Abstract 6465: Development of triple co-culture spheroid models of lung and ovarian carcinomas for in vitro evaluation of antitumor therapies

Tumor spheroids have emerged as superior in vitro models for studying solid tumors compared to monolayer cultures due to their ability to replicate key aspects of the tumor microenvironment (TME), including cell-cell interactions, extracellular matrix deposition, and nutrient and oxygen gradients. However, spheroids composed solely of tumor cells, i.e. monoculture spheroids, still lack the non-tumor cell components that contribute to additional in vivo TME complexity. This study focuses on developing triple co-culture spheroid models that integrate tumor cells, tissue-specific fibroblasts, and endothelial cells to better replicate the structural and functional features of the TME. Using an optimized liquid overlay technique, triple co-culture spheroids were successfully generated with drug-resistant lung and ovarian tumor cells. These spheroids demonstrated key tumor-like characteristics, such as extracellular matrix production and distinct spatial localization of cell types. Specifically, fibroblasts concentrated at the spheroid core during growth, while endothelial cells localized to the core only in the presence of fibroblasts. When treated with a liposomal formulation previously identified as selectively toxic to drug-resistant tumor cells in monolayer cultures, the triple co-culture spheroids exhibited reduced sensitivity to the treatment in a manner dependent on their composition. These results highlight the potential of triple co-culture spheroids as advanced in vitro models for investigating tumor biology and optimizing therapeutic strategies prior to in vivo evaluation. Citation Format: Rock Pulak, Shrey Shah, Yongwoon Kim, Gerard G. D’Souza. Development of triple co-culture spheroid models of lung and ovarian carcinomas for in vitro evaluation of antitumor therapies [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 6465.