Myelin-forming cell-specific cadherin-19 is a marker for minimally infiltrative glioblastoma stem-like cells

Abstract

Object: Glioblastoma stem-like cells (GSCs) exhibit stem-like properties, are highly efficient at forming tumor xenografts, and are resistant to many current therapies. Current molecular identifiers of GSCs are scarce and controversial. The authors describe differential cell-surface gene expression profiling to identify GSC-specific markers.

Methods: Independent human GSC lines were isolated and maintained in standard neural stem cell (NSC) media and were validated for self-renewal, multipotent differentiation, and tumor initiation properties. Candidate upregulated GSCspecific plasma membrane markers were identified through differential Affymetrix U133 Plus 2.0 Array gene expression profiling of GSCs, human NSCs (hNSCs), normal brain tissue, and primary/recurrent glioblastoma multiforme samples. Results were validated by using comparative quantitative reverse transcription polymerase chain reaction and Western blot analysis of GSCs, hNSCs, normal human astrocytes, U87 glioma cell line, and patient-matched serum-cultured glioblastoma multiforme samples.

Results: A candidate GSC-specific signature of 19 upregulated known and novel plasma membrane-associated genes was identified. Preferential upregulation of these plasma membrane-linked genes was validated by quantitative polymerase chain reaction. Cadherin-19 (CDH19) protein expression was enhanced in minimally infiltrative GSC lines.

Conclusions: Gene expression profiling of GSCs has shown CDH19 to be an exciting new target for drug development and study of GBM tumorigenesis.

Keywords: ACTB = β-actin; CDH = cadherin; CDH19; CNP = 2′, 3′-cyclic-nucleotide 3′-phosphodiesterase; GBM = glioblastoma multiforme; GPC3 = glypican-3; GPR17 = G protein–coupled receptor 17; GSC = glioblastoma stem-like cell; HLA = human leukocyte antigen; MCPIP = monocyte chemotactic protein 1–induced protein; MHC = major histocompatibility complex; NCBI GEO = National Center for Biotechnology Information Gene Expression Omnibus; NCI REMBRANDT = National Cancer Institute Repository of Molecular Brain Neoplasia Database; NHA = normal human astrocyte; OPC = oligodendrocyte progenitor cell; cadherin-19; gene expression profiling; glioblastoma multiforme; glioblastoma stem-like cells; hNSC = human neural stem cell; oncology; qRT-PCR = quantitative reverse transcription polymerase chain reaction.

Figure 1

Nineteen upregulated plasma membrane GSC-specific transcripts. (A): Logic diagram showing how gene expression profiles were sorted to identify GSC-specific transcripts (*). Normal human brain (n=21), primary GBM (n=21), and recurrent GBM (n=22) gene expression profiles were obtained from NCI REMBRANDT (Table 1). GSCs (n=2) were isolated from two patients at University of Wisconsin. hNSC (n=2) were isolated from two human fetal brains and obtained courtesy of Dr. Clive Svendsen. (B): Heat map with accompanying color key of raw intensity values from gene expression profiles generated from Affymetrix U133 Plus 2.0 arrays and processed with Geospiza GeneSifter (PerkinElmer). Data was filtered for plasma membrane transcripts and gated for GSC values greater than 10-fold change above all other samples. Acronyms correspond to official gene symbols found online at the National Center for Biotechnology Information. Multiple probe sets shown to demonstrate internal reproducibility.

Figure 2

Thirteen downregulated plasma membrane GSC-specific transcripts. Heat map with accompanying color key of raw intensity values from gene expression profiles generated from Affymetrix U133 Plus 2.0 arrays and processed with Geospiza GeneSifter (PerkinElmer). Data was filtered for plasma membrane transcripts and gated for GSC values less than 10-fold change below all other samples. Acronyms correspond to official gene symbols found online at the National Center for Biotechnology Information. Multiple probe sets shown to demonstrate internal reproducibility.

Figure 3

Cadherin-19 is specifically expressed in minimally infiltrative GSCs. (A): Heat map of qRT-PCR validation of all candidate GSC cell surface markers. Additional GSC lines, 33 & 44 GSCs, were evaluated compared to hNSCs, normal human astrocytes (NHA), U87 glioma cells, and patient matched, serum-cultured GBM tumor cells 22T and 33T. Data is representative of two experimental repetitions normalized to hNSC, and reported as a fold change value with accompanying color key. (B): Western blot of cadherin-19 (CDH19) specifically expressed in 12.1 and 22 GSCs. Ms-pAb-anti-CDH19 (Abnova, H00028513-B01P) at 1:250, identified at ∼114k kDa. Rb-pAb-anti-β-actin (Abcam, ab8227) at 1:2000 was used as a loading control.

Figure 4

Cadherin-19 transcript found in olfactory bulb and dorsal root ganglia from human tissue in BioGPS. Compiled from gene expression profiles (probe ID: 206898_at). Accessed June 5, 2013 (http://biogps.org).

Figure 5

Cadherin-19 upregulation in GBM tissue from NCI REMBRANDT shows higher survival probability. (A): Kaplan-Meir survival probability curve for CDH19 (probe ID: 206898_at, highest geometric mean intensity). Upregulation and downregulation was designated at a 2-fold difference in expression. (B): Statistics generated using a log-rank test. Upregulated vs. intermediate group is statistically significant for difference in survival (p=0.03). Accessed June 5, 2013 (https://caintegrator.nci.nih.gov/rembrandt).