Intratumor heterogeneity of breast cancer is driven by extrinsic factors from the tumor microenvironment (TME) as well as tumor cell–intrinsic parameters including genetic, epigenetic, and transcriptomic traits. The extracellular matrix (ECM), a major structural component of the TME, impacts every stage of tumorigenesis by providing necessary biochemical and biomechanical cues that are major regulators of cell shape/architecture, stiffness, cell proliferation, survival, invasion, and migration. Moreover, ECM and tissue architecture have a profound impact on chromatin structure, thereby altering gene expression. Considering the significant contribution of ECM to cellular behavior, a large body of work underlined that traditional two-dimensional (2D) cultures depriving cell–cell and cell–ECM interactions as well as spatial cellular distribution and organization of solid tumors fail to recapitulate in vivo properties of tumor cells residing in the complex TME. Thus, three-dimensional (3D) culture models are increasingly employed in cancer research, as these culture systems better mimic the physiological microenvironment and shape the cellular responses according to the microenvironmental cues that will regulate critical cell functions such as cell shape/architecture, survival, proliferation, differentiation, and drug response as well as gene expression. Therefore, 3D cell culture models that better resemble the patient transcriptome are critical in defining physiologically relevant transcriptional changes. This review will present the transcriptional factor (TF) repertoire of breast cancer in 3D culture models in the context of mammary tissue architecture, epithelial-to-mesenchymal transition and metastasis, cell death mechanisms, cancer therapy resistance and differential drug response, and stemness and will discuss the impact of culture dimensionality on breast cancer research.

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