CD133-positive Glioma Stem Cells Research Articles

CD163, a novel therapeutic target, regulates the proliferation and stemness of glioma cells via casein kinase 2.

Glioma is a devastating cancer with a dismal prognosis and there is an urgent need to discover novel glioma-specific antigens for glioma therapy. Previous studies have identified CD163-positive tumour cells in certain solid tumours, but CD163 expression in glioma remains unknown. In this study, via an analysis of public datasets, we demonstrated that CD163 overexpression in glioma specimens correlated with an unfavourable patient prognosis. CD163 expression was increased in glioma cells, especially primary glioma cells. The loss of CD163 expression inhibited both cell cycle progression and the proliferation of glioblastoma multiforme (GBM) cell lines and primary glioma cells. CD163 interacted directly with casein kinase 2 (CK2) and CD163 silencing reduced AKT/GSK3β/β-catenin/cyclin D1 pathway activity via CK2. Moreover, CD163 was upregulated in CD133-positive glioma stem cells (GSCs), and CD163 downregulation decreased the expression of GSC markers, including CD133, ALDH1A1, NANOG and OCT4. The knockdown of CD163 impaired GSC stemness by inhibiting the CK2/AKT/GSK3β/β-catenin pathway. Finally, a CD163 antibody successfully induced complement-dependent cytotoxicity against glioma cells. Our findings indicate that CD163 contributes to gliomagenesis via CK2 and provides preclinical evidence that CD163 and the CD163 pathway might serve as a therapeutic target for glioma.

Abstract 4764: Activation of estrogen receptor beta signaling reduces stemness and promotes differentiation of glioma stem cells

Abstract Glioblastoma (GBM) are the most common and deadliest tumors of the central nervous system. GBM have poor prognosis due to tumor recurrence and resistance to current therapies. Glioma Stem Cells (GSCs) are implicated in the tumor initiation and therapy resistance of GBM. Agents that can specifically reduce stemness of GSCs are urgently needed for the effective treatment of GBM. Estrogen play a crucial role during brain development, differentiation and in neuroprotection. However, its role in promoting differentiation of GSCs remain unknown. Estrogen effects are mediated by two estrogen receptors (ESR1 and ESR2) and ESR2 functions as a tissue-specific tumor suppressor. Our recent studies discovered that GSCs preferably express ESR2 with low or undetectable levels of ESR1. The objective of this study is to test whether ESR2 agonists modulate stemness of GSCs and to determine their mechanism(s) of action. We have tested the hypothesis using GSCs isolated from established and patient derived GBM cells using stem cell markers CD133 and CD15. Knockout of ESR2 using CRISPR/Cas9 system increased the CD133 positive GSCs and overexpression of ESR2 reduces the CD133-positive population in GBM cells. Treatment of GSCs with ESR2 agonists (Liquiritigenin and LY500307) significantly inhibited the neurosphere formation, self-renewal ability and proliferation. Further ESR2 agonist treatment resulted in the loss of stemness and induction of differentiation and apoptosis of GSCs. Western blot analysis and RT-qPCR analysis revealed reduced expression of stemness markers such as Nestin and Sox-2 and increased expression of differentiation markers GFAP and tuj-1 in GSCs. RNA sequencing analysis of ESR2 agonist treated and untreated GSCs revealed modulation of pathways related to apoptosis, cell cycle, stemness and differentiation. Further, ESR2 agonists treatment significantly reduced the GSCs mediated tumor growth in orthotopic models and improved the mice overall survival. Immunohistochemical studies demonstrated that ESR2 agonists reduces the expression of proliferation of marker Ki67 and induced the apoptosis in tumors. Together, our results established ESR2 agonists liquiritigenin and LY500307 as novel therapeutic agents for elimination of GSCs. Since ESR2 agonists has good blood-brain barrier permeability and less neuronal toxicity, they can be readily transferred to clinical use with current radiation and chemotherapies, thereby providing an additional tool for enhancing survival in GBM patients. Citation Format: Gangadhara Reddy Sareddy, Jinyou Liu, Suryavathi Viswanadhapalli, Rajeshwar R. Tekmal, Andrew Brenner, Ratna K. Vadlamudi. Activation of estrogen receptor beta signaling reduces stemness and promotes differentiation of glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4764. doi:10.1158/1538-7445.AM2017-4764

