Spheroid cultures reveal cancer stem cell enrichment and distinct chemotherapeutic response in triple-negative breast cancer

Three-dimensional (3D) cancer spheroid models provide physiologically relevant platforms for studying tumor biology and therapeutic response. Using the liquid overlay method, we optimized conditions for reproducible spheroid generation from MDA-MB-231 breast cancer cells and extended this approach to 10 human cancer cell lines. Growth outcomes varied, yielding compact spheroids, loose aggregates, or no spheroids, with compactness strongly associated with high breast cancer stem cell (BCSC) content (≥20%). Transcriptomic profiling revealed distinct gene expression programs between spheroid-forming and non-forming cells, highlighting pathways in extracellular matrix remodeling, differentiation, and developmental lineage specification. Notably, PROM1, HOXB4, BMP5, and TENM4 emerged as key regulators, with TENM4 consistently upregulated across compact spheroid models. Drug response assays demonstrated increased chemoresistance in 3D spheroids compared to 2D cultures, with triple-negative breast cancer (TNBC) spheroids exhibiting reduced sensitivity to bortezomib+nedaplatin despite context-dependent synergy. Collectively, these findings establish optimized experimental parameters for spheroid culture, define molecular features linked to spheroid formation and stemness, and underscore the importance of 3D models for evaluating therapeutic efficacy.