GRIK1 promotes glioblastoma malignancy and is a novel prognostic factor of poor prognosis.

Primary tumors of the central nervous system (CNS) are classified into over 100 different histological types. The most common type of glioma is derived from astrocytes, and the most invasive glioblastoma (WHO IV) accounts for over 57% of these tumors. Glioblastoma (GBM) is the most common and fatal tumor of the CNS, with strong growth and invasion capabilities, which makes complete surgical resection almost impossible. Despite various treatment methods such as surgery, radiotherapy, and chemotherapy, glioma is still an incurable disease, and the median survival time of patients with GBM is shorter than 15 months. Thus, molecular mechanisms of GBM characteristic invasive growth need to be clarified to improve the poor prognosis. Glutamate ionotropic receptor kainate type subunit 1 (GRIK1) is essential for brain function and is involved in many mental and neurological diseases. However, GRIK1's pathogenic roles and mechanisms in GBM are still unknown. Single-nuclear RNA sequencing of primary and recurrent GBM samples revealed that GRIK1 expression was noticeably higher in the recurrent samples. Moreover, immunohistochemical staining of an array of GBM samples showed that high levels of GRIK1 correlated with poor prognosis of GBM, consistent with The Cancer Genome Atlas database. Knockdown of GRIK1 retarded GBM cells growth, migration, and invasion. Taken together, these findings show that GRIK1 is a unique and important component in the development of GBM and may be considered as a biomarker for the diagnosis and therapy in individuals with GBM.

Glioblastoma-instructed microglia transition to heterogeneous phenotypic states with phagocytic and dendritic cell-like features in patient tumors and patient-derived orthotopic xenografts.

BACKGROUND: A major contributing factor to glioblastoma (GBM) development and progression is its ability to evade the immune system by creating an immune-suppressive environment, where GBM-associated myeloid cells, including resident microglia and peripheral monocyte-derived macrophages, play critical pro-tumoral roles. However, it is unclear whether recruited myeloid cells are phenotypically and functionally identical in GBM patients and whether this heterogeneity is recapitulated in patient-derived orthotopic xenografts (PDOXs). A thorough understanding of the GBM ecosystem and its recapitulation in preclinical models is currently missing, leading to inaccurate results and failures of clinical trials. METHODS: Here, we report systematic characterization of the tumor microenvironment (TME) in GBM PDOXs and patient tumors at the single-cell and spatial levels. We applied single-cell RNA sequencing, spatial transcriptomics, multicolor flow cytometry, immunohistochemistry, and functional studies to examine the heterogeneous TME instructed by GBM cells. GBM PDOXs representing different tumor phenotypes were compared to glioma mouse GL261 syngeneic model and patient tumors. RESULTS: We show that GBM tumor cells reciprocally interact with host cells to create a GBM patient-specific TME in PDOXs. We detected the most prominent transcriptomic adaptations in myeloid cells, with brain-resident microglia representing the main population in the cellular tumor, while peripheral-derived myeloid cells infiltrated the brain at sites of blood-brain barrier disruption. More specifically, we show that GBM-educated microglia undergo transition to diverse phenotypic states across distinct GBM landscapes and tumor niches. GBM-educated microglia subsets display phagocytic and dendritic cell-like gene expression programs. Additionally, we found novel microglial states expressing cell cycle programs, astrocytic or endothelial markers. Lastly, we show that temozolomide treatment leads to transcriptomic plasticity and altered crosstalk between GBM tumor cells and adjacent TME components. CONCLUSIONS: Our data provide novel insights into the phenotypic adaptation of the heterogeneous TME instructed by GBM tumors. We show the key role of microglial phenotypic states in supporting GBM tumor growth and response to treatment. Our data place PDOXs as relevant models to assess the functionality of the TME and changes in the GBM ecosystem upon treatment.

Exploring ncRNA-mediated regulation of EGFR signalling in glioblastoma: From mechanisms to therapeutics.

