An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.

Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.

Extracellular vesicles throughout development: A potential roadmap for emerging glioblastoma therapies.

Extracellular vesicles (EVs) are membrane-delimited vesicular bodies carrying different molecules, classified according to their size, density, cargo, and origin. Research on this topic has been actively growing through the years, as EVs are associated with critical pathological processes such as neurodegenerative diseases and cancer. Despite that, studies exploring the physiological functions of EVs are sparse, with particular emphasis on their role in organismal development, initial cell differentiation, and morphogenesis. In this review, we explore the topic of EVs from a developmental perspective, discussing their role in the earliest cell-fate decisions and neural tissue morphogenesis. We focus on the function of EVs through development to highlight possible conserved or novel processes that can impact disease progression. Specifically, we take advantage of what was learned about their role in development so far to discuss EVs impact on glioblastoma, a particular brain tumor of stem-cell origin and poor prognosis, and how their function can be hijacked to improve current therapies.

UTX inhibition suppresses proliferation and promotes apoptosis in patient-derived glioblastoma stem cells by modulating periostin expression.

Glioblastoma stem cells (GSCs) exert a crucial influence on glioblastoma (GBM) development, progression, resistance to therapy, and recurrence, making them an attractive target for drug discovery. UTX, a histone H3K27 demethylase, participates in regulating multiple cancer types. However, its functional role in GSCs remains insufficiently explored. This study aims to investigate the role and regulatory mechanism of UTX on GSCs. Analysis of TCGA data revealed heightened UTX expression in glioma, inversely correlating with overall survival. Inhibiting UTX suppressed GBM cell growth and induced apoptosis. Subsequently, we cultured primary GSCs from three patients, observing that UTX inhibition suppressed cell proliferation and induced apoptosis. RNA-seq was performed to analyze the gene expression changes after silencing UTX in GSCs. The results indicated that UTX-mediated genes were strongly correlated with GBM progression and regulatory tumor microenvironment. The transwell co-cultured experiment showed that silencing UTX in the transwell chamber GSCs inhibited the well plate cell proliferation. Protein-protein interaction analysis revealed that periostin (POSTN) played a role in the UTX-mediated transcriptional regulatory network. Replenishing POSTN reversed the effects of UTX inhibition on GSC proliferation and apoptosis. Our study demonstrated that UTX inhibition hindered POSTN expression by enhancing the H3K27me2/3 level, eventually resulting in inhibiting proliferation and promoting apoptosis of patient-derived GSCs. Our findings may provide a novel and effective strategy for the treatment of GBM.

The Heterogeneous Cellular States of Glioblastoma Stem Cells Revealed by Single Cell Analysis.

Glioblastoma stem cells (GSCs) contributed to the progression, treatment resistance, and relapse of glioblastoma (GBM). However, current researches on GSCs were performed usually outside the human tumor microenvironment, ignoring the importance of the cellular states of primary GSCs. In this study, we leveraged single-cell transcriptome sequencing data of 6 independent GBM cohorts from public databases, and combined lineage and stemness features to identify primary GSCs. We dissected the cell states of GSCs and correlated them with the clinical outcomes of patients. As a result, we constructed a cellular hierarchy where GSCs resided at the center. In addition, we identified and characterized 2 different and recurrent GSCs subpopulations: proliferative GSCs (pGSCs) and quiescent GSCs (qGSCs). The pGSCs showed high cell cycle activity, indicating rapid cell division, while qGSCs showed a quiescent state. Then we traced the processes of tumor development by pseudo-time analysis and tumor phylogeny, and found that GSCs accumulated throughout the whole tumor development period. During the process, pGSCs mainly contributed to the early stage and qGSCs were enriched in the later stage. Finally, we constructed an 8-gene prognostic signature reflecting pGSCs activity and found that patients whose tumors were enriched for the pGSC signature had poor clinical outcomes. Our study highlights the primary GSCs heterogeneity and its correlation to tumor development and clinical outcomes, providing the potential targets for GBM treatment.

Extracellular vesicles produced by irradiated endothelial or Glioblastoma stem cells promote tumor growth and vascularization modulating tumor microenvironment.

