Ferroptosis in Glioblastoma: Mechanisms, Therapeutic Strategies, and Future Directions

Ferroptosis in Glioblastoma: Mechanisms, Therapeutic Strategies, and Future Directions This systematic review examines the role of ferroptosis in glioblastoma through analysis of 95 studies spanning 2018–2025. Key regulators such as GPX4, ROS, and NCOA4 emerge as potential therapeutic targets, while innovative strategies include gene editing and nanotechnology-based combinations to induce ferroptosis. Despite its potential, challenges like small sample sizes, in vitro reliance, and tumor microenvironment complexity hinder clinical application. Addressing adaptive resistance, validating biomarkers, and employing multimodal approaches in diverse in vivo models are essential for advancing ferroptosis-based glioblastoma therapies. Below are three illustrative findings of many in the review. FTL upregulation fosters glioblastoma progression through immunosuppressive M2 macrophage polarization; its inhibition enhances T cell recruitment and antiPD1 sensitivity, offering promising immunotherapy strategies. GPX7 silencing elevates ferroptosis-related oxidative stress via lipid peroxidation and Fe²⁺ increases, presenting an effective target to enhance ferroptosis sensitivity and suppress glioblastoma development. Iron oxide nanoparticles synergize with paclitaxel to induce ferroptosis by modulating autophagy pathways, combining nanotechnology and chemotherapeutics to overcome resistance and boost treatment efficacy. 5172 Records screened 95 Full-text studies included Jan 2018–Oct 2025 Coverage window Table of Contents 1. Methods 2. Results 2.1. Ferroptosis and Immune Modulation: Mechanisms and Therapeutic Strategies in Glioblastoma 2.1.1. Signaling Networks and Subtype Transition in Glioblastoma 2.1.2. Therapeutic Targets and Drug Interactions 2.2. Ferroptosis in Glioblastoma: Pathways, Immune Interactions, and Overcoming Resistance 2.2.1. Mechanisms of Ferroptosis in Glioblastoma 2.2.2. Prognostic Markers and Risk Models 2.2.3. Therapeutic Strategies and Treatment Outcomes 2.3. CD95 Gene Deletion: Reducing Malignancy in Glioblastoma Cells 2.4. Iron Oxide Nanoparticles and Paclitaxel: Inducing Ferroptosis via Autophagic Pathways in Glioblastoma 3. Discussion and Interpretation of Findings 3.1. Ferroptosis and Immune Modulation: Mechanisms and Therapeutic Strategies in Glioblastoma 3.2. Ferroptosis in Glioblastoma: Pathways, Immune Interactions, and Overcoming Resistance 3.3. CD95 Gene Deletion: Reducing Malignancy in Glioblastoma Cells 3.4. Iron Oxide Nanoparticles and Paclitaxel: Inducing Ferroptosis via Autophagic Pathways in Glioblastoma 4. Discussion of Limitations 4.1. Ferroptosis and Immune Modulation: Mechanisms and Therapeutic Strategies in Glioblastoma 4.2. Ferroptosis in Glioblastoma: Pathways, Immune Interactions, and Overcoming Resistance 5. Conclusions 6. References 1. Methods Go to Annex 1: Methods 2. Results 2.1. Ferroptosis and Immune Modulation: Mechanisms and Therapeutic Strategies in Glioblastoma 2.1.1. Signaling Networks and Subtype Transition in Glioblastoma Selected findings FTL upregulation in tumor-associated macrophages fosters an immunosuppressive environment by inducing M2 polarization, promoting glioblastoma progression, and enhancing angiogenesis. Its inhibition reprograms the tumor microenvironment, increases T cell recruitment, and significantly improves sensitivity to anti-PD1 therapy, offering a promising strategy for immunotherapy in glioblastoma. Overexpression of FOSL1 in glioblastoma cells leads to differential expression of 493 genes, including upregulated genes associated with poor prognosis and downregulated genes linked to improved outcomes. These findings highlight FOSL1's role in ferroptosis and glioblastoma subtype transition, presenting opportunities for prognostic biomarker development and therapeutic targeting. NRF2 upregulation in TMZ-resistant glioblastoma cells enhances ferroptosis sensitivity by promoting glutathione efflux and lipid peroxidation, correlating with increased tumor aggressiveness and reduced survival rates. This underscores the therapeutic potential of ferroptosis induction to overcome drug resistance and improve glioblastoma treatment outcomes. Table 1. Signaling Networks and Subtype Transition in Glioblastoma Study ID Length of intervention Population of intervention Intervention Intervention details Primary outcome Secondary outcome 1 Guo S et al. (2025)72 hours Nude mice aged 6–8 weeks, male and female, subcutaneous implantation of PDX tumor tissue cubes Transfection of DNA constructs to overexpress FOSL1 tagged with GFP Transfection performed at 50–75% cell confluency, lipofectamine 3000 reagent used, RNA extraction at 72 h using TRIzol reagent, overexpression assessed at 48 or 72 h posttransfection, studies conducted in triplicates Role of FOSL1 in regulating biological processes and signaling networks during proneural to mesenchymal subtype transition in glioblastoma, including the identification of upregulated_genes and downregulated_genes. Identification of 8 upregulated and 4 downregulated genes, pathway implications for glioblastoma prognosis Go to Annex 2: Table 1. Signaling Networks and Subtype Transition in Glioblastoma The intricate relationship between ferroptosis—an iron-dependent cell death mechanism characterized by lipid peroxidation and oxidative stress—and immune modulation in glioblastoma has garnered increasing attention due to its potential therapeutic implications. Recent findings have elucidated mechanisms by which ferroptosis influences immune responses, including tumor-associated macrophage polarization, immune evasion, and cytokine release, as well as the role of key ferroptosis-related genes in shaping the tumor microenvironment. These insights underscore the significance of targeting ferroptosis pathways to enhance immunebased strategies and improve treatment outcomes in glioblastoma, offering promising avenues for innovative therapeutic modalities in managing this aggressive malignancy. Previous studies demonstrated that ferroptosis represents a promising therapeutic strategy for glioblastoma, leveraging the tumor's unique iron metabolism and lipid peroxidation characteristics to overcome drug resistance and reduce tumor growth [7, 8, 9, 10, 11]. Compounds such as DHI, DHA, ALZ003, and PAB effectively induced ferroptosis; yet, challenges persist in validating ferroptosis biomarkers for prognosis and personalized treatment [12, 13]. Autophagy was identified as a critical mechanism influencing glioblastoma progression and therapeutic resistance, with protective autophagy limiting drug efficacy and cytotoxic autophagy offering potential for tumor suppression [14, 15]. However, the interplay between ferroptosis and immune modulation, as well as the impact of genetic alterations and feedback mechanisms like the PERK-ATF4-HSPA5-GPX4 cascade, remains poorly understood, highlighting the need for further investigation into these pathways [16]. The role of ferroptosis in glioblastoma progression is further exemplified by the differential gene expression linked to FOSL1 overexpression. Overexpression of FOSL1 in glioblastoma cells results in 493 differentially expressed genes, with 152 upregulated and 341 downregulated (adjusted p < 0.05) according to Guo S et al. (2025). Functional enrichment analyses confirm its role in ferroptosis, NF-κB signaling, and cell proliferation, aligning with key pathways previously identified in glioblastoma progression. Survival analysis highlights poor prognosis associated with FOSL1-upregulated genes, such as ITGA5 and STEAP3, while FOSL1-downregulated genes, including ARL3 and BEX1, correlate with improved outcomes. Experimental validation using qPCR and IHC confirms significant differential expression of identified genes at both mRNA and protein levels (p < 0.05, p < 0.01, p < 0.001). Building on the connection between ferroptosis and immune modulation, FTL emerges as a key regulator of tumor-associated macrophage polarization and glioblastoma progression. Li H et al. (2023) FTL upregulation in tumor-associated macrophages fosters an immunosuppressive environment by inducing M2 polarization, promoting glioblastoma progression, and enhancing angiogenesis. FTL inhibition reprograms the tumor microenvironment by reducing M2 macrophage polarization, attenuating angiogenesis, and increasing CD3+ and CD8+ T cell recruitment, which significantly improves sensitivity to anti-PD1 therapy. Median survival in glioma-bearing mice increases from 24 days to 34.5 days with this combined treatment (p < 0.05). Expanding on the therapeutic implications of ferroptosis, NRF2 upregulation highlights its potential in overcoming drug resistance in glioblastoma. NRF2 upregulation in TMZ-resistant glioblastoma cells enhances ferroptosis sensitivity by promoting ABCC1-mediated glutathione efflux and lipid peroxidation (de Souza I et al. 2022). Clinical data indicate high ABCC1 expression significantly correlates with increased tumor aggressiveness and reduced survival rates (5-year OS: 18% vs. 68%, HR = 1.10, CI = 1.06–1.14, p < 0.001). These findings align with the therapeutic potential of ferroptosis induction to address drug resistance in glioblastoma. 2.1.2. Therapeutic Targets and Drug Interactions Selected findings 35G8 induces ferroptosis in glioblastoma cells through PDI inhibition, ER stress activation, and disruption of redox homeostasis, while crossing the bloodbrain barrier for therapeutic feasibility. This finding highlights a novel drug candidate with significant potential for glioblastoma treatment by targeting ferroptosis pathways and overcoming delivery challenges. Modulation of autophagy and ferroptosis in glioblastoma stem-like cells significantly impacts tumor growth, patient survival, and susceptibility to temozolomide. This finding provides critical insights into the therapeutic interplay of ferroptosis and autophagy, enabling targeted strategies for improving glioblastoma treatment outcomes. Table 2. Therapeutic Targets and Drug Interactions Study ID Length of intervention Population of intervention Control Intervention Intervention details Primary outcome Secondary outcome Kyani A et al. (2018) 72 hours, 6 hours, 24 hours Human glioblastoma cell lines U87MG, U118MG, NU04, A172 Treatment of cells with compounds including 35G8, NAC, Z-VAD-FMK, Necrostatin-1, H2O2, catalase, DTT Cells seeded in 96-well plates, compounds added at specific time intervals, incubation at 37°C and 5% CO2, MTT assay performed for cell viability, PDI activity assessed via reduction reaction, thermal shift assay conducted using microplates and ThermoFluor instrument, Western blotting performed for protein analysis Validation of PDI as a therapeutic target for glioblastoma, cytotoxicity of pyrimidotriazinedione 35G8 Upregulation of heme oxygenase 1 and SLC7A11, repression of TXNIP and EGR1, induction of autophagy and ferroptosis, activation of Nrf2 antioxidant response and ER stress response Go to Annex 3: Table 2. Therapeutic Targets and Drug Interactions The therapeutic relevance of ferroptosis is reinforced by the potent efficacy of 35G8 as a targeted inducer in glioblastoma models. 35G8 demonstrates potent therapeutic efficacy for glioblastoma, validated as a PDI inhibitor (IC50 = 0.17 ± 0.01 μM) with cytotoxicity in glioblastoma cells (IC50 = 1.1 ± 0.2 μM in U87MG), as reported by Kyani A et al. (2018). It induces ferroptosis by upregulating HMOX1 (19-fold) and SLC7A11 (63-fold), repressing TXNIP (−7.40-fold) and EGR1 (−5.65fold), and activating ER stress markers such as DDIT3 (4-fold) and GRP78 (8-fold). Additionally, 35G8 disrupts proteostasis and redox homeostasis, confirmed by reduced potency in the presence of deferoxamine, and crosses the blood-brain barrier, supporting its feasibility for glioblastoma treatment. The interplay between ferroptosis and autophagy provides further insights into glioblastoma stem-like cell survival and therapeutic strategies. Buccarelli M et al. (2018) 2 Modulation of autophagy and ferroptosis significantly affects glioblastoma stem-like cell (GSC) growth and survival. Low autophagic levels correlate with improved overall survival in GBM patients (p = 0.0012; HR = 0.3634; 95% CI: 0.1967–0.6715), while autophagy inhibition enhances GSC susceptibility to TMZ in vitro (p < 0.05). Induction of ferroptosis increases lipid peroxidation (p < 0.01), decreases GSH levels (p < 0.01), promotes mitochondrial ROS accumulation (p < 0.01), and reduces GPx activity under ferroptosis inhibition (p < 0.01). Despite increased LC3 expression (p < 0.0001) and ferroptosis markers (p < 0.01), adjunctive treatment with quinacrine does not significantly enhance TMZ's antitumor efficacy in vivo. Building on the role of ferroptosis modulation in glioblastoma treatment, combinatorial approaches such as Coix and TMZ showcase therapeutic potential through ferroptosis activation and lipid pathway regulation. The combination of Coix (2 mg/ml) and TMZ (0.1 mg/ml) demonstrates a synergistic effect (CI < 1) in inhibiting U251 glioblastoma cell proliferation, with a drug reduction index for TMZ (DRI = 3.266) (Zhao Z et al. 2023). This synergy involves Coix's modulation of interferon-related genes (e.g., RSAD2: 2.37-fold down-regulation in Coix, 2.66-fold up-regulation in TMZ, and 2.14-fold up-regulation in the combination group) and enrichment of lipidrelated pathways, including cholesterol metabolism. Ferroptosis activation via ANGPTL4 up-regulation and ABCA1 down-regulation contributes to therapeutic efficacy. RNA-seq and qRT-PCR analyses confirm these mechanisms with statistical significance (FDR < 0.05), and the combination therapy reduces TMZ dosage (IC50: Coix 17.82 mg/ml, TMZ 0.358 mg/ml) while maintaining efficacy. Expanding on therapeutic strategies targeting ferroptosis and lipid pathways, interventions such as STAT3 depletion provide insights into modulating autophagydependent cell death mechanisms in glioblastoma cells. Recent findings by Remy J et al. (2022) demonstrate that STAT3 depletion mitigates lysosomal membrane permeabilization and reduces cathepsin-dependent cell death in glioblastoma cells under Pimozide treatment, significantly enhancing cell survival (p < 0.0001, TwoWay ANOVA). Additionally, STAT3 depletion significantly reduces autophagy-dependent cell death mechanisms (p < 0.01, p < 0.0001), aligning with the understanding of targeted approaches to modulate cell death pathways in apoptosis-resistant glioblastoma cells. The presented data delineate the interplay between ferroptosis and immune modulation in glioblastoma, highlighting molecular mechanisms and therapeutic approaches explored in the study. 2.2. Ferroptosis in Glioblastoma: Pathways, Immune Interactions, and Overcoming Resistance 2.2.1. Mechanisms of Ferroptosis in Glioblastoma Selected findings GPX7 silencing significantly enhances ferroptosis-related oxidative stress in glioblastoma, as evidenced by reduced glutathione levels, increased lipid peroxidation, and elevated Fe²⁺ concentrations. This finding suggests that targeting GPX7 and its regulatory pathways could improve ferroptosis sensitivity and suppress glioblastoma progression both in vitro and in vivo, offering a promising therapeutic avenue. CDKN2A deletion in glioblastoma leads to lipidomic remodeling, redistributing polyunsaturated fatty acids to membrane phospholipids, increasing lipid peroxidation, and priming tumors for ferroptosis. This discovery provides a molecular basis for exploiting lipid metabolism alterations as a therapeutic strategy, particularly through GPX4 inhibition in CDKN2A-null glioblastoma models. Hypoxia-induced resistance to ferroptosis in glioblastoma is mediated by SLC7A11 upregulation via the PI3K/AKT/HIF-1α pathway, which reduces lipid peroxidation and increases IC50 values for ferroptosis inducers. Targeting HIF-1α with PX-478 or inhibiting AKT reverses this resistance and enhances sulfasalazine-induced ferroptosis, presenting a synergistic anticancer therapeutic approach. Quiescent astrocyte-like glioma cells exhibit mitochondrial dysfunction and lipid peroxidation, rendering them highly sensitive to GPX4 inhibition and ferroptosis (Banu MA et al. 2024). These findings provide a targeted therapeutic strategy to selectively eliminate resistant glioblastoma cell populations by exploiting mitochondrial vulnerabilities. Hypoxia-induced ferroptosis resistance in glioblastoma is mediated by SLC7A11 upregulation via the PI3K/AKT/HIF-1α pathway (Sun S et al. 2022). Targeting HIF-1α with PX-478 or AKT inhibition reverses resistance, enhancing the efficacy of ferroptosis inducers and improving survival in preclinical models. METTL16 knockdown induces ferroptosis in glioma cells by destabilizing NFE2L2 mRNA and reducing antioxidant defenses, while also modulating immune cell infiltration and checkpoint expression (Yang Y et al. 2024). This dual role in ferroptosis regulation and immune interactions highlights METTL16 as a promising therapeutic target in glioblastoma. CDKN2A deletion redistributes polyunsaturated fatty acids to membrane phospholipids, increasing lipid peroxidation and ferroptosis susceptibility in glioblastoma cells (Minami JK et al. 2023). GPX4 inhibition in CDKN2A-null models demonstrates improved survival, validating lipidomic alterations as a therapeutic vulnerability. Table 3. Mechanisms of Ferroptosis in Glioblastoma Study ID Length of intervention Population of intervention Control Intervention Intervention details Primary outcome Secondary outcome Chen Y et al. (2019) 14 days Patients with malignant glioma undergoing surgical treatment; specific pathogen-free athymic nude BALB/c mice injected with glioma cells Use of antibodies, chemical compounds (DHA, ferrostatin-1, liproxstatin-1, GSK2606414, EGCG, deferoxamine), siRNA transfections Transfections via Lipofectamine 2000, compound treatments, flow cytometry staining for ROS and lipid ROS, biochemical assays for glutathione and GPX4 activity, incubations at 37°C (4 hours for assay, 30 minutes for ROS staining, 10 minutes for lipid ROS staining), tumor generation through cell injections (2×106 cells in 0.2 ml PBS per mouse), protocols followed based on manufacturer’s recommendations Induction of ferroptosis in glioma cells by DHA, characterized by irondependent cell death, ROS generation, and lipid peroxidation Activation of protective PERK-ATF4HSPA5-GPX4 pathway, increased ferroptosis sensitivity upon pathway inhibition Go to Annex 4: Table 3. Mechanisms of Ferroptosis in Glioblastoma Ferroptosis, a regulated form of cell death driven by iron-dependent lipid peroxidation, has emerged as a promising therapeutic target in glioblastoma. This chapter explores the molecular pathways governing ferroptosis, including iron metabolism, lipid peroxidation, and antioxidant systems, alongside its interactions with the immune microenvironment and mechanisms of therapeutic resistance. Advances in understanding these processes have highlighted opportunities to modulate ferroptosis, enhance immune system engagement, and overcome therapeutic barriers. Integrating these insights offers promising strategies for improving glioblastoma outcomes amidst its aggressive nature and resistance to conventional therapies. 