Genomic and Expression Profiling of Glioblastoma Stem Cell–Like Spheroid Cultures Identifies Novel Tumor-Relevant Genes Associated with Survival

Abstract

Purpose: Glioblastoma spheroid cultures are enriched in tumor stem-like cells and therefore may be more representative of the respective primary tumors than conventional monolayer cultures. We exploited the glioma spheroid culture model to find novel tumor-relevant genes. Experimental Design: We carried out array-based comparative genomic hybridization of spheroid cultures derived from 20 glioblastomas. Microarray-based gene expression analysis was applied to determine genes with differential expression compared with normal brain tissue and to nonneoplastic brain spheroids in glioma spheroid cultures. The protein expression levels of three candidates were determined by immunohistochemistry on tissue microarrays and correlated with clinical outcome. Functional analysis of PDPN was done. Results: Genomic changes in spheroid cultures closely resembled those detected in primary tumors of the corresponding patients. In contrast, genomic changes in serumgrown monolayer cultures established from the same patients did not match well with the respective primary tumors. Microarray-based gene expression analysis of glioblastoma spheroid cultures identified a set of novel candidate genes being upregulated or downregulated relative to normal brain. Quantitative real-time PCR analyses of 8 selected candidate genes in 20 clinical glioblastoma samples validated the microarray findings. Immunohistochemistry on tissue microarrays revealed that expression of AJAP1, EMP3, and PDPN was significantly associated with overall survival of astrocytic glioma patients. Invasive capacity and RhoA activity were decreased in PDPN-silenced spheroids. Conclusion: We identified a set of novel candidate genes that likely play a role in glioblastoma pathogenesis and implicate AJAP1, EMP3, and PDPN as molecular markers associated with the clinical outcome of glioma patients. (Clin Cancer Res 2009;15(21):6541-50) Glioblastoma is the most common and most malignant primary brain tumor and has one of the worst survival rates among all human cancers. Despite aggressive multimodal treatment, the median survival time after diagnosis has improved only marginally and is still <1 year in population-based studies (1). A better understanding of the complex molecular and cellular mechanisms leading to glioblastoma is an important prerequisite to

Tumor-infiltrating lymphocytes (TILs) from patients with glioma

“…39 Tumor cells with "stem cell like properties" have been identified in cancer and "stem cell-associated targets," including CD133, may represent clinically relevant TAA in some brain tumors [40][41][42] leading to target-specific T-cell responses. 43 A dense infiltration of CD8 C TIL into CNS tumors has been reported to be correlated with increased survival 44 making TIL infusion an attractive therapy option for adoptive cellular therapy.…”

Stem cell characteristics in glioblastoma are maintained by the ecto-nucleotidase E-NPP1

“…Phenotypic and genotypic characterisations of the GSC lines were carried out in previous studies. 16,52 Patient characteristics of all samples used for RNA expression analysis are summarised in Supplementary Table S5. For passaging and plating, NCH421k and NCH441 cells were dissociated using accutase (Sigma-Aldrich, St. Louis, MO, USA) and NCH644 cells by the use of a trypsin-EDTA solution (Life Technologies, Darmstadt, Germany).…”

Transient treatment with epigenetic modifiers yields stable neuroblastoma stem cells resembling aggressive large-cell neuroblastomas

“…SKNAS iCSCs expressed elevated levels of genes involved in stem cell pathways (NOTCH/DELTA, BMPR/BMP, and FZD/WNT) and genes highly expressed in other CSC types. These include ALDH1B1 (colon carcinoma (20); BIN1, COL1A1, COL1A2, ENPP2, and THY1 (breast CSC-like cells (21); and PDPN (glioblastoma sphere cultures) (22) (Fig. S5 A and B).…”