Brain Tumor Stem-like Cells Research Articles

Epigenetic modulators for brain cancer stem cells: Implications for anticancer treatment.

Primary malignant brain tumors are a major cause of morbidity and mortality in both adults and children, with a dismal prognosis despite multimodal therapeutic approaches. In the last years, a specific subpopulation of cells within the tumor bulk, named cancer stem cells (CSCs) or tumor-initiating cells, have been identified in brain tumors as responsible for cancer growth and disease progression. Stemness features of tumor cells strongly affect treatment response, leading to the escape from conventional therapeutic approaches and subsequently causing tumor relapse. Recent research efforts have focused at identifying new therapeutic strategies capable of specifically targeting CSCs in cancers by taking into consideration their complex nature. Aberrant epigenetic machinery plays a key role in the genesis and progression of brain tumors as well as inducing CSC reprogramming and preserving CSC characteristics. Thus, reverting the cancer epigenome can be considered a promising therapeutic strategy. Three main epigenetic mechanisms have been described: DNA methylation, histone modifications, and non-coding RNA, particularly microRNAs. Each of these mechanisms has been proven to be targetable by chemical compounds, known as epigenetic-based drugs or epidrugs, that specifically target epigenetic marks. We review here recent advances in the study of epigenetic modulators promoting and sustaining brain tumor stem-like cells. We focus on their potential role in cancer therapy.

Riluzole: a potential therapeutic intervention in human brain tumor stem-like cells.

A small subpopulation of tumor stem-like cells has the capacity to initiate tumors and mediate radio- and chemoresistance in diverse cancers hence also in glioblastoma (GBM). It has been reported that this capacity of tumor initiation in the brain is mainly dependent on the body’s nutrient supply. This population of so-called brain tumor initiating or brain tumor stem-like cells (BTSCs) is able to extract nutrients like glucose with a higher affinity. Riluzole, a drug approved for treating amyotrophic lateral sclerosis (ALS), was reported to possess anticancer properties, affecting the glutamate metabolism. We report that riluzole treatment inhibits the growth of brain tumor stem-like cells enriched cultures isolated from two human glioblastomas. The effects of riluzole on these cells were associated with an inhibition of a poor prognostic indicator: glucose transporter 3 (GLUT3). A decrease in GLUT3 is associated with a decrease in the p-Akt/HIF1α pathway. Further, downregulation of the DNA (Cytosine-5-)-methyltransferase 1 (DNMT1) gene that causes hypermethylation of various tumor-suppressor genes and leads to a poor prognosis in GBM, was detected. Two hallmarks of cancer cells—proliferation and cell death—were positively influenced by riluzole treatment. Finally, we observed that riluzole reduced the tumor growth in in vivo CAM assay, suggesting it could be a possible synergistic drug for the treatment of glioblastoma.

Resistance to Hypoxia-Induced, BNIP3-Mediated Cell Death Contributes to an Increase in a CD133-Positive Cell Population in Human Glioblastomas In Vitro

In addition to intrinsic regulatory mechanisms, brain tumor stemlike cells (BTSCs), a small subpopulation of malignant glial tumor-derived cells, are influenced by environmental factors. Previous reports showed that lowering oxygen tension induced an increase of BTSCs expressing CD133 and other stem cell-related genes and more pronounced clonogenic capacity in vitro. We investigated the mechanisms responsible for hypoxia-dependent induction of CD133-positive BTSCs in glioblastomas. We confirmed that cultures exposed to lowered oxygen levels showed a severalfold increase of CD133-positive BTSCs. Both the increase of CD133-positive cells and deceleration of the growth kinetics were reversible after transfer to normoxic conditions. Exposure to hypoxia induced BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3)-dependent apoptosis preferentially in CD133-negative cells. In contrast, CD133-positive cells proved to be more resistant to hypoxia-induced programmed cell death. Application of the demethylating agent 5'-azacitidine resulted in an increase of BNIP3 expression levels in CD133-positive cells. Thus, epigenetic modifications led to their better survival in lowered oxygen tension. Moreover, the, hypoxia-induced increase of CD133-positive cells was inhibited after 5'-azacitidine treatment. These results suggest the possible efficacy of a novel therapy for glioblastoma focused on eradication of BTSCs by modifications of epigenetic regulation of gene expression.

