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

2. Results

2.1. Ferroptosis and Immune Modulation: Mechanisms and Therapeutic Strategies in Glioblastoma

2.1.1. 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 immune-based 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

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 (−5.65-fold), 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) 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 lipid-related 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 autophagy-dependent 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, Two-Way 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

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.

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/HIF-1α 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 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

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 3- and 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 high- and 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 FRG-related risk scores are strongly correlated with glioma prognosis, effectively categorizing patients into high- and 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).

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. Up-regulation 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 short- and 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 high- and 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 high-risk 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 5-year 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 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

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 by increasing Teff cells (23.1%, p < 0.01), decreasing Treg cells (5.29%, p < 0.01), and enhancing dendritic cell maturation (CD11c+CD80+CD86+ cells: up to 68.2% in vitro and 77.31% with IFN-γ, p < 0.01). Additionally, the nanoparticles facilitate M1 microglial polarization (highest M1/M2 ratio observed under magnetic field, p < 0.01), contributing to prolonged survival (median survival: 45.00 days in Fe3O4-siPD-L1@M-BV2+magnet group, p < 0.01) and safety without organ damage or abnormal serum markers.

Complementing nanoparticle-based strategies, CircLRFN5 has been shown to induce ferroptosis and suppress glioblastoma stem cell viability, offering additional therapeutic potential. Jiang Y et al. (2022) CircLRFN5 suppresses glioblastoma stem cell viability, proliferation, neurosphere formation, and tumorigenesis by inducing ferroptosis through PRRX2 degradation and GCH1 downregulation. The study demonstrates statistically significant effects (p < 0.05, p < 0.01, p < 0.001) in both in vitro and in vivo models, validating its role as a tumor suppressor.

Building on the role of ferroptosis in glioblastoma suppression, apatinib is presented as another agent targeting distinct pathways to induce this cell death mechanism. Apatinib significantly reduces glioma cell viability and tumor growth by inducing ferroptosis through the VEGFR2/Nrf2/Keap1 pathway, as evidenced by reduced tumor volume and weight, increased levels of ROS, MDA, and Fe, and decreased GSH, GPX4, and SLC7A11 (p < 0.05; p < 0.01), as reported by Xia L et al. (2022). Overexpression of Nrf2 counteracts these effects in glioblastoma U251 and U87 cells, restoring cell viability most significantly at 72 hours (p < 0.01), reversing changes in ferroptosis markers, and normalizing VEGFR2/Nrf2/Keap1 pathway components (p < 0.05 or p < 0.01).

Therapeutic strategies targeting ferroptosis are further examined, with HSP27 silencing shown to enhance ferroptosis and improve survival outcomes. Silencing HSP27 significantly increases ferroptosis in glioblastoma cells, as evidenced by elevated ROS production mediated by Fe2+ accumulation and enhanced ACSL4 stability (Zhang K et al. 2023). This results in reduced intracranial tumor growth rates and improved survival times in xenograft models, with statistical significance indicated across multiple metrics (p < 0.05 to p < 0.0001). Additionally, the knockdown correlates with reduced Ki67 staining intensity and increased ferroptosis markers such as Fe2+ and MDA levels.

Ferroptosis modulation continues to be pivotal in glioblastoma research, with RBMS1 silencing highlighting its impact on tumor progression and epithelial-mesenchymal transition. Liang X et al. (2024) RBMS1 silencing significantly inhibits glioblastoma cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT), while promoting apoptosis through ferroptosis pathways. This is demonstrated by marked increases in TBARS production (p < 0.001), lipid ROS levels (p < 0.001), and total iron levels (p < 0.001), alongside decreased expression of MMP2, MMP9, N-cadherin, Vimentin, and Snail, and increased E-cadherin levels (p < 0.01, p < 0.001). Treatment with the ferroptosis inhibitor Fer-1 partially reverses these effects (p < 0.01 and p < 0.001), indicating a protective role of RBMS1 in glioblastoma progression via ferroptosis modulation.

Expanding the scope of ferroptosis-based therapies, cobalt-doped iron oxide nanoparticles demonstrate promising anticancer effects through oxidative stress induction. Cobalt-doped iron oxide nanoparticles (Co40-MION) exhibit a significant therapeutic effect by reducing glioblastoma tumor spheroid volumes by approximately 40% at 60 µg/mL and achieving enhanced cytotoxicity with an EC-50 of 0.2 µg/mL (p < 0.0001) (Carvalho SM et al. 2023). Their anticancer activity is mediated by hydroxyl radical-induced oxidative stress, triggering ferroptosis and apoptosis pathways, and demonstrating comparable efficacy to doxorubicin in both 2D and 3D in vitro models. Building on the role of ferroptosis in glioblastoma treatment, alternative therapeutic approaches such as combination treatments have also demonstrated efficacy in targeting tumor growth and cell death pathways. Combined treatment with chloramphenicol (CAP) and 2-deoxy-d-glucose (2-DG) significantly inhibits glioblastoma cell growth under normal glucose conditions (1000 mg/L) and hypoxic conditions (O2 = 1%), as evidenced by statistical significance (p < 0.001, p < 0.0001) (Miki K et al. 2023). The mechanism involves ferroptosis, supported by increased expression of markers PTGS2, CHAC1, HO-1, and FTH1, and the inhibition of cell death through deferoxamine (DFO).

Extending the focus on ferroptosis, neuronal reprogramming has emerged as another promising strategy for glioblastoma treatment, offering insights into cellular differentiation and survival mechanisms. Wang H et al. (2023) found that NeuroD4 overexpression induces neuronal reprogramming in glioblastoma cells, leading to terminal differentiation, a significant reduction in proliferation (EdU+ cells decreased from 38.6% to 2.1% in U251 cells and from 37.4% to 1.5% in KNS89 cells at 14 dpi, Ki67 protein nearly absent, p < 0.001), and prolonged survival in tumor-bearing mice (up to 60 days, p < 0.001). This process is closely tied to ferroptosis, as evidenced by reduced SLC7A11 and GPX4 expression, with ferrostatin-1 blocking the reprogramming. Quantitative data further reveal TUJ1+ rates decrease from 87.6% to 18.4% and 13.6%, and Ki67+ rates increase from 13.3% to 48.1% and 30.3% upon SLC7A11 and GPX4 overexpression, demonstrating inhibition of NeuroD4-induced reprogramming (p < 0.001).

