Traditional two-dimensional (2D) cell culture methodologies exhibit significant limitations, notably their failure to replicate the intricate three-dimensional (3D) milieu of human tissue architecture. This discrepancy has catalyzed the exploration of advanced drug screening platforms that more faithfully mimic the complex 3D biological environment. To this end, precision medicine research has increasingly used 3D in vitro models that emulate the tissue microenvironment of the human body. In this study, breast cancer cell lines (MDA-MB-231 and SK-BR-3) were co-cultured with adipose-derived stem cells (ADSCs), mimicking the in vitro 3D tumor microenvironment with the multicellular and heterogeneous nature of solid tumors compared to a 2D cancer cell only system. Additionally, collagen type I was used to replicate the microenvironment within a 3D spheroid platform, enhancing the accuracy of the platform in mimicking human tissue characteristics. Significantly, the interactions between the ADSCs and two breast cancer lines markedly influenced factors such as cell elongation, molecular expression, migration patterns, and drug sensitivity. The integration of ADSCs was pivotal in simulating the cancer microenvironment, highlighting that even within the same cancer cell line, varying microenvironmental contexts can lead to vastly different experimental results. Thus, this study provides insights into the role of factors such as cellular substrates and stem cells in simulating a 3D tumor microenvironment more akin to the human body when constructing a 3D in vitro system based on breast cancer cells.