Combined Effect of Osilodrostat and Mitotane in Adrenocortical Carcinoma 3D models

Systemic treatment of adrenocortical carcinoma (ACC) relies on mitotane. Osilodrostat, an 11β-hydroxylase inhibitor used to treat Cushing’s syndrome, is also used to manage the ACC-associated hypercortisolism. Currently, the combination of osilodrostat and mitotane remains poorly investigated, even though it represents a treatment strategy in ACC with combined hypercortisolism. We assessed the effects of osilodrostat and mitotane, both individually and in combination, using the NCI-H295R cell line in two-dimensional (2D) cultures and three-dimensional (3D) spheroid models, which more closely recapitulate the in vivo tumour microenvironment. Osilodrostat showed no significant cytotoxicity in either the 2D or 3D models (IC₅₀ >100 μM). By contrast, mitotane reduced cell viability in a dose-dependent way, with IC₅₀ values of 25.92 μM in 2D and 44.98 μM in 3D, indicating lower sensitivity in the 3D context. Drug interaction was studied using a dose-dose matrix and the Highest Single Agent (HSA) model. In 3D models, the drug combination produced greater inhibition of cell viability than mitotane alone; at 50 μM mitotane, inhibition increased from 21.84% (alone) to 42.89% (combined with 100 μM osilodrostat). The HSA analysis showed a mean synergy score of 5.97 (P = 1.35 × 10⁻⁶), reaching a maximum score of 18.12 for the combination of 50 μM mitotane and 100 μM osilodrostat. Morphological analysis also showed a significant reduction in spheroid area after the administration of the drug combination compared to mitotane alone at 50 μM (−45.6% vs −21.2%; P = .0076). Moreover, calcein AM/DAPI fluorescent staining revealed a significant increase in the ratio of non-viable to viable cells (MFIb/g from 0.84 with the combination to 1.79 with 50 μM of mitotane; P < .0001). Cell counting confirmed a 72% reduction in viability with the combination, compared with a 46% reduction observed with 50 μM mitotane alone. Docking analysis of CYP11B1 identified a primary catalytic site (C1) for both compounds. However, they displayed distinct orientations and steric incompatibilities that precluded simultaneous binding to the same site. Sequential docking revealed an alternative, lower-affinity cavity (C4) for mitotane when osilodrostat occupied C1, suggesting a potential concentration-dependent multi-site effect. Overall, osilodrostat does not exert a direct cytotoxic effect but significantly enhances mitotane’s adrenolytic activity in 3D ACC models. These findings provide a preclinical rationale for combination strategies and support further translational and clinical studies integrating steroidogenesis inhibition with adrenolytic therapy in ACC.