Abstract 5217: Microenvironmental influences on glioma stem cell migration

Abstract Glioblastoma (GBM) is the most aggressive of the primary brain tumors and carries a poor prognosis due to its highly infiltrative nature and tendency to progress. Glioma stem cells are a small tumor-initiating population in GBM whose properties are incompletely understood. Autocrine and paracrine factors associated with specific microenvironmental niches, such as the perivascular space and hypoxic regions, are thought to attract and support this stem cell component. Precise mechanisms guiding the homing to these niches have not been defined. Glioma stem cells express the receptor CXCR4, which initiates cell migration in the presence of the chemokine, stromal cell-derived factor-1α (SDF-1α). Both hypoxic and vascular regions in the brain express SDF-1α. Therefore, we hypothesize that glioma stem cells transplanted into the brain are driven by SDF-1α chemoattraction towards vascular and hypoxic niches. Cells were sorted for the stem cell marker CD133 using MACS for two explanted human GBM cell lines grown as neurospheres (N08-74 and N08-30). Sorted cells were compared to unsorted cells by Western blot for expression of CD133, hypoxia inducible factor-1α (HIF-1α), CXCR4 and stem cell markers under normoxia and hypoxia. Two million CD133-sorted cells suspended in PBS were delivered into the nasal mucosa of NOD/SCID mice to test cellular migration into the brain via intranasal transplantation. Coronal sections of brain tissue were analyzed at 12 hours after intranasal cell delivery. In both GBM cell lines (N08-30 and N08-74), we confirmed that sorted cells expressed more CD133 than unsorted cells and that both cell lines expressed CXCR4. Further, CXCR4 expression was significantly greater in the CD133-sorted N08-30 cells with a trend in an increase of c-Myc expression compared to unsorted cells. Hypoxic exposure of N08-74 CD133-sorted cells showed a trend of increased HIF-1α and c-Myc expression. In mice, we demonstrated that CD133-sorted cells can migrate into the brain when delivered intranasally. To show the potential for migration towards the vasculature, we show that SDF-1α is expressed in the brain vasculature in vivo. These data demonstrate that GBM cells can be sorted for CD133-positive glioma stem cells, respond to hypoxia, and express the SDF-1α receptor, CXCR4. In one cell line, CXCR4 expression increased after CD133 cell sorting. With intranasal delivery, CD133-sorted cells can migrate into the brain. Understanding the migration behavior of glioma stem cells relative to the diverse microenvironments has implications in treatment for targeting certain migratory populations or microenvironments. These data prepare the conditions to test the migratory potential of glioma stem cells in hypoxic and vascular environments in the brain. Citation Format: Monica J. Chau, Myles R. McCrary, Subhas Mukherjee, Daniel J. Brat. Microenvironmental influences on glioma stem cell migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5217. doi:10.1158/1538-7445.AM2015-5217

RT-40 * THE DOWN-REGULATION OF H-FERRITIN AS AN ADJUVANT THERAPY IN HUMAN GLIOMA

Cancer cells generally exhibit increased iron requirements and heightened iron metabolism. As the major iron storage protein, ferritin expression is elevated in many types of cancer. In this study, we report that the sensitization of H-ferritin down-regulation in glioma cells against radiation and suggest the potential of H-ferritin down-regulation as an adjuvant therapy in human glioma. In human glioma cells, down-regulation of H-ferritin performed through a nanotechnology-based transfection platform led to a transient decrease in transferrin receptor level, suggesting a release of intracellular iron with loss of ferritin. This iron release in turn produced intracellular oxidative stress, demonstrated by protein oxidation damage, as well as a decrease in the stability of hypoxia-inducible factors (HIFs), which is an indicator of radioresistance. In glioma cells, exposure to radiation resulted in protein oxidation which could be exacerbated by H-ferritin down-regulation. Previously we have demonstrated a DNA protection role for H-ferritin. Here we expand this concept by showing that the down-regulation of H-ferritin is associated with increased DNA vulnerability when cells were exposed to ionizing radiation. The activation of DNA repair mechanisms was impaired when H-ferritin was absent. The in vitro data suggest a synergistic effect of radiation and H-ferritin down-regulation. Additionally, we expand our research into CD133-positive glioma stem cells (GSCs), which are notoriously resistant to anti-cancer treatment. Down-regulation of H-ferritin in GSCs inhibited cell proliferation in vitro, presumably through apoptosis induction. With an intracranial glioma model established by GSC implantation, we demonstrated that the survival was significantly prolonged by H-ferritin siRNA transfection through intravenous injection, in tumor bearing mice treated with first-line drug Temodar. This study supports the potential of H-ferritin siRNA as an adjuvant therapy in glioma treatment.

