Glioma Stem Cells Population Research Articles

Identification of a peptide that interacts with Nestin protein expressed in brain cancer stem cells

Glioma stem cells (GSCs) are presumably major culprits for brain tumor initiation, progression, and recurrence after conventional therapies. Thus, selective targeting and eradication of GSCs may provide a promising and effective therapeutic approach. Here, we isolated a GSC-targeting (GSCT) peptide that demonstrated selective binding affinity for many undifferentiated GSCs using in vitro phage display technology. This GSCT peptide binds to isotypes of Nestin proteins specifically expressed in GSCs, enabling it to target Nestin-positive cells in human glioblastoma tissues. In human glioblastoma tissue specimens, the fluorescence-conjugated GSCT peptide could visualize putative GSC populations, showing its possible use as a diagnostic agent. GSCT peptide is also internalized into undifferentiated GSCs specifically in vitro, and moreover, intravenously injected GSCT peptide effectively penetrated into tissues, specifically accumulated in gliomas that arise from subcutaneous and orthotopic implantation, and predominantly targeted Nestin-positive cells in these tumors. Thus, our GSCT peptide may be useful for the development of more promising therapeutic and diagnostic modalities that target GSCs in brain tumors.

Temozolomide decreases invasion of glioma stem cells by down-regulating TGF-β2

Gliomas are characterized by excessive proliferation, diffuse infiltration and immunosuppression. Recent studies implicate a key role for a restricted population of glioma stem cells (GSCs) in glioma invasive growth and recurrence. Transforming growth factor (TGF)-β2 is a mediator of immunosuppression associated with malignant glioma and also influences pro-invasive functions. Temozolomide (TMZ), is a new alkylating agent with promising antitumour efficacy for malignant gliomas, and the effect of TMZ on GSCs invasion has not been known. To address this issue, we developed studies aimed at neurospheres from primary cultured glioma cells, due to the fact that since neurospheres can be enriched in GSCs, we could examine whether TMZ inhibits the invasion of GSCs. TMZ reduced the TGF-β2-mediated invasion, and down-regulated TGF-β2 expression at the mRNA and protein levels. Thus, these results indicate that TMZ, as a chemotherapeutic agent, can reduce the invasion of GSCs and their immunosuppressive activity. TMZ may be used as an immunomodulating agent for glioma therapy.

HMSC-mediated Concurrent Delivery of Endostatin and Carboxylesterase to Mouse Xenografts Suppresses Glioma Initiation and Recurrence

Glioma stem cells (GSCs) are known to be maintained within a "vascular niche"; thereby, disruption of this microenvironment using antiangiogenesis agents is a promising therapeutic modality. However, this regimen leads to treatment failure and tumor recurrence in patients with glioblastoma multiforme (GBM). Therefore, more effective therapeutic approaches that can eradicate GSCs and the bulk tumors are needed. Toward this goal, we examined the antitumor effects of an antiangiogenesis approach combined with conventional chemotherapy on suppressing glioma xenograft growth. We established three genetically engineered mesenchymal stem cell (MSC) lines (GE-AF-MSCs) by stably transducing the gene encoding endostatin (an antiangiogenesis factor), the gene encoding secretable form of carboxylesterase 2 (sCE2, a prodrug-activating enzyme), or a mixture of both genes. Among the three GE-AF-MSC cell lines, injection of amniotic fluid (AF)-MSCs-endostatin-sCE2 cells into U87MG-EGFRvIII-driven orthotopic brain tumor and postsurgery tumor recurrence models, and subsequent CPT11 treatment yielded the strongest antitumor responses, including diminished angiogenesis, increased cell death, and a reduced Nestin-positive GSC population. Therefore, our antitumor strategy provides a novel basis for designing stem cell-mediated therapeutic approaches to target and eradicate GSCs and the bulk tumors.

