A CD133-related gene expression signature identifies an aggressive glioblastoma subtype with excessive mutations Xiaowei Yana, Li Maa, Danielle Yia, Jae-geun Yoonb, Alan Diercksa, Gregory Foltza,b, Nathan D. Pricec, Leroy E. Hooda,1, and Qiang Tiana,d,1 a Institute for Systems Biology, Seattle, WA 98103; bSwedish Neuroscience Institute, Seattle, WA 98122; cDepartment of Chemical and Biomolecular Engineering, Institute for Genomic Biology, University of Illinois, Urbana–Champaign, Urbana, IL 61801; and dDepartment of Medicine/Division of Hematology, Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, WA 98195
Contributed by Leroy E. Hood, December 16, 2010 (sent for review November 10, 2010)
molecular profiling
| systems biology
T
he identification of tumor-initiating cancer stem cell (CSC) subpopulations in leukemia (1) and subsequently in breast cancer (2) has led to the hypothesis that tumor proliferation arises largely from these stem/progenitor-like cells and raises the possibility that a similar hypothesis can be applied to a number of solid tumor types (3) including glioblastoma multiforme (GBM) (4), the most deadly form of brain cancer with a diverse cellular phenotype and genetic heterogeneity (5–7). A CD133+ cell population has been isolated from brain as well as other cancers and has been shown to possess stem-cell properties (4, 8), to be more tumorigenic than the CD133− cells in xenografted animal models (4, 8, 9), and to confer radiation resistance (10). Gene-expression signatures derived from both tumorigenic and normal stem cells have been correlated to expression profiles of patient samples in an attempt to stratify these patients according to their disease aggressiveness (presumably more CSCs indicate greater aggressiveness). For instance, an invasiveness gene signature derived from tumorigenic breast CSCs has been related to poor survival in breast cancer patients, suggesting that CSCs may contribute to tumor invasion and metastasis (11). A recent computational analysis linking an ES cell-like gene expression signature to the poorly differentiated states of aggressive human tumors, including breast cancer and GBM, further strengthens the putative stem-cell origins of cancer cell proliferation (12). However, this ES cell signature was unable to stratify the tumor-initiating CSCs from the nontumorigenic cells in breast cancer (11, 12), implying that tissue www.pnas.org/cgi/doi/10.1073/pnas.1018696108
type-specific information embedded in the tumor of origin may be lacking in the ES cell signature. Furthermore, CD133− tumor cells also reportedly are capable of initiating tumors in xenografted animal models (13–15). Thus, the stem-cell origin of cancer, as viewed through the window of CD133+ tumor cell isolation, and the clinical implications remain to be fully elucidated. The Cancer Genome Atlas (TCGA) Network recently characterized genomic abnormalities in more than 200 GBM patients (6, 16), laying the foundation for systematic integration of genetic variations, molecular profiles, and clinical phenotypes. We here established a GBM CD133 gene expression signature and conducted extensive computational analyses to relate it to a wide collection of stem-cell and cancer-cell profiles including the TCGA dataset, providing key initial insights to an understudied subpopulation of cells with critical medical importance. We were able to identify a molecular subtype of GBM reflecting aggressive clinical behavior and hypermutated genetic background, both characteristics of CSCs. Because of the difficulty in obtaining pure CD133+ cells in sufficient volume from patients to perform transcriptomic profiling, no such data linking this key cell population to genomic mutations exist to date. Results Establishment of a GBM CD133+/− Cell Gene Expression Signature. To
explore the underlying molecular difference between phenotypically distinct CD133+ and CD133− subpopulations in GBM and the stem-cell relatedness of each population, we conducted gene expression profiling experiments comparing the two cell populations by using DNA microarrays. Freshly excised GBM tumor samples were enzymatically dispersed into single-cell suspensions and subjected to immunostaining with an anti-CD133 antibody (Miltenyi Biotec Inc.) and FACS analysis (Fig. 1A). Both CD133+ and CD133− cell populations were isolated from each tumor tissue. We processed 21 fresh GBM tumor samples over the course of 18 mo and observed CD133+ populations (>0.05%) in 15 samples. Sufficient total RNA was obtained from five patient samples for DNA microarray analysis. To derive the GBM CD133 gene signature, we first applied a Wilcoxon rank-sum test to the microarray data with a cutoff P value of 0.05; we then retained genes exhibiting at least twofold difference between the CD133+ and the CD133− cells. After we removed lower-abundance genes (those with the sum of all expression values below 10), we obtained a final list consisting of the 214 most differentially expressed genes, which
Author contributions: G.F., L.E.H., and Q.T. designed research; X.Y., L.M., D.Y., J.-g.Y., A.D., and Q.T. performed research; X.Y. contributed new reagents/analytic tools; X.Y., N.D.P., and Q.T. analyzed data; and X.Y., G.F., N.D.P., L.E.H., and Q.T. wrote the paper. The authors declare no conflict of interest. 1
To whom correspondence may be addressed. E-mail:
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This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1018696108/-/DCSupplemental.
PNAS | January 25, 2011 | vol. 108 | no. 4 | 1591–1596
MEDICAL SCIENCES
Cancer cells are heterogeneous and, it has been proposed, fall into at least two classes: the tumor-initiating cancer stem cells (CSC) and the more differentiated tumor cells. The transmembrane protein CD133 has been widely used to isolate putative CSC populations in several cancer types, but its validity as a CSC marker and hence its clinical ramifications remain controversial. Here, we conducted transcriptomic profiling of sorted CD133+ and CD133− cells from human glioblastoma multiforme (GBM) and, by subtractive analysis, established a CD133 gene expression signature composed of 214 differentially expressed genes. Extensive computational comparisons with a compendium of published gene expression profiles reveal that the CD133 gene signature transcriptionally resembles human ES cells and in vitro cultured GBM stem cells, and this signature successfully distinguishes GBM from lower-grade gliomas. More importantly, the CD133 gene signature identifies an aggressive subtype of GBM seen in younger patients with shorter survival who bear excessive genomic mutations as surveyed through the Cancer Genome Atlas Network GBM mutation spectrum. Furthermore, the CD133 gene signature distinguishes higher-grade breast and bladder cancers from their lower-grade counterparts. Our systematic analysis provides molecular and genetic support for the stem cell-like nature of CD133+ cells and an objective means for evaluating cancer aggressiveness.
A
B
CD133 Neg
CD133 Pos
CD133-down
CD133-up
An-CD133
6.84%
Isotype
0.05%
Fig. 1. Isolation of CD133+/− GBM cells and establishment of a CD133 geneexpression signature. (A) Gating used to isolate CD133+/− cells. Because the samples exhibited significant autofluorescence, gating was performed in the 530/30 (FL1) vs. 580/40 (FL2) plane. Gating was set so that
