Project description:Analysis of Hoechst 33342 dye-effluxing side population cells (SP cells defined as glioma stem cells, GSCs) and dye-retaining main population cells (MP cells defined as non-GSCs) that were FACS-sorted from the C6 glioma cell line stably expressing EGFP (C6-eGFP). ECM-related genes, such as Col4a1 and Col4a2, and the iron carrier gene Tf are upregulated in MP cells. Results provide the insight into molecular basis underlying the maintenance of GSCs by non-GSCs. Gene expression profiles were compared between SP and MP cells just after FACS-sorting from the whole C6-eGFP cells based on their Hoechst-effluxing abilities.
Project description:Analysis of Hoechst 33342 dye-effluxing side population cells (SP cells defined as glioma stem cells, GSCs) and dye-retaining main population cells (MP cells defined as non-GSCs) that were FACS-sorted from the C6 glioma cell line stably expressing EGFP (C6-eGFP). ECM-related genes, such as Col4a1 and Col4a2, and the iron carrier gene Tf are upregulated in MP cells. Results provide the insight into molecular basis underlying the maintenance of GSCs by non-GSCs.
Project description:Glioblastomas show heterogeneous histological features. These distinct phenotypic states are thought to be associated with the presence of glioma stem cells (GSCs), which are highly tumorigenic and self-renewing sub-population of tumor cells that have different functional characteristics. To investigate Glioblastomas (GBMs) show heterogeneous histological features. These distinct phenotypic states are thought to be originated by the glioma stem cells (GSCs), which are highly tumorigenic and self-renewing sub-population of tumor cells. Notch signaling has been shown to be important for maintenance of GSC population both in vitro and in vivo. A recent study showed that NOTCH -triggered oncogenic activity can be due to not only its ability to regulate coding genes but also long non-coding RNAs (lncRNAs). Here, we investigated the molecular effects of lncRNA in GSC, whose expression is induced by Notch signaling. We searched for downregulated lncRNAs in GSCs by treatment of small interfering RNA (siRNA) targeting Notch1 and JAG1 (si-Notch1 and si-JAG1).
Project description:Abnormal activation of stemness factors is a crucial signature of cancer stem cells (CSCs), a highly tumorigenic subpopulation in malignant tumors. However, it is unclear whether multi-signaling pathways are activated in CSCs, as like normal stem cells. I would like to report that an inhibitor of differentiation 1 (ID1) activates intracellular multi-signaling involved in proliferation, genesis, and maintenance of glioma stem cells (GSCs) by suppression of Cullin3, an E3 ubiquitin ligase that degrades Cyclin E and components of SHH and WNT signaling. ID1 inhibits BMP-dependent differentiation of GSCs by activation of BMPR2-targeting miR17/20a. ID1HIGH-Cullin3LOW signature correlates with a poor prognosis of GBM patients with a significant association to gene signatures enriched in EGF, WNT, SHH, and BMP signaling. Combinational inhibition of GSC intracellular multi-signaling network increases tumor-bearing mice survival. These results provide insights on molecular and cellular basis of GSC biology, and also suggest necessity of multi-signaling inhibition for GSCs therapy. Two human primary glioma stem cells (GSCs) such as GSC2 and GSC8 were isolated from two individual primary human glioma specimens. The GSCs were directly transfected with pSuper-GFP-ID1-shRNA and pSuper-GFP-Scrambled-shRNA using FuGENE 6 reagent (Roche). The RNA extraction in these cells was used to analyze gene expression.
Project description:he unique prolyl isomerase Pin1 that promotes protein conformational changes is a pivotal therapeutic target in many cancers, but little is known about the regulation of Pin1 protein stability. We previously found that Pin1 was highly expressed in glioma stem cells (GSCs) relative to non-stem tumor cells (NSTCs). Interestingly, treatment of the proteasome inhibitor MG132 markedly restored Pin1 protein expression in NSTCs to a level similar to GSCs. We therefore sought to identify the molecular regulators controlling the proteasomal degradation of Pin1 by mass spectrometry analysis of Pin1-interacting proteins in GSCs. The results showed that Pin1 interacts with a deubiquitinase USP34 in GSCs, suggesting that Pin1 may be stabilized by USP34.
