Heffernan, M. | bioRxiv Science

bioRxiv2026

Physioxia Reprograms Glioblastoma Cells Enhancing Migration and Altering Therapeutic Sensitivity

Background/ObjectivesGlioblastoma is an aggressive primary brain tumor that develops within a chronically low-oxygen microenvironment, yet most preclinical studies are performed under atmospheric oxygen conditions that poorly reflect in vivo physiology. This study investigated how sustained culture under physiological oxygen tension (physioxia; 5% O{square}) influences glioblastoma cell behavior, signaling, and therapeutic response. MethodsMultiple patient-derived glioblastoma models were cultured under normoxia (21% O{square}) or sustained physioxia (5% O{square}) for at least seven days before experimentation. Cell migration, proliferation, cell cycle distribution, expression of the epithelial-to-mesenchymal transition-associated transcription factor Slug (SNAI2), PDGFR{beta}-associated signaling, and sensitivity to 5-fluorouracil were evaluated using transwell migration assays, cell counting, flow cytometry, RT-qPCR, immunoblotting, and BrdU incorporation assays. Additional patient-derived cultures established and maintained continuously under physioxia were used to examine the effects of oxygen history. ResultsSustained physioxia consistently increased migration across all glioblastoma models while reducing proliferation in normoxia-adapted cell lines through increased G0/G1 cell cycle arrest. Physioxia significantly increased Slug expression in all models and enhanced PDGFR{beta}, AKT, and ERK phosphorylation in a cell line-dependent manner. Therapeutic sensitivity to 5-fluorouracil was also altered, with physioxia conferring increased resistance in selected glioblastoma models but not universally. Patient-derived cultures maintained continuously under physioxia retained enhanced migratory capacity and exhibited increased proliferation compared with normoxia, indicating that prior oxygen exposure influences proliferative responses while the pro-migratory phenotype remains conserved. ConclusionsPhysiological oxygen tension is a major regulator of glioblastoma cell behavior, influencing migration, proliferation, signaling, and therapeutic response. These findings demonstrate that conventional normoxic culture conditions can obscure biologically relevant phenotypes and support incorporating physioxia into experimental design to improve the physiological and translational relevance of preclinical glioblastoma research.

cancer biology↗

bioRxiv2026

A cellular midbrain mechanism for executing fast and reliable escape

Escape from threat is one of the most ancient and conserved sensorimotor transformations and must satisfy two competing demands. It must be fast and reliable, because failing to escape genuine threats risks death, yet also selective, because escaping indiscriminately costs energy and missed resources 1,2. Speed and reliability favour a system with a low threshold that responds to the slightest indication of danger, whereas selectivity demands a high threshold that filters out innocuous stimuli -- yet both must be achieved simultaneously. While previous work has identified mechanisms for implementing selectivity 3-6, it is not known how the mammalian brain achieves speed and reliability once genuine threats have been identified. Here we use whole-cell recordings from dorsal periaqueductal gray (dPAG) neurons in behaving mice to show that the escape circuit resolves this tension via high intrinsic excitability at the single cell level. We find that while the membrane potential of dPAG neurons does not reach action potential threshold during exploratory behaviour, they require only a small amount of current to fire. Threat stimuli cause a sparse increase in synaptic input rate that, because of the high excitability, produces a sustained depolarizing voltage step that drives spiking and escape behaviour. Individual dPAG neurons respond similarly on escape and non-escape trials --what determines escape is the fraction of the dPAG population that is recruited, a finding we confirm with single-unit recordings in freely moving mice. The membrane voltage step response is also invariant to threat type, in contrast to superior colliculus neurons where membrane potential dynamics reflect stimulus identity. These findings reveal a single neuron mechanism for fast and reliable escape, in which the high intrinsic excitability of dPAG neurons transforms sparse, threat-evoked synaptic input into a rapid and stereotyped voltage signal, converting diverse threat representations into a uniform escape command.

neuroscience↗