Disrupting glioblastoma progression through connexin hemichannels inhibition

Glioblastoma multiforme (GBM) is one of the most aggressive primary brain tumors, characterized by extreme invasiveness, high cellular heterogeneity, and limited therapeutic options. The poor prognosis underscores the urgent need for novel therapies, while a deeper understanding of glioma–neuron interactions and glioma-driven signaling within the tumor microenvironment is critical to uncover mechanisms sustaining tumor growth and comorbidities such as epilepsy. Connexin (Cx) hemichannels (HCs) have been implicated in GBM progression by mediating the release of gliotransmitters such as ATP and glutamate, thereby enhancing proliferation, invasiveness, and resistance to apoptosis. Here, we evaluated abEC1.1, a monoclonal antibody that selectively inhibits Cx26, Cx30, and Cx32 hemichannels with nanomolar potency, previously shown to be effective in mouse models of HC-related diseases. Mice bearing intracranial GL261 gliomas were treated with abEC1.1 through multiple strategies, including AAV-mediated gene transfer and intra-tumoral convection-enhanced delivery. Using these approaches, we demonstrated that abEC1.1 achieves widespread brain distribution, co-localizes with Cx26 in the tumor periphery, reduces glioma volume, decreases invasiveness and proliferation, and extends survival. In parallel, we developed and validated an in vivo multiphoton imaging system capable of simultaneously monitoring cytosolic Ca²⁺ and extracellular glutamate dynamics in GBM cells engineered to express the cytosolic Ca²⁺ indicator Rex-GECO1 and the membrane-bound glutamate biosensor iGluSnFr, together with Ca²⁺ activity in cortical neurons expressing jGCaMP7b. This system was combined with electroencephalographic (EEG) recordings to assess peritumoral neuronal function. Preliminary data in AAV8-abEC1.1–treated mice implanted with engineered GL261 cells demonstrate the utility of this platform for evaluating pharmacological effects in intercellular signalling in GBM complex microenvironment and they indicate that Cx HC blockade by abEC1.1 may reduce neuronal hyperexcitability in GL261-implanted animals. These findings support the therapeutic potential of abEC1.1 for GBM to counteract tumor progression and associated epilepsy.