Glioblastoma Stem Research Articles

A Tumor-Homing Peptide Platform Enhances Drug Solubility, Improves Blood-Brain Barrier Permeability and Targets Glioblastoma.

Simple SummaryGlioblastoma (GBM) is a fatal and incurable brain cancer, and current treatment options have demonstrated limited success. Here, we describe the use of a dg-Bcan-Targeting-Peptide (BTP-7) that has BBB-penetrating properties and targets GBM. Conjugation of BTP-7 to an insoluble anti-cancer drug, camptothecin (CPT), improves drug solubility in aqueous solution, retains drug efficacy against patient-derived GBM stem cells (GSC), enhances BBB permeability, and enables therapeutic targeting to intracranial patient-derived GBM xenograft in mice, leading to higher toxicity in GBM cells compared to normal brain tissues and prolonged animal survival. This work demonstrates a proof-of-concept for BTP-7 as a tumor-targeting peptide for therapeutic delivery to GBM. Background: Glioblastoma (GBM) is the most common and deadliest malignant primary brain tumor, contributing significant morbidity and mortality among patients. As current standard-of-care demonstrates limited success, the development of new efficacious GBM therapeutics is urgently needed. Major challenges in advancing GBM chemotherapy include poor bioavailability, lack of tumor selectivity leading to undesired side effects, poor permeability across the blood–brain barrier (BBB), and extensive intratumoral heterogeneity. Methods: We have previously identified a small, soluble peptide (BTP-7) that is able to cross the BBB and target the human GBM extracellular matrix (ECM). Here, we covalently attached BTP-7 to an insoluble anti-cancer drug, camptothecin (CPT). Results: We demonstrate that conjugation of BTP-7 to CPT improves drug solubility in aqueous solution, retains drug efficacy against patient-derived GBM stem cells (GSC), enhances BBB permeability, and enables therapeutic targeting to intracranial GBM, leading to higher toxicity in GBM cells compared to normal brain tissues, and ultimately prolongs survival in mice bearing intracranial patient-derived GBM xenograft. Conclusion: BTP-7 is a new modality that opens the door to possibilities for GBM-targeted therapeutic approaches.

Crosstalk between β-Catenin and CCL2 Drives Migration of Monocytes towards Glioblastoma Cells.

Isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) is a fast growing and highly heterogeneous tumor, often characterized by the presence of glioblastoma stem cells (GSCs). The plasticity of GSCs results in therapy resistance and impairs anti-tumor immune response by influencing immune cells in the tumor microenvironment (TME). Previously, β-catenin was associated with stemness in GBM as well as with immune escape mechanisms. Here, we investigated the effect of β-catenin on attracting monocytes towards GBM cells. In addition, we evaluated whether CCL2 is involved in β-catenin crosstalk between monocytes and tumor cells. Our analysis revealed that shRNA targeting β-catenin in GBMs reduces monocytes attraction and impacts CCL2 secretion. The addition of recombinant CCL2 restores peripheral blood mononuclear cells (PBMC) migration towards medium (TCM) conditioned by shβ-catenin GBM cells. CCL2 knockdown in GBM cells shows similar effects and reduces monocyte migration to a similar extent as β-catenin knockdown. When investigating the effect of CCL2 on β-catenin activity, we found that CCL2 modulates components of the Wnt/β-catenin pathway and alters the clonogenicity of GBM cells. In addition, the pharmacological β-catenin inhibitor MSAB reduces active β-catenin, downregulates the expression of associated genes and alters CCL2 secretion. Taken together, we showed that β-catenin plays an important role in attracting monocytes towards GBM cells in vitro. We hypothesize that the interactions between β-catenin and CCL2 contribute to maintenance of GSCs via modulating immune cell interaction and promoting GBM growth and recurrence.