BACKGROUND: Glioblastoma (GBM) is the most lethal primary brain tumor in adult, characterized by highly aggressive and infiltrative growth. The current therapeutic management of GBM includes surgical resection followed by ionizing radiations and chemotherapy. Complex and dynamic interplay between tumor cells and tumor microenvironment drives the progression and contributes to therapeutic resistance. Extracellular vesicles (EVs) play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment. METHODS: In this study, we isolated by ultracentrifugation EVs from GBM stem-like cell (GSC) lines and human microvascular endothelial cells (HMVECs) exposed or not to ionizing irradiation. After counting and characterization, we evaluated the effects of exposure of GSCs to EVs isolated from endothelial cells and vice versa. The RNA content of EVs isolated from GSC lines and HMVECs exposed or not to ionizing irradiation, was analyzed by RNA-Seq. Periostin (POSTN) and Filamin-B (FLNB) emerged in gene set enrichment analysis as the most interesting transcripts enriched after irradiation in endothelial cell-derived EVs and GSC-derived EVs, respectively. POSTN and FLNB expression was modulated and the effects were analyzed by in vitro assays. RESULTS: We confirmed that ionizing radiations increased EV secretion by GSCs and normal endothelial cells, affected the contents of and response to cellular secreted EVs. Particularly, GSC-derived EVs decreased radiation-induced senescence and promoted migration in HMVECs whereas, endothelial cell-derived EVs promoted tumorigenic properties and endothelial differentiation of GSCs. RNA-Seq analysis of EV content, identified FLNB and POSTN as transcripts enriched in EVs isolated after irradiation from GSCs and HMVECs, respectively. Assays performed on POSTN overexpressing GSCs confirmed the ability of POSTN to mimic the effects of endothelial cell-derived EVs on GSC migration and clonogenic abilities and transdifferentiation potential. Functional assays performed on HMVECs after silencing of FLNB supported its role as mediator of the effects of GSC-derived EVs on senescence and migration. CONCLUSION: In this study, we identified POSTN and FLNB as potential mediators of the effects of EVs on GSC and HMVEC behavior confirming that EVs play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.

4,5-Dimethoxycanthin-6-one Inhibits Glioblastoma Stem Cell and Tumor Growth by Inhibiting TSPAN1 Interaction with TM4SF1.

Glioblastoma stem cells (GSCs) have been implicated in the self-renewal and treatment resistance of glioblastoma (GBM). Our previous study found that 4,5-dimethoxycanthin-6-one has the potential to inhibit GBM cell proliferation. This current study aims to elucidate the molecular mechanism underlying the effects of 4,5-dimethoxycanthin-6-one in GBM development. The effect of 4,5-dimethoxycanthin-6-one on GSC formation and differentiation was explored in human GBM cell lines U251 and U87. Subsequently, 4,5-dimethoxycanthin-6-one binding to tetraspanin 1 (TSPAN1) / transmembrane 4 L six family member 1 (TM4SF1) was analyzed by molecular simulation docking. Co-immunoprecipitation (Co-IP) and immunofluorescence (IF) were used to assess the interactions between TSPAN1 and TM4SF1 in GSCs. Cell proliferation was detected by cell counting kit-8 (CCK-8) and colony formation assay. To evaluate cell migration, invasion and apoptosis, we employed wound healing assay, transwell and flow cytometry, respectively. Furthermore, subcutaneous xenograft tumor models in nude mice were constructed to evaluate the impact of 4,5-dimethoxycanthin-6-one on GSCs in vivo by examining tumor growth and histological characteristics. 4,5-Dimethoxycanthin-6-one inhibited GSC formation and promoted stem cell differentiation in a concentration-dependent manner. Molecular docking models of 4,5-dimethoxycanthin-6-one with TM4SF1 and TSPAN1 were constructed. Then, the interaction between TSPAN1 and TM4SF1 in GSC was clarified. Moreover, 4,5-dimethoxycanthin-6-one significantly inhibited the expressions of TM4SF1 and TSPAN1 in vitro and in vivo. Overexpression of TSPAN1 partially reversed the inhibitory effects of 4,5-dimethoxycanthin-6-one on GSC formation, proliferation, migration and invasion. 4,5-Dimethoxycanthin-6-one inhibited GBM progression by inhibiting TSPAN1/TM4SF1 axis. 4,5-Dimethoxycanthin-6-one might be a novel and effective drug for the treatment of GBM.

Targeting sphingolipid metabolism with the sphingosine kinase inhibitor SKI-II overcomes hypoxia-induced chemotherapy resistance in glioblastoma cells: effects on cell death, self-renewal, and invasion.