The modulation of ferroptosis pathways is further highlighted by evidence of epigenetic regulation, which impacts glioblastoma cell survival and resistance. KAT6B overexpression epigenetically activates STAT3 expression, enhancing ferroptosis resistance in glioma cells by modulating cell viability, apoptosis, lipid ROS, and iron levels, with statistically significant effects (p < 0.01) (Liu Y et al. 2022). Depletion of STAT3 reverses these changes, confirming its critical role in the pathway.

Expanding on the role of prognostic markers, circRNF10 highlights ferroptosis regulation as a key mechanism in glioblastoma progression and therapeutic intervention. Wang C et al. (2023) CircRNF10 upregulation in glioblastoma enhances tumorigenic efficacy and ferroptosis defense by stabilizing ZBTB48, transcriptionally upregulating HSPB1, and forming a circRNF10/ZBTB48/IGF2BP3 positive feedback loop, which significantly correlates with poor prognosis (p < 0.05, p < 0.01, p < 0.001). Silencing circRNF10 significantly extends survival rates (Kaplan-Meier analysis, p < 0.05, p < 0.01, p < 0.001), disrupts the circRNF10/ZBTB48/IGF2BP3 feedback loop, remodels iron metabolism via HSPB1, induces ferroptosis in glioblastoma stem cells, and reduces tumor burden in vivo.

Building on the exploration of ferroptosis mechanisms, the role of S670 in inducing ferroptosis and modulating autophagy offers further insights into glioblastoma treatment strategies. Yang YH et al. (2024) S670 demonstrates dose-dependent inhibition of glioblastoma cell proliferation, with IC50 values of 5.961 μM at 24 hours for U87 cells and 8.023 μM at 24 hours for U251 cells. It induces ferroptosis through mitochondrial ROS generation, lipid peroxidation, and GPX4 inhibition, as evidenced by increased COX2 expression. In vivo studies show significant tumor growth suppression in a U87 mouse xenograft model, with tumor weight reduction at doses of 25 mg/kg and 50 mg/kg (p < 0.01, p < 0.0001). Additionally, S670 modulates autophagy by promoting Nrf2 activation and TFEB nuclear translocation while impairing autophagosome-lysosome fusion via STX17 suppression, exacerbating ROS accumulation and enhancing ferroptosis.

The variability in ferroptosis susceptibility among glioblastoma subtypes underscores the importance of understanding subtype-specific resistance mechanisms. D'Aprile S et al. (2024) Mesenchymal glioblastoma subtype exhibits significantly enhanced resistance to ferroptosis induction compared to the proneural subtype, characterized by elevated basal GSH levels (7.43 ± 0.39 nmol vs. 5.72 ± 0.12 nmol, p = 0.0141), 260-fold upregulation of GPX4 mRNA post-FAC treatment, and increased expression of GSS (4.90 ± 0.29 vs. 4.35 ± 0.44, p = 0.0017). These antioxidant mechanisms correlate with reduced overall survival (357 vs. 455 days, p = 0.0308) and progression-free interval (164 vs. 311 days, p = 0.0027) in glioblastoma patients.

Advancing the study of ferroptosis sensitivity, imaging biomarkers such as [18F]hGTS13 provide valuable tools for monitoring therapeutic responses. [18F]hGTS13 demonstrates significant differential uptake between ferroptosis-sensitive and resistant cell lines, with HT-1080 and C6 cells exhibiting lower baseline uptake (3.0 ± 0.6 and 5.8 ± 0.6 % uptake/mg protein) and glutathione levels (4.2 ± 0.04 μM and 1.8 ± 0.05 μM) compared to resistant H460 cells (82.3 ± 0.6 % uptake/mg protein, 6.4 ± 0.6 μM, p < 0.0001) (Moses A et al. 2025). Additionally, its application in glioma-bearing rat models shows a reduced tumor-to-brain ratio following imidazole ketone erastin treatment (mean TBR: 10.5 ± 1.8 to 5.9 ± 2.2, p = 0.006), supporting its utility in tracking drug engagement and ferroptosis sensitivity. Uptake correlates strongly with cellular glutathione content across cell lines (HT-1080: r = 0.91, H460: r = 0.97, C6: r = 0.97), affirming its role as a biomarker for ferroptosis-related therapeutic strategies.

Integrating molecular markers like PGRMC1 highlights their impact on therapy resistance and susceptibility to ferroptosis induction. Dumitru CA et al. (2023) PGRMC1 expression in glioblastoma (GBM) patients is significantly associated with poor overall survival (Hannover cohort: p = 0.010; Magdeburg cohort: p = 0.005) and serves as an independent prognostic biomarker (Hannover cohort: HR = 1.532, CI [95%] = 1.042–2.253, p = 0.030; Magdeburg cohort: HR = 1.462, CI = 1.039–2.057, p = 0.029). PGRMC1 enhances tumor progression by promoting proliferation, anchorage-independent growth, invasion, and immune interactions, with increased neutrophil recruitment (p < 0.001, Rho = 0.363) and elevated IL-8 production. It modulates therapy response by reducing susceptibility to temozolomide (TMZ) and increasing sensitivity to the ferroptosis inducer erastin (p < 0.05), underscoring its dual role in therapy resistance and ferroptosis susceptibility.

