Brain Glioma Stem Cells Research Articles

The tumor-enriched small molecule gambogic amide suppresses glioma by targeting WDR1-dependent cytoskeleton remodeling

Glioma is the most prevalent brain tumor, presenting with limited treatment options, while patients with malignant glioma and glioblastoma (GBM) have poor prognoses. The physical obstacle to drug delivery imposed by the blood‒brain barrier (BBB) and glioma stem cells (GSCs), which are widely recognized as crucial elements contributing to the unsatisfactory clinical outcomes. In this study, we found a small molecule, gambogic amide (GA-amide), exhibited the ability to effectively penetrate the blood-brain barrier (BBB) and displayed a notable enrichment within the tumor region. Moreover, GA-amide exhibited significant efficacy in inhibiting tumor growth across various in vivo glioma models, encompassing transgenic and primary patient-derived xenograft (PDX) models. We further performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout screen to determine the druggable target of GA-amide. By the combination of the cellular thermal shift assay (CETSA), the drug affinity responsive target stability (DARTS) approach, molecular docking simulation and surface plasmon resonance (SPR) analysis, WD repeat domain 1 (WDR1) was identified as the direct binding target of GA-amide. Through direct interaction with WDR1, GA-amide promoted the formation of a complex involving WDR1, MYH9 and Cofilin, which accelerate the depolymerization of F-actin to inhibit the invasion of patient-derived glioma cells (PDCs) and induce PDC apoptosis via the mitochondrial apoptotic pathway. In conclusion, our study not only identified GA-amide as an effective and safe agent for treating glioma but also shed light on the underlying mechanisms of GA-amide from the perspective of cytoskeletal homeostasis.

Rational design of a glycopeptide probe system based on a reconfigurable immune microenvironment.

Glioma is a highly challenging human malignancy and conventional drugs typically exhibit low blood-brain barrier (BBB) permeability as well as poor tumor targeting. To complicate matters further, recent advances in research on oncology have highlighted the dynamic and complex cellular networks within the immunosuppressive tumor microenvironment (TME) that complicate glioma treatment. Therefore, precise and efficient targeting of tumor tissue, whilst reversing immunosuppression, may provide an ideal strategy for the treatment of gliomas. Here, by using the "one-bead-one-component" combinatorial chemistry approach, we designed and screened a peptide that can specifically target brain glioma stem cells (GSCs), which was further engineered into glycopeptide-functionalized multifunctional micelles. We demonstrated that the micelles can carry DOX and effectively penetrate the BBB to achieve targeted killing of glioma cells. Meanwhile, mannose confers a unique tumor immune microenvironment modulating function to the micelles, which can activate the anti-tumor immune response function of tumor-associated macrophages and is expected to be further applied in vivo. This study highlights that glycosylation modification of targeted peptides specific to cancer stem cells (CSCs) may serve as an effective tool to improve the therapeutic outcome of brain tumor patients.

MiR-124 participates in the proliferation and differentiation of brain glioma stem cells through regulating Nogo/NgR expression.

The effect of miR-124 on the proliferation and differentiation of brain glioma stem cells and Nogo/NgR signaling pathway were investigated. miR-124 mimic, miR-124 inhibitor and miR-control expression vector were designed and produced to transfect U87 glioma stem cells. The results of transfection were tested via RT-qPCR and the expression of protein was detected by western blot analysis. Cell proliferation was detected by MTT proliferation and the proportion of CD133+ cells was detected by immunomagnetic beads to determine cell differentiation. The correlation between miR-124 and Nogo-A, and NgR protein expression was analyzed by Spearman correlation analysis. The relative expression of miR-124 in cells of miR-124 mimic group was significantly higher than that of miR-124 inhibitor and miR-control groups (P<0.05). The relative expression of Nogo-A and NgR protein in cells of the miR-124 mimic group was significantly lower than that of miR-124 inhibitor and miR-control groups (P<0.05). Absorbance values of the cells in the miR-124 mimic and miR-control groups were significantly lower than those in the miR-124 inhibitor group at each time point (P<0.05), while the values of the cells in the miR-124 mimic group were significantly lower than that in miR-control group (P<0.05). The level of CD133+ cells in miR-124 mimic group was significantly lower than that in miR-124 inhibitor and miR-control groups (P<0.05), while the level of CD133+ cells in miR-124 inhibitor group was higher than that in miR-control group (P<0.05). Correlation analysis revealed that there was a negative correlation between miR-124 and the expression of Nogo-A and NgR protein (P<0.05). miR-124 may participate in the differentiation of brain glioma stem cells through the Nogo/NgR pathway, which may bring a new direction for the clinical treatment of brain glioma.