BACKGROUND: Glioblastoma patients commonly develop resistance to temozolomide chemotherapy. Hypoxia, which supports chemotherapy resistance, favors the expansion of glioblastoma stem cells (GSC), contributing to tumor relapse. Because of a deregulated sphingolipid metabolism, glioblastoma tissues contain high levels of the pro-survival sphingosine-1-phosphate and low levels of the pro-apoptotic ceramide. The latter can be metabolized to sphingosine-1-phosphate by sphingosine kinase (SK) 1 that is overexpressed in glioblastoma. The small molecule SKI-II inhibits SK and dihydroceramide desaturase 1, which converts dihydroceramide to ceramide. We previously reported that SKI-II combined with temozolomide induces caspase-dependent cell death, preceded by dihydrosphingolipids accumulation and autophagy in normoxia. In the present study, we investigated the effects of a low-dose combination of temozolomide and SKI-II under normoxia and hypoxia in glioblastoma cells and patient-derived GCSs. METHODS: Drug synergism was analyzed with the Chou-Talalay Combination Index method. Dose-effect curves of each drug were determined with the Sulforhodamine B colorimetric assay. Cell death mechanisms and autophagy were analyzed by immunofluorescence, flow cytometry and western blot; sphingolipid metabolism alterations by mass spectrometry and gene expression analysis. GSCs self-renewal capacity was determined using extreme limiting dilution assays and invasion of glioblastoma cells using a 3D spheroid model. RESULTS: Temozolomide resistance of glioblastoma cells was increased under hypoxia. However, combination of temozolomide (48 µM) with SKI-II (2.66 µM) synergistically inhibited glioblastoma cell growth and potentiated glioblastoma cell death relative to single treatments under hypoxia. This low-dose combination did not induce dihydrosphingolipids accumulation, but a decrease in ceramide and its metabolites. It induced oxidative and endoplasmic reticulum stress and triggered caspase-independent cell death. It impaired the self-renewal capacity of temozolomide-resistant GSCs, especially under hypoxia. Furthermore, it decreased invasion of glioblastoma cell spheroids. CONCLUSIONS: This in vitro study provides novel insights on the links between sphingolipid metabolism and invasion, a hallmark of cancer, and cancer stem cells, key drivers of cancer. It demonstrates the therapeutic potential of approaches that combine modulation of sphingolipid metabolism with first-line agent temozolomide in overcoming tumor growth and relapse by reducing hypoxia-induced resistance to chemotherapy and by targeting both differentiated and stem glioblastoma cells.

Novel terpestacin derivatives with l-amino acid residue as anticancer agents against U87MG-derived glioblastoma stem cells.

Based on the natural product terpestacin, seventeen derivatives (1-17) with various l-amino acid side chains were designed and synthesized. Their anticancer activities against U87MG-derived glioblastoma stem cells (GSCs) were evaluated, and compounds 5, 11, 13 and 15 showed strong abilities to inhibit the proliferation (IC50 = 2.8-6.9 µM) and tumorsphere formation of GSCs. Besides, compounds 13 and 15 could effectively induce apoptosis and significantly inhibit the invasion of GSCs (95 and 97 % inhibition, respectively, at 2.5 µM). The levels of CD133 marker in GSCs also decreased in dose-dependent manners after the treatment of these active compounds. Compared to terpestacin and the positive control A1938, our derivatives showed stronger activities and compounds 13 and 15 are promising candidates for further development as anticancer agents by targeting GSCs.

FOSL1 promotes proneural-to-mesenchymal transition of glioblastoma stem cells via UBC9/CYLD/NF-κB axis.

Proneural (PN) to mesenchymal (MES) transition (PMT) is a crucial phenotypic shift in glioblastoma stem cells (GSCs). However, the mechanisms driving this process remain poorly understood. Here, we report that Fos-like antigen 1 (FOSL1), a component of AP1 transcription factor complexes, is a key player in regulating PMT. FOSL1 is predominantly expressed in the MES subtype, but not PN subtype, of GSCs. Knocking down FOSL1 expression in MES GSCs leads to the loss of MES features and tumor-initiating ability, whereas ectopic expression of FOSL1 in PN GSCs is able to induce PMT and maintain MES features. Moreover, FOSL1 facilitates ionizing radiation (IR)-induced PMT and radioresistance of PN GSCs. Inhibition of FOSL1 enhances the anti-tumor effects of IR by preventing IR-induced PMT. Mechanistically, we find that FOSL1 promotes UBC9-dependent CYLD SUMOylation, thereby inducing K63-linked polyubiquitination of major nuclear factor κB (NF-κB) intermediaries and subsequent NF-κB activation, which results in PMT induction in GSCs. Our study underscores the importance of FOSL1 in the regulation of PMT and suggests that therapeutic targeting of FOSL1 holds promise to attenuate molecular subtype switching in patients with glioblastomas.