Expansion of CD133-positive glioma cells in recurrent de novo glioblastomas after radiotherapy and chemotherapy

Recent evidence suggests that a glioma stem cell subpopulation may determine the biological behavior of tumors, including resistance to therapy. To investigate this hypothesis, the authors examined varying grades of gliomas for stem cell marker expressions and histopathological changes between primary and recurrent tumors. Tumor samples were collected during surgery from 70 patients with varying grades of gliomas (Grade II in 12 patients, Grade III in 16, and Grade IV in 42) prior to any adjuvant treatment. The samples were subjected to immunohistochemistry for MIB-1, factor VIII, GFAP, and stem cell markers (CD133 and nestin). Histopathological changes were compared between primary and recurrent tumors in 31 patients after radiation treatment and chemotherapy, including high-dose irradiation with additional stereotactic radiosurgery. CD133 expression on glioma cells was confined to de novo glioblastomas but was not observed in lower-grade gliomas. In de novo glioblastomas, the mean percentage of CD133-positive glioma cells in sections obtained at recurrence was 12.2% ± 10.3%, which was significantly higher than that obtained at the primary surgery (1.08% ± 1.78%). CD133 and Ki 67 dual-positive glioma cells were significantly increased in recurrent de novo glioblastomas as compared with those in primary tumors (14.5% ± 6.67% vs 2.16% ± 2.60%, respectively). In contrast, secondary glioblastomas rarely expressed CD133 antigen even after malignant progression following radiotherapy and chemotherapy. The authors' results indicate that CD133-positive glioma stem cells could survive, change to a proliferative cancer stem cell phenotype, and cause recurrence in cases with de novo glioblastomas after radiotherapy and chemotherapy.

Abstract 4436: The down-regulation of ferritin heavy chain as an adjuvant therapy in human glioma.

Abstract Increased iron requirements and heightened iron metabolism is among the key characteristics of highly proliferative cancer cells. As the major iron storage protein, ferritin expression is elevated in cancer cells and we have previously shown that ferritin down-regulation in human glioblastoma cells increases their sensitivity to chemotherapeutic agent both in vitro and in vivo. In this study, we report that the sensitization of H-ferritin down-regulation in glioma cells against radiation and suggest the potential of H-ferritin down-regulation as an adjuvant therapy in human glioma. Down-regulation of H-ferritin was performed in human glioma cell line U251 through a nanotechnology-based transfection platform and effective knockdown efficiency was achieved. A transient decrease in transferrin receptor level was also observed, suggesting a release of intracellular iron with loss of ferritin. This iron release in turn produced intracellular oxidative stress, demonstrated by protein oxidation damage, as well as a decrease in the stability of hypoxia-inducible factors (HIFs), which is an indicator of radioresistance. In U251 cells, exposure to radiation resulted in protein oxidation which could be exacerbated by H-ferritin down-regulation. Previously we have demonstrated a DNA protection role for H-ferritin. Here we report that the down-regulation of H-ferritin is associated with increased DNA vulnerability when cells were exposed to ionizing radiation. Moreover, the activation of DNA repair mechanisms was impaired when H-ferritin was absent. The in vitro data suggest a synergistic effect of radiation and H-ferritin down-regulation. Additionally, we expand our research into CD133-positive glioma stem cells (GSCs), which are notoriously resistant to anti-cancer treatment. Down-regulation of H-ferritin in these cells inhibited cell proliferation in vitro. With an intracranial glioma model established by GSC implantation, we demonstrated that the survival was significantly prolonged by H-ferritin siRNA transfection through intravenous injection, in tumor bearing mice treated with first-line drug Temodar. This study supports the potential of H-ferritin siRNA as an adjuvant therapy in glioma treatment. Citation Format: Min Pang, A.B. Madhankumar, Becky Webb, Xiaoli Liu, James R. Connor. The down-regulation of ferritin heavy chain as an adjuvant therapy in human glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4436. doi:10.1158/1538-7445.AM2013-4436

Abstract 3335: Identification of candidate proteins for the targeted imaging and therapy of glioma stem cells: Effect of antibody-conjugated magnetic nanoparticles and proteasomal inhibitor bortezomib on GBM-derived neurospheres and glioma stem cell marker CD133