Emerging Pathogenic and Prognostic Significance of Paired Box 3 (PAX3) Protein in Adult Gliomas

Gliomas present the most common type of brain tumors in adults, characterized by high morbidity and mortality. In search of potential molecular targets, members of paired box (PAX) family have been found expressed in neural crest cells, regulating their proliferation, apoptosis, migration and differentiation. Recently, PAX3 overexpression has been implicated in glioma tumorigenesis by enhancing proliferation, increasing invasiveness and inducing resistance to apoptosis of glioma cells, while maintaining brain glioma stem cells (BGSCs) stemness. Although the oncogenic potential of PAX3 in gliomas is still under investigation, experimental evidence suggests that PAX3 function is mainly mediated through the canonical and non-canonical Wnt signaling pathway as well as through its interaction with GFAP and p53 proteins. In addition, PAX3 may contribute to the chemoresistance of glioma cells and modulates the effectiveness of novel experimental therapies. Further evidence indicates that PAX3 may represent a novel diagnostic and prognostic biomarker for gliomas, facilitating personalized treatment. This review addresses the emerging role of PAX3 in glioma diagnosis, prognosis and treatment, aiming to shed more light on the underlying molecular mechanisms that could lead to more effective treatment approaches.

Transcriptional Repression of p53 by PAX3 Contributes to Gliomagenesis and Differentiation of Glioma Stem Cells.

Although there are available therapies as surgery, chemotherapy and radiation, glioblastoma (GBM) still has been considered as the most common and overwhelming primary tumor of brain. In GBM, the brain glioma stem cells (BGSCs) were identified and played a crucial role in resistance of GBM to conventional therapies described above. PAX3 was previously identified by our group as a diagnostic/prognostic marker and a therapeutic regulator in the therapy of GBM. Here, we hypothesized PAX3/p53 axis promoted the process of differentiation, regulating to the cancer stem cell properties, such as proliferation and migration. The correlation between PAX3 and p53 in GBM were first clarified. Immunofluorescence of p53 was shown activated following BGSCs differentiation. We further identified that PAX3 might specifically bind to the promoter of p53 gene, and transcriptionally repressed p53 expression. ChIP assay further confirmed that PAX3/p53 axis regulated the differentiation process of BGSCs. Then, the function of PAX3 in BGSCs were sequentially investigated in vitro and in vivo. Ectopic PAX3 expression promoted BGSCs growth and migration while PAX3 knockdown suppressed BGSCs growth, migration in vitro and in vivo. Similar to PAX3 overexpression, p53 inhibition also showed increase in growth and migration of differentiated BGSCs. Regarding the functional interaction between PAX3 and p53, PAX3 knockdown-mediated decrease in proliferation was partially rescued by p53 inhibition. Hypoxia significantly promoted the migration potential of BGSCs. In addition, hypoxia inducible factor-1α (HIF-1α) might be a potential upstream regulator of PAX3 in differentiated BGSCs under hypoxia. Our work may provide a supplementary mechanism in regulation of the BGSCs differentiation and their functions, which should provide novel therapeutic targets for GBM in future.

Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma

Development of safe, efficient nanocomplex for targeted imaging and therapy of cancer stem cells in brain glioma has become a great challenge. Herein, a low-density lipoprotein receptor-related protein and a RNA aptamer bound CD133 were used as dual-targeting ligands to prepare dual-modified cationic liposomes (DP-CLPs) loaded with survivin siRNA and paclitaxel (DP-CLPs–PTX–siRNA) for actively targeting imaging and treating CD133+ glioma stem cells after passing through the blood–brain barrier. After being administrated with DP-CLPs–PTX–siRNA nanocomplex, DP-CLPs showed a persistent target ability to bind glioma cells and brain microvascular endothelial cells (BCECs) and to deliver drugs (PTX/siRNA) to CD133+ glioma stem cells. Prepared DP-CLPs–PTX–siRNA nanocomplex showed very low cytotoxicity to BCECs, but induced selectively apoptosis of CD133+ glioma stem cells, and improved CD133+ glioma stem cells' differentiation into non-stem-cell lineages, also markedly inhibited tumorigenesis, induced CD133+ glioma cell apoptosis in intracranial glioma tumor-bearing nude mice and improved survival rates. In conclusion, prepared DP-CLPs–PTX–siRNA nanocomplex selectively induced CD133+ glioma stem cell apoptosis in vitro and in vivo exhibits great potential for targeted imaging and therapy of brain glioma stem cells.

Lipid vesicles containing transferrin receptor binding peptide TfR-T12 and octa-arginine conjugate stearyl-R8 efficiently treat brain glioma along with glioma stem cells

Surgery and radiotherapy cannot fully remove brain glioma; thus, chemotherapy continues to play an important role in treatment of this illness. However, because of the restriction of the blood-brain barrier (BBB) and the regeneration of glioma stem cells, post-chemotherapy relapse usually occurs. Here, we report a potential solution to these issues that involves a type of novel multifunctional vinblastine liposomes equipped with transferrin receptor binding peptide TfR-T12 and octa-arginine conjugate stearyl-R8. Studies were performed on brain glioma and glioma stem cells in vitro and were verified in brain glioma-bearing mice. The liposomes were transported across the BBB, killing brain glioma and glioma stem cells via the induction of necrosis, apoptosis and autophagy. Furthermore, we reveal the molecular mechanisms for treating brain glioma and glioma stem cells via functionalized drug lipid vesicles.

The functional curcumin liposomes induce apoptosis in C6 glioblastoma cells and C6 glioblastoma stem cells in vitro and in animals.

Glioblastoma is a kind of malignant gliomas that is almost impossible to cure due to the poor drug transportation across the blood–brain barrier and the existence of glioma stem cells. We prepared a new kind of targeted liposomes in order to improve the drug delivery system onto the glioma cells and induce the apoptosis of glioma stem cells afterward. In this experiment, curcumin was chosen to kill gliomas, while quinacrine was used to induce apoptosis of the glioma stem cells. Also, p-aminophenyl-α-D-mannopyranoside could facilitate the transport of liposomes across the blood–brain barrier and finally target the brain glioma cells. The cell experiments in vitro indicated that the targeted liposomes could significantly improve the anti-tumor effects of the drugs, while enhancing the uptake effects, apoptosis effects, and endocytic effects of C6 glioma cells and C6 glioma stem cells. Given the animal experiments in vivo, we discovered that the targeted liposomes could obviously increase the survival period of brain glioma-bearing mice and inhibit the growth of gliomas. In summary, curcumin and quinacrine liposomes modified with p-aminophenyl-α-D-mannopyranoside is a potential preparation to treat brain glioma cells and brain glioma stem cells.

X-ray radiosensitivity of brain glioma stem cells and U251 cells

X-ray radiosensitivity of brain glioma stem cells and U251 cells

GFAP expression is regulated by Pax3 in brain glioma stem cells.