FTO gene polymorphisms and hepatoblastoma susceptibility among Chinese children

ABSTRACT Current knowledge on the etiology of hepatoblastoma remains limited. FTO gene has been documented as a susceptibility gene for several types of cancer. However, its role has not been characterized in hepatoblastoma. Herein, we intended to explore whether FTO gene single nucleotide polymorphisms (SNPs) contribute to the risk of hepatoblastoma. A multi-center case–control study was conducted including 358 cases and 1512 controls recruited from the night hospitals in China. Odds ratios (ORs) and 95% confidence intervals (CIs), for the association of FTO gene SNPs with hepatoblastoma risk, were estimated using conditional logistic regression models, adjusted for relevant confounding variables. Four SNPs (rs1477196 G > A, rs9939609 T > A, rs7206790 C > G, and rs8047395 A > G) in the FTO gene were genotyped. We detected a significant association between rs9939609 T > A and decreased risk of hepatoblastoma (TA vs. TT: adjust OR = 0.73, 95% CI = 0.54–0.99, P = 0.041; TA/AA vs. TT: adjust OR = 0.73, 95% CI = 0.55–0.97, P = 0.032). Compared to 0–3 protective genotypes, carriers with four protective genotypes showed enough strength to protect from hepatoblastoma (adjust OR = 0.65, 95% CI = 0.47–0.91, P = 0.012). In stratification analysis, we also detected a significantly decreased risk of hepatoblastoma in subjects with rs9939609 TA/AA or with four protective genotypes in some subgroups. Our results provided some clues for an association of FTO gene SNPs with hepatoblastoma risk in Chinese children. Abbreviations: GWAS, genome-wide association study; FTO, The fat mass and obesity-associated gene; SNP, single nucleotide polymorphism; m6A, N6-methyladenosine; mRNA, messenger RNA; LD, linkage disequilibrium; HWE, Hardy-Weinberg equilibrium; OR, odds ratio; CI, confidence interval; AML, acute myeloid leukemia; GSC, glioblastoma stem(-like) cell; HER2, human epidermal growth factor receptor 2.

Purine Synthesis Inhibitor L-Alanosine Impairs Mitochondrial Function and Stemness of Brain Tumor Initiating Cells.

Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that homozygous MTAP deletion, a genetic alteration occurring in about half of all GBMs, promotes stemness in GBM cells. Exploiting MTAP loss-conferred deficiency in purine salvage, we demonstrate that purine blockade via treatment with L-Alanosine (ALA), an inhibitor of de novo purine synthesis, attenuates stemness of MTAP-deficient GBM cells. This ALA-induced reduction in stemness is mediated in part by compromised mitochondrial function, highlighted by ALA-induced elimination of mitochondrial spare respiratory capacity. Notably, these effects of ALA are apparent even when the treatment was transient and with a low dose. Finally, in agreement with diminished stemness and compromised mitochondrial function, we show that ALA sensitizes GBM cells to temozolomide (TMZ) in vitro and in an orthotopic GBM model. Collectively, these results identify purine supply as an essential component in maintaining mitochondrial function in GBM cells and highlight a critical role of mitochondrial function in sustaining GBM stemness. We propose that purine synthesis inhibition can be beneficial in combination with the standard of care for MTAP-deficient GBMs, and that it may be feasible to achieve this benefit without inflicting major toxicity.

Impact of Regorafenib on Endothelial Transdifferentiation of Glioblastoma Stem-like Cells.

