Abstract
Abstract BACKGROUND: Recent studies suggest that tumor stem cells (TSCs) possess self-renewal property and thus are responsible to tumor growth and resistance to treatments. In this study, we examined how glioblastoma TSCs and non-stem cells respond to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). METHODS AND RESULTS: TSCs-enriched neurospheres (NSCs) were generated from the earlier passages of primary cultures of glioblastomas surgically removed from patients. The NSCs were grown in serum-free medium containing epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2). Immunoflorescent staining of neurospheres revealed the presence of TSCs, as identified by nestin and CD133 positive cells. CD133+ cells were sorted from the NSC with CD133 antibody labeled magnetic microbeads and flow cytometry using fluorochrome-labeled CD133 antibody revealed approximately 7% and 73% CD133+ cells in the NSCs and sorted CD133+ cell preparation. The NSC and CD133+ cell population were characterized for the following TSC properties: the self-renewing capacity was established by neurosphere formation assay; the tumorigenic capacity was demonstrated by xenograft formation in NOD-SCID mouse brain; and the multipotent property was shown in differentiation assay in the generation of neurons as identified by neuronal marker Tuj1 and astrocytes as marked by glial fibrilary acid protein (GFAP). Of 8 sets of NSCs and CD133+ cells examined thus far, one set was sensitive to TRAIL-induced apoptosis, as evident by a bromide-based colorimetric cell viability assay and Caspase-Glo®-8 and −3/7 activity assay. Of the remaining 7 sets of TRAIL resistant NSC and CD133+ cell population, one set showed the lack of expression of caspase-8, as demonstrated by western blot. Fluorescence in situ hybridization (FISH) further revealed the loss of chromosome locus 2q33-34 of caspase-8 gene. In addition, multiple clones were generated from the TRAIL-resistant primary culture in serum culture condition and showed heterogeneity in the expression of caspase-8, as revealed by western blot, and the response to TRAIL-induced apoptosis, as shown by cleavage of caspases. G-banding karyotying analysis of chromosomes revealed heterogeneity of chromosomal alterations among TSC-enriched NSC and CD133+ population and non-stem serum-grown clones. CONCLUSION: This study establishes the heterogeneity of glioblastoma TSC enriched NSC and CD133+ cell population and non-stem serum-grown clones in the genomic status of caspase-8 locus and response to TRAIL-induced apoptosis. Genomic loss of caspase-8 in the stem and non-stem cells results in their resistance to TRAIL treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4269.
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