Abstract 3363: Brain tumor stem-like cells as a tool for testing small molecules for anti-tumor therapy

Abstract Glioblastoma multiforme (GBM) is the most common primary malignant tumor of the adult central nervous system. The highly lethal nature of this tumor partly derives from the acquisition of an invasive phenotype, which allows the tumor cells to infiltrate the surrounding brain tissues. In fact the tumor cells, leading to the recurrence of the primary neoplasm highly contribute to the lack of success in eradicating this disease. Recent evidences suggested that a rare population of stem-like cells (or tumor initiating cells) present in brain tumors may be responsible for development of highly invasive and chemoresistant recurrent tumors. It was suggested that not all the cells from a given brain tumor show the same ability to proliferate and sustain the growth. Only a relatively small fraction of those cells, termed Brain Tumor Stem-like Cells (BTSCs), possess the ability to proliferate, self-renew extensively and adapt to very different micro-environments. Further investigations have demonstrated that BTSCs also more closely mirror the phenotype and genotype of the primary tumors, in fact when used in animal models they are able to generate a tumor that recapitulates the features of the human GBMs. The absence of stem cell specific markers has posed challenges to the identification and isolation of BTSCs from the bulk tumor cells in a definitive manner. Furthermore, recent findings suggest that the Hedgehog (HH) pathway is involved in BTSCs population maintaining in GBM. We report that in a commercially available human recurrent GBM line (DBTRG-05MG) and in different patients derived GBM cells; the key components required for an active HH pathway, such as PTCH1, SMO and SHH, are present and differently expressed according to culture conditions. Cells cultured in serum containing media, have a switched-off HH pathway, while cells kept in serum free containing media supplemented with EGF and bFGF to promote the formation of floating growing neurospheres, show a functional HH signaling pathway. These GBM cells respond to the same mitogens that activate normal adult neural stem cells showing multipotent differentiation properties and expressing markers of the normal stem cells lineage, such as Nestin, SOX2, Bmi1 and Musashi1. Proliferation assays performed using GBM patient-derived neurospheres clearly showed higher sensitivity to the different SMO antagonists tested when compared to the adherent counterpart cultured in serum containing media. The effect of small molecules SMO antagonists in self-renewal assays, confirmed the implication of the HH pathway in the growth of these stem-like cells. Taken together, these data demonstrate the presence of an operational HH pathway in human GBM derived neurospheres, and the ability of those cells to be applied in a drug discovery setting as a reliable SMO antagonists phenotypic testing tool. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3363. doi:1538-7445.AM2012-3363

CD44v6 Regulates Growth of Brain Tumor Stem Cells Partially through the AKT-Mediated Pathway

Identification of stem cell-like brain tumor cells (brain tumor stem-like cells; BTSC) has gained substantial attention by scientists and physicians. However, the mechanism of tumor initiation and proliferation is still poorly understood. CD44 is a cell surface protein linked to tumorigenesis in various cancers. In particular, one of its variant isoforms, CD44v6, is associated with several cancer types. To date its expression and function in BTSC is yet to be identified. Here, we demonstrate the presence and function of the variant form 6 of CD44 (CD44v6) in BTSC of a subset of glioblastoma multiforme (GBM). Patients with CD44high GBM exhibited significantly poorer prognoses. Among various variant forms, CD44v6 was the only isoform that was detected in BTSC and its knockdown inhibited in vitro growth of BTSC from CD44high GBM but not from CD44low GBM. In contrast, this siRNA-mediated growth inhibition was not apparent in the matched GBM sample that does not possess stem-like properties. Stimulation with a CD44v6 ligand, osteopontin (OPN), increased expression of phosphorylated AKT in CD44high GBM, but not in CD44low GBM. Lastly, in a mouse spontaneous intracranial tumor model, CD44v6 was abundantly expressed by tumor precursors, in contrast to no detectable CD44v6 expression in normal neural precursors. Furthermore, overexpression of mouse CD44v6 or OPN, but not its dominant negative form, resulted in enhanced growth of the mouse tumor stem-like cells in vitro. Collectively, these data indicate that a subset of GBM expresses high CD44 in BTSC, and its growth may depend on CD44v6/AKTpathway.

