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From Luminal to Triple Negative: 3D Spheroids Reveal Molecular and Phenotypic Differences Across Breast Cancer Subtypes

by Maria Miguel Castro, Leticia Maretti, Catarina Esquível, Bárbara Sousa, Carmen Jerónimo, Andrew J Ewald, and Joana Paredes

Posted: March 31, 2026
Server: Preprints.org
DOI: 10.20944/preprints202603.2418.v1

Breast cancer is classified into distinct molecular subtypes, including Luminal A, Lu-minal B, HER2-enriched, Basal-like, and Claudin-low. While traditional studies mostly use 2D cell cultures, 3D models better mimic in vivo tumour conditions. In this study, we generated and characterized 3D multicellular tumour spheroids (MCTS) from breast cancer cell lines representing different molecular subtypes. Morphologically, spheroids were either compact (MCF-7/AZ, T47D, BT474, MDA-IBC-3, BT-20, SUM149PT) or loosely adhered (MDA-MB-468, SK-BR-3, MDA-MB-231), while retain-ing key parental subtype biomarkers. Cell viability decreased with increasing spheroid size, but apoptotic cCasp3 staining was restricted to basal-like spheroids. Compact spheroids expressed E- and/or P-cadherin, indicating epithelial or epithelial–mesenchymal transition (EMT) hybrid traits, while loose spheroids showed vimentin expression linked to a mesenchymal phenotype. Overall, EMT status, rather than mo-lecular subtype, primarily determined spheroid morphology.

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