Evaluation of Estrogenic and Anti-Estrogenic Activity of Endocrine Disruptors using Breast Cancer Spheroids: A Comparative Study of T47D and MCF7 Cell Lines in 2D and 3D Models

Katia Barbaro ^1* Elisa Innocenzi ¹ Valentina Monteleone ¹ Daniele Marcoccia ¹ Annalisa Altigeri ¹ Alessia Zepparoni ¹ Daniela Caciolo ¹ Cristian Alimonti ¹ Marta Mollari ¹ Paola Ghisellini ² Cristina Rando ² Roberto Eggenhoffner ² Maria Teresa Scicluna ¹

• ¹ Institute of Experimental Zooprophylactic of the Lazio and Tuscany Regions (IZSLT), Rome, Lazio, Italy
• ² Department of Integrated Surgical and Diagnostic Sciences, School of Medical and Pharmaceutical Sciences, University of Genoa, Genova, Italy

The estrogenic and anti-estrogenic effects of endocrine disruptors were examined in vitro using twodimensional 2D and three-dimensional 3D estrogen receptor-positive T47D and MCF7 human breast cancer cells. The in vitro model system was used to test the plasticizer Bisphenol A (BPA), a known endocrine disruptor (EDs) with estrogen-like action, aga inst 17-Estradiol (E2), the endogenous nuclear estrogen receptor (nERs) ligand, and the anti-estrogenic drug Fulvestrant (FUL). Spheroid formation and gene expression of estrogen-regulated markers (pS2 and TGFβ3) both in 2D and 3D cultures were used to establish the dose-dependent cellular effects of these substances, evaluated cell viability either by separately treating with the individual substances or in co-treatment. BPA exhibited a dose-dependent estrogenic activity in both 2D and 3D cultures, significantly influencing cell proliferation and gene expression of estrogen-regulated markers (pS2 and TGFβ3). In contrast, FUL displayed anti-estrogenic properties, effectively inhibiting the proliferative effects of E2, thereby highlighting the complex interactions between these compounds and the nERs pathways in human breast cancer cells. Our findings indicate that E2 and BPA significantly increase pS2 expression while decreasing TGFβ3, and that FUL co-treatment reverses these effects. Therefore, the in vitro model system could serve to observe the cell-mediated effects caused by the interaction of EDs with nERs.Through the use of these in vitro model systems both -2D and especially 3D, the latter of which allow better emulation of complex physiological and pathological processes occurring in vivo, the effects caused by EDs on nERs pathways can be detected and studied under various conditions. This approach could be used as a preliminary screening test for the determination of substances with similar estrogenic action and therefore in substitution of the use of experimentation conducted on animals, as well as being adopted as a possible model for better environmental and health risk assessment. This approach performs as a preliminary screening tool to identify estrogenic substances, offering the potential to reduce reliance on in vivo experiments and contributing to improved environmental and health risk assessments.