Breast Cancer Resistance Protein and P-Glycoprotein in Brain Cancer: Two Gatekeepers Team Up

Brain cancer is a devastating disease. Despite extensive research, treatment of brain tumors has been largely ineffective and the diagnosis of brain cancer remains uniformly fatal. Failure of brain cancer treatment may be in part due to limitations in drug delivery, influenced by the ABC drug efflux transporters P-gp and BCRP at the blood-brain and blood-tumor barriers, in brain tumor cells, as well as in brain tumor stem-like cells. P-gp and BCRP limit various anti-cancer drugs from entering the brain and tumor tissues, thus rendering chemotherapy ineffective. To overcome this obstacle, two strategies - targeting transporter regulation and direct transporter inhibition - have been proposed. In this review, we focus on these strategies. We first introduce the latest findings on signaling pathways that could potentially be targeted to down-regulate P-gp and BCRP expression and/or transport activity. We then highlight in detail the new paradigm of P-gp and BCRP working as a "cooperative team of gatekeepers" at the blood-brain barrier, discuss its ramifications for brain cancer therapy, and summarize the latest findings on dual P-gp/BCRP inhibitors. Finally, we provide a brief summary with conclusions and outline the perspectives for future research endeavors in this field.

Abstract LB-103: Signaling via CD44v6 is associated with the growth of CD44-expressing glioma stem-like cells

Abstract Background: Brain tumors contain stem-like cells are likely a therapeutic target in malignant glioma due to their therapeutic resistance and ability of tumor initiation. Several studies have suggested the presence of multiple types of brain tumor stem-like cells (BTSC). However, the molecular markers valuable for targeting these subpopulations of glioma cells are still unknown. CD44 is a cell surface protein linked to tumorigenesis in some cancers outside of the central nervous system (CNS). CD44 expression is recognized in various types of cancer stem cells. In particular, one of CD44 variant isoforms, CD44v6, is associated with malignant transformation of non-CNS tumor cells by inducing proliferation and migration. To date, expression and function of CD44 and CD44v6 in BTSC is not characterized yet. Here, we investigated the expression of CD44, including CD44v6, and their function in BTSC. Results: RT-PCR and Western blotting elucidated that a subset of GBM expresses high level of CD44. Immunostaining on glioma tissue microarray demonstrated that patients with high grade glioma expressing CD44 had poorer prognoses. Positive staining of CD44 on tumor cell surface was seen only in glioblastoma multiforme (GBM). FACS analysis of BTSC enriched from GBM specimens showed a subset of GBM expressed high level of CD44 in BTSC. CD44 neutralizing antibody inhibited cell growth of BTSC derived from CD44-high GBM, while BTSC from CD44-low GBM was not affected. Cultured BTSC from CD44-high GBM contains subpopulation expressing CD44v6. Knockdown of CD44v6 with siRNA inhibited in vitro growth of BTSC from CD44-high GBM but not from CD44-low GBM. This inhibition did not affect growth of cells derived from the same tumors that do not have stem-like characters. CD44 ligand osteopontin (OPN) activated AKT in BTSC from CD44-high GBM, but not in CD44-low samples. With CD44v6 knockdown, both phosphorylated AKT and S6R were under detectable level and OPN failed to activate AKT and S6R, while EGF substantially increased both molecules. When the PI3K/AKT pathway was inhibited by five different small molecules, a dose-dependent inhibition for neurosphere formation was observed for all inhibitors in BTSC from CD44-high GBM. In contrast, BTSC from CD44-low GBM were relatively resistant to the treatment. Conclusion: Our data indicate that a subset of GBM expresses high level of CD44 and CD44v6 in BTSC. GBM expressing high level of CD44 depends on CD44v6 on the growth of their BTSC. This action of CD44v6 is, at least in part, mediated through the AKT pathway. CD44 expression in high grade glioma corresponds to poorer prognosis. Immunoreactive site of CD44 on GBM cells correlates with pathological grade. 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 LB-103. doi:10.1158/1538-7445.AM2011-LB-103