Simple SummaryGlioblastomas (GBM) are the most frequent and aggressive adult brain tumours with a poor prognosis despite heavy therapy that combines surgical resection and radio-chemotherapy. There is a real therapeutic failure concerning these tumours and it highlights the need for new efficient and usable molecules in these particular and not easily reachable cancers. GBM are also characterised by abnormal and abundant vascularisation, of which some vessels originate from transdifferentiation of GBM stem cells (GSC) to tumour-derived endothelial cells (TDEC). As this mechanism was described as essential for tumour growth, we evaluated the impact of regorafenib, a multikinase inhibitor with anti-angiogenic and anti-tumourigenic activity on transdifferentiation but also directly on GSC. Regorafenib significantly decreases GSC proliferation in vitro but also in vivo. Regorafenib also inhibits transdifferentiation by decreasing phenotypic and functional endothelial features of TDEC obtained from non-irradiated GSC but also of TDEC obtained from irradiated GSC. All these results confirm that regorafenib clearly impacts GSC tumour formation and transdifferentiation and may therefore be a promising therapeutic option in combination with chemo/radiotherapy for the treatment of these highly aggressive brain tumours.Glioblastomas (GBM) are aggressive brain tumours with a poor prognosis despite heavy therapy that combines surgical resection and radio-chemotherapy. The presence of a subpopulation of GBM stem cells (GSC) contributes to tumour aggressiveness, resistance and recurrence. Moreover, GBM are characterised by abnormal, abundant vascularisation. Previous studies have shown that GSC are directly involved in new vessel formation via their transdifferentiation into tumour-derived endothelial cells (TDEC) and that irradiation (IR) potentiates the pro-angiogenic capacity of TDEC via the Tie2 signalling pathway. We therefore investigated the impact of regorafenib, a multikinase inhibitor with anti-angiogenic and anti-tumourigenic activity, on GSC and TDEC obtained from irradiated GSC (TDEC IR+) or non-irradiated GSC (TDEC). Regorafenib significantly decreases GSC neurosphere formation in vitro and inhibits tumour formation in the orthotopic xenograft model. Regorafenib also inhibits transdifferentiation by decreasing CD31 expression, CD31+ cell count, pseudotube formation in vitro and the formation of functional blood vessels in vivo of TDEC and TDEC IR+. All of these results confirm that regorafenib clearly impacts GSC tumour formation and transdifferentiation and may therefore be a promising therapeutic option in combination with chemo/radiotherapy for the treatment of highly aggressive brain tumours.

ADAR1-mediated RNA editing links ganglioside catabolism to glioblastoma stem cell maintenance.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor, containing GBM stem cells (GSCs) that contribute to therapeutic resistance and relapse. Exposing potential GSC vulnerabilities may provide therapeutic strategies against GBM. Here, we interrogated the role of adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase acting on RNA 1 (ADAR1) in GSCs and found that both ADAR1 and global RNA editomes were elevated in GSCs compared with normal neural stem cells. ADAR1 inactivation or blocking of the upstream JAK/STAT pathway through TYK2 inhibition impaired GSC self-renewal and stemness. Downstream of ADAR1, RNA editing of the 3′-UTR of GM2A, a key ganglioside catabolism activator, proved to be critical, as interference with ganglioside catabolism and disruption of ADAR1 showed a similar functional impact on GSCs. These findings reveal that RNA editing links ganglioside catabolism to GSC self-renewal and stemness, exposing a potential vulnerability of GBM for therapeutic intervention.

The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma.

A small population of self-renewing stem cells initiate tumors and maintain therapeutic resistance in glioblastoma (GBM). Given the limited treatment options and dismal prognosis for this disease, there is urgent need to identify drivers of stem cells that could be druggable targets. Previous work showed that the endosomal pH regulator NHE9 is upregulated in GBM and correlates with worse survival prognosis. Here, we probed for aberrant signaling pathways in patient-derived GBM cells and found that NHE9 increases cell surface expression and phosphorylation of multiple receptor tyrosine kinases (RTKs) by promoting their escape from lysosomal degradation. Downstream of NHE9-mediated receptor activation, oncogenic signaling pathways converged on the JAK2-STAT3 transduction axis to induce pluripotency genes Oct4 and Nanog and suppress markers of glial differentiation. We used both genetic and chemical approaches to query the role of endosomal pH in GBM phenotypes. Loss-of-function mutations in NHE9 that failed to alkalinize endosomal lumen did not increase self-renewal capacity of gliomaspheres in vitro. However, monensin, a chemical mimetic of Na+/H+ exchanger activity, and the H+ pump inhibitor bafilomycin bypassed NHE9 to directly alkalinize the endosomal lumen resulting in stabilization of RTKs and induction of Oct4 and Nanog. Using orthotopic models of primary GBM cells we found that NHE9 increased tumor initiation in vivo. We propose that NHE9 initiates inside-out signaling from the endosomal lumen, distinct from the established effects of cytosolic and extracellular pH on tumorigenesis. Endosomal pH may be an attractive therapeutic target that diminishes stemness in GBM, agnostic of specific receptor subtype.

Myeloperoxidase exerts anti-tumor activity in glioma after radiotherapy.