CD146 increases stemness and aggressiveness in glioblastoma and activates YAP signaling.

Glioblastoma (GBM), a highly malignant and lethal brain tumor, is characterized by diffuse invasion into the brain and chemo-radiotherapy resistance resulting in poor prognosis. In this study, we examined the involvement of the cell adhesion molecule CD146/MCAM in regulating GBM aggressiveness. Analyses of GBM transcript expression databases revealed correlations of elevated CD146 levels with higher glioma grades, IDH-wildtype and unmethylated MGMT phenotypes, poor response to chemo-radiotherapy and worse overall survival. In a panel of GBM stem cells (GSCs) variable expression levels of CD146 were detected, which strongly increased upon adherent growth. CD146 was linked with mesenchymal transition since expression increased in TGF-ß-treated U-87MG cells. Ectopic overexpression of CD146/GFP in GG16 cells enhanced the mesenchymal phenotype and resulted in increased cell invasion. Conversely, GSC23-CD146 knockouts had decreased mesenchymal marker expression and reduced cell invasion in transwell and GBM-cortical assembloid assays. Moreover, using GSC23 xenografted zebrafish, we found that CD146 depletion resulted in more compact delineated tumor formation and reduced tumor cell dissemination. Stem cell marker expression and neurosphere formation assays showed that CD146 increased the stem cell potential of GSCs. Furthermore, CD146 mediated radioresistance by stimulating cell survival signaling through suppression of p53 expression and activation of NF-κB. Interestingly, CD146 was also identified as an inducer of the oncogenic Yes-associated protein (YAP). In conclusion, CD146 carries out various pro-tumorigenic roles in GBM involving its cell surface receptor function, which include the stimulation of mesenchymal and invasive properties, stemness, and radiotherapy resistance, thus providing an interesting target for therapy.

Two Sides of The Same Coin: Normal and Tumoral Stem Cells, The Relevance of In Vitro Models and Therapeutic Approaches: The Experience with Zika Virus in Nervous System Development and Glioblastoma Treatment.

Neural stem cells (NSCs) were described for the first time more than two decades ago for their ability to differentiate into all neural cell lineages. The isolation of NSCs from adults and embryos was carried out by various laboratories and in different species, from mice to humans. Similarly, no more than two decades ago, cancer stem cells were described. Cancer stem cells, previously identified in hematological malignancies, have now been isolated from several solid tumors (breast, brain, and gastrointestinal compartment). Though the origin of these cells is still unknown, there is a wide consensus about their role in tumor onset, propagation and, in particular, resistance to treatments. Normal and neoplastic neural stem cells share common characteristics, and can thus be considered as two sides of the same coin. This is particularly true in the case of the Zika virus (ZIKV), which has been described as an inhibitor of neural development by specifically targeting NSCs. This understanding prompted us and other groups to evaluate ZIKV action in glioblastoma stem cells (GSCs). The results indicate an oncolytic activity of this virus vs. GSCs, opening potentially new possibilities in glioblastoma treatment.

Nitric Oxide Prevents Glioblastoma Stem Cells' Expansion and Induces Temozolomide Sensitization.