Abstract 3298: Structure-based computer-aided drug design to discover novel small molecules that target brain tumor stem cells

Abstract INTRODUCTION. Malignant glioma is one of the most lethal diseases in adulthood and remains refractory to the current therapies including chemoradiotherapies. The cancer stem cell theory dictates that brain tumor stem-like cells (BTSC) account for such intractability of malignant glioma. Recently, several molecularly-targeted therapies have demonstrated a varying degree of success in treatment of cancers including leukemia and breast cancer. In this study, we sought to determine the key kinases that regulate survival, self-renewal, and proliferation of BTSC, and to design novel small molecules by using the structure-based computantional modeling system. RESULTS. With the neurosphere cultures derived from our glioma patients, we identified 3 kinases that play vital roles in survival and/or proliferation of BTSC in vitro; Fibroblast Growth Factor Receptor 1, Aurora kinase B, and Maternal Embryonic Leucine-zipper Kinase. Structure-based computational modeling allowed us to perform in silico docking of various small molecule candidates to these kinases. As a result, four novel small molecules demonstrated highly specific targeting of the key structural elements of these kinases. We then synthesized these identified candidate compounds and confirmed their in vitro inhibition of the targeting kinase activities. Finally, we will also present our data about the treatment effect with these compounds on the in vitro BTSC growth and the in vivo tumor growth. CONCLUSION. Structure-based drug design will likely open up a new avenue to discover novel anti-cancer stem cell agents that can selectively target the key kinases in BTSC, leading to the growth arrest of malignant glioma. 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 3298. doi:10.1158/1538-7445.AM2011-3298

Abstract 3302: The effects of the g-quadruplex ligand telomestatin to human brain tumor stem cell survival and growth

Abstract Glioblastoma multiforme (GBM) is the leading cause of death among primary brain tumors in adults and the current therapies have only palliative effect on prognosis of patients. Recently, stem cell-like cells in GBM (brain tumor stem-like cells; BTSC) have gained substantial attention as a potential therapeutic target. Hallmarks of BTSC include their self-renewal capacity and highly migratory potential. In this study, we demonstrate that treatment of patient-derived BTSC with a G-quadruplex ligand, telomestatin (TMS), inhibits BTSC self-renewal and maintenance of their stem cell state, and induces their apoptosis in vitro and in vivo. In contrast, both normal precursors and non-stem tumor cells from the matched samples are relatively resistant to TMS treatment. Treatment with a lower dose of TMS specifically inhibits BTSC migration into normal brain. Immunofluorescence in situ hybridization with TMS-treated GBM cells displays both telomeric and non-telomeric DNA damage in BTSC but not in non-stem tumor cells. cDNA microarray analysis identifies a proto-oncogene, c-Myb, as a target of TMS in BTSC, and the pharmacodynamic analysis with TMS-treated tumor-bearing mouse brains demonstrates reduction of c-Myb expression in tumors. An elevated c-Myb expression is found in surgical specimens of GBM compared to normal brain tissues. Lastly, TMS treatment of BTSC-derived mouse intracranial tumors reduces tumor sizes in vivo without any noticeable apoptotic cells in the normal brain, and a combined treatment of TMS with radiation or temozolomide results in additive inhibitory effects on GBM sphere growth in vitro. Collectively, these data indicate a potential avenue toward BTSC-directed therapeutic strategy using TMS via telomeric DNA damage and inhibition of c-Myb, which may offer a novel therapeutic approach for GBM. 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 3302. doi:10.1158/1538-7445.AM2011-3302

Study on the proliferation and drug-resistance of human brain tumor stem-like cells.

