Prognostic relevance of high expression of kynurenine pathway markers in glioblastoma.

Glioblastoma (GBM) continues to exhibit a discouraging survival rate despite extensive research into new treatments. One factor contributing to its poor prognosis is the tumor's immunosuppressive microenvironment, in which the kynurenine pathway (KP) plays a significant role. This study aimed to explore how KP impacts the survival of newly diagnosed GBM patients. We examined tissue samples from 108 GBM patients to assess the expression levels of key KP markers-tryptophan 2,3-dioxygenase (TDO2), indoleamine 2,3-dioxygenase (IDO1/2), and the aryl hydrocarbon receptor (AhR). Using immunohistochemistry and QuPath software, three tumor cores were analyzed per patient to evaluate KP marker expression. Kaplan-Meier survival analysis and stepwise multivariate Cox regression were used to determine the effect of these markers on patient survival. Results showed that patients with high expression of TDO2, IDO1/2, and AhR had significantly shorter survival times. This finding held true even when controlling for other known prognostic variables, with a hazard ratio of 3.393 for IDO1, 2.775 for IDO2, 1.891 for TDO2, and 1.902 for AhR. We suggest that KP markers could serve as useful tools for patient stratification, potentially guiding future immunomodulating trials and personalized treatment approaches for GBM patients.

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

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

MicroRNAs as the pivotal regulators of Temozolomide resistance in glioblastoma.

Glioblastoma (GBM) is an aggressive nervous system tumor with a poor prognosis. Although, surgery, radiation therapy, and chemotherapy are the current standard protocol for GBM patients, there is still a poor prognosis in these patients. Temozolomide (TMZ) as a first-line therapeutic agent in GBM can easily cross from the blood-brain barrier to inhibit tumor cell proliferation. However, there is a high rate of TMZ resistance in GBM patients. Since, there are limited therapeutic choices for GBM patients who develop TMZ resistance; it is required to clarify the molecular mechanisms of chemo resistance to introduce the novel therapeutic targets. MicroRNAs (miRNAs) regulate chemo resistance through regulation of drug metabolism, absorption, DNA repair, apoptosis, and cell cycle. In the present review we discussed the role of miRNAs in TMZ response of GBM cells. It has been reported that miRNAs mainly induced TMZ sensitivity by regulation of signaling pathways and autophagy in GBM cells. Therefore, miRNAs can be used as the reliable diagnostic/prognostic markers in GBM patients. They can also be used as the therapeutic targets to improve the TMZ response in GBM cells.

Positive feedback loop of c-myc/XTP6/NDH2/NF-κB to promote malignant progression in glioblastoma.

BACKGROUND: Recent studies have highlighted the significant role of the NF-κB signaling pathway in the initiation and progression of cancer. Furthermore, long noncoding RNAs (lncRNAs) have been identified as pivotal regulators in sustaining the NF-κB signaling pathway's functionality. Despite these findings, the underlying molecular mechanisms through which lncRNAs influence the NF-κB pathway remain largely unexplored. METHODS: Bioinformatic analyses were utilized to investigate the differential expression and prognostic significance of XTP6. The functional roles of XTP6 were further elucidated through both in vitro and in vivo experimental approaches. To estimate the interaction between XTP6 and NDH2, RNA pulldown and RNA Immunoprecipitation (RIP) assays were conducted. The connection between XTP6 and the IκBα promoter was examined using Chromatin Isolation by RNA Purification (ChIRP) assays. Additionally, Chromatin Immunoprecipitation (ChIP) assays were implemented to analyze the binding affinity of c-myc to the XTP6 promoter, providing insights into the regulatory mechanisms at play. RESULTS: XTP6 was remarkedly upregulated in glioblastoma multiforme (GBM) tissues and was connected with adverse prognosis in GBM patients. Our investigations revealed that XTP6 can facilitate the malignant progression of GBM both in vitro and in vivo. Additionally, XTP6 downregulated IκBα expression by recruiting NDH2 to the IκBα promoter, which resulted in elevated levels of H3K27me3, thereby reducing the transcriptional activity of IκBα. Moreover, the progression of GBM was further driven by the c-myc-mediated upregulation of XTP6, establishing a positive feedback loop with IκBα that perpetuated the activation of the NF-κB signaling pathway. Notably, the application of an inhibitor targeting the NF-κB signaling pathway effectively inhibited the continuous activation induced by XTP6, leading to a significant reduction in tumor formation in vivo. CONCLUSION: The results reveal that XTP6 unveils an innovative epigenetic mechanism instrumental in the sustained activation of the NF-κB signaling pathway, suggesting a promising therapeutic target for the treatment of GBM.