Project description:Understanding the glioma stem cell (GSC) heterogeneity within IDH1 and TP53 mutant tumors may elucidate possible targets for astrocytoma treatment. We performed single-nucleus transcriptomics of 6 mutant and wild-type glioma samples sorted for Sox2 stem cell marker. Malignant states of different clusters were evaluated by the expression of the normal and hematopoietic markers. We found that mutant GSCs were characterized by collagen synthesis and CD44-high phenotype prone to migration, while wild-type GSCs had vulnerability points in ATP synthesis. Additionally, mutant GSCs displayed altered lipogenesis probably attributed to the low NADPH consumed by mutant IDH1. The collagen and lipid biosynthesis represent possible target pathways for prospective tackling GSCs with IDH1 R132H and TP53 point mutations, uncouplers of oxidative phosphorylation are promising to address wild-type GSC proliferation
Project description:Glioblastomas show heterogeneous histological features. These distinct phenotypic states are thought to be associated with the presence of glioma stem cells (GSCs), which are highly tumorigenic and self-renewing sub-population of tumor cells that have different functional characteristics. To investigate gene expression including lncRNA (long non-coding RNA) in GSC, we have performed high-throughput RNA-sequencing (RNA-seq) experiment using Illumina GAIIx.
Project description:Glioblastoma is a malignant brain tumor that is highly resistant to radiation and chemotherapy, where patients survive on average only 15 months after diagnosis. Furthering the understanding of mechanisms leading to radiation resistance of glioma is paramount to identify novel therapeutic targets. Previous studies have shown that glioma stem cells (GSCs) play an important role in promoting radiation resistance and disease recurrence. Herein we analyze the proteomic alterations occurring in patient-derived GSCs upon radiation treatment in order to identify molecular drivers of resistance. We show that proteome changes upon radiation accurately predict the resistance status of the cells, and that resistance to radiation does not correlate with glioma transcriptional subtypes. We further show that the radio-resistant cell line GSC-267 sheds microvesicles (MVs) enriched in the metabolic enzyme nicotinamide phosphoribosyltransferase (NAMPT).
Project description:Glioblastoma is the most malignant brain tumor in adults and is associated with poor survival despite multimodal treatments. Glioma stem cells (GSCs) are cells with properties reminiscent from the normal neural stem cells counterparts. They have been postulated to be the culprit of glioma chemo- and radio-resistance ultimately leading to relapse. Understanding the molecular circuits governing the GSC compartment is essential. Sox2, a critical transcription regulator of embryonic and neural stem cell function, is deregulated in GSCs, but the description of the precise molecular pathways regulated by this gene in GSCs remains poorly understood. Results: We performed a genome-wide analysis of Sox2-regulated transcripts in GSCs, using microarray. We identified a total of 135 differentially expressed coding transcripts and 261 non-coding transcripts (B value > 0). Cell adhesion and cell-cell signaling are among the most enriched terms using GO classification. The pathways altered after Sox2 down-modulation includes multiple cellular processes such as aminoacid metabolism and intercellular signaling cascades. We validate the top 5 down regulated coding genes and one of the top 5 up-regulated coding genes. We also defined and classify the set of non-coding transcripts differentially expressed regulated by Sox2 in GSCs, and validated two of them. Conclusions: We present a comprehensive analysis of the transcriptome controlled by Sox2 in GSCs, gaining insights in the understanding of the potential roles of Sox2 in glioblastoma.
Project description:Glioblastoma stem cells (GSCs) have been reported to be found near the blood vessels. We wanted to identify the perivascular GSCs by combining the label retention approach with vascular perfusion. GSCs, like most of the stem cells, are in a quiescent state. Label retention approach involves staining the cells with a uniform membrane labeling dye (PKH26) and allowing them to grow in vivo in NOD SCID mice. Slow-dividing stem cells will retain the dye to a greater extent and perivascular cells are identified by injecting the mice with vascular perfusion dye (Hoechst 33342) 5 min prior to sacrifice. Perivascular GSCs (positive for Hoechst 33342 and pKH26 dye - dubbed as H+P+) were compared with the rest of the perivascular glioma cells (H+P-).