Abstract 3393: Extrinsic uPA initiates glioma stem cell transformation under hypoxic conditions via Lhx-2

Abstract Emerging evidence indicate that the capacity of a tumor to grow and propagate resides primarily in a small population of tumor cells, which are known as cancer stem cells. The origin of these cancer stem cells remains controversial. Hypoxia promotes tumor progression through multiple mechanisms, including modification of angiogenesis, metabolic switches and invasion. Researchers have demonstrated that hypoxia is one of the factors necessary for neural stem cell tropism to glioma. Although hypoxia has been shown to maintain multiple normal stem cell populations, its role in cancer stem cells is fairly unknown. Recently, researchers have identified hypoxia as a critical microenvironmental factor in regulating self-renewal of cancer stem cells, which is partially mediated by enhancing the activity of stem cell factors such as Oct4, c-Myc and Nanog. In this study, we show that under hypoxic conditions, urokinase plasminogen activator (uPA) is one of the factors necessary for the maintenance and transformation of glioma cells to glioma stem cells. Glioma cells grown under hypoxic conditions showed an increase in expression of stem cell markers Oct-4, c-Myc, CD133 and Stro-1. Supplementing normoxic or hypoxic cells with external uPA increased Lhx-2 expression and was not influenced by the presence or absence of native uPA. Hypoxic cells down regulated for Lhx-2 did not show activation of stem cell marker proteins Oct-4, c-Myc, CD133 and Stro-1. Hypoxic cells subcutaneously implanted into nude mice developed at least 2-fold larger tumors when compared to normoxic cells. Intratumoral injection of plasmids expressing siRNA targeting uPA retarded tumor development in both normoxic and hypoxic tumors. In contrast, injection of plasmids expressing siRNA targeting Lhx-2 retarded hypoxic tumors with reduced effect on normoxic tumors. In conclusion, our results demonstrate that a hypoxic environment in a high uPA environment can induce glioma stem cell transformation mediated by Lhx-2 and suppression of uPA and Lhx-2 can specifically target glioma stem cell populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3393. doi:10.1158/1538-7445.AM2011-3393

The different HMGA1 expression of total population of glioblastoma cell line U251 and glioma stem cells isolated from U251

The high-mobility group A1 (HMGA1) protein is a non-histone architectural nuclear factor and participates in diverse biological processes, including gene transcription, embryogenesis, cell cycle regulation, apoptosis, and even neoplastic transformation. In our study, glioma stem cells (GSCs) expressing the surface marker CD133 from human glioblastoma cell line U251 were isolated using MACS column and were analyzed using immunofluorescence and flow cytometry (FCM). The different expression of HMGA1 was detected using real-time RT–PCR and Western blot at transcriptional and translational levels between U251 and isolated GSCs. The results show that GSCs were successfully isolated from U251 and cultured in serum-free medium (SMF). The percentage of GSCs in U251 was 0.32%±0.07%. HMGA1 expression was significantly higher in GSCs than in glioblastoma cells (P<0.05), up to 6.13±0.25-fold and 2.75±0.99-fold at transcriptional and translational levels, respectively. These results indicated HMGA1 is overexpressed in GSCs as compared to glioblastoma cell line U251, which points to the expression of HMGA1 being closely related to malignant proliferation, invasion, and differentiation of tumors from the prospective of tumor stem cells (TSCs). We conclude that HMGA1 may be a potential biomarker and rational therapeutic target for glioblastoma and GSC.

The utility and limitations of neurosphere assay, CD133 immunophenotyping and side population assay in glioma stem cell research.

The newly proposed glioma stem cell (GSC) hypothesis may re-model the way we diagnose and treat the tumor, which highlights the need for a complete knowledge on the genetic and epigenetic "blueprints" of GSCs. To identify the true "stemness" signatures, pure GSC populations are primarily needed. Reliable in vitro methods enriching for GSCs and thereby identifying the key stem-like characteristics constitute the preliminary step forward. We discuss in this review the current widely used methods for enriching and isolating GSCs, namely neurosphere assay, CD133 Immunophenotyping and side population assay, and detail their limitations and potential pitfalls that could complicate interpretation of corresponding results.

Abstract 3336: Upregulated inflammatory cytokines in glioma stem cells: Targets for glioma therapies

