Glioblastoma Development Research Articles

AB017. NFκB1 regulates FDX1 to facilitate elesclomol-induced cuproptosis against glioblastoma.

Ferredoxin 1 (FDX1) plays key roles in promoting elesclomol-induced cuproptosis against cancer, whether it has the potential to be a new therapeutic strategy against glioblastoma has not yet been clarified. Glioblastoma cells were treated with elesclomol (20 nM/L) and copper chloride (2 μM/L), and then cell proliferation, migration, and invasion were evaluated by CCK-8, clonogenic and transwell assay. Western blot was performed to detect the expression of cuproptosis-relating proteins FDX1, lipoylated dihydrolipoamide S-acetyltransferase (DLAT), copper transport ATPase (ATP7A), heat shock protein 70 (HSP70), apoptotic markers B cell lymphoma-2 (BCL-2) associated X protein (Bax), and BCL-2, as well as the pan-apoptotic/death markers gasdermin D (GSDMD), solute carrier family 7 member 11 (SLC7A11). The effects of knockdown and overexpression of FDX1 on cell proliferation, migration, and invasion were observed. Bioinformatic analysis was performed to predict the corresponding transcription factors regulating FDX1 expression, and verified by dual luciferase assay. The regulatory relationship between FDX1 and its transcription factors was verified by rescue experiment and further evaluated in vivo. Elesclomol had obvious inhibitory effects on the proliferation, migration, and invasion capacities of tumor cells in a dose-dependent manner. When combined with copper chloride, the inhibitory effects on tumor cells were significantly higher both in vitro and in vivo. FDX1 expression was negatively correlated with overall survival of patients. Nuclear factor κ-light-chain enhancer of activated B cell 1 (NFκB1) was the transcription factor of FDX1 verified by dual luciferase assay. Both FDX1 and NFκB1 were highly expressed in glioblastoma. Knockdown of FDX1 or NFκB1 down-regulated proliferation, migration, and invasion abilities of tumor cells, and increased after FDX1 overexpression. FDX1 expression decreased correspondingly after NFκB1 knockdown. Up-regulation of FDX1 promoted elesclomol-induced cuproptosis against glioblastoma both in vitro and in vivo. FDX1 knockdown can reverse the inhibitory effect of elesclomol on tumor cells. Elesclomol inhibits glioblastoma development via inducing cuproptosis, regulated by NFκB1/FDX1 axis.

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 4L 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.

UBE2D1 promotes glioblastoma proliferation by modulating p21 ubiquitination.

Glioblastoma (GBM) cells exhibit aberrant proliferative abilities and resistance to conventional therapies. However, the mechanisms underlying these malignant phenotypes are poorly understood. In this study, we identified ubiquitin-conjugating enzyme E2D1 (UBE2D1) as a crucial stimulator of GBM development. It is highly expressed in GBM and closely associated with poor prognosis in patients with GBM. UBE2D1 knockdown inhibits GBM cell growth and leads to G1 cell cycle arrest. Mechanistically, UBCH5A binds to p21 at the protein level and induces the ubiquitination and degradation of p21. This negative regulation is mediated by STUB1. Our findings are the first to identify UBE2D1 as a key driver of GBM growth and provide a potential target for improving prognosis and therapy.

Targeting MDM2-p53 interaction in Glioblastoma: Transcriptomic analysis and Peptide-Based inhibition strategy

MDM2 is a gene that encodes a protein involved in cell survival, growth, and DNA repair. It has been implicated in the development and progression of glioblastoma (GBM). Inhibition of the MDM2-p53 interaction has emerged as a promising strategy for treating GBM. In this study, we performed comprehensive transcriptomic expression analysis from diverse datasets and observed MDM2 overexpression in a subset of GBM cases. MDM2 negatively regulates the major onco-suppressor p53. The interaction between MDM2 and p53 is a promising target for cancer therapy, as it can trigger p53-mediated cell death in response to different stress conditions, such as oncogene activation or DNA damage. In this study, we have identified a peptide-based inhibition of MDM2 as a therapeutic strategy for GBM. We have further validated the stability of the MDM2-peptide interaction using a molecular structural dynamics approach. The major trajectories, including root mean square of deviation (RMSD), root mean square of fluctuation (RMSF), and radius of gyration (RoG), indicate that the candidate peptides have a more stable binding compared to the native ligand and control drug. The stability of the binding interaction was further estimated by MMGBSA analysis, which also suggests that MDM2 has a stable binding with both peptide molecules. Based on these results, peptides P-1843 and P-3837 could be tested further for experimental validation to confirm their targeted inhibition of MDM-2. This approach could provide a highly selective and efficient inhibitor with potentially fewer side effects and less toxicity compared to small drug-based molecules.