BackgroundHost immune response is a critical component in tumorigenesis and immune escape. Radiation is widely used for glioblastoma (GBM) and can induce marked tissue inflammation and substantially alter host immune response. However, the role of myeloperoxidase (MPO), a key enzyme in inflammation and host immune response, in tumorigenesis after radiotherapy is unclear. In this study, we aimed to determine how post-radiation MPO activity influences GBM and outcome.MethodsWe injected C57BL/6J or MPO-knockout mice with 005 mouse GBM stem cells intracranially. To observe MPO's effects on post-radiation tumor progression, we then irradiated the head with 10 Gy unfractionated and treated the mice with a specific MPO inhibitor, 4-aminobenzoic acid hydrazide (ABAH), or vehicle as control. We performed semi-quantitative longitudinal molecular MRI, enzymatic assays and flow cytometry to assess changes in inflammatory response and tumor size, and tracked survival. We also performed cell culture experiments in murine and human GBM cells to determine the effect of MPO on these cells.ResultsBrain irradiation increased the number of monocytes/macrophages and neutrophils, and boosted MPO activity by ten-fold in the glioma microenvironment. However, MPO inhibition dampened radiation-induced inflammation, demonstrating decreased MPO-specific signal on molecular MRI and attenuated neutrophil and inflammatory monocyte/macrophage recruitment to the glioma. Compared to saline-treated mice, both ABAH-treated and MPO-knockout mice had accelerated tumor growth and reduced survival. We further confirmed that MPO decreased tumor cell viability and proliferation in cell cultures.ConclusionLocal radiation to the brain initiated an acute systemic inflammatory response with increased MPO-carrying cells both in the periphery and the GBM, resulting in increased MPO activity in the tumor microenvironment. Inhibition or absence of MPO activity increased tumor growth and decreased host survival, revealing that elevated MPO activity after radiation has an anti-tumor role.

ASCL1 phosphorylation and ID2 upregulation are roadblocks to glioblastoma stem cell differentiation

The growth of glioblastoma (GBM), one of the deadliest adult cancers, is fuelled by a subpopulation of stem/progenitor cells, which are thought to be the source of resistance and relapse after treatment. Re-engagement of a latent capacity of these cells to re-enter a trajectory resulting in cell differentiation is a potential new therapeutic approach for this devastating disease. ASCL1, a proneural transcription factor, plays a key role in normal brain development and is also expressed in a subset of GBM cells, but fails to engage a full differentiation programme in this context. Here, we investigated the barriers to ASCL1-driven differentiation in GBM stem cells. We see that ASCL1 is highly phosphorylated in GBM stem cells where its expression is compatible with cell proliferation. However, overexpression of a form of ASCL1 that cannot be phosphorylated on Serine–Proline sites drives GBM cells down a neuronal lineage and out of cell cycle more efficiently than its wild-type counterpart, an effect further enhanced by deletion of the inhibitor of differentiation ID2, indicating mechanisms to reverse the block to GBM cell differentiation.

Sox2 induces glioblastoma cell stemness and tumor propagation by repressing TET2 and deregulating 5hmC and 5mC DNA modifications

DNA methylation is a reversible process catalyzed by the ten–eleven translocation (TET) family of enzymes (TET1, TET2, TET3) that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Altered patterns of 5hmC and 5mC are widely reported in human cancers and loss of 5hmC correlates with poor prognosis. Understanding the mechanisms leading to 5hmC loss and its role in oncogenesis will advance the development of epigenetic-based therapeutics. We show that TET2 loss associates with glioblastoma (GBM) stem cells and correlates with poor survival of GBM patients. We further identify a SOX2:miR-10b-5p:TET2 axis that represses TET2 expression, represses 5hmC, increases 5mC levels, and induces GBM cell stemness and tumor-propagating potential. In vivo delivery of a miR-10b-5p inhibitor that normalizes TET2 expression and 5hmC levels inhibits tumor growth and prolongs survival of animals bearing pre-established orthotopic GBM xenografts. These findings highlight the importance of TET2 and 5hmC loss in Sox2-driven oncogenesis and their potential for therapeutic targeting.

In This Issue

In This Issue

Transcription Elongation Machinery Is a Druggable Dependency and Potentiates Immunotherapy in Glioblastoma Stem Cells.