Investigating the modulation of ferroptosis by NUPR1 extends the understanding of therapeutic resistance and potential intervention strategies in glioblastoma. CSF induces therapeutic resistance in GBM cells through NUPR1-mediated ferroptosis inhibition, as described by Stringer BW et al. (2023), with trifluoperazine demonstrating a significant reduction in resistance when combined with standard care treatments such as TMZ and irradiation. In highly responsive GBM cell lines, resistance was reduced by 100%, while less responsive cell lines showed a mean reduction of 35% (p < 0.05). Effective doses of trifluoperazine exhibited IC50 values ranging from 4–9 μM, and preclinical neuronal models confirmed the absence of neurotoxicity at these doses (p < 0.05).

Building on the therapeutic relevance of ferroptosis, combination therapies such as ABX and TMZ have demonstrated enhanced induction of this mechanism, improving treatment outcomes in glioblastoma models. Combination therapy of ABX and TMZ demonstrated significant suppression of GBM progression and prolonged survival in orthotopic xenograft nude mice (Qu S et al. (2023); Kaplan–Meier survival analysis, p < 0.05). Concurrent administration showed superior efficacy compared to sequential treatment, with enhanced DNA damage, persistent γ-H2AX levels, and impeded DNA repair mechanisms. The therapy induced ferroptosis, marked by increased iron accumulation, lipid ROS, reduced glutathione levels, and altered GPX4/HO-1 expression. It improved drug sensitivity in GBM patient-derived organoids, achieving an 80% response rate, with 37.5% showing a >2-fold inhibition rate improvement.

Selective ferroptosis induction, as demonstrated by GPR68 inhibition with OGM, introduces a promising approach to target glioblastoma cells while sparing normal tissues. Blocking GPR68 signaling with OGM induces ferroptotic cell death in glioblastoma cells with LC50 values ranging from 0.42 to 2.7 µM across 13 GBM cell lines (p < 0.01), irrespective of genetic heterogeneity or temozolomide resistance (Williams CH et al. 2024). This mechanism involves activation of the ATF4-CHAC1 pathway, leading to lipid peroxidation and glutathione depletion, selectively targeting glioblastoma cells without affecting normal tissues, such as HEK293 cells (p < 0.0001). OGM demonstrates non-toxicity in zebrafish larvae, sparing neurons and glial cells while effectively inducing ferroptosis in glioblastoma cells (p > 0.05).

Building on mechanisms that induce ferroptosis, SIRT3 inhibition emerges as another pathway to enhance lipid peroxidation and mitochondrial oxidative stress in glioblastoma cells. SIRT3 inhibition significantly enhances ferroptosis in glioblastoma cells by reducing SLC7A11 expression (p < 0.0001), cystine uptake, and GSH levels, while promoting lipid peroxidation, mitochondrial ROS, and Fe²⁺ accumulation (Li X et al. 2024). This results in suppressed tumor growth and elevated ferroptosis markers, including MDA accumulation and decreased GSH/GSSG ratio (p < 0.05, p < 0.01, p < 0.001, p < 0.0001). Additionally, SIRT3 targeting activates mitophagy (p < 0.0001), further supporting its role as a potential therapeutic target for glioblastoma treatment.

Extending the focus on ferroptosis, the knockdown of IGF2BP3 highlights the pivotal role of GPX4 regulation in glioblastoma cell survival and tumor formation. Recent findings from Deng L et al. (2024) demonstrate that the knockdown of IGF2BP3 significantly impairs glioma cell growth, survival, and tumor formation by inducing ferroptosis through reduced GPX4 protein expression. This process triggers apoptosis, oxidative damage, and cell cycle disruption. Xenograft assays validate the reduction in tumor formation, with statistical significance (p < 0.001, p < 0.01, p < 0.05). Furthermore, IGF2BP3 regulates ferroptosis by directly binding to the m6A-modified A575 site on GPX4 mRNA, stabilizing its expression and facilitating its translation into GPX4 protein. This knockdown increases lipid peroxidation, induces ferroptosis, and enhances glioma cell susceptibility to microglial phagocytosis, aligning with established roles of GPX4 in ferroptosis and glioma treatment strategies.

Further exploring GPX4 suppression, Juglone demonstrates its ability to induce ferroptosis through oxidative stress pathways, offering another therapeutic avenue for glioblastoma treatment. Guo F et al. (2024) Juglone induces ferroptosis in glioblastoma cells through activation of p38 MAPK phosphorylation and suppression of the Nrf2/GPX4 axis, resulting in significant reductions in tumor volume and weight, p < 0.05; p < 0.001, alongside decreased expression of Nrf2 and GPX4, lower GSH levels, and increased oxidative stress markers such as ROS and MDA. These findings highlight the role of ferroptosis-related pathways in inhibiting glioblastoma growth and enhancing oxidative stress in vitro and in vivo.

Transitioning from ferroptosis-related therapeutic strategies, NOX4 overexpression underscores its contribution to tumor growth and invasiveness in recurrent glioblastomas. NOX4 overexpression (p < 0.01) drives ferroptosis in endothelial cells, significantly contributing to tumor growth and invasiveness in recurrent glioblastomas (Liang B et al. 2024). Experimental knockdown of NOX4 inhibited ferroptosis and glioblastoma cell proliferation (p < 0.001), with reduced growth rates in recurrent tumors linked to decreased ferroptosis activity and increased GPX4 and SLC7A11 expression.

Complementing molecular approaches, Ce6@Cu nanoparticles introduce a novel ferroptosis-inducing strategy through targeted ROS amplification and GPX4 inhibition in glioblastoma. Carrier-free Ce6@Cu nanoparticles, activated by ultrasound irradiation, induce ferroptosis and cuproptosis in glioblastoma by depleting GSH, amplifying ROS generation, and reducing GPX4 expression, mechanisms consistent with tumor suppression processes observed in glioblastoma treatments (Zhu Y et al. 2024). These nanoparticles, with a particle size of ≈50 nm and a Cu:Ce6 molar ratio of ≈2.5:1, demonstrated significant tumor accumulation (enhanced fluorescence signal, p < 0.001) and increased Cu+ concentration within glioblastoma lesions (ICP-MS data, p < 0.001), contributing to substantial tumor suppression and improved survival rates in a glioblastoma mouse model (p < 0.001).