Glioblastomas are understood to evolve from brain glioma stem cells (BGSCs), and yet the biology underlying this model of tumorigenesis is largely unknown. Paired box3 protein (Pax3) is a member of the paired box (Pax) family of transcription factors that is normally expressed during embryonic development, but has recently been implicated in tumorigenesis. The present study demonstrated that Pax3 is differentially expressed in U87MG human glioma cell, BGSC and normal 1800human astrocyte lines. Herein, we identified that the glial fibrillary acidic protein (GFAP), a major intermediate filament protein of mature astrocytes, is directly downregulated during the differentiation of BGSCs via the binding of Pax3 to the promoter region of GFAP. Moreover, siRNA silencing of Pax3 arrested BGSC differentiation, while overexpression of Pax3 promoted the differentiation in BGSCs. Furthermore, we studied the cell proliferation, invasion, apoptosis, differentiation and expression of Pax3 and GFAP in Pax3 siRNA-knockdown and Pax3-overexpressing BGSC models by CCK-8, Transwell migration, flow cytometry and western blot assays. The results indicate that Pax3 regulates GFAP expression, and that Pax3 may contribute to the evolution of BGSCs towards malignancy.

Glioma stem cells and the occurrence of tumor blood vessels

Epithelial glioma is the most common brain cancer, accounting for 35.26%-60.69% of intracranial tumors with an average of 44.69%, and it remains the greatest challenge in the field of neurosurgery. The median survival time of patients with advanced glioma is only 12 to 18 months due to the characteristics of high aggression, and the therapeutic effect was poor though surgery, chemotherapy, and targeted drug therapy being treated. Because of the presence of heterogeneity and the differentiation disorder, only a small number of glioma cells are the source of tumor growth and metastasis, which are highly resistant to traditional treatments. They are deemed as the “seed” tumor cells as they could get rid of the effect of the treatment and reconstruct the organization of tumor. They are also termed as brain tumor stem cell (BTSC) or glioma stem cells (GSCs) since neural stem cells share similar features with them. Recent data reveal that they are directly related with invasion, angiogenesis, tolerance, chemotherapy, recurrence of glioma. Based on the research result by the team, the paper elaborates the characteristics of GSCs and the relationship with the tumor angiogenesis.

Glioblastoma and calcium signaling--analysis of calcium toolbox expression.

The characteristics of a cellular calcium signal (calcium signature) are determined, at least partly, by the expression of a subset of genes encoding proteins involved in calcium entry, calcium uptake and calcium modulation. Our aim in the present work was to characterize the set of genes involved in calcium signal generation that are differentially expressed in normal brain tissues versus brain tumor and/or glioma stem cells. Public datasets were analyzed according to a four step methodology consisting of: 1. detecting the outliers by using principal component analysis of the whole transcriptome; 2. building a calcium toolbox composed of 260 genes involved in the generation and modulation of the calcium signal; 3. analyzing the calcium toolbox transcriptome of different human brain areas and 4. detecting genes from the calcium toolbox preferentially expressed in tumor tissues or tumor cells compared to normal brain tissues. Our approach was validated on normal brain tissue. Tumor sample analysis allowed us to disclose a set of eighteen genes characteristic of glioblastoma tissues or glioma stem cells. Interpreting the set of genes highlighted in the study led us to propose that i) the mechanism of store operated calcium entry is strongly perturbed in cancer cells and tissues, ii) the process of calcium reuptake into mitochondria is more important in cancer cells and tissues than in their normal counterparts and iii) these two mechanisms may be coupled in at least one subgroup of the glioblastoma stem cells.

Multifunctional targeting daunorubicin plus quinacrine liposomes, modified by wheat germ agglutinin and tamoxifen, for treating brain glioma and glioma stem cells.