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

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

Evaluating soluble Axl as a biomarker for glioblastoma: A pilot study.

With current imaging, discriminating tumor progression from treatment effect following immunotherapy or oncolytic virotherapy of glioblastoma (GBM) is challenging. A blood based diagnostic biomarker would therefore be helpful. Axl is a receptor tyrosine kinase that is highly expressed by many cancers including GBM. Axl expression is regulated through enzymatic cleavage of its extracellular domain. The resulting fragment can be detected in serum as soluble Axl (sAxl). sAxl levels can distinguish patients with melanoma, hepatocellular carcinoma, and pancreatic ductal adenocarcinoma from healthy controls. This is a pilot study to determine if sAxl is a candidate biomarker for GBM. The sAxl levels in the serum of 40 healthy volunteers and 20 GBM patients were determined using an enzyme-linked immunosorbent assay (ELISA). Pre- and post- operative sAxl levels were obtained. Volumetric MRI evaluation provided GBM tumor volume metrics. There was no significant difference in the sAxl levels of the volunteers (30.16±1.88 ng/ml) and GBM patients (30.74±1.96 ng/ml) p = 0.27. The postoperative sAxl levels were significantly higher than preoperative levels (32.32±2.26 ng/ml vs 30.74±1.96 ng/ml, p = 0.03). We found no correlation between tumor volume and sAxl levels. Axl expression was low or absent in 6 of 11 (55%) patient derived GBM cell lines. Given the wide range of Axl expression by GBM tumors, sAxl may not be a reliable indicator of GBM. However, given the small sample size in this study, a larger study may be considered.

Lysyl oxidase-like 1 predicts the prognosis of patients with primary glioblastoma and promotes tumor invasion via EMT pathway.

Background: Lysyl oxidase enzymes (LOXs), as extracellular matrix (ECM) protein regulators, play vital roles in tumor progression by remodeling the tumor microenvironment. However, their roles in glioblastoma (GBM) have not been fully elucidated. Methods: The genetic alterations and prognostic value of LOXs were investigated via cBioPortal. The correlations between LOXs and biological functions/molecular tumor subtypes were explored in The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA). After Kaplan‒Meier and Cox survival analyses, a Loxl1-based nomogram and prognostic risk score model (PRSM) were constructed and evaluated by time-dependent receiver operating characteristic curves, calibration curves, and decision curve analyses. Tumor enrichment pathways and immune infiltrates were explored by single-cell RNA sequencing and TIMER. Loxl1-related changes in tumor viability/proliferation and invasion were further validated by CCK-8, western blot, wound healing, and Transwell invasion assays. Results: GBM patients with altered LOXs had poor survival. Upregulated LOXs were found in IDH1-wildtype and mesenchymal (not Loxl1) GBM subtypes, promoting ECM receptor interactions in GBM. The Loxl1-based nomogram and the PRSM showed high accuracy, reliability, and net clinical benefits. Loxl1 expression was related to tumor invasion and immune infiltration (B cells, neutrophils, and dendritic cells). Loxl1 knockdown suppressed GBM cell proliferation and invasion by inhibiting the EMT pathway (through the downregulation of N-cadherin/Vimentin/Snai1 and the upregulation of E-cadherin). Conclusion: The Loxl1-based nomogram and PRSM were stable and individualized for assessing GBM patient prognosis, and the invasive role of Loxl1 could provide a promising therapeutic strategy.

