Chen, Jianan

Glioblastoma (GBM) remains one of the most lethal brain tumors, characterized by extensive immune evasion and a macrophage-dominated tumor microenvironment (TME). However, the molecular determinants governing tumor-associated macrophage (TAM) states and their immunoregulatory functions remain poorly understood. We integrated bulk- and single-cell transcriptomic datasets (TCGA, CGGA, Ivy GAP, and Brain Immune Atlas) to systematically characterize the expression, prognostic relevance, and immune contexture of the myeloid biomarker membrane-spanning 4-domain A6A, MS4A6A, in GBM. Differential expression, survival, and pathway enrichment analyses were performed. Single-cell mapping and CellChat modeling delineated MS4A6A-associated TAM subpopulations, intercellular communication networks, and ligand–receptor signaling dynamics. Spatial transcriptomic validation and pharmacogenomic modeling were conducted to assess anatomic enrichment and therapeutic vulnerabilities. High MS4A6A expression predicted unfavorable survival and correlated with increased stromal and immune infiltration. Single-cell analyses localized MS4A6A predominantly to TAMs, especially Regulatory- and Ribo-TAM states enriched for antigen presentation, T-cell regulation, and ribosomal biogenesis pathways. CellChat analysis revealed that MS4A6A-high TAMs exhibited markedly enhanced communication with CD4+ T cells and Tregs through upregulated PGE2–PTGER2/PTGER4, PECAM1–CD38, and THBS1–CD36 signaling axes, implicating MS4A6A in prostaglandin-driven immune suppression. Spatial profiling confirmed preferential localization of MS4A6A within perivascular and angiogenic niches. Pharmacogenomic prediction indicated that MS4A6A-high tumors were more sensitive to ERK, mTOR, and CDK4/6 inhibition. MS4A6A defines a macrophage-centered, immunosuppressive ecosystem in GBM, mediated by the activation of the PGE2 signaling axis. These findings position MS4A6A both as a prognostic biomarker and as a potential therapeutic node linking myeloid reprogramming to actionable pathway vulnerabilities in glioblastoma.

Abstract Glioblastoma (GBM) is a highly treatment-resistant malignancy characterized by its poor prognosis and intrinsic resistance to therapy, driven largely by glioblastoma stem cells (GSCs) and an immunosuppressive tumor microenvironment (TME). This dual resistance plays a significant role in the failure of current treatment strategies. Consequently, there is an urgent need for novel therapeutic approaches that can effectively target GSCs while simultaneously reshaping the TME to promote anti-tumor immunity. In this study, we investigated the dual therapeutic potential of Xevinapant, a lead SMAC mimetic, through a combination of in vitro, in vivo, and single-cell transcriptomic analyses. We found that Xevinapant demonstrated potent anti-tumor activity through inducing dose-dependent apoptosis and significantly reduced cell viability in both human and mouse GSCs. In vivo administration of Xevinapant led to significant tumor suppression and extended overall survival in orthotopic models. Notably, our single-cell RNA sequencing result revealed that Xevinapant activated immune effector cell responses, contributing to the reprogramming of the immunosuppressive TME. Immune cell activation (both lymphoid and myeloid lineages) was further validated by flow cytometry using tumor-derived cells. To overcome potential therapeutic resistance, we derived Xevinapant-specific gene expression signatures and queried the LINCS database to identify small molecules with synergistic effects. Our analysis identified a promising compound ST-059620 that, when used in combination with Xevinapant, enhanced therapeutic efficacy and overcame resistance. Taken together, our findings highlight the dual mechanism of action of Xevinapant in targeting both tumor-intrinsic and microenvironmental resistance mechanisms. These results support the continued clinical development of Xevinapant and provide a strong rationale for combination strategies aimed at improving treatment efficacy in GBM.