3 Previous studies have demonstrated the therapeutic potential of ferroptosis in glioblastoma, highlighting the efficacy of GPX4 inhibition and lipid metabolism modulation in inducing ferroptosis and overcoming treatment resistance [40, 41]. Natural compounds and combination therapies integrating ferroptosis-based approaches with chemotherapy and radiotherapy have shown promise in preclinical models [42, 43]. However, challenges such as glioblastoma heterogeneity, the immunosuppressive tumor microenvironment, poor blood-brain barrier penetration, and the toxicity of ferroptosis inducers remain significant barriers to clinical translation [44]. While biomarkers like GPX4 and SLC7A11 play crucial roles in ferroptosis sensitivity, the lack of standardized markers and mechanistic understanding of ferroptosis regulation necessitates further investigation [45, 46]. The molecular pathways of ferroptosis offer insights into how DHA induces ferroptosis and how resistance mechanisms, such as the PERK/ATF4/HSPA5 pathway, modulate therapeutic efficacy. DHA effectively induces ferroptosis in glioblastoma cells through iron-dependent mechanisms, including ROS generation, lipid peroxidation, and increased MDA levels (p < 0.05, p < 0.01, p < 0.001) according to Chen Y et al. (2019). Resistance mediated by the PERK/ATF4/HSPA5 pathway, which enhances GPX4 expression and activity, is significantly reduced by pathway inhibition, resulting in enhanced ferroptosis sensitivity and anticancer effects in vitro and in vivo (p < 0.01, p < 0.001, n = 3). Building upon resistance mechanisms, GPX7 silencing emerges as another critical factor influencing ferroptosis sensitivity and oxidative stress in glioblastoma. GPX7 silencing significantly enhances ferroptosis-related oxidative stress in glioblastoma, as evidenced by reduced glutathione (GSH) levels (p < 0.001), increased lipid peroxidation (p < 0.001), and elevated Fe2+ concentrations (p < 0.05) (Zhou Y et al. 2021). It increases sensitivity to erastin, suppressing glioblastoma proliferation, migration, invasion, and tumor growth both in vitro and in vivo, while promoting apoptosis (p < 0.001). Additionally, miR-29b-mediated suppression of GPX7 expression further amplifies erastin-induced ferroptosis sensitivity, marked by increased lipid peroxidation (p < 0.001), reduced GSH levels (p < 0.001), and elevated apoptosis rates (p < 0.001), alongside significant reductions in glioblastoma cell proliferation, migration, and invasion. The role of GPX7 in ferroptosis regulation transitions into broader interactions within the p62/p53/NRF2 axis, highlighting mutation-specific effects on glioblastoma vulnerability. Yuan F et al. (2022) p62 demonstrates a dual role in ferroptosis regulation in glioblastoma, influenced by p53 mutation status. In p53-mutant glioblastoma, p62 promotes ferroptosis by decreasing SLC7A11 expression and enhancing mutant-p53/NRF2 interaction, while in p53-wild-type glioblastoma, it inhibits ferroptosis through classical p62-NRF2 activation and increased SLC7A11 expression. Therapeutic interventions targeting the p62/p53/NRF2 axis, such as APR-246, show promise, with clinical data indicating poorer survival in p53 wild-type glioblastomas with high p62 expression (p < 0.05). Additionally, p62 knockout enhances ferroptosis and improves survival in p53-wild-type glioblastoma models, whereas APR-246 reverses p62-mediated ferroptosis enhancement in p53-mutant glioblastoma by reactivating mutant p53 and restoring SLC7A11 expression. Expanding on ferroptosis regulation, the ATF4-CHAC1 pathway demonstrates its significance in modulating ferroptosis induction in glioma cells. Sevoflurane induces ferroptosis in glioma cells through the activation of the ATF4-CHAC1 pathway, as demonstrated by Xu Y et al. (2022) and dose-dependent increases in Fe²⁺ levels, ROS generation, and suppression of cell viability (p < 0.05, p < 0.01, p < 0.001). These findings align with established roles of iron metabolism and oxidative stress in ferroptosis susceptibility, further highlighting the pathway's significance. Suppression of ATF4 interrupts ferroptosis induction, while the ferroptosis inducer Erastin restores ATF4-CHAC1 activity and ferroptosis, reinforcing the pathway's critical function in regulating glioma cell death. Insights into ferroptosis pathways culminate in observations of heightened susceptibility in recurrent glioblastoma multiforme tumors, emphasizing the interplay of key molecular markers. Kram H et al. (2022), recurrent glioblastoma multiforme tumors demonstrate heightened susceptibility to ferroptosis, as evidenced by a notable increase in ACSL4 expression by 4.58 IRS points (p < 0.001) and a decrease in GPX4 expression by 4.36 IRS points (p < 0.001). Furthermore, GPX4+/GFAP+ cells exhibit a 38.0% reduction (p < 0.001), while ACSL4+/GFAP+ cells show a 29.3% increase (p < 0.001), indicating elevated ferroptosis vulnerability in GFAP-positive cells. In addition, recurrent tumors present significantly higher ALDH1A3 expression (+3.94 IRS points, p < 0.001), with a potential trend toward reduced overall survival observed in cases with increases exceeding 2.00 IRS points, though this correlation lacks statistical significance (p = 0.166). While Myrislignan demonstrates efficacy in targeting ferroptosis, hypoxia-induced resistance presents a critical challenge mediated by SLC7A11 and the PI3K/AKT/HIF1α pathway. Hypoxia-induced resistance to ferroptosis in glioblastoma is mediated by upregulation of SLC7A11 expression through the PI3K/AKT/HIF-1α pathway, as reported by Sun S et al. (2022), leading to significantly increased IC50 values and reduced lipid peroxidation (p < 0.05, p < 0.01). Targeting HIF-1α with PX-478 or inhibiting AKT effectively reverses this resistance, enhances susceptibility to sulfasalazine-induced ferroptosis, and improves anticancer efficacy in vivo (p < 0.05, p < 0.01). PX-478 demonstrates significant therapeutic potential by suppressing SLC7A11 expression, promoting lipid peroxidation, and overcoming hypoxia-induced resistance, resulting in reduced tumor growth and improved survival rates in glioma xenograft models (p < 0.05, p < 0.01). Building on the role of FANCD2 in ferroptosis resistance, FHOD1 emerges as another regulator influencing glioblastoma growth and prognosis through hypomethylation and redox modulation. FHOD1 promotes ferroptosis resistance in glioblastoma by upregulating HSPB1 through hypomethylation, enhancing glioma cell growth and reducing ferroptosis sensitivity (Zhang F et al. 2023). Knockdown of FHOD1 significantly inhibits glioma cell proliferation, improves ferroptosis sensitivity (p < 0.01), and reduces tumor volume and weight in xenograft models (p < 0.01). FHOD1 is significantly upregulated in glioma tissues, correlating with poor prognosis and increased recurrence risk. Enhanced ferroptosis sensitivity and reduced tumor growth through FHOD1 knockdown align with the observed increase in ROS and Fe²⁺ levels (p < 0.01), suggesting the FHOD1-HSPB1 axis as a promising therapeutic target. Expanding on ferroptosis-related mechanisms, CDKN2A deletion highlights lipidomic alterations that increase susceptibility to oxidative stress and therapeutic potential via GPX4 inhibition. CDKN2A deletion in glioblastoma significantly redistributes polyunsaturated fatty acids (PUFAs) from triacylglycerides to membrane phospholipids, resulting in increased lipid peroxidation and heightened ferroptosis susceptibility (Minami JK et al. (2023); p < 0.05). Lipidomic profiling across 84 patient tumors, 29 xenografts, and 43 gliomasphere models quantified 1,020 lipid species within 15 subclasses, revealing statistically significant alterations in lipid composition and peroxidation levels (e.g., p < 0.05, p < 0.001, p < 0.0001). GPX4 inhibition demonstrated improved survival in CDKN2A-null glioblastoma models (p < 0.05), validating its potential as a therapeutic target. Further exploring ferroptosis sensitivity, quiescent astrocyte-like glioma cells exhibit mitochondrial dysfunction and lipid peroxidation, offering insights into selective therapeutic targeting. Banu MA et al. (2023) Quiescent astrocyte-like glioma cells exhibit mitochondrial dysfunction, oxidative stress, and lipid peroxidation, showing significant sensitivity to GPX4 inhibition and ferroptosis (p < 0.0001 in drug screens). Selective depletion of these cells was demonstrated in murine organotypic glioblastoma slices, with Clu+ cell populations significantly reduced (p = 0.0185), and in human glioma slices treated with RSL3, where normalized enrichment scores indicated specific cell state depletion (FDR-corrected p < 0.05). These findings underscore the therapeutic potential of targeting mitochondrial vulnerabilities in resistant glioblastoma cell populations. Continuing with astrocyte-like glioma cell vulnerabilities, mitochondrial dysfunction and redox imbalance remain central to ferroptosis sensitivity and pharmacologic interventions. Quiescent astrocyte-like glioma cells exhibit selective vulnerability to GPX4 inhibition and ferroptosis, driven by mitochondrial dysfunction, lipid peroxidation, and redox imbalance (Banu MA et al. 2024). Pharmacologic GPX4 inhibition with RSL3 significantly reduces viability (p < 0.05) and depletes astrocyte-like markers in murine and human glioma models, with partial rescue observed using Ferrostatin-1. Increased ROS production and redox imbalance highlight mitochondrial dysfunction as a critical factor in this sensitivity, validated by FDR-corrected p-values in experimental models. Shifting focus to regulatory mechanisms, METTL16 knockdown reveals ferroptosis induction and immune interactions as critical factors in glioma progression and therapeutic strategies. Recent findings demonstrate that METTL16 knockdown effectively inhibits glioma progression by inducing ferroptosis, as evidenced by 4 increased malondialdehyde (MDA) and reactive oxygen species (ROS) levels, decreased glutathione (GSH) levels, and reduced NFE2L2 mRNA stability and expression (p < 0.05) (Yang Y et al. 2024). METTL16 was identified as a regulator that promotes glioma progression by stabilizing NFE2L2 mRNA through m6A modification, with NFE2L2 further associated with immune cell infiltration and immune checkpoint expression in gliomas. In lower-grade gliomas, METTL16 expression negatively correlates with CD8+ T lymphocytes (r = -0.17, p = 0.025), whereas NFE2L2 expression shows positive correlations with M2 macrophages (r = 0.24, p = 0.0019), neutrophils (r = 0.19, p = 0.011), activated memory CD4+ T cells (r = 0.22, p = 0.0034), and immune checkpoints TNFSF4, PDCD1, CD244, and ICOS. These findings expand the understanding of the molecular mechanisms underlying glioma progression and immune interactions. Ferroptosis remains central to glioblastoma treatment strategies, with procyanidin B1 emerging as another agent capable of inducing this cell death pathway. Procyanidin B1 induces ferroptosis in glioblastoma by suppressing NRF2 expression through PSMC3-mediated ubiquitin-dependent degradation, disrupting antioxidant defenses and enhancing H₂O₂ accumulation (Gao W et al. 2024). Quantitative results demonstrate significant tumor size reduction (p < 0.01) and improved survival rates in GBM-bearing mice (50% survival until day 54 vs. control group death by day 44, p < 0.05). These findings are consistent with observed mechanisms of ferroptosis induction and its therapeutic effects in glioblastoma models. Building on the therapeutic potential of ferroptosis induction, creatine kinase inhibition offers additional avenues for oxidative stress modulation and glioblastoma treatment. Katz JL et al. (2024), creatine kinase inhibition (CKi) significantly reduces glioblastoma multiforme (GBM) cell migration and invasion, as demonstrated by a near-complete inhibition of U251 wound closure at 20 µM CKi (p < 0.001). CKi induces oxidative stress, evidenced by elevated intracellular ROS levels and upregulated glutathione biosynthesis-related genes such as SLC7A11, TXNRD1, and HMOX1 (p < 0.001). Furthermore, combining CKi with glutathione inhibition or ferroptosis activation synergistically enhances GBM cell death, achieving IC50 values as low as 12 nM (e.g., GBM39, 12 nM for RSL3 + CKi; p < 0.001). These findings underscore the potential of CKi to disrupt promigratory mechanisms and anti-ferroptotic pathways in GBM, while combinatorial strategies with ferroptosis inducers and glutathione inhibitors improve therapeutic efficacy. The role of ferroptosis in glioblastoma progression is further underscored by C5aR1 knockdown, which enhances lipid peroxidation and suppresses tumor growth. Knockdown of C5aR1 in glioblastoma cells induces ferroptosis, evidenced by significantly decreased GPX4 protein levels (p < 0.01, p < 0.001) and increased lipid peroxidation markers, including 4-HNE (p < 0.01) and MDA (p < 0.05), as reported by Meng X et al. (2024). Inhibition of C5aR1 with PMX205 suppresses GPX4 expression, enhances lipid peroxidation, and significantly inhibits glioblastoma progression in a mouse intracranial xenograft model, as demonstrated by reduced tumor size (p < 0.01) and prolonged survival (p = 0.0141). Expanding on the role of ferroptosis in glioblastoma treatment, knockdown of SLC39A14 emerges as a strategy to further enhance ferroptosis markers and suppress glioma progression. Knockdown of SLC39A14 significantly suppresses glioma progression by reducing cGMP levels, inhibiting cGMP-PKG pathway-associated proteins (sGC, PKG1, PKG2), and enhancing ferroptosis markers, including increased MDA and Fe2+ levels and decreased GSH, GPX4, NRF2, and SLC7A11 protein levels (Zhang Y et al. 2023). These results demonstrate statistically significant correlations with glioma progression inhibition (p < 0.01; p < 0.001). While previous strategies focused on ferroptosis induction, KCNA1 is identified as a regulator that inhibits ferroptosis and promotes glioblastoma progression through mitochondrial protection. Wang W et al. (2024) KCNA1 upregulates SLC7A11, inhibits ferroptosis, and confers mitochondrial protection, reducing oxidative stress and mitochondrial damage, which contributes to glioblastoma progression and invasion. Knockdown of KCNA1 significantly decreases tumor growth and invasion both in vitro and in vivo, with statistical significance (p < 0.05, p < 0.01, p < 0.001, p < 0.0001). In vivo analysis further demonstrates reduced tumor size and extended survival times in mouse models following KCNA1 knockdown (p < 0.01). Returning to the therapeutic approach of ferroptosis induction, SIRT1 activation reveals a pathway to amplify glioblastoma cell sensitivity and promote cell death. SIRT1 activation enhances glioma cell sensitivity to RSL3-induced ferroptosis by promoting NAD+ depletion and ATF3 activation, which suppresses SLC7A11 and GPX4 expression (Chen X et al. 2024). This suppression leads to significant lipid peroxidation, depletion of cysteine and glutathione, and glioma cell death, p < 0.01, n=5. NAD+ depletion further triggers ATF3 activation, resulting in ferroptosis through increased ferrous iron accumulation, lipid peroxidation, and cysteine/GSH depletion. ROS-dependent upregulation of AROS sustains SIRT1 activation despite NAD+ depletion, amplifying the process, p < 0.01. Mechanisms regulating ferroptosis, such as Acsl4-mediated lipid peroxidation, provide deeper insights into therapeutic strategies for gliomas. Miao Z et al. (2022) found that the regulation of Drp1 phosphorylation at Ser637 by Hsp90 through calcineurin stabilizes Acsl4 expression, which is pivotal for promoting ferroptosis in gliomas. This mechanism enhances lipid peroxidation, as evidenced by elevated levels of 12-HETE and 15-HETE, and reduces glutathione (GSH) levels, while inducing mitochondrial morphological changes that increase sensitivity to erastin. In vitro and in vivo experiments demonstrate significant reductions in tumor growth and prolonged survival in mouse models, with statistical validation (p < 0.05, p < 0.01, p < 0.001). Low Acsl4 expression is associated with diminished ferroptosis sensitivity and poorer survival outcomes, emphasizing the critical role of Acsl4-mediated lipid peroxidation and mitochondrial alterations in enhancing erastin efficacy. Tumor heterogeneity and ferroptosis susceptibility, driven by PN-GSCs, underscore the complexity of glioblastoma progression and treatment responses. Vo VTA et al. (2022) PN glioblastoma stem cells (PN-GSCs) regulate tumor heterogeneity by secreting dopamine (DA) and transferrin (TF), which promote MES glioblastoma stem cell (MES-GSC) proliferation via iron uptake and Src-ERK signaling, while increasing ferroptosis susceptibility through elevated intracellular ROS generation and lipid peroxidation. MES-GSC proliferation in PN-GSC-conditioned media showed a significant increase (p < 0.05), and DA treatment amplified ROS levels and lipid peroxidation (p < 0.05). Combined DA and ferroptosis inducer treatments improved survival rates in orthotopic glioblastoma mouse models (p < 0.05). Additionally, GBM patients with high TFRC and DRD5 expression exhibited poor prognosis (p < 0.05), supporting the relevance of these pathways in disease progression. 