Breast cancer remains challenging to treat due to poor understanding of the complex crosstalk between the tumor microenvironment (TME) and cancer cells. As the most predominant component in TME, cancer-associated fibroblasts (CAFs) play a key role in tumor progression. CAFs are also known to participate in activating the tumor-associated macrophages (TAMs) into M2 macrophages which has pro-tumorigenic properties. What is still missing, however, is the interaction among cancer cells, CAFs, and the macrophages activation mechanisms. Thereby, a pre-established 3D co-culture in vitro model with breast cancer cells, CAFs, and monocytes was used to investigate the phenotypic changes of the monocytes.