EXPLORATION OF INTERCELLULAR NETWORKS IN EPENDYMOMA REVEALS POTENTIAL THERAPEUTIC VULNERABILITIES

BACKGROUND Aggressive ependymoma (EPN) exhibit primary and secondary radio- and chemotherapy resistance with the poorest prognosis in the pediatric groups PF-EPN-A and ST-EPN-ZFTA. Recently, tumor cell networks formed by membrane protrusions, such as tunneling nanotubes (TNT) or tumor microtubes (TM), have been identified in other glial brain tumors. These structures facilitate intercellular hierarchical Ca2+-communication and contribute to therapy resistance. Previous data from our group suggested enhancer-regulated Ca2+-communication to be essential in EPN. This study aims to further characterize intratumoral interactions and to understand potential correlations between tumor cell connections and treatment resistance in EPN. METHODS Intercellular connections in EPN patients and model systems were characterized by scanning electron microscopy, immunofluorescence and ultra-high content imaging. Transcriptomic and proteomic data were analyzed to identify potential targetable vulnerabilities within network dynamics. Candidates were functionally validated in cell culture models applying Ca2+ live cell imaging. RESULTS Connectivity-related terms such as microtube-based movement or microtube bundle formation showed increased expression in EPN transcriptome data compared to healthy controls. EPN networks were found to be build of TM- and TNT-like structures with a higher abundance of TNT-like structures in PF-EPN-A compared to ST-EPN-ZFTA. All networks showed strong Nestin-positivity in cells, mouse models and human tumor tissue of ST-EPN-ZFTA and PF-EPN-A. Thrombospondin-1, a matricellular protein enhancing TMs in glioma cells, was strongly enriched in ST-EPN-ZFTA patients. The gap junction channel connexin 43, connecting glioma cells, was highly upregulated in PF-EPN-A. Active electrochemical communication was observed between ST-EPN-ZFTA cells performing Ca2+ imaging. Moreover, inhibition of T-type Ca2+ channels impaired intercellular signaling, leading to network perturbation in vitro. CONCLUSIONS Our study revealed the importance of gap junction-coupled networks for communication of aggressive EPN cells. Targeted disconnection of the EPN-specific network as demonstrated for Ca2+ channel inhibition suggests a new avenue for therapeutic strategies in EPN to overcome resistance.