Building on the mechanisms of ferroptosis and ROS amplification, the inhibition of GPR68 further demonstrates the therapeutic potential of targeting glioblastoma cells by reducing tumor burden and enhancing lipid peroxidation. Inhibition of GPR68 in glioblastoma cells significantly reduces tumor burden and induces ferroptosis in zebrafish xenografts, as demonstrated by decreased tumor area, fluorescence intensity, and intensity/area ratio (U87: p < 0.0005; U138: p < 0.0005) (Neitzel LR et al. 2024). Treatment with OGM or shRNA-mediated knockdown further enhances lipid peroxidation and reduces tumor burden (p < 0.025), with minimal neurotoxicity observed after a 24-hour recovery period.

The induction of ferroptosis through lipid peroxidation and ROS elevation in glioblastoma cells is similarly observed with DE-FeONPs nanocomplex, which effectively targets glioblastoma stem cells and their resistant variants. Abu-Serie MM et al. (2024) DE-FeONPs nanocomplex exhibits potent inhibition of glioblastoma stem cells (GSCs) and their radioresistant variants (GSCs-RR), achieving up to 6.6-fold improvement in chemosensitivity and 8.7-fold improvement in radiosensitivity (p < 0.001). It suppresses self-renewal and cancer repopulation, as evidenced by a 64.2% reduction in sphere count, and downregulates stemness gene expression by 2–13-fold (p < 0.001). The nanocomplex induces ferroptosis through lipid peroxidation increase (2.7–5.0-fold; p < 0.001) and elevated ROS levels (up to 2.9-fold; p < 0.001), while demonstrating optimal blood-brain barrier penetration (LogP = 2.105).

Complementing the strategies of ferroptosis induction, targeting MS4A4A highlights the role of the immune microenvironment in glioblastoma therapy by modulating macrophage infiltration and enhancing T-cell activation. Targeting MS4A4A in glioblastoma enhances ferroptosis in tumor-associated macrophages, reduces M2 macrophage infiltration, and increases CD8+ T-cell activation, aligning with findings from Shao G et al. (2024) on the role of ferroptosis in modulating the tumor immune microenvironment. High MS4A4A expression is significantly associated with poor prognosis (log-rank p = 0.04), while its inhibition improves PD-1 immunotherapy efficacy, alleviates T-cell exhaustion, and delays tumor growth, as demonstrated by experimental data and survival analysis.

Extending the focus on ferroptosis as a therapeutic mechanism, Erianin demonstrates efficacy in overcoming TMZ resistance in glioma stem cells by targeting molecular pathways associated with cell viability and tumor suppression. Mansuer M et al. (2024) Erianin enhances TMZ sensitivity in glioma stem cells resistant to TMZ by inducing ferroptosis through REST downregulation and promoting SLC40A1 ubiquitination and degradation. This mechanism significantly reduces cell viability, proliferation, neurosphere formation, and tumor size, with effects observed in a time- and concentration-dependent manner (IC50 established at 24 hours). In vivo studies confirm its ability to suppress stemness markers, reduce tumor formation, and improve survival, demonstrating statistical significance (p < 0.05; p < 0.01; p < 0.001).

Integrating the concept of ferroptosis inhibition, FOXP3 emerges as a critical regulator of glioblastoma progression, with therapeutic approaches such as epirubicin showing promise in reversing its effects on tumor growth. FOXP3 has been identified as a key regulator of glioblastoma progression by inhibiting ferroptosis through the linc00857/miR-1290/GPX4 axis (Cao W et al. 2024). The transcriptional promotion of GPX4 and linc00857 by FOXP3 enhances glioblastoma cell proliferation and tumor growth. Knockdown of FOXP3 significantly induces ferroptosis and reduces glioblastoma proliferation both in vitro and in vivo (p < 0.01). Furthermore, treatment with the FOXP3 inhibitor epirubicin demonstrated notable suppression of glioblastoma growth and colony formation (p < 0.01) by increasing ROS, MDA, and iron levels, while reducing GSH and GPX activity (p < 0.01; p < 0.001). In vivo, epirubicin treatment significantly reduced tumor growth and weight (p < 0.01) while modulating key molecular markers, including downregulation of FOXP3, KI67, PCNA, linc00857, and GPX4, alongside upregulation of miR-1290.

Building upon the molecular mechanisms of glioblastoma progression, necrosis and ferroptosis-related pathways have emerged as critical factors influencing tumor development and survival outcomes. Recent studies, including Lu T et al. (2024), demonstrate that myeloperoxidase inhibition using 4-ABAH reduces necrosis formation by 33% and prolongs mouse survival by 12.5% (p < 0.05). Similarly, Vps34 depletion decreases necrosis by 43% and 57% while extending survival by 31% and 44% (p < 0.05), highlighting the role of LAP and ferroptosis-related mechanisms in necrosis development and glioblastoma progression.

Advancing from prognostic insights, innovative therapeutic strategies leveraging oxidative stress and ferroptosis demonstrate promising anti-tumor efficacy in glioblastoma. Chen H et al. (2025) Lpo@Cu₂Se-GOx nanocomposites exhibit potent anti-tumor effects in glioblastoma by inducing oxidative stress, generating reactive oxygen species (ROS), and triggering ferroptosis and immunogenic cell death (ICD). Key quantitative findings include H₂O₂ generation exceeding 350 µM, significant intracellular ROS levels, and dose-dependent reductions in GL261 cell viability, alongside ferroptosis markers such as decreased GSH and GPX4 activity (p < 0.05, p < 0.01, p < 0.001). Immunogenic effects were evidenced by ~26.9% dendritic cell maturation, 2.2-fold higher CD8+ T-cell infiltration, and reduced Treg cell levels to 4.2%. In vivo studies demonstrated significant tumor growth inhibition and immune activation, with negligible toxicity, while efficient blood-brain barrier penetration and immunogenic cell death mechanisms further underscore their therapeutic potential.

