Abstract
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. 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. Genomic changes in spheroid cultures closely resembled those detected in primary tumors of the corresponding patients. In contrast, genomic changes in serum-grown 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. 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.
Highlights
Glioblastoma spheroid cultures are enriched in tumor stem-like cells and may be more representative of the respective primary tumors than conventional monolayer cultures
We show here that genomic and mRNA profiling of glioblastoma spheroid cultures identified adherens junction-associated protein 1 (AJAP1), epithelial membrane protein 3 (EMP3), and PDPN as aberrantly expressed candidate genes whose protein expression levels, when immunohistochemically assessed in primary glioma tissue samples, turned out as molecular markers that are associated with the overall survival of glioma patients
Glioblastoma spheroid cultures were found to retain genetic hallmarks of human glioblastoma both at the mRNA expression and DNA copy number levels, including for example amplification of the EGFR locus, which is seen in 30% to 40% of primary glioblastoma but rarely conserved in established cell lines grown under standard conditions [19]
Summary
Glioblastoma spheroid cultures are enriched in tumor stem-like cells and may be more representative of the respective primary tumors than conventional monolayer cultures. Immunohistochemistry on tissue microarrays revealed that expression of AJAP1, EMP3, and PDPN was significantly associated with overall survival of astrocytic glioma patients. 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. Glioma-derived primary cultures and established cell lines grown in serum-containing medium are commonly used as in vitro models. We show here that genomic and mRNA profiling of glioblastoma spheroid cultures identified AJAP1, EMP3, and PDPN as aberrantly expressed candidate genes whose protein expression levels, when immunohistochemically assessed in primary glioma tissue samples, turned out as molecular markers that are associated with the overall survival of glioma patients. Such glioma stem cell–like cells display increased tumorigenicity and resistance to radiation treatment both in cell culture and in the brains of immunocompromised mice [5]
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