Modeling the blood–brain tumor barrier (BBTB) in vitro remains a major challenge due to the structural and functional complexity of the brain microvasculature and its dynamic interactions with glioma cells. Here, we present 3D microvascular structures fabricated by two-photon polymerization (2PP) that mimic capillary architecture and enable multicellular models for studying the BBTB. Immunofluorescence and scanning electron microscopy confirm that these structures support homogenous colonization by both human umbilical vein endothelial cells (HUVECs) and human cerebral microvascular endothelial cells (hCMEC/D3), forming tubular endothelial monolayers with polarized nuclear morphology and alignment, comparable to in vivo conditions. Additionally, endothelial cells show increased expression of cytoskeletal (tubulin, F-actin) and barrier markers (ZO-1, CD31) compared to 2D cultures. The engineered model responds to TNF-α stimulation and supports co- and tri-cultures with pericytes and glioma cells. Incorporation of glioma cells leads to reduced CD31 and elevated PLVAP expression, indicating barrier destabilization. The µPCs are also integrated into commercially available microfluidic chips via in-chip 2PP, enabling stable perfusion and providing access to both luminal and abluminal sides of the endothelium. In summary, our model provides a biomimetic and adaptable platform for studying endothelial integrity, tumor-vascular crosstalk, and broad applicability in barrier biology studies.

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 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.