Gut microbiota's role in glioblastoma risk, with a focus on the mediating role of metabolites.

This study employed Mendelian randomization (MR) analysis to systematically investigate the potential connections between gut microbiota and the risk of glioblastoma (GBM). We identified 12 microbial groups closely associated with the incidence risk of GBM. Subsequently, MR analysis was conducted on 1,091 blood metabolites and 309 metabolite ratios, revealing 19 metabolites that exert an impact on the occurrence of GBM. Hypothesizing that gut microbiota may influence the risk of glioblastoma multiforme by modulating these metabolites, we performed MR analyses, considering each microbial group as exposure and each metabolite as an outcome. Through these analyses, we constructed a regulatory network encompassing gut microbiota, metabolites, and GBM, providing a novel perspective for a deeper understanding of the role of the gut-brain axis in the pathogenesis of GBM. This research offers crucial insights into how gut microbiota may affect the risk of GBM by regulating specific metabolites. The identified regulatory network of the gut-brain axis may play a significant role in the formation and development of GBM, providing valuable information for future research and therapeutic interventions.

Matrix metalloproteinase 9 expression and glioblastoma survival prediction using machine learning on digital pathological images

This study aimed to apply pathomics to predict Matrix metalloproteinase 9 (MMP9) expression in glioblastoma (GBM) and investigate the underlying molecular mechanisms associated with pathomics. Here, we included 127 GBM patients, 78 of whom were randomly allocated to the training and test cohorts for pathomics modeling. The prognostic significance of MMP9 was assessed using Kaplan–Meier and Cox regression analyses. PyRadiomics was used to extract the features of H&E-stained whole slide images. Feature selection was performed using the maximum relevance and minimum redundancy (mRMR) and recursive feature elimination (RFE) algorithms. Prediction models were created using support vector machines (SVM) and logistic regression (LR). The performance was assessed using ROC analysis, calibration curve assessment, and decision curve analysis. MMP9 expression was elevated in patients with GBM. This was an independent prognostic factor for GBM. Six features were selected for the pathomics model. The area under the curves (AUCs) of the training and test subsets were 0.828 and 0.808, respectively, for the SVM model and 0.778 and 0.754, respectively, for the LR model. The C-index and calibration plots exhibited effective estimation abilities. The pathomics score calculated using the SVM model was highly correlated with overall survival time. These findings indicate that MMP9 plays a crucial role in GBM development and prognosis. Our pathomics model demonstrated high efficacy for predicting MMP9 expression levels and prognosis of patients with GBM.

The unfolded protein response machinery in glioblastoma genesis, chemoresistance and as a druggable target.

The role of the unfolded protein response (UPR) has been progressively unveiled over the last decade and several studies have investigated its implication in glioblastoma (GB) development. The UPR restores cellular homeostasis by triggering the folding and clearance of accumulated misfolded proteins in the ER consecutive to endoplasmic reticulum stress. In case it is overwhelmed, it induces apoptotic cell death. Thus, holding a critical role in cell fate decisions. This article, reviews how the UPR is implicated in cell homeostasis maintenance, then surveils the evidence supporting the UPR involvement in GB genesis, progression, angiogenesis, GB stem cell biology, tumor microenvironment modulation, extracellular matrix remodeling, cell fate decision, invasiveness, and grading. Next, it concurs the evidence showing how the UPR mediates GB chemoresistance-related mechanisms. The UPR stress sensors IRE1, PERK, and ATF6 with their regulator GRP78 are upregulated in GB compared to lower grade gliomas and normal brain tissue. They are activated in response to oncogenes and are implicated at different stages of GB progression, from its genesis to chemoresistance and relapse. The UPR arms can be effectors of apoptosis as mediators or targets. Recent research has established the role of the UPR in GB pathophysiology and chemoresistance. Targeting its different sensors have shown promising in overcoming GB chomo- and radioresistance and inducing apoptosis.