Expanding on therapeutic approaches, genetic modulation of ferroptosis sensitivity highlights the interplay between molecular regulation and glioblastoma progression. Knockdown of ALKBH5 significantly reduces U251 glioblastoma cell proliferation, invasion, and tumor growth both in vitro and in vivo (p < 0.01) while enhancing ferroptotic sensitivity through regulation of MUC1 expression (Jamali AW et al. 2025). In vivo results show decreased tumor growth over 21 days and reduced Ki-67 expression in tumor tissues. Conversely, ALKBH5 overexpression markedly increases glioblastoma cell proliferation, invasion, tumor growth, and MUC1 expression (p < 0.01). The inhibitory effects of ALKBH5 knockdown are reversed by MUC1 overexpression (p < 0.01, p < 0.01), demonstrating a synergistic relationship between ALKBH5 and MUC1 in glioblastoma progression.

Further exploration of therapeutic strategies reveals the potential of enhancing radiosensitivity through ferroptosis and DNA damage mechanisms in glioblastoma treatment. RSL3 enhances glioma radiosensitivity to ionizing radiation by promoting DNA double-strand breaks, evidenced by increased γ-H2AX foci, longer comet tails, and elevated p-ATM levels (Wang X et al. 2024). Synergy scores of 25.53, 33.45, and 29.36 were observed for U87, U251, and LN229 cells, respectively, p < 0.05 or p < 0.01. Additionally, RSL3 suppresses epithelial-mesenchymal transition by downregulating N-cadherin and vimentin while upregulating E-cadherin, p < 0.05. In vitro and in vivo studies demonstrated significant tumor growth reduction, with synergy scores consistent across cell lines and substantial tumor volume and weight decrease, p < 0.05, without notable side effects.

Ferroptosis emerges as another key mechanism influencing glioma progression and therapeutic sensitivity, complementing the radiosensitizing effects of RSL3. Liu T et al. (2022) found that ferroptosis emerges as a key programmed cell death mechanism in gliomas, with high ferroptosis scores significantly correlating with reduced survival and poor prognosis (p < 0.0001). Alterations in ferroptosis-related genes are linked to glioma progression, with 15% of these genes independently associated with poor survival (hazard ratio > 1, p < 0.05). Inhibition of ferroptosis enhances immunological profiles and sensitizes glioblastoma to immune checkpoint blockade therapy, as demonstrated by improved survival outcomes in GBM murine models (log-rank test, p < 0.05) and increased CD8+ T-cell infiltration, macrophage polarization, and T-cell activation.

The identification of genes associated with ferroptosis and radiosensitivity further elucidates their roles in glioma prognosis and survival prediction. The study identified seven radiosensitivity- and ferroptosis-associated genes (MAPK1, ZEB1, MAP1LC3A, HSPB1, CA9, STAT3, and TNFAIP3) as prognostic biomarkers in glioma, validated using TCGA and CGGA datasets (Xie Y et al. 2022). A risk signature model based on these genes demonstrated significant survival prediction capabilities with AUC values of 0.705, 0.764, and 0.775 at 1, 2, and 3 years, respectively (p < 0.001). High-risk groups showed shorter survival (HR = 3.43, p = 0.004 in TCGA; HR = 1.44, p < 0.001 in CGGA) and distinct immune cell infiltration patterns, including higher infiltration of macrophages, Tregs, DCs, and TILs. Experimental evidence confirmed that erastin-induced ferroptosis enhances radiosensitivity in glioma cells, with statistically significant results (p < 0.01, p < 0.001).

MicroRNA-mediated ferroptosis modulation highlights additional therapeutic avenues for enhancing glioblastoma treatment efficacy. The miR-147a mimic induces ferroptosis in glioblastoma cells by targeting SLC40A1, leading to iron accumulation, mitochondrial dysfunction, and increased lipid peroxidation (Xu P et al. 2022). This process reduces cell viability and enhances sensitivity to chemotherapy, as demonstrated by elevated LDH release and statistically significant outcomes (N=6, p < 0.05). Conversely, the miR-147a inhibitor mitigates ferroptosis and improves cell viability under erastin or RSL3 treatment by reducing ROS generation, lipid peroxidation, and intracellular iron levels, with consistent statistical significance (N=6, p < 0.05).

Building on the molecular mechanisms of ferroptosis and chemotherapeutic sensitivity, SQLE emerges as another key regulator of glioblastoma progression and treatment response. Yao L et al. (2022) SQLE modulates temozolomide (TMZ) sensitivity and suppresses metastasis in glioblastoma multiforme (GBM) by inhibiting ERK phosphorylation, with low expression correlating with poor prognosis (p < 0.05). Its expression is associated with WHO grade and 1p/19q co-deletion, while negatively correlating with tumor-infiltrating lymphocytes, immunomodulators, and MHC molecules (Spearman rho = −0.472, p = 1.97e−10). Overexpression enhances TMZ sensitivity, reduces migration and invasion, and influences immune responses, emphasizing its role in overcoming chemoresistance.

Continuing the focus on therapeutic resistance, genome-wide screening identifies GSS as a pivotal factor in radiotherapy outcomes and potential intervention strategies. Genome-wide CRISPR screening highlighted GSS as a key factor in radiotherapy resistance (Liu X et al. 2023), with targeted disruption using Ang/TAT-sgGSS-EVs achieving mutation frequencies of 61.8% in LN229-bearing mice and 57.2% in patient-derived glioma stem cell xenografts (p < 0.0001). Elevated GSS expression was significantly associated with poor prognosis in glioblastoma patients (p < 0.0001). The therapeutic approach enhanced radiosensitivity, suppressed tumor growth, and improved survival outcomes, demonstrating minimal off-target effects (<0.5%) and no significant toxicity.