Glioblastoma (GBM) is the most lethal primary brain cancer characterized by therapeutic resistance, which is promoted by GBM stem cells (GSC). Here, we interrogated gene expression and whole-genome CRISPR/Cas9 screening in a large panel of patient-derived GSCs, differentiated GBM cells (DGC), and neural stem cells (NSC) to identify master regulators of GSC stemness, revealing an essential transcription state with increased RNA polymerase II-mediated transcription. The YY1 and transcriptional CDK9 complex was essential for GSC survival and maintenance in vitro and in vivo. YY1 interacted with CDK9 to regulate transcription elongation in GSCs. Genetic or pharmacologic targeting of the YY1-CDK9 complex elicited RNA m6A modification-dependent interferon responses, reduced regulatory T-cell infiltration, and augmented efficacy of immune checkpoint therapy in GBM. Collectively, these results suggest that YY1-CDK9 transcription elongation complex defines a targetable cell state with active transcription, suppressed interferon responses, and immunotherapy resistance in GBM. SIGNIFICANCE: Effective strategies to rewire immunosuppressive microenvironment and enhance immunotherapy response are still lacking in GBM. YY1-driven transcriptional elongation machinery represents a druggable target to activate interferon response and enhance anti-PD-1 response through regulating the m6A modification program, linking epigenetic regulation to immunomodulatory function in GBM.This article is highlighted in the In This Issue feature, p. 275.

Up-Regulation of Cyclooxygenase-2 (COX-2) Expression by Temozolomide (TMZ) in Human Glioblastoma (GBM) Cell Lines.

TMZ-resistance remains a main limitation in glioblastoma (GBM) treatment. TMZ is an alkylating agent whose cytotoxicity is modulated by O6-methylguanine-DNA methyltransferase (MGMT), whose expression is determined by MGMT gene promoter methylation status. The inflammatory marker COX-2 has been implicated in GBM tumorigenesis, progression, and stemness. COX-2 inhibitors are considered a GBM add-on treatment due to their ability to increase TMZ-sensitivity. We investigated the effect of TMZ on COX-2 expression in GBM cell lines showing different COX-2 levels and TMZ sensitivity (T98G and U251MG). β-catenin, MGMT, and SOX-2 expression was analyzed. The effects of NS398, COX-2 inhibitor, alone or TMZ-combined, were studied evaluating cell proliferation by the IncuCyte® system, cell cycle/apoptosis, and clonogenic potential. COX-2, β-catenin, MGMT, and SOX-2 expression was evaluated by RT-PCR, Western blotting, and immunofluorescence and PGE2 by ELISA. Our findings, sustaining the role of COX-2/PGE2 system in TMZ-resistance of GBM, show, for the first time, a relevant, dose-dependent up-regulation of COX-2 expression and activity in TMZ-treated T98G that, in turn, correlated with chemoresistance. Similarly, all the COX-2-dependent signaling pathways involved in TMZ-resistance also resulted in being up-modulated after treatment with TMZ. NS398+TMZ was able to reduce cell proliferation and induce cell cycle arrest and apoptosis. Moreover, NS398+TMZ counteracted the resistance in T98G preventing the TMZ-induced COX-2, β-catenin, MGMT, and SOX-2 up-regulation.

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.

Transitioning pre-clinical glioblastoma models to clinical settings with biomarkers identified in 3D cell-based models: A systematic scoping review.

