P01.47.A IMPACT OF CHOLESTEROL SYNTHESIS INHIBITION IN G7 GLIOBLASTOMA STEM-LIKE CELLS CULTURED AS 3D SPHEROIDS

Abstract BACKGROUND Glioblastoma (GBM) stem-like cells (GSCs) are a treatment-resistant subpopulation of cells that underlie tumour recurrence. Emerging evidence indicates that GSCs have elevated cholesterol synthesis rates and may confer protection against standard-of-care therapies. We investigated the effects of inhibiting cholesterol synthesis at two points: upstream via simvastatin, an HMG-CoA reductase (HMGCR) inhibitor, and downstream via AY9944, which targets dehydrocholesterol 7-reductase (DHCR7). MATERIAL AND METHODS Patient-derived G7 GSC spheroids were treated with simvastatin or AY9944 across a concentration range of 10 to 0.316 µmol/L with or without cholesterol supplementation. DMSO was used as vehicle control. Spheroids were monitored over 14 days, measured using FIJI (Image J), and statistically analysed using a two-way ANOVA. Spheroid viability was assessed using calcein-AM and propidium iodide to label live/dead cells, respectively, followed by imaging with confocal microscopy, and analysed using a one-way ANOVA. To evaluate GSC frequency, a tumoursphere formation assay using extreme limiting dilution analysis was performed after 21 days of treatment at a concentration range of 3.16 to 0.316 to µmol/L. RESULTS Simvastatin reduced G7 spheroid proliferation in a concentration-dependent manner, ranging from 28% (95% CI: 3.35, 53.25; p.adj < 0.05) at 0.316 µmol/L to 60% (95% CI: 34.92, 84.81; p.adj < 0.001) at 10 µmol/L, compared to vehicle-treated controls. Cholesterol supplementation partially rescued spheroid proliferation at lower concentrations (0.316 and 1 µmol/L; p.adj > 0.05), but not at 3.16 or 10 µmol/L (p.adj < 0.001). Simvastatin did not affect spheroid viability at 3.16 or 10 µmol/L at day 14, but GSC frequency was reduced compared to vehicle treated GSCs (0.194, 0.114, 0.074, 0.045 for DMSO, 0.316, 1 and 3.16 µmol/L, respectively; p < 0.001). AY9944 decreased G7 spheroid proliferation and viability but exhibited lower potency than simvastatin. While 10 µmol/L reduced spheroid fold-change by 50.66% (95% CI: 20.28, 81.03; p < 0.001), 3.16 and 10 µmol/L decreased spheroid viability by 50% (95% CI: 32.92, 69.66; p.adj < 0.05) and 62% (95% CI: 43.74, 80.47; p.adj < 0.001), respectively, at day 14. These effects were rescued by cholesterol supplementation (p.adj < 0.05). AY9944 reduced GSC frequency at all tested concentrations compared to vehicle treated GSCs (0.194, 0.124, 0.097, 0.020 for DMSO, 0.316, 1 and 3.16 µmol/L, respectively; p < 0.001). CONCLUSION Cholesterol synthesis inhibition reduces G7 spheroid proliferation and self-renewal. Upstream inhibition exerts cytostatic, partially cholesterol-independent effects while downstream inhibition is cytotoxic and cholesterol-dependent. These findings highlight HMGCR and DHCR7 inhibition as potential treatment targets against GSC-driven GBM recurrence.