Most anticancer drugs are not able to cross the blood-brain barrier (BBB) effectively while surgery and radiation therapy cannot eradicate brain glioma cells and glioma stem cells (GSCs), hence resulting in poor prognosis with high recurrence rates. In the present study, a kind of multifunctional targeting daunorubicin plus quinacrine liposomes was developed for treating brain glioma and GSCs. Evaluations were performed on in-vitro BBB model, murine glioma cells, GSCs, and GSCs bearing mice. Results showed that the multifunctional targeting daunorubicin plus quinacrine liposomes exhibited evident capabilities in crossing the BBB, in killing glioma cells and GSCs and in diminishing brain glioma in mice. Action mechanism studies indicated that the enhanced efficacy of the multifunctional targeting drugs-loaded liposomes could be due to the following aspects: evading the rapid elimination from blood circulation; crossing the BBB effectively; improving drug uptake by glioma cells and GSCs; down-regulating the overexpressed ABC transporters; inducing apoptosis of GSCs via up-regulating apoptotic receptor/ligand (Fas/Fasl), activating apoptotic enzymes (caspases 8, 9 and 3), activating pro-apoptotic proteins (Bax and Bok), activating tumor suppressor protein (P53) and suppressing anti-apoptotic proteins (Bcl-2 and Mcl-1). In conclusion, the multifunctional targeting daunorubicin plus quinacrine liposomes could be used as a potential therapy for treating brain glioma and GSCs.

Isolation, cultivation and identification of brain glioma stem cells by magnetic bead sorting.

This study describes a detailed process for obtaining brain glioma stem cells from freshly dissected human brain glioma samples using an immunomagnetic bead technique combined with serum-free media pressure screening. Furthermore, the proliferation, differentiation and self-renewal biological features of brain glioma stem cells were identified. Results showed that a small number of CD133 positive tumor cells isolated from brain glioma samples survived as a cell suspension in serum-free media and proliferated. Subcultured CD133 positive cells maintained a potent self-renewal and proliferative ability, and expressed the stem cell-specific markers CD133 and nestin. After incubation with fetal bovine serum, the number of glial fibrillary acidic protein and microtubule associated protein 2 positive cells increased significantly, indicating that the cultured brain glioma stem cells can differentiate into astrocytes and neurons. Western blot analysis showed that tumor suppressor phosphatase and tensin homolog was highly expressed in tumor spheres compared with the differentiated tumor cells. These experimental findings indicate that the immunomagnetic beads technique is a useful method to obtain brain glioma stem cells from human brain tumors.

Expression and correlation of Bcl-2 with pathological grades in human glioma stem cells

The anti-apoptotic gene, B-cell lymphoma-2 (Bcl-2), has been reported to be overexpressed in gliomas and is related to tumor prognosis, suggesting a potential therapeutic target. Additionally, recent studies have demonstrated the existence of brain glioma stem cells (BGSCs) which are tumorigenic, self-renewable and dominate the biological behavior of gliomas. Currently BGSCs are committed as a new target of glioma therapies. However, few studies have focused on the expression of Bcl-2 in BGSCs. We performed a series of experiments to culture BGSCs from eight clinical specimens, followed by real-time RT-PCR and immunoassays to compare the expression levels of Bcl-2 in BGSCs and their corresponding primary glioma cells (PGCs). The results showed that Bcl-2 mRNA and protein expression levels are higher in BGSCs compared to their counterparts, and the expression levels are related to glioma malignancies. As an anti-apoptotic gene, Bcl-2 assigns immortality characteristics to cells, which coincide with the pivotal biological feature of BGSCs. The experimental results indicated that BGSCs would evade apoptosis for higher Bcl-2 expression, and may interpret the drug resistance of glioma to cytotoxic drugs and other pro-apoptotic agents. New therapies targeting Bcl-2 must induce apoptosis in BGSCs, thus, resulting in treatment or even eradication of glioma.