Brain tumor stem-like cells (BTSLCs) have been implied to play an important role in genesis and development of glioma. However, their characteristics on proliferation and drug-resistance are uncertain thoroughly. In this experiment, some of the biological characteristics about BTSLCs were explored. Twenty cases of different grades of human glioma tissues were obtained from clinic. The primary glioma cells were collected and CD133(+) cells from them were purified by magnetic cell sorting assay. The BTSLCs were identified by testing the expression of CD133, Nestin, NSE, and GFAP, along with the culture process. WST-8 assay kit was used to evaluate the proliferating situation of CD133(+) cells in the different grade gliomas, and to compare the drug-resistance between the CD133(+) and CD133( - ) cells in the medium containing different concentrations of teniposide (VM-26). The results showed that the CD133(+) cells could regenerate by self-renewal, then generate and different into NSE(+) and GFAP(+) cells, respectively. CD133(+) cells in the high grade of gliomas showed the faster generation than the ones in the low grade. The number of survived CD133(+) cells in the medium containing VM-26 was much more than the CD133(-) ones in it. Therefore, it was implied that the CD133(+) BTSLCs existed in the glioma tissues possessed the more tolerant ability to the VM-26, and could proliferate much more easily in the high-grade glioma.

Abstract 5148: A neural stem cells marker fatty acid binding protein 7 (FABP7) is involved in proliferation and invasion of glioblastoma cells

Abstract Malignant brain tumours are among the most deadly cancers. Current treatment strategies, such as surgery, chemotherapy and radiotherapy only modestly improve patient survival. The limited success of these strategies is largely due to a high incidence of tumor recurrence after the treatment. Recent evidences suggested that a rare population of stem-like cells (or tumour initiating cells) present in brain tumours might be responsible for the aggressiveness and recurrence of these tumours. These cells, named brain tumor stem-like cells (BTSC), share many properties of normal neural stem cells, including self renewal, extended proliferation, formation of neurospheres (when cultured in vitro in the presence of growth factors) and potential to differentiate into neurons and glial cells. Further investigations have suggested that studying this BTSC population in glioblastoma (GBM) is a more relevant system for exploring glioma biology than the adherently growing glioma cell lines. In the current study we identified target genes and proteins that are differently expressed between adherent glioma cell lines and sphere growing BTSC, using microarray and differential proteomics, respectively. Fatty acid-binding protein 7 (FABP7) was identified as overexpressed in BTSC compared to glioma adherent cell lines both at the gene as well as at the protein level. FABP proteins are a family of small, highly conserved, cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. FABPs are thought to play important roles in fatty acid uptake, transport, and metabolism. Functionally, FABPS were described to play a role in gene regulation, cell signaling, cell growth and differentiation. The functional role of FABP7 in glioblastoma was investigated by assessing the effect of silencing FABP7 on migration and cell proliferation of glioblastoma cells. We observed that down-regulating FABP7 expression significantly reduced in vitro cell proliferation and even more the migration of BTSC. Since increased tumour infiltration was reported as a consequence of radiotherapy, the potential involvement of FABP7 in this process was evaluated. Irradiated BTSCs displayed significantly higher FABP7 protein levels and therefore we suggest that FABP7 may contribute to the radiotherapy-induced aggressive phenotype of BTSCs. Moreover, further mechanistic investigations indicated that directly or indirectly reducing the expression of FABP7 significantly impacts on the behaviour of glioblastoma derived BTSC. 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 5148.

Maintenance of critical properties of brain tumor stem-like cells after cryopreservation.

