Optimization of 3D Spheroid Models of Ovarian, Breast, and Gastric Cancer for Comparative Screening of Drug Candidates

Abstract Three-dimensional (3D) spheroid models are increasingly used in cancer research because they reproduce several features of solid tumors that are not represented in conventional two-dimensional (2D) monolayer cultures. In the present study, we developed and optimized spheroid culture conditions for a panel of breast, ovarian, and gastric cancer cell lines grown on low-attachment plates. Spheroid formation, morphology, and growth behavior were assessed during culture, and the resulting models were used for the comparative testing of reference anticancer agents. The established assay framework was further applied to newly developed cyclin-dependent kinase 7 (CDK7) inhibitor candidates from our in-house compound library. Anticancer activity was evaluated by using AlamarBlue or PrestoBlue viability assays after prolonged drug exposure. Reproducible spheroid formation conditions were identified for the studied cell lines, although notable cell line-specific differences in morphology, growth behavior, and assay duration were observed. Comparative drug testing revealed differences in apparent sensitivity between 2D and 3D cultures depending on the cell line and compound. Several tested CDK7 inhibitor candidates reduced viability in the spheroid models, with iCDK7_1 showing the strongest activity across the evaluated panel. Overall, the study describes and applies a cell line-adapted 3D spheroid assay workflow for comparative evaluation of antiproliferative effects in breast, ovarian, and gastric cancer models.