2.2.2. Prognostic Markers and Risk Models Selected findings Up-regulation of FANCD2 correlates with poor prognosis in glioblastoma and promotes temozolomide resistance by attenuating ferroptosis (Song L et al. 2022). This highlights the therapeutic potential of targeting FANCD2 to overcome chemoresistance and enhance ferroptosis-based strategies in glioblastoma treatment. The 25-gene ferroptosis-related risk signature effectively stratifies glioma patients into prognostic groups, demonstrating high predictive accuracy for survival outcomes validated across multiple cohorts (Zhuo S et al. 2020). This provides a robust tool for clinical decision-making and personalized treatment planning in glioblastoma management. Paeoniflorin induces ferroptosis in glioma cells by upregulating NEDD4L and suppressing Nrf2 and GPX4 expression, with enhanced efficacy when combined with RSL3 (Nie XH et al. 2022). This combination therapy offers a promising approach to improve glioblastoma treatment outcomes through synergistic ferroptosis induction. The 25-gene ferroptosis-related risk signature effectively stratifies glioblastoma patients into distinct prognostic groups, demonstrating significant associations with survival outcomes and immune-related features validated across TCGA and CGGA cohorts. This model enhances patient stratification and clinical decision-making by integrating survival predictions and immune checkpoint therapy responsiveness. Paeoniflorin induces ferroptosis in glioblastoma cells by upregulating NEDD4L and suppressing GPX4 expression, leading to significant inhibition of tumor 5 growth in vitro and in vivo. This compound offers potential for synergistic therapeutic strategies in glioblastoma, particularly in combination with traditional chemotherapeutics like RSL3. FANCD2 upregulation correlates with poor prognosis in glioblastoma patients and contributes to temozolomide resistance by attenuating ferroptosis. Targeting FANCD2 could improve therapeutic outcomes and survival, particularly in recurrent glioblastoma cases resistant to standard treatments. The five-gene risk model (OSMR, G0S2, IGFBP6, IGHG2, FMOD) demonstrates significant prognostic value in glioblastoma patients, stratifying them into highand low-risk groups with distinct survival outcomes. This model enables precise risk stratification and therapeutic decision-making, advancing personalized treatment approaches for glioblastoma. FANCD2 upregulation correlates with poor prognosis and temozolomide resistance in glioblastoma patients, while its knockdown enhances ferroptosis sensitivity and immune response. Targeting FANCD2 offers a promising strategy to overcome therapeutic resistance and improve outcomes in glioblastoma management. Table 4. Prognostic Markers and Risk Models Study ID Length of intervention Population of intervention Control Intervention Intervention details Primary outcome Secondary outcome Liu HJ et al. (2020) U87MG glioma cell line, U251MG glioma cell line, temozolomideresistant U87TR and U251TR glioblastoma sub-lines Use of Erastin, a ferroptosis activator, to treat glioma cell lines Treated for 24 hours with varying Erastin concentrations, fresh medium replaced, CCK8 solution added, migration assay using Transwell system, cells pretreated with Erastin or without Erastin, upper chamber with serumfree medium, lower chamber with 10% FBS and Erastin, migrated cells analyzed using microscopy and Image J Identification of ferroptosis-specific markers and their relationship with glioma progression, including risk score model development and predictive metrics. Predictive accuracy of the gene signature, association with temozolomide resistance, autophagy, glioma cell migration, overall survival differences Go to Annex 5: Table 4. Prognostic Markers and Risk Models The modulation of ferroptosis pathways underscores the prognostic utility of gene signatures, such as the identified 19 ferroptosis-related gene signature. The identification of a 19 ferroptosis-related gene signature offers a validated prognostic tool for glioma progression and survival, as reported by Liu HJ et al. (2020), with predictive accuracy supported by AUC values ranging from 0.653 to 0.903 and statistically significant p-values (p < 0.001 for univariate Cox regression and p < 0.05 for multivariate Cox regression). The associated risk score model demonstrates strong correlations with glioma grade, malignancy, and therapeutic resistance, while functional annotation reveals its involvement in tumorigenesis, immune response, cell migration, and ferroptosis-related pathways. Survival differences are underscored by hazard ratios (HR = 1.212, 95% CI: 1.174–1.251, p < 0.001), highlighting its reliability as a prognostic indicator. Advancements in ferroptosis-related prognostic tools are exemplified by the development of the 25-gene risk signature, which further refines glioma patient stratification. Zhuo S et al. (2020), the 25-gene ferroptosis-related risk signature effectively stratifies glioma patients into distinct prognostic groups, demonstrating significant independent associations with overall survival outcomes. High-risk scores correlate with poorer survival, validated across CGGA and TCGA cohorts (HR = 3.654, 95% CI = 2.701–4.944, p < 0.001 for univariate analysis; HR = 1.917, 95% CI = 1.341–2.738, p < 0.001 for multivariate analysis). Predictive accuracy is highlighted by a 5-year AUC of 0.882 and clinical classification performance (cluster classification, AUC = 0.944). The nomogram, integrating this signature, forecasts 3and 5-year survival rates with a C index of 0.789, underscoring its potential clinical utility in glioma prognosis. The refinement of ferroptosis-based prognostic models continues with the introduction of the 11-gene signature, demonstrating strong predictive accuracy for survival outcomes. The 11-gene ferroptosis-related signature demonstrated strong predictive accuracy for overall survival in glioma patients, with AUC values of 0.879 at 1 year, 0.903 at 2 years, and 0.919 at 3 years in the TCGA cohort (p < 0.001), and consistent validation in the CGGA cohort (AUC = 0.790 at 1 year, 0.875 at 2 years, and 0.878 at 3 years, p < 0.001) (Chen Z et al. 2021). Furthermore, Cox regression analyses confirmed the signature as an independent prognostic factor for overall survival, with hazard ratios of 3.107 (95% CI = 2.506–3.853, p < 0.001) in the TCGA cohort and 1.943 (95% CI = 1.737–2.174, p < 0.001) in the CGGA cohort. These findings also revealed strong correlations between the signature and iron-related molecular functions, immune-related biological processes, and immune cell infiltration, reinforcing its relevance in glioblastoma prognosis. The exploration of ferroptosis-related prognostic tools continues with the development of FRSig, which further stratifies glioma patients based on survival outcomes and immune-related features. The ferroptosis-related risk signature (FRSig), developed using 10 prognostic ferroptosis regulators as described by Hu Y et al. (2021), stratifies glioma patients into distinct risk subgroups with significant differences in overall survival across all grades. High-risk gliomas identified by FRSig show worse survival outcomes, with predictive accuracy for 1-, 3-, and 5-year survival exceeding AUC > 0.800 (p < 0.05). FRSig correlates with immune-related indexes, tumor mutation burden (TMB), copy number alterations (CNA), and immune checkpoint expression, validated as an independent prognostic factor for overall survival (C-index = 0.762 in CGGA, C-index = 0.846 in TCGA). Its integration into a nomogram model alongside age and WHO grade improves prognostic predictions with high calibration accuracy. Insights into the molecular mechanisms of ferroptosis and apoptosis are complemented by studies linking ferroptosis-related genes to survival outcomes and immune activity in glioblastoma patients. Survival differences in glioblastoma patients are significantly associated with ferroptosis-related gene clusters and PCA score subgroups, with higher PCA scores correlating with improved prognosis (p = 0.002, p = 0.013, p < 0.001) (Peng X et al. 2023). Positive correlations are observed between PCA and gene scores derived from ferroptosis-related genes and immune cell infiltration, as well as immune pathway activity (p < 0.05, p = 0.002). Activated CD8+ T cells, CD4+ T cells, and Treg cells show significant positive associations with PCA and gene scores, while pathways such as TGF beta, JAK–STAT, and NK cell-mediated cytotoxicity exhibit higher activity in the high-score subgroup (p < 0.001). Building upon the role of CYBB and SOD2 in modulating ferroptosis sensitivity, proteomic analyses have further identified key regulators associated with glioblastoma prognosis and survival. Wang X et al. (2023) proteome-based analyses identified five ferroptosis regulators—HSPB1, GPX4, ACSL3, IL33, ELAVL1—as prognostic biomarkers significantly correlated with overall survival in glioblastoma multiforme, validated across multiple datasets with an adjusted p < 0.01. The five-protein signature stratified patients into highand low-risk groups with significant survival differences (adjusted p < 0.01), while ipatasertib demonstrated ferroptosis-inducing effects by targeting HSPB1 phosphorylation in high-risk glioma cells, exhibiting lower IC50 values. Expanding from ferroptosis induction, the prognostic relevance of ferroptosis-related gene signatures in glioma patients is explored. The study highlights that FRGrelated risk scores are strongly correlated with glioma prognosis, effectively categorizing patients into highand low-risk groups with distinct survival outcomes (Zuo Z et al. (2022); p < 0.001). The predictive performance of the risk scores is demonstrated by AUC values of 0.899, 0.917, and 0.930 for 1-, 2-, and 3-year survival predictions in the training cohort, and 0.765, 0.834, and 0.826 in the validation cohort. Additionally, the 3DResCNN deep learning network showed reliable diagnostic accuracy in identifying FRG signatures, achieving an average AUC of 0.781, average accuracy scores of 0.842 (TC-mask) and 0.825 (WT-mask), and average F1 scores of 0.843 (TC-mask) and 0.830 (WT-mask). 6 Investigating another therapeutic avenue, paeoniflorin is identified as a compound capable of inducing ferroptosis through distinct molecular mechanisms. Paeoniflorin induces ferroptosis in glioma cells by upregulating NEDD4L, leading to STAT3 ubiquitination and suppression of Nrf2 and GPX4 expression (Nie XH et al. 2022). It significantly increases intracellular ROS levels (p < 0.05, p < 0.01, p < 0.001), inhibits glioma cell proliferation (p < 0.01), and suppresses tumor growth in vivo (p < 0.01). The combination of paeoniflorin and RSL3 further enhances ferroptosis, as evidenced by elevated intracellular ROS, MDA, and Fe2+ levels (p < 0.01), highlighting its synergistic therapeutic potential in glioblastoma treatment. The discussion shifts to ferroptosis resistance mechanisms, with FANCD2 up-regulation linked to poor prognosis and therapeutic challenges in glioblastoma. Upregulation of FANCD2 correlates with poor prognosis in glioblastoma patients, significantly reducing overall survival across various glioma grades and recurrence statuses (HR > 1, p < 0.0001 for primary glioma of all grades, p = 0.0016 for recurrent glioma of all grades, p = 0.00025 for primary glioma of WHO grade III, and p = 0.01 for recurrent glioma of WHO grade III) (Song L et al. 2022). FANCD2 contributes to TMZ resistance by attenuating ferroptosis, while its knockdown increases reactive oxygen species (ROS) levels, inhibits cell survival, and correlates with immune features and cancer-associated pathways, further linking ferroptosis to glioblastoma progression. Insights into ferroptosis mechanisms contribute to the development of prognostic models that integrate molecular markers with survival and therapeutic outcomes in glioblastoma patients. The FRGPRS model demonstrates significant prognostic efficacy in predicting overall survival (OS) and progression-free survival (PFS) in GBM patients, validated through TCGA and GEO datasets (PFS p = 5.4E−03; OS p = 6.5E−03; AUC = 0.69) (Xiao D et al. 2021). As an independent risk factor (HR = 1.13; 95% CI [1.037, 1.23]; p = 0.005), it correlates with immune infiltration patterns, including M0 macrophages and CD8+ T cells (p < 2.2E−16), tumor tissue proportions such as stromal and immune scores (p = 7.1E−10, p = 2.9E−12), and chemotherapeutic response to temozolomide and cisplatin (p = 4.9E−03, p = 2E−05). High FRGPRS values are associated with increased tumor purity (p = 4.9E−12), reduced immune checkpoint therapy response, and chemotherapeutic resistance, while low FRGPRS values indicate improved temozolomide and cisplatin sensitivity, enhanced CD8+ T cell infiltration, and better atezolizumab response rates (p = 0.0017). Refining prognostic tools, gene-based models incorporating autophagy and ferroptosis pathways offer enhanced predictive accuracy and clinical relevance. The prognostic risk model utilizing five autophagy-ferroptosis-related genes (MTOR, BID, HSPA5, CDKN2A, and GABARAPLA2) demonstrated robust predictive accuracy, with C-index values of 0.72 in the training group and 0.74 in the verification group (p < 0.001) according to Zhou L et al. (2021). Kaplan-Meier survival analysis confirmed its significant efficacy (p < 0.001), and ROC analysis validated its strong predictive values for both shortand long-term survival. The nomogram exhibited high calibration accuracy and was effective across various clinical factors, except for WHO grade II glioma (p > 0.05). The prognostic utility of autophagy-ferroptosis-related genes extends further with the AD-FRG signature, which incorporates survival prediction alongside insights into immunological characteristics in glioma patients. The autophagy-dependent ferroptosis-related gene (AD-FRG) signature enhances survival prediction in glioma patients, as demonstrated by Sun W et al. (2022), with AUC values of 0.870, 0.922, and 0.869 for 1-, 3-, and 5-year survival rates, respectively, while confirming significant survival differences between highand low-risk groups (p < 0.001). High-risk glioblastoma patients identified through this signature exhibit an immunosuppressive tumor microenvironment, with increased macrophage infiltration (M0 and M1), elevated immune checkpoints (CD274, CTLA4, LAG3, PDCD1; p < 0.0001), and enhanced immunotolerance, correlating with reduced overall survival (p < 0.001). Building on the role of autophagy-ferroptosis-related genes in glioma prognosis, FRGPI offers additional predictive accuracy while highlighting associations with treatment responses and immune dynamics. FRGPI demonstrates high predictive accuracy for overall survival in glioma patients with 1-year AUC = 0.86, 3-year AUC = 0.88, and 5-year AUC = 0.84 (p < 0.001), according to Cai Y et al. (2021). It is negatively correlated with temozolomide IC50 (Spearman: r = -0.180, p < 0.001), indicating enhanced chemotherapy sensitivity, and positively associated with immune checkpoint inhibitor therapy response (Spearman: r = 0.300, p < 0.001). Additionally, FRGPI correlates significantly with immune cell infiltration (stromalScore: r = 0.670, p < 0.001; immuneScore: r = 0.670, p < 0.001), tumor mutational burden (r = 0.440, p < 0.001), and PD-L1 expression (r = 0.650, p < 0.001), while showing a negative correlation with microsatellite instability (r = -0.410, p < 0.001). Expanding on the prognostic relevance of ferroptosis-related signatures, the 3-FRLs signature provides robust stratification of glioma patients and reveals mechanisms underlying ferroptosis inhibition and tumor immunity. The 3-FRLs signature, comprising AL133415.1, LINC01426, and AC009227.1, stratifies glioma patients into highrisk and low-risk groups, demonstrating stable prognostic accuracy for overall survival with AUC values of 0.837 for 1-year OS, 0.837 for 3-year OS, and 0.790 for 5year OS in the training cohort (p < 0.001) (Huang L et al. 2022). Validation confirmed its robustness, particularly in LGG and IDH-mutant patients, and revealed correlations with tumor immunity, metastasis, and biological metabolism. LINC01426 functions as a ferroptosis inhibitor, with knockdown significantly enhancing ferroptosis by increasing ROS, MDA, and Fe²⁺ levels and