It would be very useful to be able to classify brain tumor stem cells (BTSCs) by certain criteria to afford the design of specific or individualized treatment. Here, we studied two BTSC lines with differing biological and molecular features and whose respective features were well preserved after cryopreservation as single cells in SFM or 90% serum with 10% DMSO, a method not previously reported. The resuscitated BTSCs shared properties indistinguishable from their respective parental cells, including tumor sphere forming potentials, growth and differentiation properties, and tumorigenesis in vivo. The two cell lines also had differing molecule profiles, which can be well preserved after cryopreservation, similar to that of their respective primary tumors. Therefore, BTSCs from different patients, that have their own properties, were well retained by the present cryopreservation method, which might be a useful and reliable method for preserving BTSCs for long-term studies, such as classification and specific therapy design.

B7-H1 is correlated with malignancy-grade gliomas but is not expressed exclusively on tumor stem-like cells

Human glioblastoma is well known for its capacity to interfere with effective antitumor immune responses. B7-H1 is the third member of the B7 family that plays important roles in tumor immune evasion. Recent studies have shown that brain tumor stem-like cells (TSCs) contribute to tumorigenesis and radioresistance. However, the relationship between B7-H1 and the clinical behavior of brain TSCs remains unclear. In the present study, we report that B7-H1 is correlated with the malignancy grade of astrocyte tumors. B7-H1 was significantly upregulated at the growing edge of the tumors. Immunostaining and flow cytometric analysis indicate that B7-H1 was expressed primarily by Ki67-negative tumor cells. In vitro, tumors cultured under medium favoring the growth of neural stem cells were able to form spheres, along with expression of neural stem/progenitor cell markers. These cells were able to differentiate into different neural lineages when cultured in differentiation medium, indicating that these cells have TSC characteristics. We also found that B7-H1 was expressed, but not exclusively on CD133-positive stem cells. Interestingly, we found that CD133-negative tumor cells also had the capacity to form brain tumors. Our data establish a correlation between the expression of the negative costimulatory molecule B7-H1 and the malignancy grade of human gliomas, suggesting that B7-H1 may be a novel tumor marker and target for therapy, although it is not expressed exclusively on brain TSCs.

Brain Tumor Stem-Like Cells Identified by Neural Stem Cell Marker CD15

In recent years, a small number of cells that have stem cell properties were identified in human gliomas called brain tumor stem cells (BTSCs), which were thought to mainly contribute to the initiation and development of gliomas and could be identified by the surface marker CD133. However, recent studies indicated that the expression of CD133 might be regulated by environmental conditions such as hypoxia and that there might be CD133- BTSCs. Genetic mouse models demonstrated that some gliomas originated from transformed neural stem cells (NSCs). Therefore, we investigated the expression of CD15, a surface marker for NSCs, in tumor spheres derived from astrocytoma and ependymoma. CD15+ cells isolated from these tumor spheres had properties of BTSCs including self-renewal, multidifferentiation, and the ability to recapitulate the phenocopy of primary tumors. CD15 exhibited stable expression in long-term cultured tumor spheres, which sustained BTSCs properties, whereas CD133 expression decreased significantly in late passages. Furthermore, CD15+CD133- cells isolated from early or late passages of tumor spheres showed similar characteristics of BTSCs. Examination of glioma samples by immunohistochemistry showed that CD15 was expressed in a subset of human brain tumors. Therefore, CD15 can be used as a marker of stem-like cells derived from brain tumors that might contain CD133- BTSCs.

Effect of Notch inhibition on radiation in an explant model of glioblastoma multiforme