Multi-scale signaling and tumor evolution in high-grade gliomas.

Although genomic anomalies in glioblastoma (GBM) have been well studied for over a decade, its 5-year survival rate remains lower than 5%. We seek to expand the molecular landscape of high-grade glioma, composed of IDH-wildtype GBM and IDH-mutant grade 4 astrocytoma, by integrating proteomic, metabolomic, lipidomic, and post-translational modifications (PTMs) with genomic and transcriptomic measurements to uncover multi-scale regulatory interactions governing tumor development and evolution. Applying 14 proteogenomic and metabolomic platforms to 228 tumors (212 GBM and 16 grade 4 IDH-mutant astrocytoma), including 28 at recurrence, plus 18 normal brain samples and 14 brain metastases as comparators, reveals heterogeneous upstream alterations converging on common downstream events at the proteomic and metabolomic levels and changes in protein-protein interactions and glycosylation site occupancy at recurrence. Recurrent genetic alterations and phosphorylation events on PTPN11 map to important regulatory domains in three dimensions, suggesting a central role for PTPN11 signaling across high-grade gliomas.

Preoperative prediction of MGMT promoter methylation in glioblastoma based on multiregional and multi-sequence MRI radiomics analysis.

O6-methylguanine-DNA methyltransferase (MGMT) has been demonstrated to be an important prognostic and predictive marker in glioblastoma (GBM). To establish a reliable radiomics model based on MRI data to predict the MGMT promoter methylation status of GBM. A total of 183 patients with glioblastoma were included in this retrospective study. The visually accessible Rembrandt images (VASARI) features were extracted for each patient, and a total of 14676 multi-region features were extracted from enhanced, necrotic, "non-enhanced, and edematous" areas on their multiparametric MRI. Twelve individual radiomics models were constructed based on the radiomics features from different subregions and different sequences. Four single-sequence models, three single-region models and the combined radiomics model combining all individual models were constructed. Finally, the predictive performance of adding clinical factors and VASARI characteristics was evaluated. The ComRad model combining all individual radiomics models exhibited the best performance in test set 1 and test set 2, with the area under the receiver operating characteristic curve (AUC) of 0.839 (0.709-0.963) and 0.739 (0.581-0.897), respectively. The results indicated that the radiomics model combining multi-region and multi-parametric MRI features has exhibited promising performance in predicting MGMT methylation status in GBM. The Modeling scheme that combining all individual radiomics models showed best performance among all constructed moels.

The CEBPB+ glioblastoma subcluster specifically drives the formation of M2 tumor-associated macrophages to promote malignancy growth.

Rationale: The heterogeneity of tumor cells within the glioblastoma (GBM) microenvironment presents a complex challenge in curbing GBM progression. Understanding the specific mechanisms of interaction between different GBM cell subclusters and non-tumor cells is crucial. Methods: In this study, we utilized a comprehensive approach integrating glioma single-cell and spatial transcriptomics. This allowed us to examine the molecular interactions and spatial localization within GBM, focusing on a specific tumor cell subcluster, GBM subcluster 6, and M2-type tumor-associated macrophages (M2 TAMs). Results: Our analysis revealed a significant correlation between a specific tumor cell subcluster, GBM cluster 6, and M2-type TAMs. Further in vitro and in vivo experiments demonstrated the specific regulatory role of the CEBPB transcriptional network in GBM subcluster 6, which governs its tumorigenicity, recruitment of M2 TAMs, and polarization. This regulation involves molecules such as MCP1 for macrophage recruitment and the SPP1-Integrin αvß1-Akt signaling pathway for M2 polarization. Conclusion: Our findings not only deepen our understanding of the formation of M2 TAMs, particularly highlighting the differential roles played by heterogeneous cells within GBM in this process, but also provided new insights for effectively controlling the malignant progression of GBM.