inhibiting cell proliferation (p < 0.001). Stratification by the 3-FRL signature highlighted differences in cellular immunity, immune cell counts, immune-related gene expression, and somatic mutation rates, such as IDH-1 mutation at 25% in high-risk versus 94% in low-risk groups, and ATRX mutation at 18% in high-risk versus 43% in low-risk groups. Focusing on individual ferroptosis-related genes, their prognostic significance and therapeutic implications further underscore the complex interplay between ferroptosis and tumor progression in glioblastoma. Zhang X et al. (2022) found that ferroptosis-related genes, including STEAP3, HSPB1, MAP1LC3A, SOCS1, LOX, CAPG, CP, GDF15, CDKN1A, and CD44, exhibit significant prognostic value in glioblastoma, with higher expression levels correlating with poorer survival outcomes (p < 0.05, HR > 1). Predictive accuracy was confirmed through ROC analysis (AUC > 0.6 for all genes, > 0.7 for several), while a GSVA-based prognostic model demonstrated strong accuracy for predicting one-, two-, and three-year survival (AUC ~ 0.7). Immune analysis revealed associations with macrophage M2 infiltration (r = -0.32, p = 0.000052) and modulators of immune response. Therapeutic exploration identified Lumacaftor (DB09280, docking affinity -10.1 kcal/mol) and Lifirafenib (DB14773) as potential drugs targeting CAPG, CP, and CD44. The protective role of IRF2 against ferroptosis aligns with its contribution to glioma progression, emphasizing the therapeutic potential of targeting ferroptosis-related pathways. IRF2 expression is strongly associated with glioma progression, as higher levels correlate with advanced tumor grade and poor survival prognosis (p = 0.0019) (Tong S et al. 2022). Functional studies reveal that IRF2 protects glioma cells from ferroptosis by reducing reactive oxygen species (ROS) levels, decreasing lipid peroxidation, and increasing glutathione (GSH) content (p < 0.05). Additionally, IRF2 promotes glioma cell proliferation (p < 0.01), migration (p < 0.01), and invasion (p < 0.01) through epithelial-mesenchymal transition (EMT)-related pathways, highlighting its role in tumor progression and resistance mechanisms. These findings reinforce the importance of targeting ferroptosis-related pathways in glioma treatment strategies. Ferroptosis-related pathways and prognostic markers extend beyond IRF2, as evidenced by the association between ICD-related risk scores and glioblastoma prognosis. ICD-related risk scores were significantly associated with poor prognosis in GBM patients, including decreased overall survival (OS), progression-free survival (PFI), and disease-specific survival (DSS) (p < 0.05), as reported by Feng S et al. (2022). These scores correlated with enriched immune regulation pathways, increased immune cell infiltration, elevated ferroptosis regulators, and potential benefit from anti-PD1 therapy, as indicated by increased IPS and decreased TIDE scores. The prognostic efficacy of the risk signature was validated through AUC analysis for 1-, 3-, and 5-year survival. MYD88 was identified as a key biomarker linked to poor prognosis and immune-related pathways. The risk signature also demonstrated significant predictive performance for GBM subtypes, with the mesenchymal subtype exhibiting the highest risk score, and high-risk scores were associated with worse survival outcomes and potential benefit from anti-PD1 therapy (p < 0.05). Building on the predictive models of ICD-related risk scores, gene-based risk models further refine glioblastoma survival predictions and therapeutic strategies. Su J et al. (2022) The prognostic risk score model based on 12 DE-MRGs demonstrated robust predictive capabilities for glioblastoma patient survival, with AUC values of 0.75, 0.81, and 0.902 for 1-, 3-, and 5-year OS predictions in the TCGA GBM cohort. Kaplan–Meier analysis highlighted significantly improved survival outcomes for low-risk patients (p < 0.0001). Additionally, SSBP1 knockdown enhanced temozolomide sensitivity in glioblastoma cells by inducing ferroptosis, marked by increased 7 mitochondrial ROS production (p < 0.0001), altered mitochondrial morphology, reduced GPX4 and FTH1 expression, and shifts in iron and glutathione levels. Gene-based risk stratification continues to provide valuable insights into glioblastoma prognosis, as demonstrated by the five-gene risk model's predictive and therapeutic implications. Wu Y et al. (2025) The five-gene risk model (OSMR, G0S2, IGFBP6, IGHG2, FMOD) demonstrates significant prognostic value in glioblastoma patients by effectively stratifying them into high-risk and low-risk groups with distinct survival outcomes (p < 0.05). High-risk patients exhibit significantly lower survival rates compared to low-risk patients, as validated in both the training (TCGA) and testing (CGGA) cohorts. The model accurately predicts 1-, 2-, and 3-year survival rates and shows strong correlations with immune infiltration and pathways such as NOD-like receptor and JAK/STAT. Knockdown of OSMR significantly suppresses glioblastoma growth both in vitro and in vivo by promoting ferroptosis, enhancing CD8+ T cell activity, and shifting macrophage polarization toward an anti-tumor phenotype. In vivo experiments demonstrated prolonged survival in OSMR-deficient tumor-bearing mice (C57: 56 vs. 40 days, p = 0.0039; NSC: 37 vs. 32 days, p = 0.0273), reduced iron accumulation (Fe2+), and increased anti-tumor cytokines (IFN-γ, TNF-α, p < 0.05), emphasizing its therapeutic potential. Prognostic biomarkers such as MFAP4 offer additional diagnostic and therapeutic potential, complementing gene-based risk models in glioblastoma research. Lv Y et al. (2025) MFAP4 serves as an independent prognostic indicator for glioma, with significant associations with adverse clinicopathological features, including WHO grade (OR = 3.478), IDH wild-type status (OR = 0.125), and 1p/19q non-codeletion (OR = 0.272, all p < 0.001). It demonstrates high diagnostic value (ROC AUC = 0.833) and predictive efficacy for 1-, 3-, and 5-year survival (AUC > 0.7). Functional studies reveal that MFAP4 knockdown reduces glioma cell proliferation, migration, and invasion, highlighting its role in glioblastoma progression and its potential as a therapeutic target. The role of molecular mechanisms in glioblastoma progression extends to prognostic biomarkers, which provide valuable insights into patient survival and disease management. Circulating MDH1 and RNH1 biomarkers were identified as independent prognostic factors for survival in IDH-wildtype glioblastoma patients, with elevated levels correlating with reduced overall survival (13.9 months vs. 22.3 months, p = 0.002) and progression-free survival (6.0 months vs. 8.7 months, p = 0.033) (Clavreul A et al. 2024). A prognostic blood score integrating MDH1 and RNH1 levels showed high predictive accuracy, achieving hazard ratios of 2.49 (p = 0.002) for overall survival and 1.93 (p = 0.019) for progression-free survival, with AUC values of 0.80 for OS and 0.84 for PFS at 4 years. Furthermore, low tumor expression of FABP7 was significantly associated with shorter overall survival (p = 0.037), reinforcing the role of molecular markers in survival prediction. Extending the exploration of therapeutic targets, MXRA8 further demonstrates its relevance in glioblastoma progression through ferroptosis regulation and immune modulation. MXRA8 is a validated prognostic indicator in glioma progression, significantly associated with ferroptosis regulation and immune microenvironment modulation (Xu Z et al. 2022). Quantitative analysis demonstrates its predictive accuracy for survival outcomes, with AUC values of 0.780, 0.772, and 0.754 for 3-, 5-, and 10-year survival predictions (p = 0.000). Elevated MXRA8 expression correlates with unfavorable survival outcomes and immune-related factors, while knockdown studies reveal enhanced ferroptosis sensitivity, improved temozolomide efficacy, and reduced M2 macrophage infiltration, reinforcing its role in glioma progression and treatment resistance. 2.2.3. Therapeutic Strategies and Treatment Outcomes Selected findings CYP2E1 downregulation in glioblastoma correlates with ferroptosis activation, immune microenvironment alterations, and poor prognosis, supported by multivariate Cox regression analyses and high diagnostic accuracy (e.g., AUC = 0.982 for glioma diagnosis). This finding highlights CYP2E1 as a prognostic biomarker and therapeutic target, with molecular docking studies identifying compounds for potential intervention. The TMEM161B-AS1-hsa-miR-27a-3p-FANCD2/CD44 axis regulates glioblastoma progression and temozolomide resistance by promoting ferroptosis and apoptosis, with silencing TMEM161B-AS1 significantly inhibiting tumor growth and enhancing drug sensitivity in vivo (p < 0.001). This discovery provides a molecular target for overcoming chemoresistance and advancing ferroptosis-based therapeutic strategies. The combination of AF and plasma therapy induces ferroptosis and apoptosis synergistically, significantly reducing tumor growth and improving survival in glioblastoma-bearing mice (p ≤ 0.05 for tumor reduction, p ≤ 0.01 for survival). This approach demonstrates the potential of multimodal oxidative stressbased therapies to enhance ferroptosis and immunogenic cell death for glioblastoma treatment. Silencing HSP27 promotes ferroptosis in glioblastoma cells by increasing ROS production and Fe²⁺ accumulation, leading to reduced tumor growth and improved survival outcomes in xenograft models. This finding underscores the therapeutic potential of targeting HSP27 to enhance ferroptosis and improve glioblastoma treatment strategies. CircRNF10 upregulation in glioblastoma enhances tumorigenic efficacy and ferroptosis defense via a positive feedback loop involving ZBTB48 and HSPB1, while its silencing disrupts this loop and remodels iron metabolism, inducing ferroptosis. Targeting CircRNF10 offers a promising avenue for glioblastoma therapy by overcoming ferroptosis resistance and improving survival outcomes. Mesenchymal glioblastoma subtypes exhibit significantly enhanced resistance to ferroptosis induction compared to proneural subtypes, driven by elevated antioxidant defense mechanisms such as increased GPX4 and GSH levels. Understanding subtype-specific resistance mechanisms is critical for developing tailored ferroptosis-based therapies for glioblastoma. Ferroptosis sensitivity in glioblastoma cells is significantly enhanced by SIRT3 inhibition, which reduces SLC7A11 expression, promotes lipid peroxidation, and activates mitophagy pathways (Li X et al. 2024). This finding provides a promising molecular target for developing ferroptosis-based therapies to overcome glioblastoma resistance and improve treatment efficacy. The TMEM161B-AS1-hsa-miR-27a-3p-FANCD2/CD44 axis modulates glioblastoma progression and temozolomide resistance by regulating apoptosis and ferroptosis (Chen Q et al. 2021). Targeting this axis could enhance temozolomide sensitivity and ferroptosis induction, offering a novel therapeutic strategy for glioblastoma management. Combination therapy of ABX and TMZ significantly enhances ferroptosis induction in glioblastoma models, improving survival and drug sensitivity through persistent DNA damage and lipid ROS accumulation (Qu S et al. 2023). This approach underscores the potential of integrating ferroptosis mechanisms into combination treatments for better clinical outcomes in glioblastoma. Ce6@Cu nanoparticles activated by ultrasound irradiation induce ferroptosis and cuproptosis in glioblastoma cells by depleting GSH, amplifying ROS generation, and reducing GPX4 expression, leading to significant tumor suppression and improved survival in mouse models (Zhu Y et al. 2024). This finding highlights the potential of nanoparticle-based strategies to overcome treatment resistance and enhance therapeutic efficacy in glioblastoma. RSL3 enhances glioma radiosensitivity by promoting DNA double-strand breaks and suppressing epithelial-mesenchymal transition, leading to significant tumor growth reduction and improved survival in preclinical models (Wang X et al. 2024). This approach underscores the therapeutic promise of combining ferroptosis inducers with radiotherapy to improve glioblastoma treatment outcomes. Targeting MS4A4A in glioblastoma reduces M2 macrophage infiltration and enhances CD8+ T-cell activation, improving the efficacy of PD-1 immunotherapy and delaying tumor growth in mice (Shao G et al. 2024). This study suggests that modulating the tumor immune microenvironment through ferroptosis could augment immunotherapeutic strategies for glioblastoma. 8 RSL3 enhances glioblastoma radiosensitivity through DNA double-strand break induction and suppression of epithelial-mesenchymal transition (Wang X et al. 2024). This finding could inform combination therapies leveraging ferroptosis inducers to improve radiotherapy outcomes in glioblastoma patients. Ferroptosis modulation through nanoparticle-based strategies, such as Fe3O4-siPD-L1@M-BV2, enhances immune reactivation and tumor suppression in drug-resistant glioblastoma (Liu B et al. 2022). These approaches may pave the way for integrating ferroptosis-targeting nanomedicine with immunotherapy for glioblastoma treatment. The mesenchymal glioblastoma subtype exhibits significant resistance to ferroptosis compared to the proneural subtype, with elevated antioxidant mechanisms linked to poor survival outcomes (D'Aprile S et al. 2024). Understanding subtype-specific ferroptosis resistance could guide personalized therapeutic strategies targeting glioblastoma heterogeneity. Table 5. Therapeutic Strategies and Treatment Outcomes Study ID Length of intervention Population of intervention Control Intervention Intervention details Primary outcome Secondary outcome Ye L et al. (2021) 24 months Patients with gliomas (lowgrade glioma WHO grade II–III and GBM WHO grade IV), collected at Renmin Hospital of Wuhan University, Wuhan, China, no chemotherapy or radiotherapy before surgery Patients with cerebral hemorrhage, 6 participants, no treatment control group Investigation of CYP2E1 mRNA expression levels in glioma tissues, analysis of its clinical significance, and molecular docking of TCM compounds targeting CYP2E1 RNA extracted using TRIzol reagent, cDNA synthesized using PrimeScript RT Reagent Kit, CYP2E1 mRNA levels detected using SYBR Premix Ex Taq II and Bio-Rad real-time PCR Systems, relative Ct method used for comparison, gene expression analyzed using R packages, molecular docking performed using AutoDock 4.2 and PyMOL software, correlation analysis conducted for immune checkpoints, miRNA prediction performed using MiDRB and TargetScan, functional enrichment analyzed using GO and KEGG pathways Prognostic significance of CYP2E1 expression in glioma patients, correlation with poor prognosis, clinical features, survival outcomes, and statistical significance Involvement of CYP2E1 in lipid metabolism, ferroptosis, tumor immune microenvironment, correlation with methylation levels and copy number variation, miRNA targeting by hsamiR-527, identification of compounds targeting CYP2E1 Go to Annex 6: Table 5. Therapeutic Strategies and Treatment Outcomes These findings on ferroptosis vulnerability align with the molecular mechanisms influencing ferroptosis activation, such as CYP2E1 downregulation in glioma patients. CYP2E1 expression is significantly downregulated in glioma patients, with multivariate Cox regression analysis confirming its role as an independent prognostic factor (p < 0.001) (Ye L et al. 2021). ROC analysis demonstrates high diagnostic accuracy, with AUC values of 0.982 for glioma diagnosis, and predictive AUCs for overall survival (OS) at 0.810 (1-year), 0.798 (3-year), and 0.763 (5-year) in the TCGA cohort, and 0.668 (1-year), 0.671 (3-year), and 0.676 (5-year) in the CGGA cohort. Downregulation correlates with immune microenvironment alterations (e.g., rho = 0.34 for activated NK cells, p < 0.001), lipid metabolism inactivity, and ferroptosis activation (p < 0.001). Mechanisms influencing CYP2E1 downregulation include hsa-miR-527 targeting (p < 0.001), DNA hypomethylation (Pearson's r = -0.36, p < 0.0001), and copy number variation (Pearson's r = 0.61, p < 0.0001). Molecular docking studies have identified compounds such as 18beta-glycyrrhetinic acid and colchicine as potential CYP2E1-targeting agents. The role of CYP2E1 in ferroptosis activation provides a foundation for understanding how molecular axes like TMEM161B-AS1 regulate ferroptosis and tumor progression. The TMEM161B-AS1-hsa-miR-27a-3p-FANCD2/CD44 axis regulates glioblastoma progression and temozolomide resistance by modulating proliferation, migration, invasion, apoptosis, and ferroptosis (Chen Q et al. 2021). Silencing TMEM161B-AS1 and/or overexpressing hsa-miR-27a-3p significantly inhibits tumor growth, reduces FANCD2 and CD44 expression, promotes apoptosis and ferroptosis, and enhances temozolomide sensitivity, as shown by in vitro and in vivo experiments (p < 0.001). Building on prognostic insights, therapeutic strategies targeting ferroptosis emerge as promising approaches to combat glioblastoma progression. Van Loenhout J et al. (2021) The sequential combination therapy of AF (15 mg/kg orally for 14 days) and plasma (10 s direct application for 5 consecutive days) significantly reduced tumor growth kinetics (p ≤ 0.05) and prolonged survival (p ≤ 0.01) in SB28 glioblastoma-bearing mice. This treatment synergistically induced apoptosis and ferroptosis by inhibiting TrxR activity (p ≤ 0.05) and depleting GSH, leading to intracellular ROS