFP+-derived DP cells form intracellular vacuolar structures in collagen gel, characteristic of endothelial cells. d, Immunohistochemistry of CD133+/CD144-derived endothelial cells (n = 3). e, f, Single cell clonal analysis of GFP-labelled CD133+/CD144 cells. GFP+ clones derived from single cells are seeded under neural or endothelial conditions. Normal endothelial precursor cultures (EPC) and human dermal fibroblasts (HDF) were used as controls. Under endothelial conditions, all cells except HDF express endothelial but not neural markers. Under neural conditions, cells from the same GFP/CD133+ clone are positive for GFAP and nestin but not endothelial markers, while controls are negative for all markers. Scale bar, 50μm. Errors are s.d.

  4. Cancer stem-like cells and endothelial progenitors give rise to tumour and endothelial cells in vivo.
    Figure 4: Cancer stem-like cells and endothelial progenitors give rise to tumour and endothelial cells in vivo.

    a, Representative magnetic resonance imaging (MRI) images from mice that received injection of DN, CD133+/CD144 or DP cells from primary GBM specimens. T2 sequences demonstrate infiltrative tumours except in the DN group. Tumours were hypercellular on haematoxylin and eosin (H&E), showed high proliferation rates (Ki67) and nestin expression. Immunostaining for human-specific CD31 demonstrates the presence of vessels of human origin within the tumours. NA, human nuclear antigen. b, FACS plots (left) and quantitative analysis (right) for endothelial marker expression in xenograft tumours (GFP+/CD133+/CD144 cells) and controls (DN). (n = 3, s.d.). 7-AAD, 7-aminoactinomycin. FL-1 and 2, fluorescent channels 1 and 2; mIgG, mouse immunoglobulin G. c, Xenograft derived GFP+/CD133+/CD144 cells express endothelial markers upon in vitro differentiation (arrows). d, Uptake of systemic lectin in tumour xenografts demonstrates blood vessels that co-label with human endothelial markers (CD31 and CD105). e, Confocal microscopy of xenograft microvasculature. Scale bars, 100μm in a; 50μm in c; 140μm in d; 10μm in e.

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Corrected online 09 December 2010
A definition was completed in the first paragraph of the text.

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Author information

Affiliations

  1. Department of Neurosurgery, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Rong Wang,
    • Urszula Kowalik,
    • Koos E. Hovinga,
    • Adam Geber,
    • Boris Fligelman,
    • Cameron Brennan &
    • Viviane Tabar
  2. Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Rong Wang &
    • Viviane Tabar
  3. Brain Tumor Center, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Rong Wang,
    • Cameron Brennan &
    • Viviane Tabar
  4. Molecular Cytogenetics, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Kalyani Chadalavada &
    • Margaret Leversha
  5. Flow Cytometry Core, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Jennifer Wilshire
  6. Neurosurgical Center Amsterdam, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands

    • Koos E. Hovinga
  7. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Cameron Brennan

Contributions

R.W. and V.T. conceived the project, analysed the data and wrote the manuscript. R.W. and remaining authors performed experiments and analysed data.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Microarray and CGH data are deposited in NCBI's Gene Expression Omnibus (GSE24244, GSE24446, GSE24452, GSE24557 and GSE24558).

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