Highlighting advancements in treatment efficacy, combination therapies such as temozolomide with FR054 leverage ferroptosis mechanisms to overcome therapeutic resistance. Combination therapy of TMZ and FR054 significantly enhanced therapeutic outcomes in glioblastoma models, as evidenced by reduced tumor fluorescence intensity (p < 0.001) and prolonged survival in orthotopic xenograft mice (Ye R et al. 2025). In GBM cells, FR054 increased TMZ sensitivity by reducing protein O-GlcNAcylation and inducing ferroptosis, as demonstrated by HMOX1 upregulation, GPX4 downregulation, and substantial reductions in TMZ IC50 values, such as A172-TR TMZ IC50 decreasing from 1132 μM to 40 μM (p < 0.001). Minimal side effects were observed across in vivo and organoid models.

Expanding on ferroptosis-based therapies, FLD nanoparticles offer a promising approach to glioblastoma treatment through enhanced tumor targeting and biosafety. Liu S et al. (2025) FLD nanoparticles effectively traverse the blood-brain barrier (PBBB = 3.72 ± 1.34 × 10⁻⁶ cm/s) and accumulate at glioblastoma sites, amplifying ferroptosis through lipid peroxidation, GSH depletion (p < 0.001), Fe²⁺ elevation, and ROS generation. In vitro, FLD NPs demonstrated a 2.8-fold reduction in IC50 compared to FL NPs, while in vivo studies confirmed significant tumor suppression (p < 0.001) with confirmed biosafety and negligible adverse effects. Enhanced ferroptosis mechanisms include HO-1 upregulation, photothermal acceleration of the Fenton reaction, and improved tumor targeting via multimodal imaging techniques (PAI/PTI), supported by renal and hepatobiliary clearance pathways.

Building on nanoparticle-based ferroptosis strategies, alternative approaches such as high-dose ascorbate also exploit iron-dependent cytotoxicity to target glioblastoma cells. High-dose ascorbate (≥1 mM) induces significant ferroptosis-like cytotoxicity in glioblastoma cells, reducing viability to below 20% within 24 hours (p ≤ 0.001) and increasing intracellular ROS levels, as reported by Piotrowsky A et al. (2024). Ferroptosis markers, including decreased GPX4 and increased TfR1 expression, confirm the mechanism, while cell death remains caspase-3-independent. Pre-incubation with 100 µM FeCl3 amplifies the cytotoxic effects, reducing viability to 63% at 0.4 mM ascorbate and further elevating ROS levels compared to ascorbate alone (p ≤ 0.001). DFO inhibition (p ≤ 0.01) supports the iron-dependent nature of cell death, distinguishing it from apoptosis or autophagy pathways.

The results emphasize the interactions between ferroptosis pathways and glioblastoma resistance mechanisms, offering valuable insights into the dynamics of the immune system in this context.

2.3. CD95 Gene Deletion: Reducing Malignancy in Glioblastoma Cells

Table 6. CD95 Gene Deletion: Reducing Malignancy in Glioblastoma Cells

The deletion of the CD95 gene has been associated with reduced malignancy in glioblastoma cells, offering a promising avenue for therapeutic intervention. This genetic alteration mitigates the pro-tumorigenic effects of CD95 signaling, which are known to enhance cell survival, migration, immune evasion, and resistance to ferroptosis in glioblastoma. Emerging evidence suggests that targeting CD95 may disrupt key pathways driving glioblastoma progression, including inflammation and cellular survival mechanisms. These findings provide critical insights into therapeutic strategies aimed at diminishing tumor aggressiveness while addressing the challenges posed by the dual role of CD95 signaling in cancer biology.

Previous studies highlighted the paradoxical role of CD95 signaling in glioblastoma, contributing to tumor survival, immune regulation, and resistance to apoptosis, while therapeutic inhibition of CD95 signaling demonstrated clinical promise, with the CD95 inhibitor APG101 improving progression-free survival in patients [114, 115, 116, 117, 118, 119]. DISE-based approaches also emerged as a selective strategy for inducing cancer cell death, though challenges such as specificity and safety remain [120]. Despite these advances, the complexity of CD95 interactions with other pathways and limitations of exogenous CD95 agonists complicate therapeutic development [120]. Recent findings suggest that CD95 gene deletion reduces clonogenic growth, sphere-forming capacity, and invasiveness of glioblastoma cells independently of CD95 ligand, underscoring the need for further exploration of ligand-independent mechanisms.

Building upon the therapeutic implications of CD95 deletion, experimental studies have explored its impact on glioblastoma-initiating cells and tumor progression. Quijano-Rubio C et al. (2022) demonstrated that CD95 deletion in glioblastoma-initiating cells reduces resistance to CD95L-induced apoptosis, significantly decreases clonogenic growth (p < 0.05), sphere-forming capacity (p < 0.05), and invasiveness (p < 0.0001) in vitro, independent of CD95L expression. Despite these promising in vitro findings, survival outcomes in xenograft models showed no significant differences among groups, with median survival times in the S-24 model recorded as 193, 209, and 202 days, and in the ZH-161 model as 18.5, 20, and 20 days (p > 0.05). These results highlight the role of CD95 signaling in glioblastoma biology, emphasizing its influence on malignancy traits such as clonogenic growth, sphere formation, invasiveness, and apoptosis resistance.

2.4. Iron Oxide Nanoparticles and Paclitaxel: Inducing Ferroptosis via Autophagic Pathways in Glioblastoma

Table 7. Iron Oxide Nanoparticles and Paclitaxel: Inducing Ferroptosis via Autophagic Pathways in Glioblastoma

The induction of ferroptosis in glioblastoma through autophagic pathways has been explored using iron oxide nanoparticles and paclitaxel, highlighting their synergistic potential as therapeutic agents. Iron oxide nanoparticles facilitate intracellular iron accumulation, while paclitaxel enhances autophagy, collectively triggering ferroptosis. This approach leverages the unique properties of nanoparticles to enhance drug delivery and promote ferroptotic cell death, addressing the challenges of tumor resistance and recurrence. By integrating nanotechnology with chemotherapeutic strategies, this novel mechanism underscores advancements in targeted glioblastoma treatment, offering a promising avenue to overcome resistance and improve therapeutic efficacy.

