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

Abstract

Three-dimensional (3D) cancer spheroids mimic key morphological and biological features of solid tumors, providing a valuable model for evaluating drug responses. Here, we investigated whether intrinsic cancer stem cell composition and tumor subtype determine spheroid formation capacity, transcriptional profiling, and therapeutic response in breast cancer cell lines. Using the liquid overlay technique, we optimized conditions for reproducible spheroid generation using 10 human cancer cell lines derived from breast cancer, glioblastoma, and malignant melanoma. Spheroid morphology and growth dynamics were assessed over a 20-day period, while breast cancer stem cell abundance was evaluated in 2D monolayer cultures and 3D spheroids using immunohistochemistry and flow cytometry. RNA sequencing (RNA-seq) transcriptomic profiling and drug responses to bortezomib+nedaplatin following 24- and 72-hour exposure were compared between both culture systems. Optimized conditions yielded compact spheroids (5/10 cell lines), loose aggregates (3/10), and no spheroid formation (2/10). Compact spheroids (mean ± SEM, 576 ± 18 μm) and those derived from triple-negative breast cancers (TNBCs; 550 ± 19 μm) were significantly smaller than loose aggregates (822 ± 36 μm) and non-TNBC-derived spheroids (934 ± 41 μm), respectively. RNA-seq demonstrated that transcriptional variation was primarily driven by intrinsic differences between cell lines rather than culture conditions. However, spheroid-forming cells induced distinct gene expression patterns enriched in extracellular matrix remodeling, developmental pathways, and stemness-related genes. In total, 1478 differentially expressed genes were associated with spheroid formation. TENM4 (Teneurin Transmembrane Protein 4) was the only consistently deregulated gene in both spheroid-forming breast cancer models, showing up to 4.9-fold and 2.6-fold higher expression in HCC1806 and BT-474 spheroids, respectively. Consistent with the transcriptomic findings, immunohistochemical analysis demonstrated increased TENM4 protein expression, predominantly localized to the spheroid cores. Drug screening further demonstrated increased chemoresistance in 3D spheroids, with up to 7.5-fold higher viability in TNBC spheroids following bortezomib+nedaplatin treatment. Taken together, these findings highlight molecular and cellular determinants of spheroid formation in breast cancer cell lines, including stemness-associated features and upregulation of TENM4. Our results further demonstrate that intrinsic tumor subtype shapes spheroid morphology, transcriptional profiles, and drug response, providing novel insight into the mechanisms underlying 3D growth and chemoresistance that may help inform future therapeutic strategies.