Comparative cross-methodological analysis of the IDH-wildtype glioblastoma tumor microenvironment.

purposeGlioblastoma, IDH-wildtype is a highly aggressive and often recurrent brain malignancy characterized by a profoundly immunosuppressive and heterogeneous tumor microenvironment. In this study, we aimed to systematically compare commonly used immune profiling methodologies.

methodsWe conducted a cross-platform comparison using matched primary and recurrent tumor samples analyzed by immunohistochemistry, multiplex immunofluorescence, AI-driven image analysis, DNA methylation profiling, and bulk RNA sequencing. A total of 72 samples from 36 patients were evaluated to assess cross-method concordance, cell-type resolution, and each platform’s ability to capture TME dynamics throughout disease progression.

resultsAcross modalities, monocyte/macrophage-lineage cells were the most consistently identified and quantifiable population. Image-based techniques, including immunohistochemistry, multiplex immunofluorescence, and AI-driven quantification, demonstrated strong concordance for B cell and macrophage detection, whereas T cell quantification showed greater inter-method variability, particularly in recurrent tumors. RNA sequencing-based deconvolution captured broader spectrum of immune and neoplastic states, including microglial enrichment, but aligned only moderately with protein-level measurements. DNA methylation-based approaches performed robustly for myeloid cell estimation but limited accuracy for lymphocyte populations.

conclusionThis study highlights the complementary strengths and limitations of current immune profiling modalities in GB. An integrative, method-aware framework facilitates more accurate immune cell quantification and deeper biological insights into TME evolution, ultimately informing the development of precision immunotherapeutic strategies for recurrent GB.