Hypoxia within the glioblastoma tumor microenvironment: a master saboteur of novel treatments.

Glioblastoma (GBM) tumors are the most aggressive primary brain tumors in adults that, despite maximum treatment, carry a dismal prognosis. GBM tumors exhibit tissue hypoxia, which promotes tumor aggressiveness and maintenance of glioma stem cells and creates an overall immunosuppressive landscape. This article reviews how hypoxic conditions overlap with inflammatory responses, favoring the proliferation of immunosuppressive cells and inhibiting cytotoxic T cell development. Immunotherapies, including vaccines, immune checkpoint inhibitors, and CAR-T cell therapy, represent promising avenues for GBM treatment. However, challenges such as tumor heterogeneity, immunosuppressive TME, and BBB restrictiveness hinder their effectiveness. Strategies to address these challenges, including combination therapies and targeting hypoxia, are actively being explored to improve outcomes for GBM patients. Targeting hypoxia in combination with immunotherapy represents a potential strategy to enhance treatment efficacy.

A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells.

Natural killer (NK) cells hold promise in cancer treatment due to their ability to spontaneously lyse cancer cells. For clinical use, high quantities of pure, functional NK cells are necessary. Combining adherence-based isolation with specialized media showed the unreliability of the isolation method, but demonstrated the superiority of the NK MACS® medium, particularly in suboptimal conditions. Neither human pooled serum, fetal calf serum (FCS), human platelet lysate, nor chemically defined serum replacement could substitute human AB serum. Interleukin (IL-)2, IL-15, IL-21, and combined CD2/NKp46 stimulation were assessed. IL-21 and CD2/NKp46 stimulation increased cytotoxicity, but reduced NK cell proliferation. IL-15 stimulation alone achieved the highest proliferation, but the more affordable IL-2 performed similarly. The RosetteSep™ human NK cell enrichment kit was effective for isolation, but the presence of peripheral blood mononuclear cells (PBMCs) in the culture enhanced NK cell proliferation, despite similar expression levels of CD16, NKp46, NKG2D, and ICAM-1. In line with this, purified NK cells cultured in NK MACS® medium with human AB serum and IL-2 demonstrated high cytotoxicity against primary glioblastoma stem cells.

Suppressing PD-L1 Expression via AURKA Kinase Inhibition Enhances Natural Killer Cell-Mediated Cytotoxicity against Glioblastoma.

Immunotherapies have shown significant promise as an impactful strategy in cancer treatment. However, in glioblastoma multiforme (GBM), the most prevalent primary brain tumor in adults, these therapies have demonstrated lower efficacy than initially anticipated. Consequently, there is an urgent need for strategies to enhance the effectiveness of immune treatments. AURKA has been identified as a potential drug target for GBM treatment. An analysis of the GBM cell transcriptome following AURKA inhibition revealed a potential influence on the immune system. Our research revealed that AURKA influenced PD-L1 levels in various GBM model systems in vitro and in vivo. Disrupting AURKA function genetically led to reduced PD-L1 levels and increased MHC-I expression in both established and patient-derived xenograft GBM cultures. This process involved both transcriptional and non-transcriptional pathways, partly implicating GSK3ß. Interfering with AURKA also enhanced NK-cell-mediated elimination of GBM by reducing PD-L1 expression, as evidenced in rescue experiments. Furthermore, using a mouse model that mimics GBM with patient-derived cells demonstrated that Alisertib decreased PD-L1 expression in living organisms. Combination therapy involving anti-PD-1 treatment and Alisertib significantly prolonged overall survival compared to vehicle treatment. These findings suggest that targeting AURKA could have therapeutic implications for modulating the immune environment within GBM cells.

Photophysical Characterization and In Vitro Evaluation of α-Mangostin-Loaded HDL Mimetic Nano-Complex in LN-229 Glioblastoma Spheroid Model.