Previous studies identified lipid peroxidation, iron metabolism disruption, and GPX4 inactivation as key mechanisms driving ferroptosis in glioblastoma, with nano-drug delivery systems emerging as promising therapeutic strategies by exploiting tumor vulnerabilities such as high iron accumulation and lipid synthesis [122, 123, 124, 125]. Advanced nanoparticles, including FeGd-HN@LF/RGD2 and GOD-Fe3O4@DMSNs, demonstrated potential for overcoming the blood-brain barrier and inducing ferroptosis effectively, though limitations such as biological safety concerns, tumor heterogeneity, and inefficient peroxide production under weakly acidic conditions remain unresolved [126]. Recent findings suggest iron oxide nanoparticles synergize with paclitaxel to induce ferroptosis through autophagic pathways, highlighting the potential of combining ferroptosis-based strategies with novel delivery systems to improve therapeutic efficacy.

Building on the concept of ferroptosis induction via nanotechnology, experimental studies have elucidated the cellular and molecular mechanisms underlying its therapeutic impact in glioblastoma. IONP@PTX significantly inhibits glioblastoma cell viability (64.24±0.53% in NCI-H446 cells; 70.90±2.68% in M059K cells, p < 0.001) and induces ferroptosis by increasing ROS, lipid peroxidation, and intracellular iron levels (p < 0.05), as reported by Nie Q et al. (2023). These effects are mediated through autophagy-related pathways, marked by altered expression of proteins such as Beclin 1, LC3-II/I, and HDAC6 (p < 0.05), and are further amplified by rapamycin. This research highlights the synergistic interaction between iron oxide nanoparticles and paclitaxel in 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

The investigation into ferroptosis and immune modulation in glioblastoma has yielded significant insights, particularly regarding the interplay of various molecular players such as LIP, DMT1, NCOA4, ROS, lipid peroxidation, GPX4, GSH, xCT, and NRF2. A pivotal study by Guo et al. (2025) established that overexpression of FOSL1 is linked to altered gene expression associated with ferroptosis and glioblastoma progression, highlighting the potential for targeted therapeutic strategies. The correlation between ferritin light chain (FTL) upregulation and immunosuppression, as demonstrated by Li et al. (2023), further underscores the relevance of labile iron in promoting tumor growth. This study uniquely emphasizes macrophage-mediated iron dynamics, suggesting that targeting these interactions could enhance the efficacy of anti-PD1 therapies and potentially improve survival rates in glioblastoma patients.

The role of DMT1 as a critical regulator of iron uptake has been reinforced through various studies, which collectively underscore its importance in mediating ferroptosis, particularly in the context of temozolomide (TMZ) treatment. While the recent literature does not directly address DMT1, it elucidates related mechanisms such as GPX4 regulation and the differential sensitivity of glioblastoma cell lines to TMZ, as noted by de Souza et al. (2022). This variability in TMZ sensitivity may influence DMT1-mediated ferroptosis, necessitating further exploration of its regulatory pathways. NCOA4's function in ferritinophagy has emerged as a crucial mechanism for promoting ferroptosis, with recent studies validating its role in iron release and autophagy. The findings by Buccarelli et al. (2018) and de Souza et al. (2022) highlight the interplay between lipid peroxidation and GPX4 inhibition, reinforcing the established connection between these pathways in glioblastoma. Moreover, the identification of FTL as a prognostic marker by Li et al. (2023) adds a new dimension to understanding the immune modulation in ferroptosis, suggesting that therapeutic strategies targeting NCOA4 may also influence immune responses.

Reactive oxygen species (ROS) have been recognized as key mediators in inducing ferroptosis, with studies revealing their complex role in glioblastoma cell death. The work by Kyani et al. (2018) emphasizes the interaction between autophagy and ferroptosis, aligning with earlier findings that link ROS accumulation to ferroptotic cell death. However, the variability in ROS modulation across different therapeutic interventions, as highlighted by Chen et al. (2015), indicates the need for a nuanced understanding of ROS dynamics in glioblastoma treatment strategies. Lipid peroxidation remains a hallmark of ferroptosis, driven by iron-dependent oxidative degradation of polyunsaturated fatty acids. Recent studies have reinforced the significance of GPX4 in suppressing lipid peroxidation, with deficiencies leading to increased cell death. The findings by Buccarelli et al. (2018) and de Souza et al. (2022) further elucidate the relationship between lipid peroxidation and ferroptosis sensitivity, suggesting that high MRP1 expression in TMZ-resistant glioblastoma cells may present a potential target for therapeutic intervention.

The role of GPX4 as a critical regulator of ferroptosis has been consistently supported, with evidence linking its inhibition to enhanced cell death in glioblastoma. The studies by Buccarelli et al. (2018) and de Souza et al. (2022) have expanded on this by exploring the interaction between GPX4 and NRF2, revealing that NRF2 overexpression can confer resistance to ferroptosis through elevated GSH levels. This highlights the complexity of ferroptosis regulation and the potential for targeting these pathways to improve therapeutic outcomes. Glutathione (GSH) has been identified as a vital antioxidant that maintains redox balance and prevents lipid peroxidation, thereby regulating ferroptosis sensitivity. The new studies corroborate earlier findings that GSH depletion exacerbates ferroptosis, with interventions aimed at preserving GSH levels showing promise in enhancing treatment efficacy. Notably, de Souza et al. (2022) introduced a novel approach by quantifying GSH levels in specific glioblastoma cell lines, providing deeper insights into cell-specific antioxidant mechanisms.