accumulation and oxidative stress (p < 0.0001). Additionally, immunogenic cell death was elicited through increased CRT expression, ATP release, and HMGB1 secretion (p ≤ 0.05), coupled with dendritic cell maturation and reduced tumor volume in vivo, significantly improving survival in GBM-bearing mice (p ≤ 0.05). The therapeutic focus on ferroptosis expands with investigations into its synergy with apoptosis-inducing agents to enhance glioblastoma cell death. Recent findings from Moujalled D et al. (2022) show that dual targeting of pro-survival proteins MCL-1 and BCL-XL using BH3 mimetic drugs (S63845 and A1331852) significantly enhances apoptosis and cell death in glioblastoma (GBM) cell lines, with IC50 values in the low nanomolar range (e.g., IC50 < 100 nM for U251 cells) and robust activation of apoptosis markers cleaved caspase-3 and PARP1 (p < 0.05 to p < 0.0001). Additionally, ferroptosis inducers (erastin, IC50 = 2.5 μM; RSL3, IC50 = 64.5 nM) synergize with these BH3 mimetic drugs to significantly enhance glioblastoma cell killing (e.g., U251), via ferroptosis and apoptosis pathways, as evidenced by reduced cell viability, activation of apoptosis markers, and partial dependence on intrinsic apoptosis effectors BAX and BAK. Cell death is mitigated by ferroptosis inhibitors liproxstatin-1 or deferoxamine (p < 0.0001), underscoring the interplay between ferroptosis and apoptosis mechanisms. Further elucidating molecular pathways, interactions between CYBB and Nrf2 reveal mechanisms regulating ferroptosis sensitivity and therapeutic resistance in glioblastoma. CYBB interacts with Nrf2 to activate the SOD2 mitochondrial antioxidant axis, reducing oxidative stress and ferroptosis sensitivity in glioblastoma cells (Su IC et al. 2023). Elevated CYBB expression is associated with poor progression-free survival (hazard ratio = 1.6; 95% CI = 1.05–2.4; p = 0.029). Knockdown of CYBB or SOD2 enhances the sensitivity of glioblastoma cells to temozolomide and erastin-induced ferroptosis (p < 0.05, p < 0.001, p < 0.0001). Furthermore, in vivo suppression of SOD2 significantly improves the efficacy of erastin analogs (p < 0.001, p < 0.0001), demonstrating the potential for targeting SOD2 to overcome temozolomide resistance. Expanding on the identification of ferroptosis regulators, recent findings highlight Myrislignan as a promising agent for glioblastoma therapy through ferroptosis induction and EMT inhibition. Zhou Y et al. (2023) found that Myrislignan effectively suppresses glioblastoma progression by targeting ferroptosis induction and epithelial-mesenchymal transition (EMT) inhibition via the Slug-SLC7A11 pathway. Significant anti-tumor activity was observed at doses of 5–15 μg/mL in vitro and 5 mg/kg in vivo, with tumor specificity at therapeutic doses sparing normal brain tissue and survival improvement in xenograft mouse models (p < 0.05, p < 0.01, p < 0.001). To overcome such resistance mechanisms, innovative approaches such as Fe3O4-siPD-L1@M-BV2 nanoparticles have emerged, enhancing ferroptosis and immune activation in drug-resistant glioblastoma. Liu B et al. (2022) found that Fe3O4-siPD-L1@M-BV2 nanoparticles significantly enhance ferroptosis in drug-resistant glioblastoma by increasing ROS, LPO, and H2O2 levels (p < 0.01), reducing GPX4 and xCT protein expression, and depleting GSH. They promote immune reactivation 9 ferroptosis, and inflammation, highlighting its therapeutic potential within cancer biology. Quijano-Rubio et al. (2022) demonstrated that CD95 deletion significantly reduces glioblastoma cell clonogenic growth (p < 0.05), sphere-forming capacity (p < 0.05), and invasiveness (p < 0.0001), independent of CD95L expression. However, survival analysis in xenograft models revealed no significant differences in median survival times across groups in both S-24 and ZH-161 models (p > 0.05), suggesting therapeutic challenges in translating these cellular benefits to in vivo outcomes. The primary outcomes related to CD95 receptor expression in glioblastoma provide critical insights into its dual role in apoptosis signaling and tumor cell survival. Previous studies collectively highlighted the paradoxical nature of CD95, demonstrating its capacity to mediate apoptosis while simultaneously promoting tumor invasion and survival under compromised apoptotic conditions. Notably, studies by Sharma S et al. (2019) and Rossin A et al. (2015) revealed mechanisms regulating CD95 surface levels, such as endosomal trafficking via ENTR1 and post-translational modifications like palmitoylation, which prevent lysosomal degradation. These findings underscore the complexity of CD95 signaling and its regulatory pathways in glioblastoma. In corroboration, Quijano-Rubio et al. (2022) confirmed CD95 expression at both mRNA and protein levels in human glioblastoma initiating cell (GIC) lines, providing additional evidence for its presence in glioblastoma cells. However, this study does not address the functional implications of CD95 expression, such as its role in apoptosis resistance or tumor invasion, which were extensively discussed in earlier research by Wisniewski P et al. (2010), Fujita H et al. (2002), and Sharma S et al. (2019). Furthermore, it does not explore regulatory mechanisms like those identified in studies by Sharma S et al. (2019) and Rossin A et al. (2015), leaving gaps in understanding the factors modulating CD95 receptor levels. Discrepancies arise regarding the impact of CD95 gene deletion on malignancy reduction. While Hadji A et al. (2014) suggested that cancer cannot form in the absence of CD95, earlier studies indicated that CD95 can promote tumor invasion and survival under specific conditions. Quijano-Rubio et al. (2022) neither supports nor contradicts these findings, as it focuses solely on expression levels without addressing functional outcomes. The strength of evidence in Quijano-Rubio et al. (2022) lies in its robust quantitative measurement of CD95 expression across multiple GIC lines, enhancing the reproducibility of findings. However, the lack of exploration into functional consequences limits its contribution to understanding the broader implications of CD95 in ferroptosis and glioblastoma malignancy. In terms of Caspase-3 activity, Quijano-Rubio et al. (2022) provide significant evidence regarding the impact of CD95 gene deletion, demonstrating a clear abrogation of DEVD-amc peptide-cleaving activity, which is a hallmark of Caspase-3 activation. This finding aligns with earlier studies by Muzio M et al. (1996), Milhas D et al. (2005), and Song JJ et al. (2008), which emphasized the functional involvement of Caspase-3 in apoptosis induction and its regulation by upstream signaling pathways. Specifically, Quijano-Rubio et al. (2022) strengthen the understanding of CD95’s role in canonical apoptotic signaling, as previously suggested by Muzio M et al. (1996) and Milhas D et al. (2005), which highlighted the interaction between death receptors and caspase activation. However, discrepancies arise when comparing Quijano-Rubio et al. (2022) with Sánchez-Osuna M et al. (2016), which noted incomplete apoptosis in glioblastoma cells despite correct executioner caspase activation. The new data suggest that CD95 deletion effectively disrupts apoptotic signaling, whereas Sánchez-Osuna M et al. (2016) indicated that apoptosis may remain incomplete even with caspase-3 activation. This difference underscores the complexity of apoptotic mechanisms in glioblastoma and suggests that CD95 deletion may uniquely influence apoptosis progression. Methodologically, Quijano-Rubio et al. (2022) advance the field by employing CD95 knockout clonal sublines and combining exogenous CD95L stimulation with cycloheximide sensitization, providing a robust experimental design that enhances the specificity and reliability of the findings. This contrasts with earlier studies, such as Song JJ et al. (2008) and Muzio M et al. (1996), which focused on broader regulatory mechanisms like MAPK cleavage or Fas palmitoylation without directly targeting CD95 deletion. Overall, the study contributes novel insights into the mechanistic role of CD95 in Caspase-3 activity and apoptosis in glioblastoma, offering stronger evidence for the therapeutic potential of targeting CD95 to reduce malignancy. While the findings are consistent with most previous studies, the divergence from Sánchez-Osuna M et al. (2016) highlights the need for further exploration of downstream apoptotic processes in glioblastoma. 3.4. Iron Oxide Nanoparticles and Paclitaxel: Inducing Ferroptosis via Autophagic Pathways in Glioblastoma Selected findings The combination of iron oxide nanoparticles and paclitaxel significantly inhibits glioblastoma cell viability while inducing ferroptosis through increased ROS production, lipid peroxidation, and intracellular iron levels. This finding highlights a novel therapeutic strategy that leverages the synergistic effects of ROS modulation and iron delivery to overcome tumor resistance in glioblastoma. Enhanced autophagic flux induced by IONP@PTX, as evidenced by upregulation of autophagy-related proteins and downregulation of p62 and mTORC1, demonstrates a mechanistic link between autophagy and ferroptosis in glioblastoma. This insight provides a pharmacological foundation for targeting autophagic pathways to amplify ferroptosis and improve glioblastoma treatment outcomes. The exploration of ferroptosis induction via autophagic pathways in glioblastoma through the use of iron oxide nanoparticles (IONPs) in combination with paclitaxel (PTX) provides a promising therapeutic strategy to address tumor resistance and recurrence. The study by Nie Q et al. (2023) demonstrates that this combination significantly inhibits glioblastoma cell viability while inducing ferroptosis, as evidenced by increased reactive oxygen species (ROS), lipid peroxidation, and elevated intracellular iron levels. These findings highlight the critical role of ROS as pivotal mediators of ferroptosis and oxidative stress, corroborating previous research that has established the importance of ROS generation in cancer cell death mechanisms (Shen Z et al. (2018); Huo M et al. (2017)). The synergistic enhancement of ROS production observed in the current study aligns with earlier evidence and reinforces the therapeutic relevance of ROS modulation in glioblastoma treatment. Furthermore, the quantification of lipid peroxidation using a C11-BODIPY™ fluorescent probe in Nie Q et al. (2023) showcases a methodological advancement over previous studies, which primarily focused on genetic or chemical interventions to modulate lipid peroxidation (Alborzinia H et al. (2022); Bao Z et al. (2021)). This direct quantification underscores the therapeutic potential of IONP@PTX in promoting lipid peroxidation, a hallmark of ferroptosis. While discrepancies in experimental models exist—Nie Q et al. (2023) relying on in vitro analyses compared to the animal trials and cohort studies of prior research—its focused approach contributes significantly to our understanding of ferroptosis mechanisms in glioblastoma. Moreover, the study elucidates the impact of iron concentration in driving ferroptosis through enhanced ROS production. The introduction of iron via nanoparticles not only aligns with previous findings regarding the role of iron in ferroptosis induction (Shen Z et al. 2018) but also represents a distinct therapeutic strategy that differs from previous approaches focused on modulating iron uptake or storage mechanisms. The reliance on in vitro models, while limiting generalizability, provides a novel perspective on the targeted delivery of iron to glioblastoma cells, enhancing therapeutic precision. In terms of autophagic flux, Nie Q et al. (2023) further expands on earlier studies by demonstrating that IONP@PTX significantly enhances autophagic activity, as indicated by the upregulation of autophagy-related proteins such as Beclin 1, LC3-II/I, and HDAC6, alongside the downregulation of p62 and mTORC1. This evidence supports the hypothesis that autophagy plays a crucial role in ferroptosis induction, particularly within glioblastoma contexts. The amplification of these effects through the addition of rapamycin reinforces the mechanistic link between autophagy and ferroptosis, providing a pharmacological approach to enhance autophagic flux for therapeutic benefit. Lastly, the evaluation of cell viability in the study underscores the synergistic effect of IONP@PTX in reducing glioblastoma cell survival rates, complementing broader 16 vulnerabilities identified in previous research regarding ROS detoxification pathways (Floros KV et al. 2021) and ferroptosis-related molecular targets (Bao Z et al. 2021). The methodological advances utilized in Nie Q et al. (2023), including the CCK-8 assay and combination index analyses, enhance our understanding of drug synergy and its implications for cell survival, marking a significant step forward in the development of ferroptosis-related therapies for glioblastoma. In summary, the findings from Nie Q et al. (2023) not only align with existing literature but also introduce novel therapeutic approaches that leverage the synergistic effects of iron oxide nanoparticles and paclitaxel to induce ferroptosis through enhanced ROS production, lipid peroxidation, and autophagic pathways. These insights pave the way for further exploration of targeted interventions that could improve treatment outcomes for glioblastoma patients. 4. Discussion of Limitations 4.1. Ferroptosis and Immune Modulation: Mechanisms and Therapeutic Strategies in Glioblastoma The recent advancements in ferroptosis and immune modulation research in glioblastoma (GBM) have substantially addressed several limitations identified in prior reviews, yet they underscore the need for further exploration to fully resolve these challenges. One significant limitation highlighted previously was the difficulty in effectively utilizing cytotoxic autophagy to eliminate GBM cells or inhibit protective autophagy [144]. New findings have demonstrated that inhibiting protective autophagy enhances the cytotoxic effects of temozolomide (TMZ) on glioblastoma stem-like cells (GSCs), suggesting ferroptosis as a potential underlying mechanism [18]. This study, conducted on adult GBM patients, employed various autophagy modulators and revealed that lower autophagy levels correlate with improved overall survival (p = 0.0012) and increased sensitivity of GSCs to TMZ (p < 0.05). However, the short intervention duration and limited differentiation between cytotoxic and protective autophagy mechanisms restrict the study's ability to fully resolve this limitation. Future research must focus on expanding intervention periods and refining mechanistic insights to establish clinical applicability and long-term outcomes. Closely related is the challenge of ameliorating autophagy-related drug tolerance in glioma treatments, which was another limitation identified in prior reviews [144]. Recent studies have explored strategies to modulate autophagy and enhance GSC sensitivity to TMZ. For instance, the use of trehalose and quinacrine in both in vitro and in vivo models significantly improved GSC susceptibility to TMZ, with low autophagy levels correlating with better overall survival (p = 0.0012) [18]. Another study targeted STAT3 via genetic manipulation, demonstrating that STAT3 depletion enhances cell survival under drug treatment while reducing autophagy-dependent cell death (p < 0.0001) [20]. Despite these promising findings, limitations such as short intervention durations and restricted population diversity hinder comprehensive resolution. Future studies should prioritize extended treatment protocols and larger, more diverse cohorts to fully address drug tolerance in GBM therapies. A critical limitation identified in earlier reviews was the insufficient understanding of ferroptosis in GBM progression, particularly its interaction with oxidative stress, ER stress, and metabolic pathways [144]. Recent evidence has shown that TMZ treatment induces ferroptotic cell death in GSCs, marked by increased lipid peroxidation and reduced GPX4 activity, highlighting its link to oxidative stress [18]. Additionally, autophagy modulation was found to influence GSC susceptibility to TMZ, providing valuable insights into the interplay between ferroptosis and other cellular death modalities. Nonetheless, the study's short intervention duration, limited population diversity, and lack of comprehensive mechanistic exploration indicate that further research is required to elucidate the complex interactions among ferroptosis, oxidative stress, and metabolism in GBM progression. Another significant limitation was the protective feedback pathways, particularly the PERK-ATF4-HSPA5-GPX4 cascade, which shield glioblastoma cells from ferroptosis and limit the efficacy of compounds like DHA [16]. Recent research investigated the role of NRF2 and ABCC1 in ferroptosis sensitivity within glioblastoma cell lines U251MG and T98G [6]. Despite high NRF2 expression, T98G cells exhibited sensitivity to ferroptosis-inducing agents such as Erastin and RSL3, suggesting a disruption in protective feedback mechanisms. The study revealed that high levels of NRF2 and ABCC1 could facilitate glutathione (GSH) depletion, enhancing ferroptosis sensitivity. However, the lack of direct investigation into the entire feedback cascade and absence of in vivo validation limits the study's effectiveness. Future research should aim to comprehensively explore these pathways and validate findings in clinical settings to address drug resistance in GBM. Finally, the lack of specificity in ferroptosis biomarkers, which hampers their predictive utility for tumor prognosis and individualized treatment outcomes, was identified as a significant limitation [16]. New findings demonstrated that elevated NRF2 and ABCC1 expression in glioblastoma cells correlates with increased sensitivity to ferroptosis and poor patient outcomes, suggesting their potential as predictive biomarkers [6]. Specifically, T98G cells exhibited a 12-fold increase in NRF2 expression and sensitivity to ferroptosis inducers, while silencing NRF2 increased resistance. Despite these promising results, the study was limited by its use of only two glioma cell lines without a control group, restricting its generalizability. Future validation across diverse cancer types and clinical samples is essential to fully address the biomarker specificity limitation. In summary, while recent studies have made notable progress in addressing the limitations of ferroptosis and immune modulation in glioblastoma, several challenges remain unresolved. These include extending intervention durations, increasing population diversity, conducting comprehensive mechanistic investigations, and validating findings in clinical settings. Addressing these gaps will be critical to translating these promising findings into effective therapeutic strategies for glioblastoma. 