3D cell culture is a highly effective method to collect breast cancer stem cells in vitro

Introduction: Breast cancer continues to be one of the most common malignancies in females, with mortality rates among the highest worldwide. Despite the availability of numerous clinical tools for managing breast cancer, recurrence, metastasis, and drug resistance remain significant barriers for clinical experts. Scientists consider these tumorigenic processes to be closely associated with breast cancer stem cells (BCSCs). While extensive research focuses on breast cancer cell lines in vitro, replicating the intricate dynamics of the internal tumor microenvironment remains a challenge. This microenvironment is influenced not only by biochemical factors but also by biomechanical cues, such as ECM stiffness and shear stress, which regulate cancer cell behavior through mechanotransduction pathways. Recognizing these limitations, our team has drawn upon years of cancer stem cell research to establish a practicable method. This method aims to facilitate a series of experiments exploring drug resistance mechanisms and to provide deeper insights into the role of BCSCs in tumorigenesis and progression in vivo. Materials and methods: 3-dimensional (3D) mammosphere culture method was established to enrich the breast cancer stem-like cells in vitro. Mammospheres forming assay was performed and mammosphere forming efficiency was calculated. Flow cytometry Analysis was used to detect the subpopulation of CD44+CD24−, meanwhile, the biomarkers of BCSCs were detected by western blot. All these indicates the success establishment of 3D cell culture method and the breast cancer stem-like cells are enriched and collected. Furtherly, western blots were performed to detect the conjectures of the BCSCs that the Notch signaling pathway and MAPK-ERK signaling have the crosstalk in breast cancer microenvironment and the positive feedback loops could be activated by the enriching of BCSCs. All these data were analyzed with GraphPad® Prism 9 software and Wilcoxon rank-sum test, nonlinear regression analysis, unpaired t tests were used. Results: MCF-7 breast cancer stem-like cells were observed as substantially distinct from native breast cell lines under 20x microscope and the mammosphere forming efficiency of MCF-7 breast cancer stem-like cells were higher than the native MCF-7 group. The subpopulation of CD44+CD24− was significantly increased in BCSC-like group and the EMT (Epithelial-Mesenchymal Transition) markers of BCSC which includes Nanog; Vimentin; OCT3/4; Slug and Sox2 were significantly increased. Lastly, Cyclin D3 and Hes1 which play important roles in the Notch signaling pathway and ERK protein were all significantly increased. Conclusion: The three-dimensional (3D) mammosphere culture method is a highly effective approach for collecting breast cancer stem cells (BCSCs) in vitro. Unlike traditional 2D cultures, this method replicates key physiological conditions of the tumor microenvironment (TME) and captures phenotypic heterogeneity. By promoting cell-cell and cell-ECM interactions, the 3D system mimics essential biomechanical cues, such as ECM stiffness and spatial gradients, which regulate BCSC behavior. This method reliably supports investigations into the molecular mechanisms of tumorigenesis. BCSCs enriched through this approach drive processes such as epithelial-mesenchymal transition (EMT) and activate signaling pathways like Notch and MAPK-ERK, which are closely linked to the TME and play critical roles in tumor progression and resistance. The 3D mammosphere culture method thus provides a robust tool for advancing our understanding of cancer biology and therapeutic development.