Triple co-culture spheroid models of lung and ovarian carcinoma cell lines for the in vitro testing of antitumor liposomes

Tumor cells cultured as spheroids have been shown to be superior to tumor cells cultured in monolayers as in vitro models of solid tumors because they exhibit features of the tumor microenvironment (TME) such as cell-cell interactions, extracellular matrix and diffusional 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, explored the development of triple co-culture spheroid models incorporating tumor cells, tissue specific fibroblasts, and endothelial cells to mimic more of the features of the in vivo TME. Using a modified liquid overlay technique, triple co-culture spheroids were successfully generated for both drug resistant lung tumor cells as well as drug resistant ovarian tumor cells. The triple co-culture models exhibited several characteristics of in vivo tumors, including extracellular matrix (ECM) production and distinct spatial locations of cell types. Notably, fibroblasts remained in the core as the spheroid grew, while endothelial cells were found in the core only in the presence of fibroblasts. A liposomal formulation previously shown in monolayer cultures to have selective toxicity toward multiple drug resistant tumor cell types was significantly less toxic and showed composition-dependent levels of toxicity in spheroid cultures with multiple cell types. These findings demonstrate that triple co-culture spheroids can serve as in vitro models that more closely mimic in vivo tumor characteristics to facilitate the optimization of antitumor therapies prior to in vivo testing.