Abstract 5478: From 2D to 3D: Advancing preclinical platforms in cancer models

Abstract Solid tumors account for about 90% of all adult human cancers. These tumors have a heterogeneous cellular composition that includes cancer cells, stromal cells, immune cells, and more. Developing effective treatments relies on experimental models that can recapitulate the original tumor and effectively predict clinical outcomes. Two-dimensional (2D) cell cultures have been extensively used as tools to predict promising outcomes before proceeding to more expensive animal studies. However, results from 2D models often fail to replicate in vivo conditions, necessitating the development of 3D multi-cellular spheroids that better mimic the physiology, architecture, and native microenvironment of parental tumors. In this study, we highlight the differences in drug response between 2D and 3D cancer cell line cultures and how 3D cultures better simulate the response observed in vivo.Human (MDA-MB-231) and mouse (EMT6 & 4T1) cancer cell lines were cultured in 2D as monolayers or in 3D utilizing a species- and histotype-specific extracellular matrices (ECM). Cultures were treated with increasing doses of small molecule drugs (Cisplatin, Paclitaxel, or Gemcitabine) and cell viability was quantified using CellTiter-GloTM luminescent cell viability assay and/or CelltoxTM Green viability dye along with serial imaging quantification. Half-maximal inhibitory concentration (IC50) curves were plotted and quantified using GraphPad PrismTM software.Microscopic imaging revealed morphological differences between 2D and 3D cultures. Unlike 2D cultures, 3D cultures formed either compact or loose spheroids, allowing for cell-cell and cell-matrix interactions that mimic tumor morphology. MDA-MB-231 and EMT6 breast cancer cells showed higher cytotoxicity to Cisplatin in 2D cultures compared to 3D cultures (IC50 of 3.5 µM for 2D and 13.47 µM for 3D; IC50 of 21.28 µM for 2D and 42.36 µM for 3D respectively). This corresponds to in vivo MDA-MB-231 and EMT6 tumors, which show slight tumor growth inhibition post Cisplatin treatment. However, 4T1 cancer cells showed similar Cisplatin cytotoxicity in both 2D and 3D cultures (IC50 of 4.21 µM and 5.73 µM respectively), which also aligns with in vivo response, where Cisplatin resulted in significant reduction in tumor volume. These in vitro results were further confirmed by viability analysis using serial imaging.These findings underscore the efficacy and predictability of this 3D platform in drug development. Not only does this platform help in decreasing the use of laboratory animals, but it also allows to reproduce key aspects of the tumor physiology and architecture. Consequently, 3D cultures have the potential to better predict treatment response and help identify drug resistance mechanisms. Citation Format: Hussein Chehade, Amber Rowse, David Draper, Anita J. Zaitouna, Daniel Saims, Sheri Barnes, Heidi Nielsen, Scott Wise. From 2D to 3D: Advancing preclinical platforms in cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5478.