Glioblastoma multiforme (GBM) has high mortality and recurrence rates. Malignancy resilience is ascribed to Glioblastoma Stem Cells (GSCs), which are resistant to Temozolomide (TMZ), the gold standard for GBM post-surgical treatment. However, Nitric Oxide (NO) has demonstrated anti-cancer efficacy in GBM cells, but its potential impact on GSCs remains unexplored. Accordingly, we investigated the effects of NO, both alone and in combination with TMZ, on patient-derived GSCs. Experimentally selected concentrations of diethylenetriamine/NO adduct and TMZ were used through a time course up to 21 days of treatment, to evaluate GSC proliferation and death, functional recovery, and apoptosis. Immunofluorescence and Western blot analyses revealed treatment-induced effects in cell cycle and DNA damage occurrence and repair. Our results showed that NO impairs self-renewal, disrupts cell-cycle progression, and expands the quiescent cells' population. Consistently, NO triggered a significant but tolerated level of DNA damage, but not apoptosis. Interestingly, NO/TMZ cotreatment further inhibited cell cycle progression, augmented G0 cells, induced cell death, but also enhanced DNA damage repair activity. These findings suggest that, although NO administration does not eliminate GSCs, it stunts their proliferation, and makes cells susceptible to TMZ. The resulting cytostatic effect may potentially allow long-term control over the GSCs' subpopulation.

Role of Luteolin as Potential New Therapeutic Option for Patients with Glioblastoma through Regulation of Sphingolipid Rheostat.

Glioblastoma (GBM) is the most aggressive brain tumor, still considered incurable. In this study, conducted on primary GBM stem cells (GSCs), specifically selected as the most therapy-resistant, we examined the efficacy of luteolin, a natural flavonoid, as an anti-tumoral compound. Luteolin is known to impact the sphingolipid rheostat, a pathway regulated by the proliferative sphingosine-1-phosphate (S1P) and the proapoptotic ceramide (Cer), and implicated in numerous oncopromoter biological processes. Here, we report that luteolin is able to inhibit the expression of SphK1/2, the two kinases implicated in S1P formation, and to increase the expression of both SGPL1, the lyase responsible for S1P degradation, and CERS1, the ceramide synthase 1, thus shifting the balance toward the production of ceramide. In addition, luteolin proved to decrease the expression of protumoral signaling as MAPK, RAS/MEK/ERK and PI3K/AKT/mTOR and cyclins involved in cell cycle progression. In parallel, luteolin succeeded in upregulation of proapoptotic mediators as caspases and Bcl-2 family and cell cycle controllers as p53 and p27. Furthermore, luteolin determined the shutdown of autophagy contributing to cell survival. Overall, our data support the use of luteolin as add-on therapy, having demonstrated a good ability in impairing GSC viability and survival and increasing cell sensitivity to TMZ.

Patient-individual phenotypes of glioblastoma stem cells are conserved in culture and associate with radioresistance, brain infiltration and patient prognosis.

Identification of prognostic or predictive molecular markers in glioblastoma resection specimens may lead to strategies for therapy stratification and personalized treatment planning. Here, we analyzed in primary glioblastoma stem cell (pGSC) cultures the mRNA abundances of seven stem cell (MSI1, Notch1, nestin, Sox2, Oct4, FABP7 and ALDH1A3), and three radioresistance or invasion markers (CXCR4, IKCa and BKCa ). From these abundances, an mRNA signature was deduced which describes the mesenchymal-to-proneural expression profile of an individual GSC culture. To assess its functional significance, we associated the GSC mRNA signature with the clonogenic survival after irradiation with 4 Gy and the fibrin matrix invasion of the GSC cells. In addition, we compared the molecular pGSC mRNA signature with the tumor recurrence pattern and the overall survival of the glioblastoma patients from whom the pGSC cultures were derived. As a result, the molecular pGSC mRNA signature correlated positively with the pGSC radioresistance and matrix invasion capability in vitro. Moreover, patients with a mesenchymal (>median) mRNA signature in their pGSC cultures exhibited predominantly a multifocal tumor recurrence and a significantly (univariate log rank test) shorter overall survival than patients with proneural (≤median mRNA signature) pGSCs. The tumors of the latter recurred predominately unifocally. We conclude that our pGSC cultures induce/select those cell subpopulations of the heterogeneous brain tumor that determine disease progression and therapy outcome. In addition, we further postulate a clinically relevant prognostic/predictive value for the 10 mRNAs-based mesenchymal-to-proneural signature of the GSC subpopulations in glioblastoma.

GZ17-6.02 Inhibits the Growth of EGFRvIII+ Glioblastoma.

