ABSTRACT
Although mesenchymal stem cells (MSCs) have been implicated as stromal components of several cancers, their ultimate contribution to tumorigenesis and their potential to drive cancer stem cells, particularly in the unique microenvironment of human brain tumors, remain largely undefined. Consequently, using established criteria, we isolated glioma-associated-human MSCs (GA-hMSCs) from fresh human glioma surgical specimens for the first time. We show that these GA-hMSCs are nontumorigenic stromal cells that are phenotypically similar to prototypical bone marrow-MSCs. Low-passage genomic sequencing analyses comparing GA-hMSCs with matched tumor-initiating glioma stem cells (GSCs) suggest that most GA-hMSCs (60%) are normal cells recruited to the tumor (group 1 GA-hMSCs), although, rarely (10%), GA-hMSCs may differentiate directly from GSCs (group 2 GA-hMSCs) or display genetic patterns intermediate between these groups (group 3 GA-hMSCs). Importantly, GA-hMSCs increase proliferation and self-renewal of GSCs in vitro and enhance GSC tumorigenicity and mesenchymal features in vivo, confirming their functional significance within the GSC niche. These effects are mediated by GA-hMSC-secreted interleukin-6, which activates STAT3 in GSCs. Our results establish GA-hMSCs as a potentially new stromal component of gliomas that drives the aggressiveness of GSCs, and point to GA-hMSCs as a novel therapeutic target within gliomas.
Subject(s)
Cell Proliferation , Cytokine Receptor gp130/metabolism , Glioma/metabolism , Interleukin-6/metabolism , Mesenchymal Stem Cells/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Female , Glioma/pathology , Humans , Male , Mesenchymal Stem Cells/pathologyABSTRACT
OBJECT: Apoptosis, a key cellular response to therapeutic agents is often inactivated in tumor cells. In this study, we evaluated the expression of the tumor necrosis family of death receptors, DR4 and DR5, in medulloblastoma tumor samples and cell lines to determine if epigenetic modulation of gene expression could sensitize tumor cell lines to TRAIL-mediated apoptosis. METHODS: Human medulloblastoma samples and cell lines were analyzed for DR4 and DR5 expression by quantitative PCR and immunofluorescence assays. Cell lines with downregulated expression of one or both genes were treated with the histone deacetylase inhibitor, MS-275, and the expression of DR4 and DR5 measured by quantitative PCR, Western blotting, flow cytometry and chromatin immunoprecipitation assays. Induction of apoptosis in the presence of MS-275 was evaluated by TUNEL assay and its ability to augment TRAIL-mediated cytotoxicity was determined by MTT assays, Western blotting and flow cytometry. RESULTS: Compared to normal cerebellum, DR4, but not DR5 expression was consistently downregulated in medulloblastoma tumor samples and in Daoy and D283 cell lines. Interestingly, MS-275 decreased cell growth and induced apoptosis in Daoy and D283 cells. In Daoy cells, this coincided with increased histone H3 and H4 acetylation at the DR4 promoter and enhanced DR4 gene and protein expression as well as elevated Caspase-8 activity. The involvement of DR4 in the cellular response to MS-275 was further confirmed by the observation that knockdown of DR4 and FADD abrogated apoptosis. Further, addition of TRAIL to MS-275 treated cells resulted in an enhancement of apoptosis, suggesting that the upregulated death receptors were functional. CONCLUSION: Our study provides an understanding of the role of DR4 in apoptosis of medulloblastoma cell lines and suggests a potential contribution of aberrant histone deacetylation to the resistance of medulloblastoma cells to therapeutic death.
Subject(s)
Apoptosis/physiology , Cerebellar Neoplasms/metabolism , Medulloblastoma/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Apoptosis/drug effects , Benzamides/pharmacology , Blotting, Western , Caspase 8/metabolism , Cell Line, Tumor , Cerebellar Neoplasms/genetics , Enzyme Inhibitors/pharmacology , Fas-Associated Death Domain Protein/drug effects , Fas-Associated Death Domain Protein/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression , Histone Deacetylase Inhibitors , Humans , Immunoprecipitation , In Situ Nick-End Labeling , Medulloblastoma/genetics , Pyridines/pharmacology , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Reverse Transcriptase Polymerase Chain Reaction , TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/pharmacologyABSTRACT
A new method for preparing cells for microscopic examination is presented in which cell mixtures are fractionated by dielectrophoretic forces and simultaneously collected into characteristic zones on slides. The method traps cells directly from the suspending medium onto the slide, reducing cell loss. Furthermore, it exploits differences in the dielectric properties of the cells, which sensitively reflect their morphology. Because different cell types are trapped in characteristic zones on the slide, the technique represents an advance over existing methods for slide preparation, such as centrifugation and smears where cells are randomly distributed. In particular, the new method should aid in the detection of rare and anomalous cell subpopulations that might otherwise go unnoticed against a high background of normal cells. As well as being suitable for traditional microscopic examination and automated slide scanning approaches, it is compatible with histochemical and immunochemical techniques, as well as emerging molecular and proteomic methods. This paper describes the rationale and design of this so-called electrosmear instrumentation and shows experimental results that verify the theory and applicability of the method with model cell lines and normal peripheral blood subpopulations.
