Dose–response characterization and LD50 determination of X-ray–induced radiosensitivity in PC3-EMT Prostate Cancer cells

Purpose: Radiotherapy utilizes high-dose ionizing radiation to eliminate malignant cells. However, clinical efficacy is often dictated by the intrinsic biological response of the tumor rather than the physical dose alone. This study evaluates the impact of Epithelial-Mesenchymal Transition on radiation response by characterizing the dose-response relationship to determine the lethal dose, 50% (LD₅₀) of X-ray–induced radiosensitivity in a mesenchymal-like prostate cancer model (PC3-EMT) . Methods: Cells were irradiated using a MultiRad 350 cabinet irradiator at 130 kVp, 2 mA with a 0.5 mm Al filter (HVL ≈ 1.8 mm Al), delivering a dose rate of 3.279 Gy/min. To establish a reliable radiobiological profile, three independent biological replicates were performed with protocol optimizations and three technical replicates for each dose. The initial biological replicate (550 cells/well) utilized doses up to 4 Gy. Subsequent replicates optimized seeding densities (200–400 cells/well) and expanded dose ranges up to 8 Gy. The final biological replicate followed a direct-aliquot seeding protocol to maximize volumetric precision. Following a 14-day incubation, surviving fractions (SF) were normalized to plating efficiencies, and LD₅₀ values were determined via nonlinear regression analysis in Python. Results: All biological replicates demonstrated a consistent dose-dependent decrease in survival. The optimizations yielded highly reproducible LD₅₀ values: 2.25 (Replicate 1), 1.93 Gy (Replicate 2), and 2.24 Gy (Replicate 3). The low inter-experimental variance (s2=0.033) and coefficient variation of 8.50% confirms a stable radiosensitivity profile despite variations in seeding protocols. Pooled analysis indicates a moderate response at low doses (0–1 Gy), followed by a significant decline in proliferative capacity at doses ≥2 Gy. Conclusion: The findings quantitatively characterize the intrinsic radiosensitivity of PC3-EMT cells, with a consolidated LD₅₀ of 2.14 ± 0.18 Gy. The consistent reproducibility of LD₅₀ across the three biological replicates establishes a baseline for future research into EMT-driven radiation resistance in prostate cancer.