Glioblastoma (GBM) remains incurable despite the overwhelming discovery of 2-dimensional (2D) cell-based potential therapeutics since the majority of them have met unsatisfactory results in animal and clinical settings. Incremental empirical evidence has laid the widespread need of transitioning 2D to 3-dimensional (3D) cultures that better mimic GBM's complex and heterogenic nature to allow better translation of pre-clinical results. This systematic scoping review analyses the transcriptomic data involving 3D models of GBM against 2D models from 22 studies identified from four databases (PubMed, ScienceDirect, Medline, and Embase). From a total of 499 genes reported in these studies, 313 (63%) genes were upregulated across 3D models cultured using different scaffolds. Our analysis showed that 4 of the replicable upregulated genes are associated with GBM stemness, epithelial to mesenchymal transition (EMT), hypoxia, and migration-related genes regardless of the type of scaffolds, displaying close resemblances to primitive undifferentiated tumour phenotypes that are associated with decreased overall survival and increased hazard ratio in GBM patients. The upregulation of drug response and drug efflux genes (e.g. cytochrome P450s and ABC transporters) mirrors the GBM genetic landscape that contributes to in vivo and clinical treatment resistance. These upregulated genes displayed strong protein-protein interactions when analysed using an online bioinformatics software (STRING). These findings reinforce the need for widespread transition to 3D GBM models as a relatively inexpensive humanised pre-clinical tool with suitable genetic biomarkers to bridge clinical gaps in potential therapeutic evaluations.

Detection of temozolomide-induced hypermutation and response to PD-1 checkpoint inhibitor in recurrent glioblastoma.

BackgroundDespite aggressive upfront treatment in glioblastoma (GBM), recurrence remains inevitable for most patients. Accumulating evidence has identified hypermutation induced by temozolomide (TMZ) as an emerging subtype of recurrent GBM. However, its biological and therapeutic significance has yet to be described.MethodsWe combined GBM patient and derive GBM stem cells (GSCs) from tumors following TMZ to explore response of hypermutant and non-hypermutant emergent phenotypes and explore the immune relevance of hypermutant and non-hypermutant states in vivo.ResultsHypermutation emerges as one of two possible mutational subtypes following TMZ treatment in vivo and demonstrates distinct phenotypic features compared to non-hypermutant recurrent GBM. Hypermutant tumors elicited robust immune rejection in subcutaneous contexts which was accompanied by increased immune cell infiltration. In contrast, immune rejection of hypermutant tumors were stunted in orthotopic settings where we observe limited immune infiltration. Use of anti-PD-1 immunotherapy showed that immunosuppression in orthotopic contexts was independent from the PD-1/PD-L1 axis. Finally, we demonstrate that mutational burden can be estimated from DNA contained in extracellular vesicles (EVs).ConclusionHypermutation post-TMZ are phenotypically distinct from non-hypermutant GBM and requires personalization for appropriate treatment. The brain microenvironment may be immunosuppressive and exploration of the mechanisms behind this may be key to improving immunotherapy response in this subtype of recurrent GBM.

A Novel Role of BIRC3 in Stemness Reprogramming of Glioblastoma.

Stemness reprogramming remains a largely unaddressed principal cause of lethality in glioblastoma (GBM). It is therefore of utmost importance to identify and target mechanisms that are essential for GBM stemness and self-renewal. Previously, we implicated BIRC3 as an essential mediator of therapeutic resistance and survival adaptation in GBM. In this study, we present novel evidence that BIRC3 has an essential noncanonical role in GBM self-renewal and stemness reprogramming. We demonstrate that BIRC3 drives stemness reprogramming of human GBM cell lines, mouse GBM cell lines and patient-derived GBM stem cells (GSCs) through regulation of BMP4 signaling axis. Specifically, BIRC3 induces stemness reprogramming in GBM through downstream inactivation of BMP4 signaling. RNA-Seq interrogation of the stemness reprogramming hypoxic (pseudopalisading necrosis and perinecrosis) niche in GBM patient tissues further validated the high BIRC3/low BMP4 expression correlation. BIRC3 knockout upregulated BMP4 expression and prevented stemness reprogramming of GBM models. Furthermore, siRNA silencing of BMP4 restored stemness reprogramming of BIRC3 knockout in GBM models. In vivo silencing of BIRC3 suppressed tumor initiation and progression in GBM orthotopic intracranial xenografts. The stemness reprograming of both GSCs and non-GSCs populations highlights the impact of BIRC3 on intra-tumoral cellular heterogeneity GBM. Our study has identified a novel function of BIRC3 that can be targeted to reverse stemness programming of GBM.

Matrix Hyaluronic Acid and Hypoxia Influence a CD133+ Subset of Patient-Derived Glioblastoma Cells.

