Os05.1.a Molecular Characterization of Pacemaker Cells in Glioblastoma

BACKGROUND Glioblastoma form resistant, multicellular networks driven by a small subset of autonomous, rhythmically and highly active Ca2+ pacemaker cells. Elimination of pacemaker cells - via inhibition of the potassium channel KCa3.1 - disrupts global network activity, impairs tumor growth in mice, and prolongs survival. This dependence on pacemaker cells represents a therapeutic vulnerability. However, KCa3.1 remains the only identified molecular marker and drug target of these cells. MATERIAL AND METHODS We used Caprola, a novel genetic sensor for longitudinal recording of Ca2+ activity with single-cell resolution, that allows transcriptomic profiling within heterogeneous populations. By overlaying the genetic signatures of two established pacemaker cell characteristics - highly Ca2+-active cells identified by Caprola and KCa3.1-positive cells found in human glioblastoma datasets - we aimed to achieve a molecular characterization of pacemaker cells. Highly upregulated and pharmacologically targetable molecules were validated using Ca2+ imaging, viability, proliferation, and morphological assays in vitro and in human brain cerebral organoids. RESULTS As shown before, the top 5% glioblastoma cells with the highest Ca2+ activity showed increased coactivity and were enriched with pacemaker cells. Caprola-enabled identification of these cells confirmed increased connectivity- and calcium-signaling-signatures, with upregulation of KCa3.1 expression. Crucially, the pacemaker-enriched population exhibited highly upregulated expression of novel, druggable targets: the calcium-activated potassium channels KCa1.1 and KCa2.3, as well as the water channel Aquaporin 4 (AQP4). Pharmacological inhibition of these targets significantly reduced proliferation in two patient-derived glioblastoma cell lines at low molecular concentrations without compromising tumor cell viability. AQP4 inhibition led to reduced global Ca2+ activity and coactivity without primarily affecting pacemaker cell activity, suggesting that its antiproliferative effect results from broader disruption of calcium signaling or network formation rather than direct pacemaker interference. In contrast, inhibition of KCa1.1 and KCa2.3 selectively and strongly suppressed pacemaker activity, leading to a pronounced decrease in global Ca2+ signaling and tumor network communication. It also reduced pacemaker cell frequency, disrupting the tumor’s distinct rhythmic activity and its frequency-depended activation of MAPK and NF-κB pathways, key drivers of glioblastoma proliferation and survival. CONCLUSION We identified KCa1.1 and KCa2.3 as novel, druggable markers of glioblastoma pacemaker cells. Given the tumor network’s susceptibility to pacemaker cell disruption, our findings reveal promising targeted therapeutics against the resistant glioblastoma network.