Evaluation of secretome biomarkers in glioblastoma cancer stem cells: A bioinformatics analysis.

Glioblastoma (GBM) is a malignant brain tumor that frequently occurs alongside other central nervous system (CNS) conditions. The secretome of GBM cells contains a diverse array of proteins released into the extracellular space, influencing the tumor microenvironment. These proteins can serve as potential biomarkers for GBM due to their involvement in key biological processes, exploring the secretome biomarkers in GBM research represents a cutting-edge strategy with significant potential for advancing diagnostic precision, treatment monitoring, and ultimately improving outcomes for patients with this challenging brain cancer. This study was aimed to investigate the roles of secretome biomarkers and their pathwayes in GBM through bioinformatics analysis. Using data from the Gene Expression Omnibus and the Cancer Genome Atlas datasets-where both healthy and cancerous samples were analyzed-we used a quantitative analytical framework to identify differentially expressed genes (DEGs) and cell signaling pathways that might be related to GBM. Then, we performed gene ontology studies and hub protein identifications to estimate the roles of these DEGs after finding disease-gene connection networks and signaling pathways. Using the GEPIA Proportional Hazard Model and the Kaplan-Meier estimator, we widened our analysis to identify the important genes that may play a role in both progression and the survival of patients with GBM. In total, 890 DEGs, including 475 and 415 upregulated and downregulated were identified, respectively. Our results revealed that SQLE, DHCR7, delta-1 phospholipase C (PLCD1), and MINPP1 genes are highly expressed, and the Enolase 2 (ENO2) and hexokinase-1 (HK1) genes are low expressions. Hence, our findings suggest novel mechanisms that affect the occurrence of GBM development, growth, and/or establishment and may also serve as secretory biomarkers for GBM prognosis and possible targets for therapy. So, continued research in this field may uncover new avenues for therapeutic interventions and contribute to the ongoing efforts to combat GBM effectively.

Glioblastoma development following stereotactic radiosurgery for an arteriovenous malformation: A case report

Stereotactic Radiosurgery (SRS) is a non-invasive treatment modality used for various intracranial pathologies including Arteriovenous Malformations (AVMs), high and low-grade gliomas, and brain metastases. This report presents a very rare case of a 29-year-old male who initially presented with headache and insomnia. Subsequent diagnostic investigations revealed a large right temporal lobe AVM that was 45.48 cc in volume, supplied by the superior and inferior truncus of the Middle Cerebral Artery (MCA), as well as anterior and posterior choroidal arteries. The patient underwent volume-staged SRS with a marginal dose of 15 Gy for each stage, conducted in two stages six months apart. The patient achieved near-complete obliteration initially. However, three years later from the last stage presented with a headache that was diagnosed as a subarachnoid hemorrhage, leading to surgical removal of the residual AVM. Subsequently patient presented with seizures and further imaging studies revealed a contrast-bearing mass at the site previously operated. A second operation revealed the mass to be a glioblastoma. Despite chemoradiotherapy patient succumbed to death. Malignant transformations following SRS, although rare, remain one of the most feared and devastating outcomes.

The role of the ubiquitin ligase UBE4B in neural tumors in adults and children.

