Preclinical evaluation of progesterone combined with EDP-M scheme in 2D and 3D models of adrenocortical carcinoma

Background: Adrenocortical carcinoma (ACC) is a rare malignancy with limited therapeutic options. The current standard for advanced disease, the EDP-M regimen (etoposide, doxorubicin, cisplatin, plus mitotane), demonstrates modest efficacy and substantial toxicity, underscoring the need for improved strategies. Previous data from our group suggest that progesterone (Pg) has antitumor activity in ACC. Megestrol acetate, a Pg analog used in supportive oncology, may enhance both efficacy and tolerability of EDP-M. This study investigated the effects of combining Pg with EDP-M in preclinical ACC models (2D and 3D). Methods: Two ACC cell lines (NCI-H295R, TVBF-7) and three primary cultures (ACC-172, ACC-173, ACC-174) were exposed for 96 h to etoposide, doxorubicin, cisplatin and mitotane to establish IC50 values. Pg (7.5-100 μM) was then tested alone or in combination with EDP-M (1/12 ×, 1/6 ×, 1/4 ×, 1/2 × of IC50) in both 2D and 3D cultures. 3D spheroid models were further characterized by Steroidogenic Factor 1 expression and hormone secretion. Results: EDP-M and Pg each demonstrated concentration-dependent cytotoxicity. Pg at concentrations < 50 μM showed minimal additional benefit to EDP-M, except at higher EDP-M doses (1/2 IC50). By contrast, Pg ≥ 50 μM significantly enhanced EDP-M cytotoxicity across models (p < 0.001). In some 3D models, higher EDP-M concentrations (IC50 and 2 × IC50) were required to observe a similar effect, emphasizing the translational relevance of 3D models. Conclusions: Overall, these findings confirm the potential role of Pg in improving EDP-M cytotoxic effects in 2D and 3D ACC models.