The cystine/glutamate antiporter xCT has emerged as a key regulator of ferroptosis sensitivity, with recent studies reinforcing its role in cysteine uptake and glutathione synthesis. Kyani et al. (2018) and de Souza et al. (2022) highlight the importance of xCT in modulating ferroptosis, while also suggesting that compensatory mechanisms, such as transsulfuration, may confer resistance to ferroptosis-inducing agents. This finding emphasizes the need for comprehensive strategies that target multiple pathways to overcome adaptive resistance in glioblastoma. Finally, NRF2 continues to be a critical player in modulating redox balance and ferroptosis resistance. While recent studies reaffirm its role in antioxidant responses, further exploration into its therapeutic modulation is necessary to enhance our understanding of its impact on glioblastoma treatment outcomes. Overall, the integration of these findings underscores the complexity of ferroptosis in glioblastoma and the necessity for multi-targeted therapeutic approaches to improve treatment efficacy against this challenging malignancy.

3.2. Ferroptosis in Glioblastoma: Pathways, Immune Interactions, and Overcoming Resistance

In recent investigations into ferroptosis mechanisms within glioblastoma, we observe a multifaceted interplay of various molecular regulators that significantly influences therapeutic outcomes. Central to this discussion is Glutathione Peroxidase 4 (GPX4), an antioxidant enzyme crucial for maintaining cellular redox balance. Recent studies reaffirm GPX4's pivotal role in inhibiting lipid peroxidation and its dependency on glutathione, thus highlighting its importance in glioblastoma resistance to ferroptosis inducers (Battaglia AM et al. (2022), Costa I et al. (2023), Chen X et al. (2021)). Evidence from cohort and animal studies further supports the notion that GPX4 downregulation correlates with tumor recurrence and enhances sensitivity to ferroptosis (Kram H et al. (2022), Xia L et al. (2022)). This aligns with earlier findings suggesting that GPX4 inhibition sensitizes glioblastoma cells to ferroptosis (Zhao H et al. (2017), Yi R et al. (2020)). However, the emerging perspective on GPX4-independent mechanisms indicates a more complex regulatory landscape, with studies suggesting that GPX4's role may be indispensable, particularly in the context of resistance to ferroptosis inducers (Wang H et al. (2023), Minami JK et al. (2023)).

Reactive oxygen species (ROS) are also critical mediators in the ferroptotic process, as they accumulate during ferroptosis and lead to oxidative stress and lipid peroxidation. Recent studies have elucidated the relationship between mitochondrial dysfunction, ROS generation, and ferroptosis regulation, reinforcing the notion that mitochondrial ROS production exacerbates ferroptosis sensitivity (Chen Y et al. (2019), Tong S et al. (2022), Su J et al. (2022)). Furthermore, the role of key regulators such as NRF2 and SLC7A11 has been emphasized, with new evidence suggesting that NRF2 activation may be context-dependent, influenced by cellular environments and p53 status (Yuan F et al. 2022). This nuanced understanding challenges previous assertions of NRF2's uniformly protective role against ferroptosis, indicating the need for tailored therapeutic strategies.

Lipid peroxidation, a hallmark of ferroptosis, has also garnered attention in recent studies. The involvement of Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) in lipid peroxidation has been reaffirmed, with new findings indicating that ACSL4 upregulation in recurrent glioblastoma tumors correlates with enhanced lipid peroxidation (Kram H et al. 2022). This highlights the progressive role of lipid peroxidation in ferroptosis and suggests potential therapeutic targets within the PERK-ATF4-HSPA5-GPX4 feedback pathway (Chen Y et al. 2019). However, the regulation of lipid peroxidation under hypoxic conditions remains contentious, as hypoxia-induced SLC7A11 upregulation has been shown to suppress ferroptosis, indicating a complex regulatory environment that necessitates context-specific therapeutic strategies.

Iron levels, particularly the dynamics of intracellular free iron, play a crucial role in ferroptosis. Recent research underscores the significance of NCOA4-mediated ferritinophagy in regulating iron availability and enhancing ferroptosis sensitivity (Chen Q et al. (2021), Zhang K et al. (2023)). While earlier studies emphasized iron accumulation as a driver of ferroptosis, newer findings illustrate the dual role of iron, with specific genetic and molecular interventions altering iron dynamics and ferroptosis sensitivity (Nie XH et al. (2022), Xu P et al. (2022)). The complexity of iron metabolism and its interaction with the tumor microenvironment necessitates further exploration to optimize therapeutic approaches targeting iron-mediated ferroptosis.

SLC7A11, a cystine/glutamate antiporter, has emerged as a significant regulator of ferroptosis resistance in glioblastoma. New findings indicate that SLC7A11 downregulation in certain glioblastoma subtypes may promote ferroptosis, contrasting its previously established role as a resistance factor (Hu Y et al. 2021). Additionally, therapeutic strategies targeting SLC7A11, such as PX-478 combined with SAS, have shown promising results in enhancing ferroptosis sensitivity (Sun S et al. 2022). These findings underscore the potential of SLC7A11 as a therapeutic target while revealing the complexity of its regulation and expression patterns. Moreover, OTUB1's role in stabilizing SLC7A11 and its implications for ferroptosis resistance have been further elucidated, with new studies confirming its influence on lipid peroxidation (Sun S et al. 2022). However, the observed discrepancies in OTUB1 expression across different glioblastoma contexts highlight the need for precise characterization of its role in tumor biology (Hu Y et al. (2021), Yang Y et al. (2024)).

In summary, the integration of these findings advances our understanding of ferroptosis in glioblastoma, emphasizing the intricate interactions between GPX4, ROS, lipid peroxidation, iron levels, SLC7A11, ACSL4, OTUB1, and NCOA4-mediated ferritinophagy. These insights not only reinforce previous conclusions but also introduce new therapeutic avenues aimed at overcoming resistance and enhancing treatment efficacy in glioblastoma.

3.3. CD95 Gene Deletion: Reducing Malignancy in Glioblastoma Cells

The deletion of the CD95 gene in glioblastoma cells is pivotal in reducing malignancy by impairing tumor-promoting mechanisms such as immune evasion, resistance to 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

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 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 context-dependent 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 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. By addressing these gaps, future studies can pave the way for more effective therapies that leverage ferroptosis in glioblastoma management.