141 Decoding the Infiltrative Margin: Structural and Molecular Characterisation of the Microtube Syncytium in Recurrent Glioblastoma

Abstract Introduction Isocitrate dehydrogenase wild-type glioblastoma (GBM) is an aggressive brain tumour with a high post-surgical recurrence rate. Poor prognosis is linked to infiltrative tumour cells forming structural and functional intercellular networks that facilitate the transport of molecular cargo. This study focuses on functional voltage-gated potassium channels (VGKCs) and tumour microtube formation, whereby we hypothesise that the molecular scaffolding and protein composition of these networks facilitate intra-tumoral communication and survival at the invasive tumour margin. Method Patient-derived GBM core (GCE) and invasive margin (GIN) cells were analysed using high-resolution confocal microscopy and Western blotting. Structural network complexity was quantified using β -tubulin III and GAP-43 immunofluorescence, utilising Amira-Avizo to map microtube branching patterns. Molecular target PSD-95, a synaptic scaffold, was assessed through an integrated density puncta count. Functional electrical properties were evaluated using the Nanion Port-a-Patch system to measure potassium channel activity. Results Skeletal analysis revealed that GIN cells exhibit significantly increased microtube branching and total network length compared to GCE cells. Western blot and immunofluorescence confirmed a marked enrichment of PSD-95 and GAP-43 in the GIN population. These findings correlate with Nanion patch-clamp data demonstrating differential inward-rectifying potassium currents between regions. Together, these results suggest a specialised molecular hub at the infiltrative margin that coordinates network connectivity. Conclusions The infiltrative GBM margin is characterised by a specialised structural and molecular signature. Enrichment of synaptic scaffolding proteins within the microtube network suggests sophisticated integration with the brain tumour microenvironment. Future work will utilise SH-SY5Y (immortalised neuron-like cells) co-culture models to explore how this machinery facilitates direct neuro-glioma interactions. Characterising these hubs may reveal novel targets for disrupting tumour connectivity to prevent or delay recurrence.