Molecular profiling uncovers KCa2.3 as key driver of pacemaker cells in glioblastoma.

Abstract

BACKGROUND: Glioblastoma is orchestrated by pacemaker cancer cells, a rare population preferentially residing at the hubs of tumor cell networks. These cells autonomously generate periodic Ca2+ oscillations that drive glioblastoma network function, resilience, and disease progression. To date, the Ca2+-activated K+ channel KCa3.1 is the only known driver of pacemaker function, calling for studies to identify additional factors - to better understand a key component of glioblastoma pathobiology, and uncover novel therapeutic targets. METHODS: A molecular Ca2+ integrator technology enabled the transcriptional profiling of highly Ca2+-active glioblastoma cells, a population enriched for pacemaker cells. Putative regulators of pacemaker function were interrogated using genetic, pharmacological, and computational approaches across complementary glioblastoma models. RESULTS: We identified a distinct transcriptional program that captures highly Ca2+ active glioblastoma cells, including pacemaker cells, across preclinical models and human glioblastoma specimens. This program comprised upregulated cholesterol biosynthesis enzymes, TM-network markers (Aquaporin-4 and Cystatin C), and the Ca2+-activated K+ channels KCa1.1 and KCa2.3. Amongst these components, in silico modeling identified KCa2.3 as the only channel compatible with executing pacemaker function. Consistently, KCa2.3 expression was increased in pacemaker cells, and both genetic and pharmacological perturbation of KCa2.3 selectively disrupted pacemaker function, resulting in reduced network-wide Ca2+ activity. Ultimately, pharmacological inhibition of KCa2.3 with edelfosine reduced tumor burden in human brain spheroids. CONCLUSIONS: We present the first experimental approach to characterize molecular factors of glioblastoma pacemaker cells in a broad and unbiased manner, and identify KCa2.3 as a novel pacemaker gene and promising therapeutic target. In glioblastoma, a brain cancer that is hard to treat, tumor cells connect into networks. A few "pacemaker" cells set the rhythm of these networks and help the tumor grow. We asked what drives these rare but important cells. We tagged the most active tumor cells, read their genes, and tested the best candidates. A channel called KCa2.3 stood out. Removing it, or blocking it with the drug edelfosine, reduced pacemaker activity and lowered tumor growth in lab-grown human brain tissue. Edelfosine has already been given to people, so it is a realistic candidate for further clinical research.

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