Development of tetraculture spheroids as a versatile 3D model for personalized breast cancer research.

The tumor microenvironment (TME) plays a pivotal role in cancer progression and drug resistance, influenced by the interaction of tumor cells with surrounding fibroblasts, immune, and endothelial cells. Developing robust multicellular tumor spheroids (MCTSs) that mimic the tumor microenvironment is crucial for studying cancer progression and therapeutic resistance. This study aimed to establish a reproducible method for generating MCTSs using a tetraculture system in four breast cancer cell lines: BT474, T47D, MDA-MB-231, and SK-BR-3. This approach incorporates primary cancer-associated fibroblasts (CAFs), macrophages (THP-1), and endothelial cells (Ea.hy926) alongside the cancer cells. MCTSs were generated using a simple method on ultra-low attachment plates, ensuring spheroid viability and uniformity across cell lines, confirmed by immunofluorescence and immunohistochemistry. MCTSs underwent extensive characterization, including invasion pattern analysis, macrophage polarization potential, cytotoxicity assay to assess chemotherapeutic resistance, and gene expression analysis to explore extracellular matrix (ECM) remodeling. The spheroids exhibited distinct morphologies, growth patterns, and cell distributions, reflecting unique microenvironment interactions and providing a reliable platform for studying TME. This versatile 3D model offers a promising platform for personalized therapy design, as it enables the incorporation of patient-derived cells regardless of tumor phenotype or inter-patient variability. Including key elements of the tumor microenvironment supports individualized drug testing and functional analysis, serving as a reproducible and ethically favorable alternative to animal models and patient-derived explants.