P01.28.b Glycolytic Condensates as a Metabolic Adaptation in Hypoxic Glioblastoma Cells

Abstract BACKGROUND Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumour, characterised by rapid proliferation, invasiveness and limited treatment options. Hypoxic regions within the tumour microenvironment drive metabolic adaptations that sustain energy production. A key mechanism in this adaptation is the upregulation of glycolysis, which is regulated by hypoxia-inducible factor-1alpha (HIF-1alpha). Under low-oxygen conditions, HIF-1α enhances the expression of glycolytic enzymes, ensuring ATP production despite oxygen limitation. However, the spatial organisation of these enzymes remains poorly understood. Recent studies have highlighted the role of biomolecular condensates, membrane-less organelles formed by liquid-liquid phase separation (LLPS), in metabolic regulation. In yeast, glycolytic enzymes cluster into glycolytic bodies (G-bodies) to facilitate efficient metabolism. We hypothesise that GBM cells use a similar strategy, forming biomolecular condensates to optimise glycolysis under hypoxia. MATERIAL AND METHODS To investigate this, we used the U251MG glioblastoma cell line and exposed it to controlled hypoxic conditions (1% O₂, 37°C). Western blotting was used to analyse protein levels. Our results show that after 24 hours of hypoxia, there is a significant increase in HIF-1α levels and upregulation of several key glycolytic enzymes, including hexokinase (HKII), enolase (ENO1) and pyruvate kinase M2 (PKM2), compared to normoxic conditions. RESULTS Preliminary fluorescence microscopy studies are underway to investigate the spatial reorganisation of glycolytic enzymes under hypoxic stress. Initial observations suggest possible changes in the distribution of enzymes such as PFKP, possibly indicating condensate formation. These findings suggest that glycolytic condensates may contribute to metabolic plasticity in GBM by optimising energy production under oxygen-limiting conditions. CONCLUSION Understanding the mechanisms of glycolytic condensate formation in GBM will provide insights into tumour metabolism and identify potential therapeutic targets to disrupt metabolic adaptation and inhibit tumour growth. This project is supported by the National Institute for Cancer Research (Program EXCELES, ID Project No. LX22NPO5102) by the European Union - Next Generation EU.