4.2. Ferroptosis in Glioblastoma: Pathways, Immune Interactions, and Overcoming Resistance The recent advancements in ferroptosis research have significantly addressed the limitations outlined in previous reviews, particularly in the context of glioblastoma (GBM). However, while notable progress has been made, several challenges remain unresolved, necessitating further investigation. The mechanistic understanding of ferroptosis in GBM, previously identified as a critical limitation, has been expanded through studies elucidating the roles of specific proteins and pathways. For instance, GPX4 has been shown to play a pivotal role in ferroptosis regulation, with IGF2BP3 stabilizing GPX4 mRNA and preventing ferroptosis [95]. Complementary findings demonstrate that silencing GPX7 enhances ferroptosis sensitivity to erastin [145], and DHA induces ferroptosis via increased ROS and lipid peroxidation [25]. Despite these advancements, the mechanistic complexities surrounding GPX4, its interactions with other proteins, and its contextdependent roles remain partially unexplored, highlighting the need for broader experimental validation and more diverse sample populations. Glioblastoma's intrinsic heterogeneity, another major challenge, complicates the development of universally effective therapies. Recent studies have made strides in addressing this limitation by investigating molecular pathways and biomarkers associated with ferroptosis. For example, GPX7 expression has been correlated with treatment response, suggesting its potential as a biomarker for personalized therapies [145]. Additionally, targeting IGF2BP3 and erianin has demonstrated efficacy across diverse glioma subtypes, despite heterogeneity [95][100]. However, the focus on specific cell lines and limited sample sizes restricts the generalizability of these findings. Future research must integrate larger cohorts and explore additional pathways to comprehensively address glioma heterogeneity. The integration of ferroptosis-based therapies with traditional modalities, such as chemotherapy and radiotherapy, has shown promise but remains underdeveloped. Combining DHA with PERK pathway inhibition has demonstrated synergistic effects in glioma cells (p < 0.001) [25], while erianin enhances TMZ sensitivity in resistant glioblastoma stem cells, inducing ferroptosis (IC50 increased by 9.4 to 11.5 times) [100]. Despite these promising results, the studies lack exploration of combinations with radiotherapy or other modalities, underscoring the need for broader investigations into multimodal therapeutic strategies. The complex interplay between glioma cells and their microenvironment also poses significant challenges to ferroptosis-based therapies. Recent studies have begun to address this limitation by exploring microenvironmental interactions. For instance, IGF2BP3 knockdown impairs glioma cell growth and enhances susceptibility to 17 microglial phagocytosis [95], while Notch signaling has been implicated in glioma cell survival and GPX4 sensitivity [21]. However, these studies focus narrowly on specific pathways, leaving other microenvironmental factors, such as immune cell dynamics, insufficiently explored. Further research should aim to unravel the multifaceted interactions within the glioma microenvironment to optimize ferroptosis-based interventions. The absence of gold-standard biomarkers for ferroptosis in GBM has been partially addressed through the identification of gene signatures, such as a 19-gene signature [50] and an 11-gene signature [51], both demonstrating strong prognostic capabilities (AUC values up to 0.919). Additionally, SLC7A11 has emerged as a potential biomarker, offering insights into ferroptosis mechanisms [22]. Despite these promising findings, the reliance on cell lines and the lack of independent cohort validation limit their clinical applicability. Further research is essential to establish universally applicable biomarkers validated across diverse glioma models. Finally, the challenge of poor blood-brain barrier penetration and compensatory mechanisms hindering ferroptosis-based therapies has been addressed through innovative approaches. The use of Lpo@Cu2Se-GOx nanocomposites has demonstrated enhanced BBB penetration and therapeutic efficacy in in vivo GBM models [103]. Additionally, Fe3O4-siPD-L1@M-BV2 nanoparticles have shown promising results in selectively inducing ferroptosis while sparing healthy tissues [74]. However, these studies do not fully explore long-term safety, toxicity, or compensatory mechanisms, indicating the need for further research to optimize these strategies. In conclusion, while the new findings represent significant progress in addressing the limitations of ferroptosis research in glioblastoma, they underscore the necessity for ongoing investigations. Future research should focus on expanding sample diversity, integrating multimodal therapies, and validating biomarkers and therapeutic strategies in clinical settings to achieve a comprehensive understanding and effective application of ferroptosis-based treatments for GBM. 5. Conclusions The exploration of ferroptosis in glioblastoma has revealed critical insights into its underlying mechanisms and therapeutic strategies. The studies collectively highlight the role of key regulators such as GPX4, ROS, and NCOA4, emphasizing their potential as therapeutic targets. Interventions like CD95 gene deletion and the use of iron oxide nanoparticles combined with paclitaxel have shown promise in reducing tumor malignancy and inducing ferroptosis, respectively. However, limitations such as reliance on in vitro models, small sample sizes, and the complexity of tumor microenvironments pose significant challenges in translating these findings to clinical applications. Future research should prioritize the development of diverse in vivo models, validation of biomarkers across broader cohorts, and exploration of multimodal treatment strategies. Moreover, understanding the adaptive resistance mechanisms that may arise from therapeutic interventions will be crucial for enhancing treatment efficacy. 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[PMC free article] [PubMed] 22 Annex 1: Methods 1.1 Approach The search strategy was designed according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines [1]. The systematic literature review was automatically generated on demand using the Synthory AI service. All components listed below were identified, extracted, assessed, and analyzed automatically as part of the review process. The review was created for research purposes. 1.2 Criteria of Inclusion and Exclusion Inclusion criteria Publications available in PubMed and PubMed Central™. Publications related to ferroptosis, glioblastoma, and associated aspects. Primary research studies, including randomized controlled trials, cohort studies, and qualitative research. Exclusion criteria Articles published before 2018/01. Systematic literature reviews, case series, case reports, expert opinions, study protocols, and any unidentified study types. Studies without full-text availability in PubMed or PubMed Central™. Articles that have been previously included and analyzed in existing reviews within the defined focus areas, to avoid duplication and ensure the inclusion of novel research findings. 1.3 Search Strategy and Screening Process The topic of the request was 'Ferroptosis in glioblastoma'. The search employed 18 keywords. The search was conducted across the PubMed and PubMed Central™ databases, covering the publication period from 2018/01 to 2025/10. 1. Identification: 5172 records were retrieved from PubMed using the inclusion criteria. 2. Screening: a. Articles were excluded based on the criteria. b. Articles of low quality risk were excluded following an Article Quality Assessment. 3. Eligibility: Assessment of alignment with defined topics. Figure 1. PRISMA 2020 Flow Diagram IdentificationScreeningIncluded Records identified from: Databases (n = 12669) Records removed before screening: Duplicates (n = 43) Ineligible publication type (reviews, protocols, etc.) (n = 7454) Quality assessment (n = 5172) Records excluded after quality assessment (n = 495) Title and abstract screening (n = 4677) Records excluded: topic mismatch (n = 4582) Full-text availability check (n = 95) Records excluded due to unavailable full-text (n = 0) Studies included in review (n = 95) A total of 95 articles were included in the final review, based on the inclusion and exclusion criteria. Ferroptosis and Immune Modulation: Mechanisms and Therapeutic Strategies in Glioblastoma – 7 Ferroptosis in Glioblastoma: Pathways, Immune Interactions, and Overcoming Resistance – 86 CD95 Gene Deletion: Reducing Malignancy in Glioblastoma Cells – 1 Iron Oxide Nanoparticles and Paclitaxel: Inducing Ferroptosis via Autophagic Pathways in Glioblastoma – 1 1.4 Data extraction Key study characteristics were extracted from the included articles. A predefined data extraction table was used to document details such as study design and key findings. 1.5 Quality Assessment The quality of the included articles was assessed as follows: 1. The Newcastle-Ottawa Scale (NOS) was used to assess the quality of non-randomized and cohort studies [2]. 2. The risk of bias assessment was used for randomized trials [3]. 1.6 Analysis The analysis proceeded in three phases: Phase 1: Identification of potential topics. Phase 2: Data extraction from relevant articles. 23 Phase 3: Analysis of the relevance of new findings. A hybrid generative and causal method was employed for data analysis and review generation, with OpenAI™ serving as the generative component. This method combines generative modeling with causal analysis, enhancing both the reliability and interpretability of the outcomes by accounting for underlying cause-effect relationships. The approach facilitated the integration of various evidence types into a coherent summary. The review process included summarizing and interpreting findings, as well as discussing the limitations identified in the included articles. Back to 1. Methods 24 Annex 2: Table 1. Signaling Networks and Subtype Transition in Glioblastoma Study ID Length of intervention Population of intervention Intervention Intervention details Primary outcome Secondary outcome Guo S et al. (2025) 72 hours Nude mice aged 6–8 weeks, male and female, subcutaneous implantation of PDX tumor tissue cubes Transfection of DNA constructs to overexpress FOSL1 tagged with GFP Transfection performed at 50–75% cell confluency, lipofectamine 3000 reagent used, RNA extraction at 72 h using TRIzol reagent, overexpression assessed at 48 or 72 h posttransfection, studies conducted in triplicates Role of FOSL1 in regulating biological processes and signaling networks during proneural to mesenchymal subtype transition in glioblastoma, including the identification of upregulated_genes and downregulated_genes. Identification of 8 upregulated and 4 downregulated genes, pathway implications for glioblastoma prognosis Li H et al. (2023) 15 days Patients with GBM histologically diagnosed according to WHO classification, underwent surgical treatment in the Department of Neurosurgery, First Affiliated Hospital of Zhengzhou University Overexpression and knockdown of FTL, overexpression of iPLA2β lentiviruses, anti-PD-1 therapy Lentiviruses constructed by Genechem and Obio Technology, transfected into THP-1-induced macrophages and RAW264.7 macrophages, anti-PD-1 Ab administered intravenously at 10 mg/kg 5 times every 3 days starting day 5 post-tumor injection, cells implanted into right corpus striatum using stereotactic frame at 0.25 μL/min via micro-infusion syringe pump FTL upregulation in TAMs promotes an immunosuppressive tumor microenvironment by inducing M2 polarization and facilitating glioblastoma progression FTL inhibition reprograms the tumor microenvironment, attenuates glioma angiogenesis, promotes T cell recruitment, sensitizes glioma to anti-PD1 therapy de Souza I et al. (2022) 24 hours, 48 hours, 72 hours, 120 hours, 10 days Human glioma cell lines U251MG and T98G Treatment of glioblastoma cells with temozolomide (TMZ), Erastin, RSL3, and Ferrostatin-1 Compounds dissolved in DMSO, applied to cultured cells in varying concentrations (5, 10, 20, 100 μM), durations of 24 h, 48 h, 72 h, 120 h, and 10 days, cells plated in plates of varying sizes (12-well, 24-well, 35-mm dishes, 6-well), washed with PBS, fixed with paraformaldehyde or formaldehyde, stained with crystal violet or specific antibodies, analyzed using flow cytometers, fluorescence measured using GSH/GSSG Ratio Detection Assay Kit NRF2's role in ferroptosis induction and its therapeutic potential in glioblastoma cells Elevated NRF2 expression correlating with chemotherapy resistance, sensitivity to ferroptosis and GSH depletion upon system xcblockage, NRF2 silencing reducing TMZ resistance and ferroptosis sensitivity, ABCC1 silencing increasing TMZ sensitivity and resistance to Erastin, positive correlation of NRF2 and ABCC1 expression with glioma aggressiveness, drug resistance, and poor survival Back to 2.1.1. Signaling Networks and Subtype Transition in Glioblastoma 25 Nie XH et al. (2022) 4 weeks, 24 hours, 72 hours 4-to-5-week-old athymic nude mice in a subcutaneous xenograft tumor model PF treatment (1.0 g/kg/day), RSL3 treatment (100 mg/kg, 2 times/week), transfection with oeNEDD4L, shNEDD4L, oeSTAT3 plasmids Cells cultured in DMEM medium with FBS and penicillin-streptomycin, transfection performed using Lipo2000 reagent, tumor models created via subcutaneous inoculation of U251 cells into athymic nude mice, PF administered daily, RSL3 administered twice weekly, tumor volumes measured every 4 days, biochemical assays and staining performed using manufacturer protocols Antitumor activity of paeoniflorin in glioma cells through ferroptosis induction via upregulation of NEDD4L and repression of Nrf2, GPX4, and STAT3 Enhanced tumor inhibition through combination therapy with PF and RSL3, correlation of low NEDD4L expression with poor glioma prognosis, increased intracellular ROS levels, elevated MDA and Fe2+ levels Song L et al. (2022) 15 days TMZ-resistant GBM cell lines (T98G-R and U118-R) Transfection using Lipofectamine 3,000 reagent and siRNAs targeting FANCD2 Performed according to manufacturer’s protocol, siRNAs sequences provided by Genepharma, cells re-inoculated in six-well plates at 1 × 10^3/well density after 24 h, incubated at 37°C with 5% CO2 for 15 days Up-regulation of FANCD2 correlates with poor prognosis in GBM and promotes TMZ resistance by attenuating ferroptosis Knockdown of FANCD2 increases ROS levels, inhibits cell survival, links to ferroptosis, correlates with immune features and cancerassociated pathways Xiao D et al. (2021) 74 patients diagnosed with grade III or grade IV gliomas, age at diagnosis 21-89 years (median 60) Grouping patients with GBM based on FRGPRS values, analysis of prognosisrelated genes influencing progression-free survival FRGPRS constructed using prognostic gene expression levels and regression coefficients from multivariate Cox proportional hazards regression analysis, univariate Cox regression models and Lasso-logistic regression used to identify prognosisrelated core genes, FRGPRS calculated using risk score formula, relationship between FRGPRS and overall survival evaluated using logrank test Prediction of overall survival and progressionfree survival in GBM patients using the FRGPRS model Associations with immunity, tumor tissue proportions, immune response, chemotherapeutic response, predictive performance during immune checkpoint therapy Zhou L et al. (2021) Patients with glioma, 660 participants Identification of autophagyferroptosis genes, construction of prognostic risk model for overall survival (OS), creation of nomogram based on prognosisrelated genes Genes screened using univariate and lasso regression analysis with p < 0.01, patients divided into high and low expression groups based on median expression levels, nomogram constructed using five prognosisrelated genes and risk score Prognostic risk model for predicting overall survival (OS) in glioma Validation of risk model efficacy through KaplanMeier survival analysis