e14034 Background: In cancers of the nervous system such as neuroblastoma (NB), the ubiquitin ligase UBE4B directly ubiquitinates growth factor receptors (GFRs), influencing their trafficking and lysosomal degradation. While the diverse functions of UBE4B are acknowledged, we hypothesize that UBE4B expression has distinct associations with outcomes in pediatric versus adult nervous system cancers. We examined the impact of UBE4B expression on glioblastoma (GBM) development, growth, and metastasis by correlating its expression with patient outcomes across age groups. Methods: Glioblastoma samples that were IDH1/2 wild type (n = 2072) underwent DNA (592-gene/whole-exome) and RNA (whole transcriptome) sequencing at Caris Life Sciences. Samples were stratified by UBE4B expression (transcripts per million – TPM) quartiles for all GBM tumors (Q4: H and Q1: L) and by patient age at the time of tissue collection (younger: < 30 years; older: > 60 years). Cell infiltration in the tumor microenvironment was inferred by quanTIseq. Gene expression profiles were analyzed for transcriptomic signature predictive of response to immunotherapy. Overall survival (OS) was derived from insurance claims data, and Kaplan-Meier estimates were calculated. Mann-Whitney U and Chi-square/Fisher-Exact tests were applied were appropriate, with p-values adjusted for multiple comparison. Results: UBE4B expression was similar between younger (n = 57) and older (n = 1115) glioblastoma patients (median: 24.7 vs. 21.6 TPM, p = 0.17). In younger patients, those with high UBE4B expression showed improved OS compared to low expressors (24.9 vs 19.2 months [mos]; HR = 0.41, 95% CI 0.15 – 1.11, p = 0.07). However, UBE4B expression was not associated with OS among older GBM patients (13.5 vs 12.9 mos; HR 1.10 95% CI 0.9 – 1.3, p = 0.33). Within the younger cohorts, higher expression of growth factor receptor genes was observed in high compared to low UBE4B expression ( EGFR: 96.57 vs. 7.07 TPM, p = 0.0092; RET: 0.99 vs. 0.30 TPM, p = 0.018; NTRK2: 236.9 vs. 29.5 TPM, p = 0.084). Concurrent mutation of PDGFRA was also exclusively observed in UBE4B-high tumors (42.9% vs. 0% in UBE4B-L, p = 0.024). Transcriptomic analysis, however, showed that UBE4B-high tumors had lower IFN-g signature scores compared to UBE4B-L (-0.62 vs. -0.41 arbitrary units, p = 0.02). M1 macrophage cell fractions were lower in UBE4B-high compared to UBE4B-low (median: 0.33% vs. 3.38%, p = 0.005), while the fraction of B cells was significantly higher (median: 9.57% vs. 8.14%, p = 0.009). Conclusions: Higher UBE4B expression in younger GBM cases correlated with improved OS suggesting it may have utility as a prognostic biomarker. Increased expression of GFRs in younger patients with high UBE4B expression may provide rationale for new treatment strategies in these patients. Future work may prioritize designing drug targets for UBE4B, offering therapeutic opportunities, especially for younger GBM patients.

Understanding the Significance of Hypoxia-Inducible Factors (HIFs) in Glioblastoma: A Systematic Review.

The study aims to investigate the role of hypoxia-inducible factors (HIFs) in the development, progression, and therapeutic potential of glioblastomas. The study, following PRISMA guidelines, systematically examined hypoxia and HIFs in glioblastoma using MEDLINE (PubMed), Web of Science, and Scopus. A total of 104 relevant studies underwent data extraction. Among the 104 studies, global contributions were diverse, with China leading at 23.1%. The most productive year was 2019, accounting for 11.5%. Hypoxia-inducible factor 1 alpha (HIF1α) was frequently studied, followed by hypoxia-inducible factor 2 alpha (HIF2α), osteopontin, and cavolin-1. Commonly associated factors and pathways include glucose transporter 1 (GLUT1) and glucose transporter 3 (GLUT3) receptors, vascular endothelial growth factor (VEGF), phosphoinositide 3-kinase (PI3K)-Akt-mechanistic target of rapamycin (mTOR) pathway, and reactive oxygen species (ROS). HIF expression correlates with various glioblastoma hallmarks, including progression, survival, neovascularization, glucose metabolism, migration, and invasion. Overcoming challenges such as treatment resistance and the absence of biomarkers is critical for the effective integration of HIF-related therapies into the treatment of glioblastoma with the aim of optimizing patient outcomes.

LC3-associated phagocytosis of neutrophils triggers tumor ferroptotic cell death in glioblastoma

Necrosis in solid tumors is commonly associated with poor prognostic but how these lesions expand remains unclear. Studies have found that neutrophils associate with and contribute to necrosis development in glioblastoma by inducing tumor cell ferroptosis through transferring myeloperoxidase-containing granules. However, the mechanism of neutrophilic granule transfer remains elusive. We performed an unbiased small molecule screen and found that statins inhibit neutrophil-induced tumor cell death by blocking the neutrophilic granule transfer. Further, we identified a novel process wherein neutrophils are engulfed by tumor cells before releasing myeloperoxidase-containing contents into tumor cells. This neutrophil engulfment is initiated by integrin-mediated adhesion, and further mediated by LC3-associated phagocytosis (LAP), which can be blocked by inhibiting the Vps34-UVRAG-RUBCN-containing PI3K complex. Myeloperoxidase inhibition or Vps34 depletion resulted in reduced necrosis formation and prolonged mouse survival in an orthotopic glioblastoma mouse model. Thus, our study unveils a critical role for LAP-mediated neutrophil internalization in facilitating the transfer of neutrophilic granules, which in turn triggers tumor cell death and necrosis expansion. Targeting this process holds promise for improving glioblastoma prognosis.