Glioblastoma (GBM) is the most prevalent and deadly primary brain tumor type, with a discouragingly low survival rate and few effective treatments. An important function of the EGFR signalling pathway in the development of GBM is to affect tumor proliferation, persistence, and treatment resistance. Advances in molecular biology in the last several years have shown how important ncRNAs are for controlling a wide range of biological activities, including cancer progression and development. NcRNAs have become important post-transcriptional regulators of gene expression, and they may affect the EGFR pathway by either directly targeting EGFR or by modifying important transcription factors and downstream signalling molecules. The EGFR pathway is aberrantly activated in response to the dysregulation of certain ncRNAs, which has been linked to GBM carcinogenesis, treatment resistance, and unfavourable patient outcomes. We review the literature on miRNAs, circRNAs and lncRNAs that are implicated in the regulation of EGFR signalling in GBM, discussing their mechanisms of action, interactions with the signalling pathway, and implications for GBM therapy. Furthermore, we explore the potential of ncRNA-based strategies to overcome resistance to EGFR-targeted therapies, including the use of ncRNA mimics or inhibitors to modulate the activity of key regulators within the pathway.

Mesenchymal glioma stem cells trigger vasectasia-distinct neovascularization process stimulated by extracellular vesicles carrying EGFR.

Targeting neovascularization in glioblastoma (GBM) is hampered by poor understanding of the underlying mechanisms and unclear linkages to tumour molecular landscapes. Here we report that different molecular subtypes of human glioma stem cells (GSC) trigger distinct endothelial responses involving either angiogenic or circumferential vascular growth (vasectasia). The latter process is selectively triggered by mesenchymal (but not proneural) GSCs and is mediated by a subset of extracellular vesicles (EVs) able to transfer EGFR/EGFRvIII transcript to endothelial cells. Inhibition of the expression and phosphorylation of EGFR in endothelial cells, either pharmacologically (Dacomitinib) or genetically (gene editing), abolishes their EV responses in vitro and disrupts vasectasia in vivo. Therapeutic inhibition of EGFR markedly extends anticancer effects of VEGF blockade in mice, coupled with abrogation of vasectasia and prolonged survival. Thus, vasectasia driven by intercellular transfer of oncogenic EGFR may represent a new therapeutic target in a subset of GBMs.

Lidocaine attenuates TMZ resistance and inhibits cell migration by modulating the MET pathway in glioblastoma cells.

Glioblastoma multiforme (GBM) is the most aggressive type of malignant brain tumor. Currently, the predominant clinical treatment is the combination of surgical resection with concurrent radiotherapy and chemotherapy, using temozolomide (TMZ) as the primary chemotherapy drug. Lidocaine, a widely used amide­based local anesthetic, has been found to have a significant anticancer effect. It has been reported that aberrant hepatocyte growth factor (HGF)/mesenchymal­epithelial transition factor (MET) signaling plays a role in the progression of brain tumors. However, it remains unclear whether lidocaine can regulate the MET pathway in GBM. In the present study, the clinical importance of the HGF/MET pathway was analyzed using bioinformatics. By establishing TMZ­resistant cell lines, the impact of combined treatment with lidocaine and TMZ was investigated. Additionally, the effects of lidocaine on cellular function were also examined and confirmed using knockdown techniques. The current findings revealed that the HGF/MET pathway played a key role in brain cancer, and its activation in GBM was associated with increased malignancy and poorer patient outcomes. Elevated HGF levels and activation of its receptor were found to be associated with TMZ resistance in GBM cells. Lidocaine effectively suppressed the HGF/MET pathway, thereby restoring TMZ sensitivity in TMZ­resistant cells. Furthermore, lidocaine also inhibited cell migration. Overall, these results indicated that inhibiting the HGF/MET pathway using lidocaine can enhance the sensitivity of GBM cells to TMZ and reduce cell migration, providing a potential basis for developing novel therapeutic strategies for GBM.