and ROC analysis, confirmation of association with clinical factors, improved accuracy in prognosis prediction using nomogram Sun W et al. (2022) Cases with gene expression profiles and clinicopathological data Construction of predictive model based on ADFRGs to evaluate glioma prognosis Screening AD-FRGs using univariate Cox analysis, predictive model established using LASSO-penalized Cox regression, patients divided into risk groups based on median risk scores, nomogram constructed for prognosis prediction, biological pathway enrichment analyzed using GSEA software, immune response assessed using ssGSEA, CIBERSORT method, and ESTIMATE algorithm, computational analysis delivered via R packages and software tools Prediction of 1-, 3-, and 5-year survival rates in glioma patients using autophagydependent ferroptosisrelated gene signature Correlation of highrisk group with immunosuppressive tumor microenvironment, macrophage infiltration, and immunotolerance Cai Y et al. (2021) 48 hours Patients with glioma, categorized into LGG and HGG Erastin treatment of human glioma cell lines, investigation of subcellular protein distribution and expression Glioma cell lines cultured in DMEM medium with 10% FBS at 37°C with 5% CO2, erastin added after 24 h, CCK-8 assay performed at 24 and 48 h, reagent-to-medium ratio of 1:9, 100 µl solution added per well, incubated for 2 h, immunofluorescence and immunohistochemistry staining performed Prognostic implications of ferroptosisrelated gene prognostic index (FRGPI) in glioma patients, including overall survival prediction, temozolomide sensitivity, and immune checkpoint inhibitor response Correlation of FRGPI with immune cell infiltration, tumor mutational burden, PD-L1 expression, and microsatellite instability 32 Huang L et al. (2022) GBM cell lines U251 and KNS-89 Infection and selection of U251 and KNS89 cell lines with puromycin Infection of cell lines, selection after 48 hours, 2 μg/ml puromycin (cat# A1113803, Thermo Fisher) applied to cell culture Development of a 3-FRLs signature to classify glioma patients into highrisk and low-risk groups with stable prognostic accuracy for overall survival Differences in cellular immunity, immune cell numbers (NK cells, CD4+, CD8+ Tcells, macrophages), immune-related gene expression, somatic mutation rates in glioma prognosis-related genes IDH1 and ATRX, inhibition of ferroptosis after LINC01426 knockdown Zhang X et al. (2022) 24 hours Human glioblastoma cell lines U87MG and U251MG Control solvent Pretreatment of U251MG and U87MG cells with ferroptosis activator erastin (10 μM) Cells cultured in DMEM medium supplemented with 10% FBS, incubated at 5% CO2 and 37°C, migration assays performed using Transwell system (24-well, 8 μm pore size polycarbonate membrane), cells fixed with 4% PFA and stained with crystal violet, migrated cells photographed with light microscope and counted using ImageJ software, experiments repeated more than three times Prognostic value of ferroptosisrelated genes associated with IDH1 status in GBM patients Association with immune-related factors, involvement in p53 signaling, senescence, autophagy, negative regulation of kinase activity, identification of therapeutic drugs Tong S et al. (2022) 24 h, two weeks Patients with gliomas, untreated before surgery Transfection of human U251 and U87 glioblastoma cells using Lip3000 and plasmids, followed by experimental assays including cell viability testing, colony formation, Edu incorporation, ROS detection, lipid peroxidation analysis, wound healing assay, transwell migration assay, and western blot Cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, transfected with 2 μg plasmids using Lip3000 for 6 h, harvested after 24 h, seeded in plates at specific densities, cultured for 24 h to two weeks, stained with paraformaldehyde and crystal violet, Edu diluted in culture medium and incubated for 2 h, fluorescence observed under microscope, cells stained with H2DCFDA dye and C11BODIPY dye, flow cytometry and microscopy used for imaging, transwell assay performed using Matrigel-coated upper wells, cells fixed and stained, western blot conducted using primary and secondary antibodies, membranes visualized with ECL detection kit Positive correlation between IRF2 expression and glioma grade, IRF2 overexpression protects glioma cells from ferroptosis, enhances invasive and migratory abilities IRF2 identified as a potential biomarker for diagnosis and treatment in glioma, novel ferroptosisrelated signature predicts prognosis Feng S et al. (2022) GBM patients receiving anti-PD-1 checkpoint inhibition therapy Evaluation of tumor immune escape and ICB therapy response using computational analysis Consensus clustering using ConcensusClusterPlus tool in R, prognostic model construction via ridge regression, immune infiltration analysis using TIMER, MCP-counter, EPIC, xCell, quanTIseq, CIBERSORT, gene enrichment analysis via GSVA and GSEA, TIDE analysis to assess CTL dysfunction and rejection Association between higher risk scores and worse prognosis in GBM patients, including overall survival, progression-free survival, and disease-specific survival Predictive performance of risk signature for 1-, 3-, and 5-year survival, GBM subtypes, IDH status, and response to ICB treatment Su J et al. (2022) Glioma cell line U87 siRNA transfection targeting SSBP1 using Lipofectamine® RNAiMAX, knockdown of SSBP1 for 72 hours, exposure to TMZ, mitochondrial ROS and MMP detection Transfection performed with Lipofectamine® RNAiMAX Reagent, siRNA sequences specified, mitochondrial ROS detected using MitoSOX™ Red, mitochondrial membrane potential assessed using MitoTracker™ Red, cells cultured at 37°C with 5% CO₂, serum-free medium in upper chambers and 20% FBS in lower chambers, incubation with MitoSOX™ Red and MitoTracker™ Red for 30 minutes, exposure to TMZ for 48 hours, cell viability measured using Cell Counting Kit-8, manufacturer’s instructions followed Prognostic risk score model based on 12 DEMRGs demonstrated excellent performance in predicting GBM patient prognosis Association of risk score with inflammatory response, extracellular matrix, immune pathways, gene mutations, immune cell infiltration, therapeutic potential of SSBP1, SSBP1 knockdown increased TMZ sensitivity by enhancing ferroptosis 33 Wu Y et al. (2025) Patients with GBM tumor tissue samples, 415 participants Control group consisted of normal brain tissue samples from the GTEx database, totaling 1152 samples. Injection of 5 × 10⁵ cells into mice brains, lentivirus transfection of cells, weekly bioluminescence imaging of intracranial tumors Injected using stereotactic device, lentivirus transfection (MOI = 10) with polybrene for 4 h followed by medium replacement after 24 h, transfection efficiency observed after 48 h using luciferase reporter genes, stable transduced lines selected with ampicillin, weekly imaging from day 7 onward, housed in pathogen-free environment at 20°C–25°C and 60%– 65% humidity, polarized macrophages collected after 24 h co-culture, analyzed using flow cytometer Prognostic value of a five-gene risk model in GBM, stratification of patients into highrisk and low-risk groups based on survival outcomes Biological significance of ferroptosis-related genes in tumor progression, therapeutic target identification, correlation analysis between gene expression and immune checkpoints, distinct gene expression patterns between GBM and normal tissues, gene distribution across cell types via scRNA-seq Lv Y et al. (2025) 38 hours, 24 hours, 2 hours, 5 days, 1 week, 24–48 hours Patients with primary and recurrent GBM after chemotherapy, 80 participants Transfection of U251 and LN229 cells with MFAP4 siRNA or siRNA negative control using Lipofectamine 3,000 siRNA mixed with Lipofectamine 3,000 in serum-free medium, added to medium with 10% FBS, incubated for 38 h under standard conditions, cell viability assessed using CCK-8 solution and absorbance at 450 nm, scratches made in confluent cell monolayers with pipette tip, cells imaged at 0, 24, and 48 h, Transwell assay conducted with regulated cell concentration in serum-free medium, incubated for 24–48 h in 37°C 5% CO2 incubator MFAP4 identified as an independent prognostic indicator, correlated with glioma progression and adverse clinicopathological features Significant associations between MFAP4 levels, immune infiltration, ferroptosis, immune checkpoint genes, and survival metrics (OS, DSS, PFI) Clavreul A et al. (2024) Patients aged ≥18 years, newly diagnosed unilateral supratentorial IDH‐ wildtype glioblastoma, underwent tumor resection, received first-line chemoradiotherapy according to the Stupp protocol, sourced from the French GB biobank Subsequent adjuvant chemotherapy with oral TMZ depending on tolerance and radiological response Administered orally, based on tolerance and radiological response, following complete concomitant chemoradiotherapy according to the Stupp protocol Prognostic utility of circulating MDH1 and RNH1 biomarkers for survival in IDHwildtype glioblastoma patients Differential expression of tumor and serum proteins associated with ROS detoxification, identification of therapeutic pathways driven by MDH1, RNH1, and FABP7 Xu Z et al. (2022) 48 h, 24 h, 14 days Glioma patients admitted for operation in Beijing Tiantan Hospital, 325 participants siRNA transfection targeting MXRA8, polarization of macrophages to M1 and M2 phenotypes, TMZ drug treatment at varying concentrations, co-incubation of macrophages and glioma cells in a Transwell plate siRNAs transfected into glioma cells using Lipofectamine 3000 for 48 h, macrophages polarized to M1 using PMA for 6 h and to M2 using IL-4 plus IL-13 for 72 h, TMZ added to medium at concentrations of 0–400 μM for 48 h, macrophages seeded in upper chamber of Transwell plate without serum for 12 h, glioma cells incubated in bottom chamber with 10% FBS for 24 h, infiltrated macrophages fixed in formalin and stained with crystal violet Identification and validation of MXRA8 as a novel prognosis indicator associated with ferroptosis and glioma progression Correlation of MXRA8 with immune infiltration cells, immune score, enrichment in immunity-related pathways, association with unfavorable survivals Back to 2.2.2. Prognostic Markers and Risk Models 34 Annex 6: Table 5. Therapeutic Strategies and Treatment Outcomes Study ID Length of intervention Population of intervention Control Intervention Intervention details Primary outcome Secondary outcome Ye L et al. (2021)24 months Patients with gliomas (lowgrade glioma WHO grade II–III and GBM WHO grade IV), collected at Renmin Hospital of Wuhan University, Wuhan, China, no chemotherapy or radiotherapy before surgery Patients with cerebral hemorrhage, 6 participants, no treatment control group Investigation of CYP2E1 mRNA expression levels in glioma tissues, analysis of its clinical significance, and molecular docking of TCM compounds targeting CYP2E1 RNA extracted using TRIzol reagent, cDNA synthesized using PrimeScript RT Reagent Kit, CYP2E1 mRNA levels detected using SYBR Premix Ex Taq II and Bio-Rad real-time PCR Systems, relative Ct method used for comparison, gene expression analyzed using R packages, molecular docking performed using AutoDock 4.2 and PyMOL software, correlation analysis conducted for immune checkpoints, miRNA prediction performed using MiDRB and TargetScan, functional enrichment analyzed using GO and KEGG pathways Prognostic significance of CYP2E1 expression in glioma patients, correlation with poor prognosis, clinical features, survival outcomes, and statistical significance Involvement of CYP2E1 in lipid metabolism, ferroptosis, tumor immune microenvironment, correlation with methylation levels and copy number variation, miRNA targeting by hsa-miR-527, identification of compounds targeting CYP2E1 Chen Q et al. (2021)45 days U87 and U251 glioblastoma cells, 4-weekold specific pathogen-free male nude mice injected with U87 and U251 cells Incubation of U87 and U251 glioblastoma cells, transfection with NC, hsa-miR-27a-3p angomir, siTMEM161B-AS1, siTMEM161B-AS1 + hsa-miR-27a-3p angomir, migration assay, invasion assay, apoptosis assay, lipid ROS detection, subcutaneous implantation of transfected cells in nude mice Cells incubated at 37°C under 5% CO2 in DMEM containing 10% FBS and 1% penicillinstreptomycin, seeded in 96-well plates, migration assay in Transwell inserts with serum-free DMEM and 10% FBS-DMEM in bottom chamber, invasion assay with Matrigel-coated membrane, apoptosis assay using PI and FITC staining and Caspase-Glo 3/7 reagent, lipid ROS detection using C11 BODIPY dye, subcutaneous injection of 4×105 transfected cells into nude mice, tumor volume measured after 45 days Regulation of malignant biological behavior (proliferation, migration, invasion, apoptosis, ferroptosis) and temozolomide resistance through the TMEM161BAS1-hsa-miR-27a3p-FANCD2/CD44 axis Tumor growth inhibition in nude mice, identification of potential therapeutic targets for glioblastoma Van Loenhout J et al. (2021) 14 days Mice inoculated subcutaneously with 1 × 10⁶ SB28 GBM cells in the shaved abdominal flank Spheroids in untreated PBS, vehicle control AF (0–10 µM) monoor combination treatments with plasma-treated PBS (pPBS) generated by atmospheric pressure plasma jet systems or microsecond-pulsed dielectric barrier discharge (DBD) system Plasma generated using kINPenIND® with argon gas or COST jet with He/5% H2O vapor mixture, plasma applied directly to spheroids or indirectly to 2D cell cultures in PBS, AF administered via oral gavage or pretreatment for 4 h, plasma applicator held above tumors for 10 s, standardized plasma jet parameters (flow rate, gap distance, pulse width, frequency), IncuCyte® system used for reagent applications, mice sedated during DBD treatments Synergistic therapeutic strategy for glioblastoma via combination of oxidative stressinducing treatments, reducing tumor growth kinetics and prolonging survival Decrease in TrxR activity and GSH levels, intracellular ROS accumulation, apoptosis and ferroptosis induction, increase in danger signals and dendritic cell maturation, inhibited phagocytotic capacity of dendritic cells 35 Moujalled D et al. (2022) 5 days, 48 hours, 24 hours Six GBM cell lines, including U251 and SNB19; 6-week-old female NOD/SCID/γc-/- mice with intracranially implanted U251-Ch-Luc GBM tumors Mice gavaged with vehicle, health status: after detectable engraftment of tumour cells Treatment with Temozolomide, JQ1, A1331852, ferroptosis/apoptosis inducing and preventing compounds, gavage of BCL-XL inhibitor A1331852 Cells treated in RPMI1640 medium supplemented with FBS, penicillin, streptomycin, and DMSO, compounds added at specified concentrations, cells treated for 48 hours and harvested by trypsinisation, mice gavaged daily for 5 consecutive days with A1331852 formulated in Phosal 50 PG, polyethylene glycol 400, ethanol, and DMSO, flow cytometry performed using Annexin V and DAPI staining, apoptosis and viability evaluated using MTT assay Enhanced apoptosis and cell killing observed in GBM cell lines with dual targeting of prosurvival proteins BCL-XL and MCL-1 using BH3 mimetic drugs compared to conventional therapies Synergistic cell killing with ferroptosis inducers and BH3 mimetic drugs, activation of apoptosis markers caspase-3 and PARP1 cleavage, dependence on intrinsic apoptosis executioners BAX and BAK Su IC et al. (2023)3 weeks Female BALB/C nude mice aged 5–6 weeks, injected with tumor xenografts (GBM cells) shScramble xenografts treated with vehicle, 5 participants, no treatment control group Induction of ferroptosis using IKE administered intraperitoneally, generation of TMZresistant U87MGR cells through increasing TMZ doses IKE administered intraperitoneally at 25 mg/kg/day for 3 weeks, TMZ-resistant cells maintained with 100 µM TMZ, cells cultured in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum and penicillin/streptomycin, subcutaneous injection of Matrigel mixture containing 1 × 10⁶ scrambled or shSOD2 U87MG-R cells, mitochondrial ROS production analyzed using MitoSOX red, immunofluorescence staining performed CYBB orchestrated mesenchymal shift and promoted TMZ resistance by modulating the Nrf2/SOD2 axis, reducing erastinmediated ferroptosis sensitivity Upregulation of CYBB and SOD2 in mesenchymal GBM subtype, association of CYBB with poor clinical outcomes, protective role of SOD2 against erastintriggered ferroptosis in TMZresistant GBM cells Zhou Y et al. (2023)21 days Male nude mice, 5 weeks old, injected with U87 glioma cells, treated with myrislignan U87-injected nude mice, DMSO-treated control group Stereotactic implantation of U87 glioma cells into the striatum, intraperitoneal injection of myrislignan (5 mg/kg) every 3 days U87 cells suspended in 3μL PBS, stereotactically implanted under isoflurane anesthesia, myrislignan administered intraperitoneally starting 7 days posttumor injection, injections repeated every 3 days for 21 days Suppression of glioblastoma growth through EMTmediated ferroptosis in a Slug-dependent manner Inhibition of NF-κB signaling, induction of ferroptosis via Slug-SLC7A11 pathway, suppression of glioblastoma progression in xenograft mouse model Liu B et al. (2022)9 days 6 weeks old C57 mice, GBM-bearing Normal saline group Use of Fe3O4-siPDL1@M-BV2 nanoparticles for PD-L1 silencing and ferroptosis induction Fe3O4-siPD-L1@MBV2 nanoparticles, siPD-L1, IFN-γ, Fer-1, DFO, APC-CD11c antibody, FITC-CD80 antibody, PE-CD86 antibody, delivered via tail vein injections every three days (4 times total) and serum-free DMEM for 48 