Tumor surface signature in glioblastoma distinguishes IDH mutant from wildtype high grade glioma and identifies novel targets for immunotherapy

Introduction: Glioblastoma remains one of the most aggressive brain malignancies, with poor patient outcomes despite current therapies. The tumor microenvironment shows complex interactions between tumor cells and immune populations, particularly immunosuppressive tumor-associated macrophages (TAMs). The composition of myeloid populations varies between IDH-mutant and IDH-wildtype tumors, with the latter showing higher proportions of monocyte-derived macrophages. While several surface markers are known, a comprehensive understanding of profiles specific to tumor cells and immunosuppressive myeloid cells remains critical for developing effective combination immunotherapies.

Methods: We analyzed 46 high-grade glioma specimens using spectral flow cytometry with a 32-marker panel for tumor cells and macrophages. Analysis included UMAP MiloR clustering, differential abundance testing, and survival correlation. Surface marker screening utilized LEGENDScreen™ technology with color barcoding on patient-derived cell lines. Marker selection and validation integrated multiple databases including TCGA and Cancer Surfaceome Atlas, with transcriptomic validation performed using cyCombine.

Results: Analysis revealed distinct clustering patterns between IDH mutant and wildtype tumors. IDH wildtype gliomas demonstrated unique immunosuppressive signatures, characterized by higher frequencies of cycling monocyte-derived macrophages expressing CD39, CD112, CD31, CD276, PDL1, and additional markers. Tumor cells displayed elevated levels of GPR56, CD271, CD71, CD112, CD323, CD146, and other surface proteins. Transcriptomic integration identified immunosuppressive perivascular macrophages expressing EREG, APOBEC3A, S100A8/9, and FCN1, while mature inhibitory macrophages showed high expression of APOE, APOC1, and C1QA. Survival analysis demonstrated that increased frequencies of exhausted CD8+ T cells correlated with improved outcomes, while elevated levels of FOLR2+CD39+CD163+ macrophages and cycling CD169+MERTK+CD163+CD206+ macrophages predicted poor survival. The study also identified a distinct population of activated regulatory T cells characterized by FOXP3, CTLA4, and other immunosuppressive markers, further contributing to the complex immune landscape.

Conclusion : Our comprehensive profiling establishes distinct surface marker signatures differentiating IDH mutant and wildtype gliomas, revealing novel therapeutic targets. These findings provide a foundation for developing targeted combination immunotherapies that address both tumor cells and the immunosuppressive microenvironment, potentially improving outcomes for patients with aggressive high-grade gliomas.