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

## TL;DR

This study compares different methods for analyzing immune cells in IDH-wildtype glioblastoma tumors to better understand their changing environment during disease progression.

## Contribution

The study provides a systematic cross-methodological comparison of immune profiling techniques in glioblastoma tumor microenvironments.

## Key findings

- Monocyte/macrophage-lineage cells were most consistently detected across methods.
- Image-based techniques showed strong agreement for B cells and macrophages but varied for T cells.
- RNA sequencing captured broader immune states but aligned moderately with protein-level data.

## Abstract

Glioblastoma, 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.

We 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.

Across 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.

This 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.

The online version contains supplementary material available at 10.1007/s00432-026-06428-6.