e22080 Background: Glioblastoma Multiforme (GBM) is highly radioresistant, possibly due to a subpopulation of Brain Tumor “Stem-like‘ Cells (BTSC) capable of repopulating the tumor. The Notch signaling pathway is essential for maintaining normal neural stem cells. We asked whether Notch inhibition can overcome radioresistance by allowing BTSCs to exit their stem cell state. Methods: To maintain the BTSCs in their original niche we used an organotypic culture method (explants). GBM explants are cultured on a semiporous membrane in an air-medium interface. They are treated with the Notch inhibitor DAPT, radiation or a combination thereof. Proliferation and neurosphere forming capacity are assessed. Results: The explant model faithfully maintained the cytoarchitecture of the tumor (preservation of blood vessels and pericytes) and its high proliferation rate (%ki67+ cells: 14.3% -17.1%). The effect of DAPT or radiation treatment alone differed among tumors (from no change to a two-fold and nine-fold decrease post DAPT or radiation respectively). However, in all tumors tested the combination of DAPT and radiation dramatically decreased the proliferation rate compared to either treatment alone (%ki67 + cells: 0.19%-2.24%). Interestingly the rate of neurosphere formation was highest following radiation alone (threefold increase). Neurosphere formation after DAPT treatment differed amongst the tumors from a decrease (fourfold) to an increase (twofold). The combination treatment resulted in a modest increase over control (twofold). Conclusions: Our data demonstrates that inhibition of proliferation does not correlate with the status of neurosphere forming ability, arguably a surrogate for cancer stem cell function. Combining radiation with notch inhibition has a profound effect on GBM proliferation, most likely due to selectively inhibiting the BTSC repopulating ability. Ongoing studies will determine the impact of this approach on tumor growth and progression in in vivo tumor models. No significant financial relationships to disclose.

Hedgehog Signaling Regulates Brain Tumor-Initiating Cell Proliferation and Portends Shorter Survival for Patients with PTEN-Coexpressing Glioblastomas

The identification of brain tumor stem-like cells (BTSCs) has implicated a role of biological self-renewal mechanisms in clinical brain tumor initiation and propagation. The molecular mechanisms underlying the tumor-forming capacity of BTSCs, however, remain unknown. Here, we have generated molecular signatures of glioblastoma multiforme (GBM) using gene expression profiles of BTSCs and have identified both Sonic Hedgehog (SHH) signaling-dependent and -independent BTSCs and their respective glioblastoma surgical specimens. BTSC proliferation could be abrogated in a pathway-dependent fashion in vitro and in an intracranial tumor model in athymic mice. Both SHH-dependent and -independent brain tumor growth required phosphoinositide 3-kinase-mammalian target of rapamycin signaling. In human GBMs, the levels of SHH and PTCH1 expression were significantly higher in PTEN-expressing tumors than in PTEN-deficient tumors. In addition, we show that hyperactive SHH-GLI signaling in PTEN-coexpressing human GBM is associated with reduced survival time. Thus, distinct proliferation signaling dependence may underpin glioblastoma propagation by BTSCs. Modeling these BTSC proliferation mechanisms may provide a rationale for individualized glioblastoma treatment.

B7-H4 is preferentially expressed in non-dividing brain tumor cells and in a subset of brain tumor stem-like cells

B7-H4, a newly discovered member of B7 family that negatively regulates T cell-mediated immunity, may facilitate tumor progression by undermining host immunity. Recent studies show that brain tumor stem-like cells (TSCs) contribute to tumorigenesis. However, the relationship between B7-H4 and the clinical behavior of brain TSCs remains unclear. In this study, we found that B7-H4 was expressed in cultured tumor cells from human gliomas (n = 5) and medulloblastomas (n = 3). Double immunostaining indicated that B7-H4 was primarily restricted to non-dividing (Ki67(-)) cultured tumor cells. Tumor cells cultured under medium conditions favoring the growth of neural stem cells were able to form primary and secondary spheres, along with expression of neural stem/progenitor cell markers. These cells differentiated into different neural lineages when cultured in differentiation medium, indicating that these cells have TSCs characteristics. Double immunostaining showed that TSCs consisted of proliferative (Ki67(+)) and quiescent (Ki67(-)) cells. We also found that B7-H4 was expressed in a small population of CD133(+) cells sorted by flow cytometry. Interestingly, both CD133(+) and CD133(-) cells were tumorigenic in SCID mice in vivo. However, CD133(+) cells-initiated glioblastomas showed a higher proliferation index, compared to CD133(-) cells-induced glioblastomas in vivo. Secondary glioma cells derived from CD133(+) or CD133(-) cell xenografts expressed B7-H4 as well. Our data suggest B7-H4 is preferentially expressed in non-dividing brain tumor cells and in a subpopulation of brain TSCs, and CD133(-) tumor cells also have the capacity to initiate brain formation in vivo.