Targeted inhibition of branched-chain amino acid metabolism drives apoptosis of glioblastoma by facilitating ubiquitin degradation of Mfn2 and oxidative stress

Glioblastoma is one of the most challenging malignancies with high aggressiveness and invasiveness and its development and progression of glioblastoma highly depends on branched-chain amino acid (BCAA) metabolism. The study aimed to investigate effects of inhibition of BCAA metabolism with cytosolic branched-chain amino acid transaminase (BCATc) Inhibitor 2 on glioblastoma, elucidate its underlying mechanisms, and explore therapeutic potential of targeting BCAA metabolism. The expression of BCATc was upregulated in glioblastoma and BCATc Inhibitor 2 precipitated apoptosis both in vivo and in vitro with the activation of Bax/Bcl2/Caspase-3/Caspase-9 axis. In addition, BCATc Inhibitor 2 promoted K63-linkage ubiquitination of mitofusin 2 (Mfn2), which subsequently caused lysosomal degradation of Mfn2, and then oxidative stress, mitochondrial fission and loss of mitochondrial membrane potential. Furthermore, BCATc Inhibitor 2 treatment resulted in metabolic reprogramming, and significant inhibition of expression of ATP5A, UQCRC2, SDHB and COX II, indicative of suppressed oxidative phosphorylation. Moreover, Mfn2 overexpression or scavenging mitochondria-originated reactive oxygen species (ROS) with mito-TEMPO ameliorated BCATc Inhibitor 2-induced oxidative stress, mitochondrial membrane potential disruption and mitochondrial fission, and abrogated the inhibitory effect of BCATc Inhibitor 2 on glioblastoma cells through PI3K/AKT/mTOR signaling. All of these findings indicate suppression of BCAA metabolism promotes glioblastoma cell apoptosis via disruption of Mfn2-mediated mitochondrial dynamics and inhibition of PI3K/AKT/mTOR pathway, and suggest that BCAA metabolism can be targeted for developing therapeutic agents to treat glioblastoma.

Small molecule drug discovery for glioblastoma treatment based on bioinformatics and cheminformatics approaches.

Background: Glioblastoma (GBM) is a common and highly aggressive brain tumor with a poor prognosis for patients. It is urgently needed to identify potential small molecule drugs that specifically target key genes associated with GBM development and prognosis. Methods: Differentially expressed genes (DEGs) between GBM and normal tissues were obtained by data mining the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Gene function annotation was performed to investigate the potential functions of the DEGs. A protein-protein interaction (PPI) network was constructed to explore hub genes associated with GBM. Bioinformatics analysis was used to screen the potential therapeutic and prognostic genes. Finally, potential small molecule drugs were predicted using the DGIdb database and verified using chemical informatics methods including absorption, distribution, metabolism, excretion, toxicity (ADMET), and molecular docking studies. Results: A total of 429 DEGs were identified, of which 19 hub genes were obtained through PPI analysis. The hub genes were confirmed as potential therapeutic targets by functional enrichment and mRNA expression. Survival analysis and protein expression confirmed centromere protein A (CENPA) as a prognostic target in GBM. Four small molecule drugs were predicted for the treatment of GBM. Conclusion: Our study suggests some promising potential therapeutic targets and small molecule drugs for the treatment of GBM, providing new ideas for further research and targeted drug development.

Abstract LB332: Spatial transcriptomic and proteomic analysis of the treatment resistant slow cycling micro environment in GBM

