CNSC-05. Exploration of intercellular networks in ependymoma to reveal potential drug vulnerabilities

Aggressive ependymoma frequently relapses, demonstrating a high degree of resistance to both radiotherapy and chemotherapy. Tumor-tumor cell networks formed by membrane protrusions, such as tunneling nanotubes (TNTs) or tumor microtubes (TMs), have been implicated in therapy resistance in other glial brain malignancies. Our previous findings suggested that enhancer-­regulated, calcium-mediated intercellular communication is critical for ependymoma tumorigenesis. This study aims to further characterize intratumoral interactions and investigate the role of tumor cell connections in mediating therapy resistance of aggressive posterior fossa group A (PFA) and ZFTA fusion-positive supratentorial (ZFTA) ependymomas. Intercellular connections were examined using scanning electron microscopy, immunofluorescence, and ultra-high content imaging in tumor models and patient-derived tumor tissues. Transcriptomic and proteomic datasets were analyzed to identify targetable vulnerabilities within these networks. Ependymoma transcriptomic data revealed increased expression of connectivity-related terms, such as microtube-based movement and microtube bundle formation, compared to non-malignant control tissues. Morphological analysis showed networks of ependymoma tumor cells formed by both TM- and TNT-like structures, with TNT-like structures being more prominent in PFA compared to ZFTA. High levels of nestin-positive neuronal protrusions were observed in cell lines, mouse models, and human tumor tissue from both PFA and ZFTA subtypes. Particularly, the gap junction protein connexin 43, known to mediate tumor network communication via TMs in glioblastoma, was found to be highly upregulated at both RNA and protein levels in ependymoma, especially in the PFA subtype. Treatment with meclofenamate, an inhibitor of gap junctions, disrupted these networks and enhanced sensitivity to irradiation, as demonstrated by live-cell imaging and by luminescent ATP quantification in vitro. These findings highlight the critical role of gap junction-coupled networks in the intratumoral communication aggressive ependymomas. Targeted disruption of ependymoma-specific intercellular networks offers a promising avenue for overcoming therapy resistance and improving treatment outcomes.