Abstract Gliomas are the most common primary brain tumors in adults. With the highly invasive growth pattern, gliomas often escape current therapeutic modalities including combinations of surgery, radiotherapy and chemotherapy. Recent studies implicate that a minority population of glioma stem cells (GSCs) are responsible for glioma maintenance and recurrence. Interleukin (IL)-1 beta, IL-6 and IL-8 are pleiotropic proinflammatory cytokines and their excessive production in the tumor microenvironment is associated with poor disease outcome in many cancer types, including glioma. These cytokines promote tumor growth via induction of proteins associated with tumor invasion and angiogenesis and anti-apoptosis. We hypothesize that GSCs acquire their chemo-resistance and invasion by excessive cytokine production. To test the hypothesis, we compared the cytokine gene expression of a human GSC-enriched population in serum-free medium (SFM) in the presence of epidermal growth factor and fibroblast growth factor, to that of glioma cells in serum-containing medium (SCM). SFM is the neural stem/progenitor cell (NPC) medium in which NPCs proliferate in neurospheres and this medium also has been used to enrich GSCs. We found that IL-1 beta and IL-6 were upregulated in GSC-enriched population along with increased expression of Nestin, a marker of neural stem cells. More interestingly, Smad interacting protein 1 (SIP1), which is thought to be involved in glioma cell migration and invasion, was also increased in the GSC-enriched population. The paralleled upregulation of IL-1 beta and IL-6 with Nestin and SIP1 in GSC-enriched population suggest that there is a possible correlation between them. The correlation is now under investigation. Since IL-1 beta is elevated in GSC enriched population, we further determined whether IL-1 beta plays a critical role in glioma growth. Glioma cells were co-cultured with mouse NPCs expressing IL-1 receptor antagonist (IL-1ra), which can bind to IL-1 receptors without transmitting activation signals and represents a competitive inhibitor of IL-1. Proliferation of glioma cells, which were modified to express Renilla luciferase, was measured using bioluminescence imaging. Our results showed that proliferation of glioma cells was inhibited in the co-culture with NPCs expressing IL-1ra, but not in the co-culture with NPCs without expression of IL-1ra, compared to glioma cells cultured alone. The results suggest that IL-1 has an important role in glioma cell growth and NPCs engineered to express IL-1ra have therapeutic potential for gliomas. 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 3336.

Abstract 4241: Glioma stem cells perform oxidative phosphorylation, while the rest of glioma cells perform aerobic glycolysis

Abstract We have previously demonstrated that glioma stem cells have low 26S proteasome activity. Therefore, in order to visualize the 26S proteasome activity in living cells we have engineered glioma primary lines to stably express a fluorescent fusion protein, ZsGreen-ODC, which is recognized by the 26S proteasome and immediately degraded in an ubiquitin-independent manner. This fusion protein, thus reports for low proteasome activity in a live cell - glioma cells that have high proteasome activity will degrade the fluorescent fusion protein (ZsGreen-negative), while the glioma stem cells with low proteasome activity will accumulate the fluorescent protein (ZsGreen-high). This system has allowed us to visualize, track and separate the glioma stem cell population from the bulk of glioma cells for further characterization. We have employed this unique feature of glioma stem cells to analyze their metabolic state. Using ATP production and glucose uptake assays, as well as analyzing the production of lactic acid and consumption of oxygen we have obtained insight into the metabolic state of glioma stem cells in established and primary glioma lines, stably expressing the ZsGreen-ODC fusion protein. These cells lines were propagated as neurospheres in serum-free conditions, supplemented with growth factors, and at the time of the experiments the ZsGreen-negative population was separated from the ZsGreen-high population by FACS sorting. We have found that glioma stem cells with low proteasome activity (ZsGreen-high) produce more ATP and consume less glucose than the rest of glioma cells with high proteasome activity (ZsGreen-negative). Furthermore, the stem cell population produces less lactic acid and consumes more oxygen, and expresses less Glut-1 protein on the surface. Interestingly, when lactic acid is added to the growth medium the number of ZsGreen-high cells declines, indicating that the amount of lactic acid produced by the bulk of glioma cells in the tumor microenvironment might affect the number of glioma stem cells in the tumor. Taken together these preliminary results suggest that glioma stem cells are performing oxidative phosphorylation, while the bulk of the tumor is performing aerobic glycolysis - thus, metabolic PET tracers designed to detect glycolytically active cells would miss the small population of glioma stem cells. Also, given that elimination of glioma stem cells is crucial for a successful glioma therapy understanding their basic metabolic needs will be very important in designing successful therapies, especially since these results indicate that the glioma stem cell population maybe metabolically quite different from the bulk of glioma cells. 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 4241.

Radioresistant glioma stem cells—Therapeutic obstacle or promising target?

Attempts to improve outcomes for patients with glioblastoma multiforme have focused largely on dose intensification and multimodal therapy and have been met with limited success. An alternative approach involves identifying and targeting the mechanisms responsible for tumour resistance. Recent data suggests that these high grade malignancies harbour a population of glioma stem cells that are both radioresistant and capable of initiating tumour regrowth. Furthermore, the radioresistant phenotype may be the consequence of constitutively upregulated and hyper-responsive cell cycle checkpoint pathways. This article considers whether these findings herald the dawn of a new era of effective, targeted therapy for this refractory tumour.