Cytotoxic activity has been reported for the xanthone α-mangostin (AMN) against Glioblastoma multiforme (GBM), an aggressive malignant brain cancer with a poor prognosis. Recognizing that AMN's high degree of hydrophobicity is likely to limit its systemic administration, we formulated AMN using reconstituted high-density lipoprotein (rHDL) nanoparticles. The photophysical characteristics of the formulation, including fluorescence lifetime and steady-state anisotropy, indicated that AMN was successfully incorporated into the rHDL nanoparticles. To our knowledge, this is the first report on the fluorescent characteristics of AMN with an HDL-based drug carrier. Cytotoxicity studies in a 2D culture and 3D spheroid model of LN-229 GBM cells and normal human astrocytes showed an enhanced therapeutic index with the rHDL-AMN formulation compared to the unincorporated AMN and Temozolomide, a standard GBM chemotherapy agent. Furthermore, treatment with the rHDL-AMN facilitated a dose-dependent upregulation of autophagy and reactive oxygen species generation to a greater extent in LN-229 cells compared to astrocytes, indicating the reduced off-target toxicity of this novel formulation. These studies indicate the potential therapeutic benefits to GBM patients via selective targeting using the rHDL-AMN formulation.

Evaluation of the Immunomodulatory Effects of Radiation for Chimeric Antigen Receptor T Cell Therapy in Glioblastoma Multiforme.

Standard-of-care treatment for Glioblastoma Multiforme (GBM) is comprised of surgery and adjuvant chemoradiation. Chimeric Antigen Receptor (CAR) T cell therapy has demonstrated disease-modifying activity in GBM and holds great promise. Radiation, a standard-of-care treatment for GBM, has well-known immunomodulatory properties and may overcome the immunosuppressive tumor microenvironment (TME); however, radiation dose optimization and integration with CAR T cell therapy is not well defined. Murine immunocompetent models of GBM were treated with titrated doses of stereotactic radiosurgery (SRS) of 5, 10, and 20 Gray (Gy), and the TME was analyzed using Nanostring. A conditioning dose of 10 Gy was determined based on tumor growth kinetics and gene expression changes in the TME. We demonstrate that a conditioning dose of 10 Gy activates innate and adaptive immune cells in the TME. Mice treated with 10 Gy in combination with mCAR T cells demonstrated enhanced antitumor activity and superior memory responses to rechallenge with IL13Rα2-positive tumors. Furthermore, 10 Gy plus mCAR T cells also protected against IL13Rα2-negative tumors through a mechanism that was, in part, c-GAS-STING pathway-dependent. Together, these findings support combination conditioning with low-dose 10 Gy radiation in combination with mCAR T cells as a therapeutic strategy for GBM.

PRMT6 facilitates EZH2 protein stability by inhibiting TRAF6-mediated ubiquitination degradation to promote glioblastoma cell invasion and migration.

Invasion and migration are the key hallmarks of cancer, and aggressive growth is a major factor contributing to treatment failure and poor prognosis in glioblastoma. Protein arginine methyltransferase 6 (PRMT6), as an epigenetic regulator, has been confirmed to promote the malignant proliferation of glioblastoma cells in previous studies. However, the effects of PRMT6 on glioblastoma cell invasion and migration and its underlying mechanisms remain elusive. Here, we report that PRMT6 functions as a driver element for tumor cell invasion and migration in glioblastoma. Bioinformatics analysis and glioma sample detection results demonstrated that PRMT6 is highly expressed in mesenchymal subtype or invasive gliomas, and is significantly negatively correlated with their prognosis. Inhibition of PRMT6 (using PRMT6 shRNA or inhibitor EPZ020411) reduces glioblastoma cell invasion and migration in vitro, whereas overexpression of PRMT6 produces opposite effects. Then, we identified that PRMT6 maintains the protein stability of EZH2 by inhibiting the degradation of EZH2 protein, thereby mediating the invasion and migration of glioblastoma cells. Further mechanistic investigations found that PRMT6 inhibits the transcription of TRAF6 by activating the histone methylation mark (H3R2me2a), and reducing the interaction between TRAF6 and EZH2 to enhance the protein stability of EZH2 in glioblastoma cells. Xenograft tumor assay and HE staining results showed that the expression of PRMT6 could promote the invasion of glioblastoma cells in vivo, the immunohistochemical staining results of mouse brain tissue tumor sections also confirmed the regulatory relationship between PRMT6, TRAF6, and EZH2. Our findings illustrate that PRMT6 suppresses TRAF6 transcription via H3R2me2a to enhance the protein stability of EZH2 to facilitate glioblastoma cell invasion and migration. Blocking the PRMT6-TRAF6-EZH2 axis is a promising strategy for inhibiting glioblastoma cell invasion and migration.