hours, enzymelinked immune analyzer and flow cytometer used, standardized concentrations (Fe3O4: 10–200 μg/mL; IFN-γ: 10 ng/mL; Fer-1: 10 μM; DFO: 100 μM), incubation at 37 ℃ Inhibition of orthotopic drugresistant GBM growth, prolonged survival time, increased siPD-L1 and Fe2+ accumulation, decreased PD-L1 protein expression, enhanced ferroptosis, immune reactivation Altered invasion-related protein expressions, ferroptosis-related protein expressions, increased effector T cell to regulatory T cell ratio, increased M1/M2 microglia ratio, maturation of dendritic cells 36 Jiang Y et al. (2022)15 days Patients diagnosed with grade IV glioblastoma, underwent surgery at Shanghai Tenth People’s Hospital Lentiviral-based infections to overexpress or knock down molecules in glioma stem cells Lentiviral vectors constructed by GeneChem, RNAi-mediated lentivirus vectors, treated with necrostatin-1, Z-VADFMK, 3-MA, ferrostatin-1, RSL, selection with 10 μg/ml puromycin for 15 days, reagents delivered at specific concentrations and incubation times, stereotaxic apparatus used for orthotopic injections into mouse brains Inhibition of glioblastoma stem cell viability, proliferation, neurosphere formation, stemness, and tumorigenesis via ferroptosis induction Potential biomarker for glioblastoma, therapeutic target for molecular or ferroptosis-dependent therapies Xia L et al. (2022)24-72 hours Human glioma U251 and U87 cell lines, female BALB/c nude mice, age 4 weeks No treatment control group Treatment of human glioma U251 and U87 cell lines with apatinib Apatinib transfection at 50% confluence, plasmid transfection at 70% confluence using Lipofectamine 2000, cells seeded at densities of 5 × 10^3 cells/well (96-well plate) or 1 × 10^5 cells/mL (6-well plate), treated for 24/48/72 hours, staining with PI solution and fixation with ethanol, flow cytometry performed, biochemical assays conducted using detection kits (ROS, MDA, GSH, LDH, iron), protein analysis with BCA Protein Assay Kit and SDSPAGE, membranes incubated with antibodies, gliomas fixed in paraformaldehyde and dehydrated with ethanol Restraint of glioma cell proliferation through induction of ferroptosis via inhibition of VEGFR2/Nrf2/Keap1 pathway. Primary outcome of the study. Counteraction of ferroptosis induction by overexpression of Nrf2 Zhang K et al. (2023) 2 weeks Human GBM and brain contusion tissue samples, BALB/c nude mice, 4 weeks old Culturing human GBM and brain contusion tissues, assessing proliferation and mortality Cells cultured in DMEM with 10% FBS, seeded in 96-well plates at 5000 cells/well, proliferation tested using CCK8 reagent at 24, 48, 72, and 96 h, mortality assessed with lactate dehydrogenase cytotoxicity assay, JC1 staining for fluorescence analysis, lentivirus-encoded SHG-140 cells injected into mice skulls, tumor size recorded using IVIS imaging system on days 7, 14, 21, and 28, brains fixed with 4% paraformaldehyde and analyzed with HE/IHC staining Promotion of ferroptosis induction and retardation of tumor growth rate in GBM cells via HSP27 deficiency Reduction in tumor growth capacity in intracranial xenograft models Liang X et al. (2024) 1 hour, 48 hours, 24 hours, 14 days T98G human glioblastoma cell line, RBMS1 expression reduced using sh-RBMS1 constructs Negative control group (sh-NC) Use of ferroptosis inhibitor Fer-1 (1 µM) to treat T98G cells Transfected using Lipofectamine 2000 reagent at 37 °C for 48 h, inoculated into 6-well plates at specified densities, incubated in serumfree medium precoated with Matrigel, exposed to C11BODIPY probe, washed with pre-cold PBS, homogenized with iron assay buffer, centrifuged at 16,000×g for 15 min at 4 °C Inhibition of GBM cell proliferation and promotion of apoptosis through RBMS1 silence, possibly mediated by ferroptosis Inhibition of migration, invasion, EMT process; promotion of ferroptosis 37 Carvalho SM et al. (2023) 7 days U87 glioblastoma cells, treated with MION, Co40-MION, and DOX at concentrations of 0.6, 6, and 60 μg mL−1 Negative control group (DMEM with 10% FBS and sterile polypropylene chips), positive control group (DMEM with 10% FBS and TBHP) Synthesis and application of magnetic iron oxide nanoparticles (MION) and cobaltdoped nanoparticles (Co40-MION) Synthesized using coprecipitation method with NH4OH in alkaline conditions, iron salts dissolved in preheated CMC solution, heated in nitrogen atmosphere, dialyzed for purification, applied to U87 cells and spheroids, spheroids treated on Day 0 and Day 3, reagents include FeSO4, FeCl3, cobalt salts, phosphate-citrate buffer, hydrogen peroxide, DMPO spin trap, MTT reagent, SDS solution, acetic acid, treatments lasting up to 24 hours Reduction in glioblastoma tumor spheroid volume and enhanced cytotoxicity through cobalt-doped iron oxide nanoparticles Comparison of nanozyme efficacy with doxorubicin, concentration-dependent cytotoxicity, role of hydroxyl radicals in anticancer activity Miki K et al. (2023) Cell lines U87MG, U373, and KNS1451, KNS1451 classified as aggressive mesenchymal glioblastoma Culturing glioblastoma cell lines under controlled conditions with specific additives and hypoxic environment DMEM with glucose, CAP, 2-DG, DFO, SS, metformin, DMEM/Ham’s F12 for stem-like cells, 6-well dishes, medium replaced every 3 days, hypoxic conditions (1% O2, 5% CO2) using personal CO2 multigas incubator and Gas Cylinder Auto Changer, Coulter counter, TC 20 automated cell counter with trypan blue, Seahorse XF24 Flux analyzer and microplates, cells seeded in triplicate, cultured for 7 days, sphere size and number evaluated on the 7th day, OCR measured after 16 h incubation Significant inhibition of glioblastoma cell growth with combined CAP and 2-DG treatment under normal glucose conditions Validation of effectiveness in hypoxic conditions, identification of ferroptosis as a potential mechanism Wang H et al. (2023)21 days Human glioblastoma cells, U251, U87, KNS89 Infection of glioblastoma cells with GFP and GFP+NeuroD4 viruses, stereotactic injection into nude mice Lentiviral vectors and packaging plasmids transfected into HEK293T cells, infected glioblastoma cells injected into mice, neuronal induction medium (DMEM, F12, neurobasal, N2, B27, forskolin, dorsomorphin) used for cultivation, medium changed every other day, virus media collected and filtered at 24 and 48 hours, supplemented with polybrene, tumor growth evaluated every 7 days, EdU administered for 2 hours, PI dye used for cell cycle analysis, dihydroethidium applied 30 minutes before flow cytometry Reprogramming glioblastoma cells into neuron-like cells induced terminal differentiation, inhibited proliferation, and prolonged survival in tumor-bearing mice Smaller tumor sizes in NeuroD4-infected xenografts, reduced SLC7A11 and GPX4 expression, ferrostatin-1 blocking NeuroD4-mediated reprogramming Liu Y et al. (2022)2 weeks Patients with glioma (astrocytoma, IDH wildtype, grades I-IV), 35 participants Negative control group Transfection of glioma cell lines U251 and LN229 with small interference RNA targeting KAT6B and STAT3 Transfected using Lipofectamine 2000 reagent, maintained in DMEM medium with 10% FBS and 1% penicillin/streptomycin, incubated overnight post-transfection, assays performed at indicated time points, lipid ROS measured by flow cytometry, iron levels measured using Iron Assay Kit, ChIP assay performed using Pierce Magnetic ChIP Kit Contribution of KAT6B to glioma progression by repressing ferroptosis via epigenetic induction of STAT3 Regulation of glioma cell viability, apoptosis, lipid ROS, iron levels, epigenetic mechanisms involving H3K23ac and RNA polymerase II enrichment on STAT3 promoter 38 Wang C et al. (2023)24-120 hours Glioma patients undergoing surgery, grades 2, 3, and 4 gliomas, 60 participants Normal brain tissue specimens from GBM patients, 10 specimens Experimental processing of glioma stem cells (GSCs) GSCs isolated and cultured in StemFlex™ medium at 37℃ with 5% CO2 atmosphere for 2 weeks until tumor spheres formed, treated with necrostatin-1, Z-VADFMK, 3-MA, ferrostatin-1, transfected with lentiviral vectors synthesized by GeneChem, injected into mice brains using stereotactic apparatus at density of 5 × 10⁴ cells in 5 μL solution, processed using RNA/protein assay kits and detection kits, tumor volume calculated using formula V = (D × d²) / 2 CircRNF10 upregulation in glioblastoma promotes ferroptosis defense and tumorigenic efficacy CircRNF10 silencing extends survival rates, establishes circRNF10/ZBTB48/IGF2BP3 feedback loop remodeling iron metabolism Yang YH et al. (2024) 12 days Female BALB/c nude mice, xenograft tumor model with U87 cells, 24 participants Female BALB/c nude mice, placebo control group (DMSO), 6 participants Administration of S670 via irrigation Administered via irrigation, once daily for 12 days, tumor volume calculated using V = 0.5 × l × w2, Ki67, LC3, and LAMP2 expression analyzed in tumor tissues Significant inhibition of tumor growth, dose-dependent inhibition of GBM cell proliferation (IC50 value for GBM cell proliferation inhibition), induction of ferroptosis via ROS generation ROS-mediated Nrf2 activation, TFEB nuclear translocation, autophagosome and lysosome biogenesis, impaired autophagosomelysosome fusion, autophagy flux inhibition, STX17 suppression D'Aprile S et al. (2024) 24-48 hours Patients diagnosed with mesenchymal or proneural GBM subtypes, 153 participants Vehicletreated cells Exposure of cells to FAC and erastin FAC prepared as 50 mM stock solution in PBS, erastin prepared as 2 mM stock solution in DMSO, cells seeded in 96-well plates, 24-well plates, and T-75 flasks at specified densities, FAC exposure at concentrations of 5– 100 µM for 24–48 h, erastin exposure at 1– 20 µM for 24–48 h, FAC exposure at 100 µM for 6 h or 24 h, cells collected at 30, 90, or 180 min, use of blocking buffer for membranes, incubation with primary antibodies overnight at 4 °C Differential response to ferroptosis induction between mesenchymal and proneural GBM subtypes Up-regulation of antioxidant defense mechanisms in mesenchymal GBM subtypes Moses A et al. (2025) 12 h Male and female Sprague Dawley rats, healthy and glioma-bearing, 15 participants Vehicle control group Ferroptosis induction using erastin2 and RSL3 Delivered through cell seeding in plates and intravenous administration in rats, monitored using Incucyte SX1 live-cell analysis instrument and PET/CT scanner, BODIPY C11 dye at 1.5 μM, erastin2 and RSL3 at specified concentrations, [18F]hGTS13 administered intravenously (~20 MBq), IKE prepared in 10% DMSO/50% PEG-400/40% saline and administered at 25 mg/kg 48 h post- [18F]hGTS13 Monitoring ferroptosis sensitivity and engagement of system xctargeted therapies using [18F]hGTS13 Distinction between sensitive and resistant cell lines, association of [18F]hGTS13 uptake with cellular glutathione content 39 Dumitru CA et al. (2023) 10 days Adult patients with newly diagnosed IDH wild type GBM Transfected cells with control plasmid Transfection of GBM cell lines with shRNA clone Transfection in antibiotics-free medium using PANFect A-plus reagent, selection with Puromycin at specified concentrations, conditioned supernatants prepared by culturing GBM cells for 24 h at 37°C, neutrophils isolated from EDTAanticoagulated blood using Pancoll and purified by sedimentation and osmotic shock, MTT assay performed with 4 h incubation, agarose-based culture setup for 10– 11 days, invasion assay using ORISTM system with rat tail collagen I matrix, neutrophil chemotaxis assessed using transwell inserts with 3 µm pores after 3 h incubation, MMP9 release analyzed by gelatine zymography, IL-8 levels measured using ELISA kit PGRMC1 levels significantly predicted poor overall survival and promoted proliferation, anchorageindependent growth, invasion, and progression of GBM Increased susceptibility of GBM cells to ferroptosis inducer erastin, decreased susceptibility to temozolomide, enhanced tumor-related inflammation, promoted recruitment of neutrophils, correlation of PGRMC1 levels with ITGB1 expression Stringer BW et al. (2023) 5 days, 7 days, 24 hours Patients with GBM undergoing surgery at Flinders Medical Centre or the Royal Adelaide Hospital, South Australia Cells without radiation, 6 replicates Drug treatments with TMZ and TFP, ionizing radiation applied to GBM cells TMZ dissolved in DMSO and diluted in cell medium, TFP dissolved in DPBS and diluted in cell medium, GBM cells seeded in 384-well CellCarrier Ultra imaging plates, treated with 2 Gy radiation daily for 5 days, TMZ treatment for 7 days at 0, 25, and 100 μM, TFP treatment for 24 hours at 0, 4, 7, and 10 μM, half-medium changes every 48 hours, neuronal cultures matured for >100 days, MEA recordings conducted using Maestro Pro system on Lumos MEA plates coated with poly-lornithine and laminin CSF induces therapeutic resistance in GBM cells through NUPR1-mediated ferroptosis inhibition, trifluoperazine enhances killing of resistant GBM cells Improved survival outcomes for GBM patients suggested by combining trifluoperazine with standard care Qu S et al. (2023) 72 h, 24 h, 2 weeks Orthotopic xenograft nude mice implanted with human GBM cells Combination treatment of ABX and TMZ to suppress GBM progression and prolong survival Delivered via intracranial injection using Hamilton syringe, conventional skin disinfection and skin suture closure performed, tumor growth monitored using imaging facility, treatments administered for 24– 72 h, organoid medium refreshed every 48 h Suppression of GBM progression (Primary Outcome) and prolonged survival (Secondary Outcome 1) in orthotopic xenograft nude mice through combination treatment of ABX and TMZ (Secondary Outcome 2, Secondary Outcome 3) Induction of sustained DNA damage, enhancement of ferroptosis, increased drug sensitivity confirmed through GBM PDOs models 40 Williams CH et al. (2024) 72 hours, 3 days Glioblastoma cell lines (13 tested), neurosphere lines, PDX models Standard nontargeting siRNA, transfection control plasmid Blocking GPR68 signaling using small molecule inhibitor OGM and genetic means Cells reverse transfected with siRNA, sgRNA, or plasmids using lipofectamine RNAiMAX or lipofectamine 3000, OGM compounds added to media at indicated concentrations, incubation under standard cell culture conditions for 72 hours, exposure to DMSO, OGM, or Erastin, addition of 2.5 µM Liperfluo resuspended in DMSO for 1-hour incubation, cells plated in 96-well plates, 12-well plates, or 100 mm cell culture dishes Robust cell death induced in glioblastoma cells by blocking GPR68 signaling, irrespective of genetic heterogeneity or temozolomide resistance Non-toxicity of OGM to zebrafish, selective sparing of non-malignant neural cells Li X et al. (2024) 24 hours, 12 hours followed by 24 hours, 8 days GBM cells including U251 and U87MG cell lines, GBM#4 cells isolated from primary surgical GBM biopsy specimens from patients treated at Renji Hospital, Shanghai, China Treatment of U251 and U87MG cells with erastin, RSL3, DMSO, DFO, NAC, 3-TYP, FerroOrange, FerroGreen, MitoFerroGreen, DCFHDA, MitoSOX solutions, injection of 3-TYP and RSL3 into tumor-bearing mice Cells treated in 96well plates with prepared solutions, incubated at 37°C in 5% CO2, solutions freshly prepared before treatments, cells washed with serum-free DMEM or PBS before treatments, compounds injected into tumors every 2 days for 8 days SIRT3 inhibition sensitized GBM cells to RSL3-induced ferroptosis both in vitro and in vivo Downregulation of SLC7A11, activation of mitophagy pathway, accumulation of ferrous iron and ROS in mitochondria Deng L et al. (2024) 1-2 weeks, 2 days, 24 hours Nude mice with subcutaneous injection of U87 IGF2BP3-KD cells Subcutaneous injection of U87 control cells IGF2BP3 knockdown in glioma cells, lentiviral infection, xenograft tumor model establishment, coculture of human microglia and glioma cells Lentivirus transfection using pLKO.1 vector, filtered through 0.45 μm filter, polybrene added for infection, puromycin selection for 2 days, xenograft model with subcutaneous injection into nude mice, tumor monitoring every two days, co-culture in 6well plates for 3 days, confocal microscopy used for phagocytosis analysis IGF2BP3 knockdown impairs glioma cell growth, survival, and tumor formation Regulation of ferroptosis via GPX4 mRNA binding, stabilization of GPX4 mRNA through m6A modification, increased susceptibility of glioma cells to microglial phagocytosis Guo F et al. (2024) 17 days, 15 days + 24 hours + 24 hours LN229 and T98G human glioblastoma cell lines, immunodeficient mice (6 total: 3 females, 3 males, age 6 weeks). Placebo control group (6 mice: 3 females, 3 males). Juglone treatment alone or combined with inhibitor applied to LN229 and T98G glioblastoma cell lines and xenograft tumor models. Cells inoculated into 96-well and 6-well plates, juglone medium maintained for 24 h with pretreatment using inhibitor for 1 h, medium replaced with normal growth medium, cells fixed with 4% polyformaldehyde and stained with crystal violet solution, total protein extracted using RIPA buffer with protease inhibitors, protein analyzed via SDS-PAGE and NC membrane transfer, primary and secondary antibodies used for detection, intracellular ROS measured with DCFDA incubation and flow cytometry, xenograft tumor model created using LN229 cell suspension injected into nude mice, treatment administered via abdominal injections every other day Induction of ferroptosis and inhibition of glioblastoma growth by juglone Activation of p38MAPK phosphorylation, negative regulation of Nrf2-GPX4 signaling pathway 41