ID #346 Tumor microtube networks in diffuse midline glioma display calcium activity across multiple model systems
Luisa Kües, Sophie Heuer, Sandra Horschitz, Ralph Sinn, Wolfgang Wick, Frank WinklerAbstract
Background
Diffuse midline gliomas (DMGs) are a highly aggressive and fatal subtype of paediatric-type diffuse high-grade gliomas, with tumour progression partially driven by intercellular signalling. In other brain tumour entities, most notably glioblastoma, extensive intercellular connectivity through tumour microtubes (TMs) – long protrusions linking tumour cells – and hierarchical rhythmic calcium signalling within these networks have been identified as key mechanisms of tumour progression and therapeutic resistance. However, whether these mechanisms of connectivity and communication extend to DMGs remains poorly understood.
Methods
TM-mediated intercellular networks were characterised using several patient-derived DMG cell lines across complementary in vitro and in vivo systems, including monolayers, spheroids, co-culture with human iPSC-derived cortical organoids and mouse xenograft models, with additional validation in human tumour specimens. Network structure and dynamics were assessed by immunostaining, live-cell calcium imaging, MRI-based tumour tracking and in vivo two-photon microscopy.
Results
DMG networks were found to consist of Nestin-positive TMs across in vitro cultures, mouse xenograft models and patient tumour samples. TM morphology and spatial organisation revealed intertumoral heterogeneity between patient-derived cell lines, with cell line–specific differences in protrusion length, branching and vascular association as well as radial glia-like, neuron-like, perivascular and OPC-like growth patterns preserved across experimental contexts. Longitudinal in vivo tracking of DMG infiltration over several weeks captured infiltrative single-cell migration patterns resembling those reported for glioblastoma. Functional calcium imaging demonstrated calcium activity within DMG networks, including calcium waves and rhythmic activity patterns, observable both in vitro and in vivo, supporting the presence of intercellular coupling.
Conclusions
Our findings provide a characterisation of TM-mediated network organisation and calcium activity in DMG across complementary model systems and highlight the potential for targeting these networks in future therapeutic strategies. Our ability to track these dynamics in vivo offers a powerful tool for understanding DMG tumour biology within the living brain.