Epidermal Growth Factor Receptor (EGFR) is amplified in over 50% of glioblastomas and promotes tumor formation and progression. However, attempts to treat glioblastoma with EGFR tyrosine kinase inhibitors have been unsuccessful thus far. The current standard of care is especially poor in patients with a constitutively active form of EGFR, EGFRvIII, which is associated with shorter survival time. This study examined the effect of GZ17-6.02, a novel anti-cancer agent undergoing phase 1 studies, on two EGFRvIII+ glioblastoma stem cells: D10-0171 and D317. In vitro analyses showed that GZ17-6.02 inhibited the growth of both D10-0171 and D317 cells with IC50 values of 24.84 and 28.28 µg/mL respectively. RNA sequencing and reverse phase protein array analyses revealed that GZ17-6.02 downregulates pathways primarily related to steroid synthesis and cell cycle progression. Interestingly, G17-6.02's mechanism of action involves the downregulation of the recently identified glioblastoma super-enhancer genes WSCD1, EVOL2, and KLHDC8A. Finally, a subcutaneous xenograft model showed that GZ17-6.02 inhibits glioblastoma growth in vivo. We conclude that GZ17-6.02 is a promising combination drug effective at inhibiting the growth of a subset of glioblastomas and our data warrants further preclinical studies utilizing xenograft models to identify patients that may respond to this drug.

HIF2α Upregulates the Migration Factor ODZ1 under Hypoxia in Glioblastoma Stem Cells.

Background: Glioblastoma (GBM) remains a major clinical challenge due to its invasive capacity, resistance to treatment, and recurrence. We have previously shown that ODZ1 contributes to glioblastoma invasion and that ODZ1 mRNA levels can be upregulated by epigenetic mechanisms in response to hypoxia. Herein, we have further studied the transcriptional regulation of ODZ1 in GBM stem cells (GSCs) under hypoxic conditions and analyzed whether HIF2α has any role in this regulation. Methods: We performed the experiments in three primary GSC cell lines established from tumor specimens. GSCs were cultured under hypoxia, treated with HIF regulators (DMOG, chetomin), or transfected with specific siRNAs, and the expression levels of ODZ1 and HIF2α were analyzed. In addition, the response of the ODZ1 promoter cloned into a luciferase reporter plasmid to the activation of HIF was also studied. Results: The upregulation of both mRNA and protein levels of HIF2α under hypoxia conditions correlated with the expression of ODZ1 mRNA. Moreover, the knockdown of HIF2α by siRNAs downregulated the expression of ODZ1. We found, in the ODZ1 promoter, a HIF consensus binding site (GCGTG) 1358 bp from the transcription start site (TSS) and a HIF-like site (CCGTG) 826 bp from the TSS. Luciferase assays revealed that the stabilization of HIF by DMOG resulted in the increased activity of the ODZ1 promoter. Conclusions: Our data indicate that the HIF2α-mediated upregulation of ODZ1 helps strengthen the transcriptional control of this migration factor under hypoxia in glioblastoma stem cells. The discovery of this novel transcriptional pathway identifies new targets to develop strategies that may avoid GBM tumor invasion and recurrence.

Upregulation of Cathepsin X in Glioblastoma: Interplay with γ-Enolase and the Effects of Selective Cathepsin X Inhibitors.

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Understanding GBM pathobiology and discovering novel therapeutic targets are critical to finding efficient treatments. Upregulation of the lysosomal cysteine carboxypeptidase cathepsin X has been linked to immune dysfunction and neurodegenerative diseases, but its role in cancer and particularly in GBM progression in patients is unknown. In this study, cathepsin X expression and activity were found to be upregulated in human GBM tissues compared to low-grade gliomas and nontumor brain tissues. Cathepsin X was localized in GBM cells as well as in tumor-associated macrophages and microglia. Subsequently, potent irreversible (AMS36) and reversible (Z7) selective cathepsin X inhibitors were tested in vitro. Selective cathepsin X inhibitors decreased the viability of patient-derived GBM cells as well as macrophages and microglia that were cultured in conditioned media of GBM cells. We next examined the expression pattern of neuron-specific enzyme γ-enolase, which is the target of cathepsin X. We found that there was a correlation between high proteolytic activity of cathepsin X and C-terminal cleavage of γ-enolase and that cathepsin X and γ-enolase were colocalized in GBM tissues, preferentially in GBM-associated macrophages and microglia. Taken together, our results on patient-derived material suggest that cathepsin X is involved in GBM progression and is a potential target for therapeutic approaches against GBM.

Sevoflurane Does Not Promote the Colony-Forming Ability of Human Mesenchymal Glioblastoma Stem Cells In Vitro.