Developmental signaling pathways in brain tumor‐derived stem‐like cells

Recently, a subpopulation of cells highly efficient in tumor initiation and growth has been isolated from brain tumors. Of interest, these brain tumor initiating cells exhibit many stem-like properties, including self-renewal, extended proliferation, and multipotency, and are both phenotypically and genetically similar to normal neural stem cells (NSCs). Aberrant expression of developmental pathways, such as WNT, Hedgehog, Notch, and transforming growth factor-beta/bone morphogenetic protein, have been demonstrated in brain tumors, and extrinsic regulation of these pathways may be used to target brain tumor stem-like cells (BTSCs) and form the basis of novel biological therapies. Because of regulatory redundancy during normal development, future therapeutic strategies to inhibit BTSC-mediated tumor growth and minimize NSC-related deleterious effects may require detailed understanding and regulation of multiple cellular mechanisms. This review analyzes the role developmental pathways play in brain tumors, focusing on the potential effects of pathway regulation on BTSC-driven tumorigenesis.

Anticancer Therapies Combining Antiangiogenic and Tumor Cell Cytotoxic Effects Reduce the Tumor Stem-Like Cell Fraction in Glioma Xenograft Tumors

Vascular endothelial cells have been identified as a critical component of the neural stem cell niche, raising the possibility that brain tumor stem-like cells (TSLC) may also rely on signaling interactions with nearby tumor vasculature to maintain their stem-like state. The disruption of such a TSLC vascular niche by an antiangiogenic therapy could result in loss of stemness characteristics associated with intrinsic drug resistance and, thus, preferentially sensitize TSLC to the effects of chemotherapy. Considering these possibilities, we investigated the impact of antiangiogenic anticancer therapy on the TSLC fraction of glioma tumors. Athymic nude mice bearing s.c. tumor xenografts of the C6 rat glioma cell line were treated with either a targeted antiangiogenic agent, antiangiogenic schedules of low-dose metronomic chemotherapy, combination therapies of antiangiogenic agents and chemotherapy, or, for the purpose of comparison, a conventional cytotoxic schedule of maximum tolerated dose chemotherapy using cyclophosphamide. Targeted antiangiogenic therapy or cytotoxic chemotherapy did not reduce the fraction of tumor sphere-forming units (SFU) in the tumor, whereas all treatment groups that combined both antiangiogenic and cytotoxic drug effects caused a significant reduction in SFU. This work highlights the possibility that selective eradication of TSLC may be achieved by targeting the tumor microenvironment (and potentially a supportive TSLC niche) rather than the TSLC directly. Furthermore, this work suggests a possible novel effect of antiangiogenic therapy, namely, as a chemosensitizer of TSLC, and thus represents a possible new mechanism to explain the ability of antiangiogenic therapy to enhance the efficacy of chemotherapy.

Neurosphere Assays: Growth Factors and Hormone Differences in Tumor and Nontumor Studies

The "no new neuron" dogma that the brain is quiescent throughout adult life has been challenged by the discovery of cells with stem cell-like qualities of self-renewal and multipotency in the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus in adults. This self-renewing capacity also makes these neural stem cells a possible source of brain tumors, which was supported by the discovery of self-sustaining brain tumor stem-like cells in cancers such as glioblastoma multiforme. Neurosphere assays are the standard for studying these stem-like cells in both normal and cancer tissues. Despite the importance of these assays, there is no standardized protocol to allow for a comparison of results because several studies use different growth factors and hormones at different concentrations. The primary objective of this study is to review the literature for both nontumor and tumor studies to assess their respective neurosphere assay components. We found significant variation in assay components, namely hormones and growth factors, as well as their respective concentrations. This illustrates the need for a standardized protocol to allow proper comparison among studies and a better assessment of the effects of different factors.