Abstract Within the Glioblastoma (GBM) microenvironment, there are micro-niches originating from independent lineages of cancer stem cells with distinct metabolic requirements that are characterized by Fast cycling cells (FCCs) and Slow cycling cells (SCC). The FCCs employ aerobic glycolysis for energy, while the treatment-resistant SCCs primarily rely on lipid metabolism. Our study allows for the molecular and spatial decoding of the heterogeneity within the GBM microenvironment, with a specific focus on decoding the dichotomy of the immune infiltrates and studying the interactions between the SCCs and the immune compartment in human GBM patients. Our research reveals a new paradigm of interaction within the tumor microenvironment (TME) of glioblastoma (GBM) patients, in which SCCs are instrumental in altering the tumor milieu to favor immune suppression. These SCCs actively recruit and harness sub-population of immune cells of myeloid origin that promote tumor growth and simultaneously provide metabolic aid to these treatment-resistant cells. Our research maps out the intricate metabolic dialogues within GBM and evaluates the potential therapeutic effect of disrupting this interaction to open up new treatment avenues. The findings based on our spatial transcriptomic and proteomic study on brain biopsies from human patients provide a deeper understanding of the critical links among the tumor's local environment, cellular metabolism, anti-tumor immunity, and potential treatment vulnerabilities. In summary, our investigation offers novel insights into how metabolic exchanges contribute to the evolution of GBM leading to the development of drug and immune resistant GBM. Our data is suggestive that therapies aimed at these metabolic interactions, especially those between SCCs and metabolically supporting immunosuppressive myeloid originating immune cells, hold potential as effective therapeutic strategy for GBM. Citation Format: Avirup Chakraborty, Diana Feier, Aryeh Silver, Changlin Yang, Miruna Anica, Avinash Pittu, Olusegun Sobanjo, Ethan Hodge, Mia Engelbart, Christina Von Roemeling, Maryam Rahman, Matthew Sarkisian, Jianping Huang, Jeffrey Harrison, Duane Mitchell, Loic Deleyrolle. Spatial transcriptomic and proteomic analysis of the treatment resistant slow cycling micro environment in GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB332.

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

BackgroundA 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.MethodsHere, 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.ResultsWe 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.ConclusionsOur 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.Graphical

Recent in vitro models and tissue engineering strategies to study glioblastoma

Glioblastoma is a highly malignant brain tumor classified as grade IV with a poor prognosis and approximately a year of survival rate. The molecular changes that trigger primary glioblastoma are usually epidermal growth factor receptor mutations and amplifications, Mouse Double Minute and TP53 mutations, p16 deletion, phosphatase and tensin homolog and telomerase promoter mutations. In the vast majority of glioblastomas, altered signaling pathways were identified as receptor tyrosine kinase/Ras/PI3K, p53. Isocitrate dehydrogenase 1/2 mutations have also been associated with poor prognosis in glioblastoma The treatment options are very limited and complicated because of the diverse composition and heterogeneity of the tumors and unresponsiveness to the treatments with the existence of barriers reaching the brain tissue. Despite new trials, drug candidates that appeared effective in cell culture or mouse models failed in the clinic. Recently, new sophisticated experimental systems, including the those that mimic the tumor microenvironment, have started being used by several research groups, which will allow accurate prediction of drug efficacy. Tissue engineering strategies are also being combined with innovative cancer models, including spheroids, tumorspheres, organotypic slices, explants, tumoroids, and organoids. Such 3D systems provide powerful tools for studying glioblastoma biology by representing the dynamic evolution of the disease from the early to the metastatic stages and enabling interaction with the microenvironment. In this review, we both enlighten the molecular mechanisms that lead to glioblastoma development and detailed information on the tissue engineering approaches that have been used to model glioblastoma and the tumor microenvironment with the advantages and disadvantages. We anticipate that these novel approaches could improve the reliability of preclinical data by reducing the need for animal models.

Abstract 5994: Neutrophils internalized through LC3-associated phagocytosis trigger tumor ferroptotic cell death in glioblastoma

Abstract Necrosis is commonly found in various solid tumors and predicts worse outcome. Chronic ischemia can initiate tumor necrosis, however, how the damaged tissue further expands is unclear. Previous studies found that neutrophils associate with necrosis and could contribute the necrosis development in glioblastoma (GBM) through transferring myeloperoxidase-containing granules into tumor cells and inducing tumor cell ferroptosis. How the neutrophilic granule transfer occurs is unknown. Here, through an unbiased small molecule screen, we found that statins can inhibit the neutrophil-induced tumor cell death through blocking the neutrophilic content transfer into tumor cells. Surprisingly, we found that neutrophils are engulfed by tumor cells before they are fragmented and release the MPO-containing contents in tumor cells. This process is through LC3-associated phagocytosis (LAP) and can be blocked by inhibiting the Vps34-UVRAG-containing PI3K complex. Inhibition of MPO or depletion of Vps34 in an orthotopic xenograft GBM mouse model showed that necrosis formation is reduced, and the tumor-bearing mice can survive longer. Therefore, this study revealed that the neutrophilic granule transfer is through LAP-mediated neutrophil internalization, which then trigger tumor ferroptotic cell death in glioblastoma. Blocking this process may improve prognosis of GBM. Citation Format: Tong Y. Lu, Patricia P. Yee, Stephen Y. Chih, Dawit G. Aregawi, Michael J. Glantz, Brad E. Zacharia, Hong-Gang Wang, Wei Li. Neutrophils internalized through LC3-associated phagocytosis trigger tumor ferroptotic cell death in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5994.