Background: Tumor-associated macrophages (TAMs) are essential regulators of the glioblastoma (GBM) microenvironment; their functional heterogeneity and interaction networks are not fully elucidated. We identify RNF135 as a novel TAM-enriched gene associated with immune activation and adverse prognosis in GBM. Methods: To evaluate RNF135’s expression profile, prognostic significance, and functional pathways, extensive transcriptome analyses from TCGA and CGGA cohorts were conducted. The immunological landscape and cellular origin of RNF135 were outlined using single-cell RNA-seq analyses and bulk RNA-seq immune deconvolution (MCP-counter, xCell and ssGSEA). Cell–cell communication networks between tumor cells and RNF135-positive and -negative tumor-associated macrophage subsets were mapped using CellChat. Results: RNF135 predicted a poor overall survival and was markedly upregulated in GBM tissues. Functional enrichment analyses showed that increased cytokine signaling, interferon response, and innate immune activation were characteristics of RNF135-high samples. Immune infiltration profiling showed a strong correlation between the abundance of T cells and macrophages and RNF135 expression. According to the single-cell analyses, RNF135 was primarily expressed in TAMs, specifically in proliferation, phagocytic, and transitional subtypes. RNF135-positive TAMs demonstrated significantly improved intercellular communication with aggressive tumor subtypes in comparison to RNF135-negative TAMs. This was facilitated by upregulated signaling pathways such as MHC-II, CD39, ApoE, and most notably, the CCL signaling axis. The CCL3/CCL3L3–CCR1 ligand–receptor pair was identified as a major mechanistic driver of TAM–TAM crosstalk. High RNF135 expression was also linked to greater sensitivity to Selumetinib, a selective MEK1/2 inhibitor that targets the MAPK/ERK pathway, according to drug sensitivity analysis. Conclusions: RNF135 defines a TAM phenotype in GBM that is both immunologically active and immunosuppressive. This phenotype promotes inflammatory signaling and communication between cells in the tumor microenvironment. Targeting the CCL–CCR1 axis or combining RNF135-guided immunomodulation with certain inhibitors could be a promising therapeutic strategies for GBM.

Glioblastoma (GBM) is an aggressive brain tumor with a highly immunosuppressive microenvironment that promotes tumor progression and therapy resistance. Tumor-associated macrophages (TAMs), comprising up to 50% of the tumor mass, are recruited via chemokine axes such as CCL2/CCR2, CX3CL1/CX3CR1, and CXCL12/CXCR4 and adopt an M2-like immunosuppressive phenotype, facilitating immune escape and angiogenesis. Key signaling pathways, including CSF1R, STAT3, NF-κB, PI3K/Akt, and HIF-1α, regulate TAM function, making them promising therapeutic targets. Strategies such as TAM depletion, reprogramming, and immune checkpoint blockade (PD-1/PD-L1, and CD47-SIRPα) have shown potential in preclinical models. Emerging approaches, including CAR-macrophage (CAR-M) therapy, nanotechnology-based drug delivery, and exosome-mediated modulation, offer new avenues for intervention. However, clinical translation remains challenging due to GBM’s heterogeneity and adaptive resistance mechanisms. Future research should integrate multi-omics profiling and AI-driven drug discovery to refine TAM-targeted therapies and improve patient outcomes. This review provides a comprehensive analysis of TAM-mediated immune regulation in GBM and explores evolving therapeutic strategies aimed at overcoming its treatment barriers.

Background: Glioblastoma is a highly malignant brain tumor with limited treatment options and a poor prognosis, largely driven by its complex immune microenvironment. This study aimed to identify and characterize an immune-related gene signature associated with prognosis and immune regulation in glioblastoma. Methods: We performed integrative analyses using bulk and single-cell RNA sequencing data to identify prognostically significant immune-related genes. A five-gene signature (THEMIS2, SIGLEC9, CSTA, LILRB3, and MS4A6A) was derived and its expression patterns were analyzed in association with immune cell infiltration and macrophage subtypes. Functional enrichment and pathway analyses were conducted, followed by drug sensitivity profiling to explore potential therapeutic implications. Results: The five-gene signature was significantly associated with worse survival outcomes and increased immune cell infiltration. Functional analyses revealed involvement in key immune pathways, including antigen presentation, cytokine signaling, and immune cell activation. Single-cell RNA sequencing demonstrated high expression of the signature in tumor-associated macrophages, particularly immune-suppressive and proliferation-associated subtypes. The high expression in proliferation TAMs suggests a role in promoting tumor angiogenesis and growth. Drug sensitivity analysis revealed distinct vulnerabilities between high- and low-risk groups based on signature expression. Conclusions: This Macrophage-Associated Prognostic Signature (MAPS) provides new insights into glioblastoma immunobiology and identifies potential biomarkers and therapeutic targets. It may serve as a valuable tool to guide personalized immunotherapy-based strategies for glioblastoma patients.