Changes Induced by P2X7 Receptor Stimulation of Human Glioblastoma Stem Cells in the Proteome of Extracellular Vesicles Isolated from Their Secretome.

Extracellular vesicles (EVs) are secreted from many tumors, including glioblastoma multiforme (GBM), the most common and lethal brain tumor in adults, which shows high resistance to current therapies and poor patient prognosis. Given the high relevance of the information provided by cancer cell secretome, we performed a proteomic analysis of microvesicles (MVs) and exosomes (EXOs) released from GBM-derived stem cells (GSCs). The latter, obtained from the brain of GBM patients, expressed P2X7 receptors (P2X7Rs), which positively correlate with GBM growth and invasiveness. P2X7R stimulation of GSCs caused significant changes in the EV content, mostly ex novo inducing or upregulating the expression of proteins related to cytoskeleton reorganization, cell motility/spreading, energy supply, protection against oxidative stress, chromatin remodeling, and transcriptional regulation. Most of the induced/upregulated proteins have already been identified as GBM diagnostic/prognostic factors, while others have only been reported in peripheral tumors. Our findings indicate that P2X7R stimulation enhances the transport and, therefore, possible intercellular exchange of GBM aggressiveness-increasing proteins by GSC-derived EVs. Thus, P2X7Rs could be considered a new druggable target of human GBM, although these data need to be confirmed in larger experimental sets.

Mesenchymal-Stem-Cell-Based Therapy against Gliomas.

Glioblastoma is the most aggressive, malignant, and lethal brain tumor of the central nervous system. Its poor prognosis lies in its inefficient response to currently available treatments that consist of surgical resection, radiotherapy, and chemotherapy. Recently, the use of mesenchymal stem cells (MSCs) as a possible kind of cell therapy against glioblastoma is gaining great interest due to their immunomodulatory properties, tumor tropism, and differentiation into other cell types. However, MSCs seem to present both antitumor and pro-tumor properties depending on the tissue from which they come. In this work, the possibility of using MSCs to deliver therapeutic genes, oncolytic viruses, and miRNA is presented, as well as strategies that can improve their therapeutic efficacy against glioblastoma, such as CAR-T cells, nanoparticles, and exosomes.

A Novel Approach for Glioblastoma Treatment by Combining Apoptosis Inducers (TMZ, MTX, and Cytarabine) with E.V.A. (Eltanexor, Venetoclax, and A1210477) Inhibiting XPO1, Bcl-2, and Mcl-1.

Adjuvant treatment for Glioblastoma Grade 4 with Temozolomide (TMZ) inevitably fails due to therapeutic resistance, necessitating new approaches. Apoptosis induction in GB cells is inefficient, due to an excess of anti-apoptotic XPO1/Bcl-2-family proteins. We assessed TMZ, Methotrexate (MTX), and Cytarabine (Ara-C) (apoptosis inducers) combined with XPO1/Bcl-2/Mcl-1-inhibitors (apoptosis rescue) in GB cell lines and primary GB stem-like cells (GSCs). Using CellTiter-Glo® and Caspase-3 activity assays, we generated dose-response curves and analyzed the gene and protein regulation of anti-apoptotic proteins via PCR and Western blots. Optimal drug combinations were examined for their impact on the cell cycle and apoptosis induction via FACS analysis, paralleled by the assessment of potential toxicity in healthy mouse brain slices. Ara-C and MTX proved to be 150- to 10,000-fold more potent in inducing apoptosis than TMZ. In response to inhibitors Eltanexor (XPO1; E), Venetoclax (Bcl-2; V), and A1210477 (Mcl-1; A), genes encoding for the corresponding proteins were upregulated in a compensatory manner. TMZ, MTX, and Ara-C combined with E, V, and A evidenced highly lethal effects when combined. As no significant cell death induction in mouse brain slices was observed, we conclude that this drug combination is effective in vitro and expected to have low side effects in vivo.