Exploring the causal relationship between antihypertensive drugs and glioblastoma by combining drug target Mendelian randomization study, eQTL colocalization, and single-cell RNA sequencing.

Recent reports indicate a potential oncogenic role of antihypertensive drugs in common cancers. However, it remains uncertain whether this phenomenon influences the risk of glioblastoma multiforme (GBM). This study aimed to assess the potential causal effects of blood pressure (BP) and antihypertensive drugs on GBM. Genome-wide association study (GWAS) summary statistics for systolic blood pressure (SBP), diastolic blood pressure (DBP), and GBM in Europeans were downloaded. To represent the effects of antihypertensive drugs, we utilized single nucleotide polymorphisms (SNPs) associated with SBP/DBP adjacent to the coding regions of different antihypertensive drugs as instrumental variables to model five antihypertensive drugs, including angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, β-receptor blockers (BBs), and thiazide diuretics. Positive control studies were performed using GWAS data in chronic heart failure. The primary method for causality estimation was the inverse-variance-weighted method. Mendelian randomization analysis showed that BBs with the β1-adrenergic receptor (ADRB1) as a therapeutic target could significantly reduce the risk of GBM by mediating DBP (OR = 0.431, 95% CI: 0.267-0.697, p < .001) and that they could also significantly reduce the risk of GBM by mediating SBP (OR = 0.595, 95% CI: 0.422-0.837, p = .003). Sensitivity analysis and colocalization analysis reinforced the robustness of these findings. Finally, the low expression of the ADRB1 gene in malignant gliomas was found by GBM data from TCGA and single-cell RNA sequencing, which most likely contributed to the poor prognosis of GBM patients. In summary, our study provides preliminary evidence of some causal relationship between ADRB1-targeted BBs and glioblastoma development. However, more studies are needed to validate these findings and further reveal the complex relationship between BP and GBM.

CD133 significance in glioblastoma development: in silico and in vitro study

BackgroundGlioblastoma multiform (GBM) is among the commonly diagnosed brain malignancies with poor prognosis. CD133 has been introduced as an oncogene in various cancers, like GBM. This study aimed to investigate the significance of CD133 in GBM development using in silico and in vitro techniques.MethodThe TCGA-GBM database was analyzed for the correlational and comparative studies. After selecting the U87MG cell line, CD133-siRNA was transfected into U87MG cells and treated with temozolomide. The cell viability, cell cycle, migration, clonogenicity, and apoptosis of groups were investigated using MTT, flow cytometry, wound-healing, colony formation, and annexin V/PI assays. Using qRT-PCR method, the mRNA expression levels of MMP16, SOX2, RAF1, MAP2K1, MAPK3, PIK3CA, AKT3, mTOR, CDK4, and BCL2 were studied.ResultsCD133 silencing improves apoptosis rate, arrests the cell cycle at the sub-G1 phase, suppresses the clonogenicity of U87MG cells, and inhibits the PI3K/Akt and MAPK pathways via downregulating the RAF1, MAP2K1, MAPK3, PIK3CA, AKT3, and mTOR expression. Besides, combining CD133 silencing with temozolomide treatment considerably inhibits the migration of U87MG cells compared to temozolomide monotherapy.ConclusionCD133 can regulate the PI3K/Akt and MAPK pathways and modulate the clonogenicity, apoptosis, and cell cycle of GBM. Combining CD133 silencing with temozolomide treatment considerably increases apoptosis, arrests the cell cycle at the sub-G1, and suppresses migration of U87MG cells compared to temozolomide monotherapy.Graphical