DNA Methylation-Based Deconvolution Sheds Light on Glioblastoma Heterogeneity and Cell-Type Composition Associated with Patient Survival

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Abstract Background IDH-wildtype glioblastoma (GBM) is an aggressive, heterogeneous brain tumor with limited treatment options. DNA methylation profiling allows detailed tumor characterization. This study applies methylation-based deconvolution to define GBM’s cellular composition and its association with patient outcomes. Methods We generated oligodendroglial precursor cells at various developmental stages from enriched human neural progenitor cultures and used their DNA methylation signatures, along with published signatures of brain tumor and tumor microenvironment–relevant cell types, to deconvolve 263 adult GBMs (Heidelberg-cohort). Tumor purity was estimated using RF_Purify. An independent cohort of 199 GBMs from TCGA and GEO, all treated with standard-of-care therapy, was similarly deconvolved, followed by Kaplan–Meier survival analysis to assess the prognostic value of neoplastic component proportions. Results Deconvolution uncovered distinct cellular compositions, consistent with single-cell RNA sequencing findings. Tumor purity analysis showed neoplastic fractions averaged 70% of tumor bulk, predominantly oligodendrocyte-like (43%), oligodendrocyte precursor-like (27%), astrocyte-like (19%), and mesenchymal stem cell-like (11%) populations. Non-neoplastic fractions were enriched for macrophages, vascular cells, and immune populations. A higher oligodendrocyte-like signature was linked to poorer survival (median survival 14.3 vs. 15.3 months; p = 0.017), while a higher astrocyte-like signature correlated with improved outcomes (15.3 vs. 13.4 months; p = 0.044). The astrocyte-to-oligodendrocyte ratio emerged as a strong prognostic marker, with a higher ratio predicting significantly longer survival (15.8 vs. 11.9 months; p < 0.00011). Conclusions Methylation-based deconvolution provides insight into GBM heterogeneity, highlighting the prognostic relevance of the astrocyte-to-oligodendrocyte ratio, which may guide personalized treatment strategies. Graphical Abstract A. We generated oligodendroglial precursor cells (OPs) at various developmental stages from enriched human neural progenitor cultures. A reference atlas of methylation signatures from 14 normal cell types, including the in vitro–generated cells, combined with published profiles of brain tumor and tumor microenvironment–relevant cell types, was applied to deconvolve 263 GBM samples based on their methylation profiles (Heidelberg cohort). Cell type proportions were analyzed with RF_Purify to estimate tumor purity and distinguish neoplastic from non-neoplastic components. B. An independent cohort of 199 GBMs from TCGA and GEO, with available clinical data and all treated with standard-of-care therapy, was similarly deconvolved based on methylation profiles. Kaplan–Meier survival analysis assessed the prognostic impact of cell type proportions, identifying the astrocyte-to-oligodendrocyte ratio as the most significant marker. (Created with BioRender.com ) Key points Methylation-based deconvolution incorporating in-vitro-derived oligodendrocyte precursor differentiation data reveals GBM heterogeneity GBM’s neoplastic fraction is dominated by oligodendrocyte-lineage cells Astrocyte/oligodendrocyte ratio is a strong prognostic marker for patient survival Importance of the study This study advances neuro-oncology research by using methylation-based deconvolution to uncover the cellular heterogeneity of glioblastoma. By constructing a comprehensive reference atlas and identifying cell-type-specific methylation signatures, it provides prognostic insights into the distinct cellular compositions of glioblastoma. Combining deconvolution results with purity analysis enables the differentiation between neoplastic and non-neoplastic tumor components. This approach complements single-cell RNA sequencing while offering greater clinical applicability, as DNA methylation profiling can be performed on FFPE or fresh frozen samples, unlike the more costly and tissue-sensitive single-cell methods. Importantly, the identification of the astrocyte-to-oligodendrocyte ratio as a strong prognostic marker highlights key cellular determinants of glioblastoma survival. These findings deepen our understanding of glioblastoma biology and offer a practical tool for patient stratification.

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