SINGLE-CELL METABOLIC PROfiLING OF TUMOUR ASTROCYTE INTERACTIONS WITHIN A CO-CULTURE MODEL OF THE GLIOBLASTOMA INVASIVE MARGIN

Abstract AIMS Glioblastoma (GBM) is an aggressive brain tumour conferring poor prognosis and one of the highest recurrence rates despite multimodal treatment. The mechanisms underlying recurrence are not fully understood, but the tumour microenvironment is known to play a key role. Astrocytes, the most abundant glial cells in the brain, have been implicated in supporting tumour progression, yet their metabolic interactions with glioblastoma invasive margin cells remain poorly understood. This study aims to investigate metabolic interactions using single-cell spatial metabolomics to identify potential pathways involved in tumour adaptation and progression. METHODS The study employed atmospheric pressure matrix-assisted laser desorption/ionisation (AP-MALDI) imaging mass spectrometry at 10 μm spatial resolution to investigate metabolic interactions between primary glioblas- toma invasive margin cells and human cortical astrocytes in a co-culture model designed to mimic post-surgical residual disease. RESULTS Metabolomic analysis revealed distinct metabolic changes in glioblastoma invasive margin (GIN) cells and hu- man cortical astrocytes (HA) under co-culture conditions. In GIN cells, pyroglutamic acid and glutamate levels were altered, along with changes in lipid metabolism, including a reduction in oleamide. In astrocytes, co- culture influenced nucleotide metabolism, with increased levels of deoxyguanosine, alongside shifts in lipid profiles. Findings indicate bidirectional metabolic interactions that may contribute to glioblastoma progres- sion. CONCLUSION By mapping metabolite spatial distributions at the single-cell level, this study provides novel insights into tumour–astrocyte metabolic interactions at the glioblastoma invasive margin. These findings highlight poten- tial metabolic dependencies that could be therapeutically targeted to disrupt glioblastoma recurrence.