[Dynamic trajectory and cell communication of different cell clusters in malignant progression of glioblastoma].

OBJECTIVE: To delve deeply into the dynamic trajectories of cell subpopulations and the communication network among immune cell subgroups during the malignant progression of glioblastoma (GBM), and to endeavor to unearth key risk biomarkers in the GBM malignancy progression, so as to provide a more profound understanding for the treatment and prognosis of this disease by integrating transcriptomic data and clinical information of the GBM patients. METHODS: Utilizing single-cell sequencing data analysis, we constructed a cell subgroup atlas during the malignant progression of GBM. The Monocle2 tool was employed to build dynamic progression trajectories of the tumor cell subgroups in GBM. Through gene enrichment analysis, we explored the biological processes enriched in genes that significantly changed with the malignancy progression of GBM tumor cell subpopulations. CellChat was used to identify the communication network between the different immune cell subgroups. Survival analysis helped in identifying risk molecular markers that impacted the patient prognosis during the malignant progression of GBM. This method ological approach offered a comprehensive and detailed examination of the cellular and molecular dynamics within GBM, providing a robust framework for understanding the disease' s progression and potential therapeutic targets. RESULTS: The analysis of single-cell sequencing data identified 6 different cell types, including lymphocytes, pericytes, oligodendrocytes, macrophages, glioma cells, and microglia. The 27 151 cells in the single-cell dataset included 3 881 cells from the patients with low-grade glioma (LGG), 10 166 cells from the patients with newly diagnosed GBM, and 13 104 cells from the patients with recurrent glioma (rGBM). The pseudo-time analysis of the glioma cell subgroups indicated significant cellular heterogeneity during malignant progression. The cell interaction analysis of immune cell subgroups revealed the communication network among the different immune subgroups in GBM malignancy, identifying 22 biologically significant ligand-receptor pairs across 12 key biological pathways. Survival analysis had identified 8 genes related to the prognosis of the GBM patients, among which SERPINE1, COL6A1, SPP1, LTF, C1S, AEBP1, and SAA1L were high-risk genes in the GBM patients, and ABCC8 was low-risk genes in the GBM patients. These findings not only provided new theoretical bases for the treatment of GBM, but also offered fresh insights for the prognosis assessment and treatment decision-making for the GBM patients. CONCLUSION: This research comprehensively and profoundly reveals the dynamic changes in glioma cell subpopulations and the communication patterns among the immune cell subgroups during the malignant progression of GBM. These findings are of significant importance for understanding the complex biological processes of GBM, providing crucial new insights for precision medicine and treatment decisions in GBM. Through these studies, we hope to provide more effective treatment options and more accurate prognostic assessments for the patients with GBM.

USP19 regulates DNA methylation damage repair and confers temozolomide resistance through MGMT stabilization.

OBJECTIVE: To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance. METHODS: Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management. RESULTS: USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients. CONCLUSION: The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response.

Inhibition of signaling protein ERN1 increases the sensitivity of serine synthesis gene expressions to glucose and glutamine deprivations in U87MG glioblastoma cells.