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

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

Targeting MS4A4A: A novel pathway to improve immunotherapy responses in glioblastoma.

INTRODUCTION: Glioblastoma (GBM) remains a challenging brain tumor to treat, with limited response to PD-1 immunotherapy due to tumor-associated macrophages (TAMs), specifically the M2 phenotype. This study explores the potential of MS4A4A (membrane spanning four domains, subfamily A, member 4A) inhibition in driving M2 macrophage polarization toward the M1 phenotype via the ferroptosis pathway to enhance the effectiveness of immunotherapy in GBM. METHODS: Single-cell RNA sequencing and spatial transcriptomic analyses were employed to characterize M2 macrophages and MS4A4A expression in GBM. In vitro studies utilizing TAM cultures, flow cytometry, and western blot validations were conducted to assess the impact of MS4A4A on the tumor immune microenvironment and M2 macrophage polarization. In vivo models, including subcutaneous and orthotopic transplantation in mice, were utilized to evaluate the effects of MS4A4A knockout and combined immune checkpoint blockade (ICB) therapy on tumor growth and response to PD-1 immunotherapy. RESULTS: Distinct subsets of GBM-associated macrophages were identified, with spatial distribution in tumor tissue elucidated. In vivo experiments demonstrated that inhibiting MS4A4A and combining ICB therapy effectively inhibited tumor growth, reshaped the tumor immune microenvironment by reducing M2 TAM infiltration and enhancing CD8+ T-cell infiltration, ultimately leading to complete tumor eradication. CONCLUSION: MS4A4A inhibition shows promise in converting M2 macrophages to M1 phenotype via ferroptosis, decreasing M2-TAM infiltration, and enhancing GBM response to PD-1 immunotherapy. These findings offer a novel approach to developing more effective immunotherapeutic strategies for GBM.

TRP-2 / gp100 DNA vaccine and PD-1 checkpoint blockade combination for the treatment of intracranial tumors.

Intracranial tumors present a significant therapeutic challenge due to their physiological location. Immunotherapy presents an attractive method for targeting these intracranial tumors due to relatively low toxicity and tumor specificity. Here we show that SCIB1, a TRP-2 and gp100 directed ImmunoBody® DNA vaccine, generates a strong TRP-2 specific immune response, as demonstrated by the high number of TRP2-specific IFNγ spots produced and the detection of a significant number of pentamer positive T cells in the spleen of vaccinated mice. Furthermore, vaccine-induced T cells were able to recognize and kill B16HHDII/DR1 cells after a short in vitro culture. Having found that glioblastoma multiforme (GBM) expresses significant levels of PD-L1 and IDO1, with PD-L1 correlating with poorer survival in patients with the mesenchymal subtype of GBM, we decided to combine SCIB1 ImmunoBody® with PD-1 immune checkpoint blockade to treat mice harboring intracranial tumors expressing TRP-2 and gp100. Time-to-death was significantly prolonged, and this correlated with increased CD4+ and CD8+ T cell infiltration in the tissue microenvironment (TME). However, in addition to PD-L1 and IDO, the GBM TME was found to contain a significant number of immunoregulatory T (Treg) cell-associated transcripts, and the presence of such cells is likely to significantly affect clinical outcome unless also tackled.