Abstract Introduction: Glioblastoma multiforme (GBM) is the most common and lethal malignant primary brain tumor in adults. Glioma stem cells (GSCs) have properties of self renewal, pluripotency and high tumorigenicity. Magnetic iron oxide nanoparticles (IONPs) have emerged as a potential multifunctional clinical tool that can provide cancer cell detection by magnetic resonance imaging (MRI) contrast enhancement as well as therapy by cancer cell targeted delivery of agents. In this study, we have studied differential activation of signaling pathways in purified CD133-positive GSCs in an effort to identify candidate proteins for the targeted imaging and therapy of GSCs by magnetic nanoparticles. We have also studied the therapeutic effect of IONPs conjugated with an EGFRvIII antibody against GSCs and the regulation of the GSC marker, CD133, by the proteasomal inhibitor bortezomib. Methods: Glioblastoma tumor specimens were used to isolate neurospheres. Neurospheres of an EGFRvIII-negative and EGFRvIII-positive GBM tumors were further enriched for CD133-positive cells using Indirect CD133 MicroBeads kit or FACS analysis to ∼90% purity. Magnetic nanoparticles were conjugated with the EGFRvIII antibody using Ocean NanoTech kit. Neurospheres were treated with 200 nM bortezomib. Results: Using Western blot analysis of CD133-positive GSCs from different patients, we identified nestin, wt EGFR, EGFRvIII, PDGFRß, carbonic anhydrase IX (CAIX) podoplanin as well as CD133 marker as potential candidates for the targeted imaging and therapy of GSCs. Hypoxia (1% O2) greatly enhanced expression of CD133 antigen in EGFRvIII positive neurospheres in a HIF-2α-independent manner. Treatment with proteasomal inhibitor bortezomib decreased expression of CD133. EGFRvIII antibody conjugated to IONPs (EGFRvIIIAb-IONPs) promoted apoptosis of not only EGFRvIII-positive stem cells, but also CD133-negative cells. Conclusion: Candidate proteins found in CD133-positive GSCs include nestin, wt EGFR, EGFRvIII, PDGFRβ, CAIX, and podoplanin. Hypoxia activated expression of CD133 and CAIX, a well-established hypoxia marker. The proteasomal inhibitor bortezomib downregulated hypoxia-induced expression of CD133 and CAIX. Antibodies against the candidate proteins, conjugated to magnetic nanoparticles, may form the basis of targeted imaging and therapy of GSCs. IONPs conjugated to an antibody specific to the EGFRvIII deletion mutant can promote apoptosis in neurospheres, CD133-positive GSCs, and CD133-negative neurospheres. In conclusion, a combination therapy in which GSCs are targeted a) with antibody-conjugated IONPs b) with proteasomal inhibitors to downregulate GSCs markers may have a synergistic effect against GSCs. 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 3335.

Hypoxia promotes expansion of the CD133-positive glioma stem cells through activation of HIF-1α

Hypoxia contributes to the progression of a variety of cancers by activating adaptive transcriptional programs that promote cell survival, motility and tumor angiogenesis. Although the importance of hypoxia and subsequent hypoxia-inducible factor-1alpha (HIF-1alpha) activation in tumor angiogenesis is well known, their role in the regulation of glioma-derived stem cells is unclear. In this study, we show that hypoxia (1% oxygen) promotes the self-renewal capacity of CD133-positive human glioma-derived cancer stem cells (CSCs). Propagation of the glioma-derived CSCs in a hypoxic environment also led to the expansion of cells bearing CXCR4 (CD184), CD44(low) and A2B5 surface markers. The enhanced self-renewal activity of the CD133-positive CSCs in hypoxia was preceded by upregulation of HIF-1alpha. Knockdown of HIF-1alpha abrogated the hypoxia-mediated CD133-positive CSC expansion. Inhibition of the phosphatidylinositol 3-kinase(PI3K)-Akt or ERK1/2 pathway reduced the hypoxia-driven CD133 expansion, suggesting that these signaling cascades may modulate the hypoxic response. Finally, CSCs propagated at hypoxia robustly retained the undifferentiated phenotype, whereas CSCs cultured at normoxia did not. These results suggest that response to hypoxia by CSCs involves the activation of HIF-1alpha to enhance the self-renewal activity of CD133-positive cells and to inhibit the induction of CSC differentiation. This study illustrates the importance of the tumor microenvironment in determining cellular behavior.

Glioma stem cells promote radioresistance by preferential activation of the DNA damage response

Ionizing radiation represents the most effective therapy for glioblastoma (World Health Organization grade IV glioma), one of the most lethal human malignancies, but radiotherapy remains only palliative because of radioresistance. The mechanisms underlying tumour radioresistance have remained elusive. Here we show that cancer stem cells contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. The fraction of tumour cells expressing CD133 (Prominin-1), a marker for both neural stem cells and brain cancer stem cells, is enriched after radiation in gliomas. In both cell culture and the brains of immunocompromised mice, CD133-expressing glioma cells survive ionizing radiation in increased proportions relative to most tumour cells, which lack CD133. CD133-expressing tumour cells isolated from both human glioma xenografts and primary patient glioblastoma specimens preferentially activate the DNA damage checkpoint in response to radiation, and repair radiation-induced DNA damage more effectively than CD133-negative tumour cells. In addition, the radioresistance of CD133-positive glioma stem cells can be reversed with a specific inhibitor of the Chk1 and Chk2 checkpoint kinases. Our results suggest that CD133-positive tumour cells represent the cellular population that confers glioma radioresistance and could be the source of tumour recurrence after radiation. Targeting DNA damage checkpoint response in cancer stem cells may overcome this radioresistance and provide a therapeutic model for malignant brain cancers.