Expression levels of Fas/Fas-L mRNA in human brain glioma stem cells

Despite advancements in research made over the past decades, the prognosis of gliomas remains dismal. Fas/Fas ligand (Fas-L)-related immunotherapy may be regarded as a treatment of choice as it can induce the apoptosis of glioma cells, and has shown promising results in experimental studies. Over the years, brain glioma stem cells (BGSCs) have been accepted as the origin of gliomas and determine their biological features. The theory of BGSCs has facilitated the study of gliomas. In this study, we conducted a series of assays to culture BGSCs from clinical samples and determined the mRNA expression levels of Fas/Fas-L in BGSCs. We also investigated the effects of Fas/Fas-L-related immunotherapy on the apoptosis of glioma cells. BGSCs were grown from samples of 8 patients suffering from gliomas and identified by the assessment of biological characteristics and immunocytochemistry. Total RNA was extracted and reverse-transcribed into cDNA, and the expression levels of Fas/Fas-L mRNA were determined by real-time RT-PCR, followed by statistical analysis. The results showed that the Fas and Fas-L mRNA expression levels in BGSCs were lower compared to those in primary cultured glioma cells, which were statistically significant (P<0.001). These results indicate that immunotherapy involving Fas/Fas-L may not eradicate the BGSCs, which would result in the relapse of glioma. However, further research is required to investigate the mechanisms involved and define the prospect of Fas-involved immunotherapy against gliomas.

Human brain glioma stem cells are more invasive than their differentiated progeny cells in vitro

Glioma, the most commonly occurring primary intracranial tumor, remains associated with a dismal outcome, despite the availability of multimodal therapies. Recently, however, the identification of brain glioma stem cells (BGSC) has opened up new avenues for research. BGSC are now accepted as the progenitor cells of gliomas and are thought to determine the biological features of the resulting tumors. Thus, the diffuse invasiveness of gliomas should also be theoretically driven by BGSC. However, little research effort has been directed at understanding the invasiveness of BGSC. In the present study, BGSC from eight surgical glioma specimens were cultured and identified. Using Matrigel invasion assays, the invasiveness of these cells was measured and compared with that of their respective differentiated progeny cells in vitro. For all eight clinical specimens, the BGSC were significantly more invasive than their differentiated progeny cells. These findings indicate a key role for BGSC in glioma infiltration and invasion. We also found that BGSC tended to aggregate and reform into new spheres after travelling into the Matrigel, preserving some of the morphological characteristics of the suspended cells. The invasiveness of the BGSC did not correlate with the pathological grade of glioma in the present study, so further investigations using larger sample sizes are needed to better understand the mechanisms underlying the invasiveness of BGSC.

A simplified and modified procedure to culture brain glioma stem cells from clinical specimens

In recent years, the theory of brain glioma stem cells (BGSCs) has facilitated the study of gliomas. BGSCs have been accepted as the origin of gliomas and determine their biological features. Numerous efforts have been made to probe into the biological characteristics and behaviors of BGSCs. However, the culturing of target cells remains the essential first step for research on BGSCs. In this study, we established a simplified procedure to culture and isolate BGSCs from samples of clinical glioma patients. Samples of 17 glioma patients were included in the study, and the processed glioma cells were grown in serum-free stem cell media. After the tumor spheres appeared, a proliferation assay, a single-cell-derived colonies formation assay and an induced differentiation assay were carried out, followed by an immunocytochemistry assay. Serial passage was used to purify the target cells, whereas neither animal experiments nor sorting techniques were included. As a result, CD133(+) BGSCs from 8 out of 17 patients were grown and maintained in a serum-free condition combined with EGF, FGF and B-27 supplements. The tumor sphere cells were serially passaged and showed pluripotency in an induced differentiation assay. Immunocytochemistry identified the committed markers (CD133, GFAP and TU-20) and confirmed the cells were BGSCs and their progeny. The results proved that CD133(+) BGSCs from resected glioma tissue may be cultured in serum-free stem cell media, and may also be purified by conditioned culture combining serial passage, which is time-saving and cost-effective, and allows the cells to be used for subsequent research. The cell sorting techniques and animal experiments of tumorigenecity are optional. Thus, this modified procedure is more practical and feasible than other available procedures.