3083 - Inhibition of Cholinergic Signaling Enhances Radiation Treatment Efficacy in Glioblastoma

Purpose/Objective(s):

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor in adults with a median survival of less than two years due to tumor recurrence despite treatment with surgery, chemotherapy, and radiation. Glioblastoma stem cells (GSCs) are thought to be one of the drivers of tumor recurrence and often, treatment resistance. Understanding the mechanisms that promote GSC resistance is imperative for developing targeted therapies to improve patient outcomes.

GBMs occur in brain regions rich in acetylcholine (ACh). ACh has been shown to regulate stemness characteristics in normal oligodendrocyte progenitor cells, suggesting a potential role in tumor biology. ACh signals through muscarinic acetylcholine receptors (mAChRs). The M3 mAChR subtype is overexpressed in some GBMs and is identified as a potential poor prognostic marker. However, the specific impact of M3 mAChR signaling on radiation resistance in GBM remains poorly understood. We hypothesized that targeting ACh signaling via M3R using benztropine (BZT), a muscarinic antagonist, may lead to radiation sensitization of GBM.

Materials/Methods:

Human GBM LN-229 cells were maintained under conditions that promote stem cell characteristics, and CRISPR/Cas9 was used to create a knockout cell line for the M3 mAChR. To assess the influence of ACh on stemness, cells were treated with either vehicle control or carbachol, an ACh mimetic. Sphere formation assays and immunoblotting for stem-like markers (nestin, vimentin, and CD44) were performed. To evaluate whether M3 mAChR inhibition and radiation could reduce stemness and tumor cell viability, cells were treated with BZT at 1 µM and 3 µM. Cells were then given a radiation fraction of 0 Gy, 4 Gy, 8 Gy, or 10 Gy. MTT assays, cell viability assessments, and immunoblotting were conducted. All experiments were performed in triplicate.

Results:

Carbachol treatment significantly increased the number of spheres (mean: 34 vs. 20, p = 0.0141), and the size of spheres (mean: 192 µM vs. 100 µM, p < 0.0001) compared to vehicle control. Immunoblotting after carbachol treatment demonstrated an increase in M3 mAChR expression and stemness markers. The M3 mAChR knockout cell line exhibited decreased cell viability after 48 hours of treatment compared to wildtype control (p < 0.0001) and reduced stem cell markers levels. BZT treatment significantly reduced cell viability compared to vehicle control in the MTT assay and cell viability counts at both concentrations (1 µM: p < 0.0001; 3 µM: p < 0.0001). There was also a significant decrease in cell viability, sphere-forming ability, and stemness markers of cells treated with BZT and radiation (p < 0.001).

Conclusion:

This study indicates that ACh signaling via M3 mAChR promotes stemness in GBM. Disruption of cholinergic signaling by knocking out the M3 receptor decreases stemness and tumor cell viability. Pharmacological inhibition of cholinergic signaling using BZT may provide a promising strategy to target GSCs, aiming to improve patient outcomes.