Objective. Glucose and glutamine supply as well as serine synthesis and endoplasmic reticulum (ER) stress are important factors of glioblastoma growth. Previous studies showed that the knockdown of ERN1 (ER to nucleus signaling 1) suppressed glioblastoma cell proliferation and modified the sensitivity of numerous gene expressions to nutrient deprivations. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of serine synthesis genes in U87MG glioblastoma cells in relation to ERN1 knockdown with the intent to reveal the role of ERN1 signaling pathway on the ER stress-dependent regulation of these gene expressions. Clarification of the regulatory mechanisms of serine synthesis is a great significance for glioblastoma therapy. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed under glucose and glutamine deprivation conditions for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine amino-transferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that the expression level of genes responsible for serine synthesis such as PHGDH, PSAT1, PSPH, and transcription factor ATF4 was up-regulated in U87MG glioblastoma cells under glucose and glutamine deprivations. Furthermore, inhibition of ERN1 significantly enhances the impact of glucose and especially glutamine deprivations on these gene expressions. At the same time, the expression of the SHMT1 gene, which is responsible for serine conversion to glycine, was down-regulated in both nutrient deprivation conditions with more significant changes in ERN1 knockdown glioblastoma cells. Conclusion. Taken together, the results of present study indicate that the expression of genes responsible for serine synthesis is sensitive to glucose and glutamine deprivations in gene-specific manner and that suppression of ERN1 signaling significantly modifies the impact of both glucose and glutamine deprivations on PHGDH, PSAT1, PSPH, ATF4, and SHMT1 gene expressions and reflects the ERN1-mediated genome reprograming introduced by nutrient deprivation condition.

ID1high/activin Ahigh glioblastoma cells contribute to resistance to anti-angiogenesis therapy through malformed vasculature.

Although bevacizumab (BVZ), a representative drug for anti-angiogenesis therapy (AAT), is used as a first-line treatment for patients with glioblastoma (GBM), its efficacy is notably limited. Whereas several mechanisms have been proposed to explain the acquisition of AAT resistance, the specific underlying mechanisms have yet to be sufficiently ascertained. Here, we established that inhibitor of differentiation 1 (ID1)high/activin Ahigh glioblastoma cell confers resistance to BVZ. The bipotent effect of activin A during its active phase was demonstrated to reduce vasculature dependence in tumorigenesis. In response to a temporary exposure to activin A, this cytokine was found to induce endothelial-to-mesenchymal transition via the Smad3/Slug axis, whereas prolonged exposure led to endothelial apoptosis. ID1 tumors showing resistance to BVZ were established to be characterized by a hypovascular structure, hyperpermeability, and scattered hypoxic regions. Using a GBM mouse model, we demonstrated that AAT resistance can be overcome by administering therapy based on a combination of BVZ and SB431542, a Smad2/3 inhibitor, which contributed to enhancing survival. These findings offer valuable insights that could contribute to the development of new strategies for treating AAT-resistant GBM.

Spatial transcriptomics reveals segregation of tumor cell states in glioblastoma and marked immunosuppression within the perinecrotic niche.

Glioblastoma (GBM) remains an untreatable malignant tumor with poor patient outcomes, characterized by palisading necrosis and microvascular proliferation. While single-cell technology made it possible to characterize different lineage of glioma cells into neural progenitor-like (NPC-like), oligodendrocyte-progenitor-like (OPC-like), astrocyte-like (AC-like) and mesenchymal like (MES-like) states, it does not capture the spatial localization of these tumor cell states. Spatial transcriptomics empowers the study of the spatial organization of different cell types and tumor cell states and allows for the selection of regions of interest to investigate region-specific and cell-type-specific pathways. Here, we obtained paired 10x Chromium single-nuclei RNA-sequencing (snRNA-seq) and 10x Visium spatial transcriptomics data from three GBM patients to interrogate the GBM microenvironment. Integration of the snRNA-seq and spatial transcriptomics data reveals patterns of segregation of tumor cell states. For instance, OPC-like tumor and NPC-like tumor significantly segregate in two of the three samples. Our differentially expressed gene and pathway analyses uncovered significant pathways in functionally relevant niches. Specifically, perinecrotic regions were more immunosuppressive than the endogenous GBM microenvironment, and perivascular regions were more pro-inflammatory. Our gradient analysis suggests that OPC-like tumor cells tend to reside in areas closer to the tumor vasculature compared to tumor necrosis, which may reflect increased oxygen requirements for OPC-like cells. In summary, we characterized the localization of cell types and tumor cell states, the gene expression patterns, and pathways in different niches within the GBM microenvironment. Our results provide further evidence of the segregation of tumor cell states and highlight the immunosuppressive nature of the necrotic and perinecrotic niches in GBM.