Background and Objectives: Clinically used concentrations of sevoflurane, an inhaled anesthetic, have been reported to significantly inhibit tumor growth. We investigated the effects of sevoflurane on sphere formation and the proliferation of human glioblastoma stem cells (GSCs) to determine whether sevoflurane exerts short- and long-term effects on human tumor cells. Materials and Methods: High-grade patient-derived GSCs (MD13 and Me83) were exposed to 2% sevoflurane. To evaluate the effect of sevoflurane on viability, proliferation, and stemness, we performed a caspase-3/7 essay, cell proliferation assay, and limiting dilution sphere formation assays. The expression of CD44, a cell surface marker of cancer stem-like cells in epithelial tumors, was evaluated using quantitative reverse transcription PCR. Differences between groups were evaluated with a one-way analysis of variance (ANOVA). Results: Sevoflurane exposure for 4 days did not significantly promote caspase 3/7 activity in MD13 and Me83, and cell proliferation was not observed after 5 days of exposure. Furthermore, prolonged exposure to sevoflurane for 6 days did not promote the sphere-forming and proliferative potential of MD13 and Me83 cells. These results suggest that sevoflurane does not promote either apoptosis, proliferative capacity, or the colony-forming ability of human mesenchymal glioblastoma stem cells in vitro. Conclusions: Sevoflurane at clinically used concentrations does not promote the colony-forming ability of human mesenchymal glioblastoma stem cells in vitro. It is very important for neurosurgeons and anesthesiologists to know that sevoflurane, a volatile anesthetic used in surgical anesthesia, would not exacerbate the disease course of GSCs.

The Importance of Tumor Stem Cells in Glioblastoma Resistance to Therapy.

Glioblastoma (GBM) is known to be the most common and lethal primary malignant brain tumor. Therapies against this neoplasia have a high percentage of failure, associated with the survival of self-renewing glioblastoma stem cells (GSCs), which repopulate treated tumors. In addition, despite new radical surgery protocols and the introduction of new anticancer drugs, protocols for treatment, and technical advances in radiotherapy, no significant improvement in the survival rate for GBMs has been realized. Thus, novel antitarget therapies could be used in conjunction with standard radiochemotherapy approaches. Targeted therapy, indeed, may address specific targets that play an essential role in the proliferation, survival, and invasiveness of GBM cells, including numerous molecules involved in signal transduction pathways. Significant cellular heterogeneity and the hierarchy with GSCs showing a therapy-resistant phenotype could explain tumor recurrence and local invasiveness and, therefore, may be a target for new therapies. Therefore, the forced differentiation of GSCs may be a promising new approach in GBM treatment. This article provides an updated review of the current standard and experimental therapies for GBM, as well as an overview of the molecular characteristics of GSCs, the mechanisms that activate resistance to current treatments, and a new antitumor strategy for treating GSCs for use as therapy.

An Update on Glioblastoma Biology, Genetics, and Current Therapies: Novel Inhibitors of the G Protein-Coupled Receptor CCR5.

The mechanisms governing therapeutic resistance of the most aggressive and lethal primary brain tumor in adults, glioblastoma, have increasingly focused on tumor stem cells. These cells, protected by the periarteriolar hypoxic GSC niche, contribute to the poor efficacy of standard of care treatment of glioblastoma. Integrated proteogenomic and metabolomic analyses of glioblastoma tissues and single cells have revealed insights into the complex heterogeneity of glioblastoma and stromal cells, comprising its tumor microenvironment (TME). An additional factor, which isdriving poor therapy response is the distinct genetic drivers in each patient's tumor, providing the rationale for a more individualized or personalized approach to treatment. We recently reported that the G protein-coupled receptor CCR5, which contributes to stem cell expansion in other cancers, is overexpressed in glioblastoma cells. Overexpression of the CCR5 ligand CCL5 (RANTES) in glioblastoma completes a potential autocrine activation loop to promote tumor proliferation and invasion. CCL5 was not expressed in glioblastoma stem cells, suggesting a need for paracrine activation of CCR5 signaling by the stromal cells. TME-associated immune cells, such as resident microglia, infiltrating macrophages, T cells, and mesenchymal stem cells, possibly release CCR5 ligands, providing heterologous signaling between stromal and glioblastoma stem cells. Herein, we review current therapies for glioblastoma, the role of CCR5 in other cancers, and the potential role for CCR5 inhibitors in the treatment of glioblastoma.