Glioblastoma (GBM) displays diffusive invasion throughout the brain microenvironment, which is partially responsible for its short median survival rate (<15 months). Stem-like subpopulations (GBM stem-like cells, GSCs) are believed to play a central role in therapeutic resistance and poor patient prognosis. Given the extensive tissue remodeling and processes such as vessel co-option and regression that occur in the tumor microenvironment, it is essential to understand the role of metabolic constraint such as hypoxia on GBM cell populations. This work describes the use of a multidimensional gelatin hydrogel to culture patient-derived GBM cells, to evaluate the influence of hypoxia and the inclusion brain-mimetic hyaluronic acid on the relative activity of GSCs versus overall GBM cells. Notably, CD133+ GBM cell fraction is crucial for robust formation of tumor spheroids in multidimensional cultures. In addition, while the relative size of the CD133+ GBM subpopulation increased in response to both hypoxia and matrix-bound hyaluronan, we did not observe cell subtype-specific changes in invasion signaling pathway activation. Taken together, this study highlights the potential of biomimetic culture systems for resolving changes in the population dynamics and behavior of subsets of GBM specimens for the future development of precision medicine applications. Impact Statement This study describes a gelatin hydrogel platform to investigate the role of extracellular hyaluronic acid and hypoxia on the behavior of a CD133+ subset of cells within patient-derived glioblastoma (GBM) specimens. We report that the relative expansion of the CD133+ GBM stem cell-like population is strongly responsive to extracellular cues, highlighting the significance of biomimetic hydrogel models of the tumor microenvironment to investigate invasion and therapeutic response.

Inhibition of Carbonic Anhydrase 2 Overcomes Temozolomide Resistance in Glioblastoma Cells.

About 95% of Glioblastoma (GBM) patients experience tumor relapse as a consequence of resistance to the first-line standard chemotherapy using temozolomide (TMZ). Recent studies reported consistently elevated expression levels of carbonic anhydrase CA2 in recurrent glioblastoma and temozolomide-resistant glioblastoma stem-like cells (GSCs). Here we show that CA2 is preferentially expressed in GSCs and upregulated by TMZ treatment. When expressed in GBM cell lines, CA2 exerts significant metabolic changes reflected by enhanced oxygen consumption and increased extracellular acidification causing higher rates of cell invasion. Notably, GBM cells expressing CA2 respond to combined treatment with TMZ and brinzolamide (BRZ), a non-toxic and potent CA2 inhibitor. Interestingly, brinzolamide was more effective than the pan-CA inhibitor Acetazolamide (ACZ) to sensitize naïve GSCs and TMZ-resistant GSCs to TMZ induced cell death. Mechanistically, we demonstrated that the combined treatment of GBM stem cells with TMZ and BRZ caused autophagy of GBM cell lines and GSCs, reflected by enhanced LC3 cleavage (LC3-II) and p62 reduction. Our findings illustrate the potential of CA2 as a chemo-sensitizing drug target in recurrent GBM and propose a combined treatment of TMZ with CA2 inhibitor to tackle GBM chemoresistance and recurrence.

Drug Repurposing for Glioblastoma and Current Advances in Drug Delivery-A Comprehensive Review of the Literature.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults with an extremely poor prognosis. There is a dire need to develop effective therapeutics to overcome the intrinsic and acquired resistance of GBM to current therapies. The process of developing novel anti-neoplastic drugs from bench to bedside can incur significant time and cost implications. Drug repurposing may help overcome that obstacle. A wide range of drugs that are already approved for clinical use for the treatment of other diseases have been found to target GBM-associated signaling pathways and are being repurposed for the treatment of GBM. While many of these drugs are undergoing pre-clinical testing, others are in the clinical trial phase. Since GBM stem cells (GSCs) have been found to be a main source of tumor recurrence after surgery, recent studies have also investigated whether repurposed drugs that target these pathways can be used to counteract tumor recurrence. While several repurposed drugs have shown significant efficacy against GBM cell lines, the blood–brain barrier (BBB) can limit the ability of many of these drugs to reach intratumoral therapeutic concentrations. Localized intracranial delivery may help to achieve therapeutic drug concentration at the site of tumor resection while simultaneously minimizing toxicity and side effects. These strategies can be considered while repurposing drugs for GBM.