RAPID resistance to BET inhibitors is mediated by FGFR1 in glioblastoma.

Bromodomain and extra-terminal domain (BET) proteins are therapeutic targets in several cancers including the most common malignant adult brain tumor glioblastoma (GBM). Multiple small molecule inhibitors of BET proteins have been utilized in preclinical and clinical studies. Unfortunately, BET inhibitors have not shown efficacy in clinical trials enrolling GBM patients. One possible reason for this may stem from resistance mechanisms that arise after prolonged treatment within a clinical setting. However, the mechanisms and timeframe of resistance to BET inhibitors in GBM is not known. To identify the temporal order of resistance mechanisms in GBM we performed quantitative proteomics using multiplex-inhibitor bead mass spectrometry and demonstrated that intrinsic resistance to BET inhibitors in GBM treatment occurs rapidly within hours and involves the fibroblast growth factor receptor 1 (FGFR1) protein. Additionally, small molecule inhibition of BET proteins and FGFR1 simultaneously induces synergy in reducing GBM tumor growth in vitro and in vivo. Further, FGFR1 knockdown synergizes with BET inhibitor mediated reduction of GBM cell proliferation. Collectively, our studies suggest that co-targeting BET and FGFR1 may dampen resistance mechanisms to yield a clinical response in GBM.

The RAS oncogene in brain tumors and the involvement of let-7 microRNA.

RAS oncogenes are master regulator genes in many cancers. In general, RAS-driven cancers have an oncogenic RAS mutation that promotes disease progression (colon, lung, pancreas). In contrast, brain tumors are not necessarily RAS-driven cancers because RAS mutations are rarely observed. In particular, glioblastomas (the most lethal brain tumor) do not appear to have dominant genetic mutations that are suitable for targeted therapy. Standard treatment for most brain tumors continues to focus on maximal surgical resection, radiotherapy and chemotherapy. Yet the convergence of genomic aberrations such as EGFR, PDGFR and NF1 (some of which are clinically effective) with activation of the RAS/MAPK cascade is still considered a key point in gliomagenesis, and KRAS is undoubtedly a driving gene in gliomagenesis in mice. In cancer, microRNAs (miRNA) are small, non-coding RNAs that regulate carcinogenesis. However, the functional consequences of aberrant miRNA expression in cancer are still poorly understood. let-7 encodes an intergenic miRNA that is classified as a tumour suppressor, at least in lung cancer. Let-7 suppresses a plethora of oncogenes such as RAS, HMGA, c-Myc, cyclin-D and thus suppresses cancer development, differentiation and progression. let-7 family members are direct regulators of certain RAS family genes by binding to the sequences in their 3'untranslated region (3'UTR). let-7 miRNA is involved in the malignant behaviour in vitro-proliferation, migration and invasion-of gliomas and stem-like glioma cells as well as in vivo models of glioblastoma multiforme (GBM) via KRAS inhibition. It also increases resistance to certain chemotherapeutic agents and radiotherapy in GBM. Although let-7 therapy is not yet established, this review updates the current state of knowledge on the contribution of miRNA let-7 in interaction with KRAS to the oncogenesis of brain tumours.

Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

BACKGROUND: Glioblastoma is a malignant and aggressive type of central nevous system malignancy characterized by many distinct biological features including extensive hypoxia. Hypoxia in glioblatoma associates with complex signaling patterns including activation of several pathways such as MAPK, PI3K-AKT/mTOR and IL-6/JAK/STAT3 with the master regulator HIF-1, which in turn drive particular tumor behaviors determining, in the end, treatment outcomes and patients fate. Thus, the present study was designed to investigate the expression of selected hypoxia related factors including STAT3 in a small set of long-term surviving glioma patients. METHODS: The expression of selected hypoxia related factors including STAT3 was evaluated in a time series of formalin fixed paraffin embedded and cryopreserved glioma samples from repeatedly resected patients. In addition, comparative studies were also conducted on primary glioma cells derived from original patient samples, stabilized glioma cell lines and tumor-xenograft mice model. Obtained data were correlated with clinical findings too. RESULTS: Glioblastoma samples of the analyzed patients displayed heterogeneity in the expression of hypoxia- related and EMT markers with most interesting trend being observed in pSTAT3. This heterogeneity was subsequently confirmed in other employed models (primocultures derived from glioblastoma tissue resections, cryopreserved tumor specimens, stabilized glioblastoma cell line in vitro and in vivo) and concerned, in particular, STAT3 expression which remained stable. In addition, subsequent studies on the role of STAT3 in the context of glioblastoma hypoxia demonstrated opposing effects of its deletion on cell viability as well as the expression of hypoxia and EMT markers. CONCLUSIONS: Our results suport the importance of STAT3 expression and activity in the context of hypoxia in malignant glioblastoma long-term surviving glioma patients while emphasizing heterogeneity of biological outcomes in varying employed tumor models.

DMRTA2 supports glioma stem-cell mediated neovascularization in glioblastoma.

Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Due to its fast proliferation, diffusive growth and therapy resistance survival times are less than two years for patients with IDH-wildtype GBM. GBM is noted for the considerable cellular heterogeneity, high stemness indices and abundance of the glioma stem-like cells known to support tumor progression, therapeutic resistance and recurrence. Doublesex- and mab-3-related transcription factor a2 (DMRTA2) is involved in maintaining neural progenitor cells (NPC) in the cell cycle and its overexpression suppresses NPC differentiation. Despite the reports showing that primary GBM originates from transformed neural stem/progenitors cells, the role of DMRTA2 in gliomagenesis has not been elucidated so far. Here we show the upregulation of DMRTA2 expression in malignant gliomas. Immunohistochemical staining showed the protein concentrated in small cells with high proliferative potential and cells localized around blood vessels, where it colocalizes with pericyte-specific markers. Knock-down of DMRTA2 in human glioma cells impairs proliferation but not viability of the cells, and affects the formation of the tumor spheres, as evidenced by strong decrease in the number and size of spheres in in vitro cultures. Moreover, the knockdown of DMRTA2 in glioma spheres affects the stabilization of the glioma stem-like cell-dependent tube formation in an in vitro angiogenesis assay. We conclude that DMRTA2 is a new player in gliomagenesis and tumor neovascularization and due to its high expression in malignant gliomas could be a biomarker and potential target for new therapeutic strategies in glioblastoma.

Nanoparticle-Based Combinational Strategies for Overcoming the Blood-Brain Barrier and Blood-Tumor Barrier.

The blood-brain barrier (BBB) and blood-tumor barrier (BTB) pose substantial challenges to efficacious drug delivery for glioblastoma multiforme (GBM), a primary brain tumor with poor prognosis. Nanoparticle-based combinational strategies have emerged as promising modalities to overcome these barriers and enhance drug penetration into the brain parenchyma. This review discusses various nanoparticle-based combinatorial approaches that combine nanoparticles with cell-based drug delivery, viral drug delivery, focused ultrasound, magnetic field, and intranasal drug delivery to enhance drug permeability across the BBB and BTB. Cell-based drug delivery involves using engineered cells as carriers for nanoparticles, taking advantage of their intrinsic migratory and homing capabilities to facilitate the transport of therapeutic payloads across BBB and BTB. Viral drug delivery uses engineered viral vectors to deliver therapeutic genes or payloads to specific cells within the GBM microenvironment. Focused ultrasound, coupled with microbubbles or nanoparticles, can temporarily disrupt the BBB to increase drug permeability. Magnetic field-guided drug delivery exploits magnetic nanoparticles to facilitate targeted drug delivery under an external magnetic field. Intranasal drug delivery offers a minimally invasive avenue to bypass the BBB and deliver therapeutic agents directly to the brain via olfactory and trigeminal pathways. By combining these strategies, synergistic effects can enhance drug delivery efficiency, improve therapeutic efficacy, and reduce off-target effects. Future research should focus on optimizing nanoparticle design, exploring new combination strategies, and advancing preclinical and clinical investigations to promote the translation of nanoparticle-based combination therapies for GBM.

