Background. Approximately half of the isocitrate dehydrogenase (IDH)-wildtype glioblastomas (GBMs) exhibit EGFR amplification. Additionally, genomic changes that occur in the extracellular domain of EGFR can lead to ligand-hypersensitivity (R108K/A289V/G598V) or ligand-independence (EGFRvIII). Unlike in lung adenocarcinoma (LUAD), clinical trials with epidermal growth factor receptor (EGFR) inhibitors showed no survival benefit for GBM and it remains unclear why. We aimed to elucidate differences in molecular mechanisms of EGFR activation and regulation between GBM and LUAD. Methods. We used RNA-sequencing (RNA-seq) data to find EGFR co-regulated genes and pathways in GBM and compare EGFR signaling patterns between GBM and LUAD. Cellular origins of expression signals were determined by analyzing single-cell RNA-seq data. Results. We identified 2 ligands (BTC/EREG) among the significant EGFR predictor genes (TCGA-GBM: n = 169, Intellance-2: n = 166). Their expression was inversely correlated with EGFR amplification and incidence of ligand-sensitive mutations. Ligands were expressed by nonmalignant cells and differed in their primary source of expression (BTC: neurons, EREG: myeloid). High expression of MDM2 and CDK4 was less common in EGFR-amplified GBMs with ligand-sensitive mutations compared with those without these mutations. Our analyses revealed distinct transcriptional profiles between GBM and LUAD when comparing tumors carrying activating mutations. Conclusions.BTC and EREG are negatively associated with EGFR expression in GBM. These findings emphasize the role of ligands in regulating EGFR, where EGFR activation seems to be modulated by the highly varying levels of EGFR amplification, the sensitivity of the receptor toward ligands, and ligand expression levels. Ligand expression levels and EGFR mutations could refine patient stratification for EGFR-targeted therapies in GBM.

A better understanding of transcriptional evolution of IDH-wild-type glioblastoma may be crucial for treatment optimization. Here, we perform RNA sequencing (RNA-seq) (n = 322 test, n = 245 validation) on paired primary-recurrent glioblastoma resections of patients treated with the current standard of care. Transcriptional subtypes form an interconnected continuum in a two-dimensional space. Recurrent tumors show preferential mesenchymal progression. Over time, hallmark glioblastoma genes are not significantly altered. Instead, tumor purity decreases over time and is accompanied by co-increases in neuron and oligodendrocyte marker genes and, independently, tumor-associated macrophages. A decrease is observed in endothelial marker genes. These composition changes are confirmed by single-cell RNA-seq and immunohistochemistry. An extracellular matrix-associated gene set increases at recurrence and bulk, single-cell RNA, and immunohistochemistry indicate it is expressed mainly by pericytes. This signature is associated with significantly worse survival at recurrence. Our data demonstrate that glioblastomas evolve mainly by microenvironment (re-)organization rather than molecular evolution of tumor cells.

A major obstacle in glioma research is the lack of in vitro models that can retain cellular features of glioma cells in vivo. To overcome this limitation, a 3D-engineered scaffold, fabricated by two-photon polymerization, is developed as a cell culture model system to study patient-derived glioma cells. Scanning electron microscopy, (live cell) confocal microscopy, and immunohistochemistry are employed to assess the 3D model with respect to scaffold colonization, cellular morphology, and epidermal growth factor receptor localization. Both glioma patient-derived cells and established cell lines successfully colonize the scaffolds. Compared to conventional 2D cell cultures, the 3D-engineered scaffolds more closely resemble in vivo glioma cellular features and allow better monitoring of individual cells, cellular protrusions, and intracellular trafficking. Furthermore, less random cell motility and increased stability of cellular networks is observed for cells cultured on the scaffolds. The 3D-engineered glioma scaffolds therefore represent a promising tool for studying brain cancer mechanobiology as well as for drug screening studies.