Exploring the combined anti-cancer effects of sodium butyrate and celastrol in glioblastoma cell lines: a novel therapeutic approach.

Glioblastoma, a highly aggressive and lethal brain cancer, lacks effective treatment options and has a poor prognosis. In our study, we explored the potential anti-cancer effects of sodium butyrate (SB) and celastrol (CEL) in two glioblastoma cell lines. SB, a histone deacetylase inhibitor, and CEL, derived from the tripterygium wilfordii plant, act as mTOR and proteasome inhibitors. Both can cross the blood-brain barrier, and they exhibit chemo- and radiosensitive properties in various cancer models. GB cell lines LN-405 and T98G were treated with SB and CEL. Cell viability was assessed by MTT assay and IC50 values were obtained. Gene expression of DNA repair, apoptosis, and autophagy-related genes was analyzed by RT-PCR. Cell cycle distribution was determined using flow cytometry. Viability assays using MTT assay revealed IC50 values of 26 mM and 22.7 mM for SB and 6.77 µM, and 9.11 µM for CEL in LN-405 and T98G cells, respectively. Furthermore, we examined the expression levels of DNA repair genes (MGMT, MLH-1, MSH-2, MSH-6), apoptosis genes (caspase-3, caspase-8, caspase-9), and an autophagy gene (ATG-6) using real-time polymerase chain reaction. Additionally, flow cytometry analysis revealed alterations in cell cycle distribution following treatment with SB, CEL and their combination. These findings indicate that SB and CEL may act through multiple mechanisms, including DNA repair inhibition, apoptosis induction, and autophagy modulation, to exert their anti-cancer effects in glioblastoma cells. This is the first study providing novel insights into the potential therapeutic effects of SB and CEL in glioblastoma.

Spatially Resolved Microglia/Macrophages in Recurrent Glioblastomas Overexpress Fatty Acid Metabolism and Phagocytic Genes.

BACKGROUND: Glioblastoma (GBM) tumors are rich in tumor-associated microglia/macrophages. Changes associated with treatment in this specific cell population are poorly understood. Therefore, we studied changes in gene expression of tumor-associated microglia/macrophages (Iba1+) cells in de novo versus recurrent GBMs. METHODS: NanoString GeoMx® Digital Spatial Transcriptomic Profiling of microglia/macrophages (Iba1+) and glial cells (Gfap+) cells identified on tumor sections was performed on paired de novo and recurrent samples obtained from three IDH-wildtype GBM patients. The impact of differentially expressed genes on patient survival was evaluated using publicly available data. RESULTS: Unsupervised analyses of the NanoString GeoMx® Digital Spatial Profiling data revealed clustering based on the transcriptomic data from Iba1+ and Gfap+ cells. As expected, conventional differential gene expression and enrichment analyses revealed upregulation of immune-function-related genes in Iba1+ cells compared to Gfap+ cells. A focused differential gene expression analysis revealed upregulation of phagocytosis and fatty acid/lipid metabolism genes in Iba1+ cells in recurrent GBM samples compared to de novo GBM samples. Importantly, of these genes, the lipid metabolism gene PLD3 consistently correlated with survival in multiple different publicly available datasets. CONCLUSION: Tumor-associated microglia/macrophages in recurrent GBM overexpress genes involved in fatty acid/lipid metabolism. Further investigation is needed to fully delineate the role of PLD phospholipases in GBM progression.