Breast cancer is considered as a worldwide disease due to its high incidence and malignant metastasis. Although numerous techniques have been developed well to conduct breast cancer therapy, the influence of micropattern-induced interfacial heterogeneity on the molecular mechanism and nuclear signalling transduction of carcinogenesis is rarely announced. In this study, PDMS stencil-assisted micropatterns were fabricated on tissue culture plates to manage cell clustering colony by adjusting initial cell seeding density and the size of microholes. The curvature of each microholes was controlled to construct the interfacial heterogeneity of MDA-MB231 cancer cells at the periphery of micropatterned colony. The distinguished focal adhesion (FA) and cytoskeleton distribution at the central and peripheral regions of the cell colony were regulated by heterogeneous properties. The interfacial heterogeneity of FA and cytoskeleton would induce the biased tension force to encourage more ezrin expression at the periphery and further promote DNA synthesis, therefore disclosing a stem-like phenotype in heterogeneous cells. This study will provide a value source of information for the development of micropattern-induced heterogeneity and the interpretation of metastatic mechanism in malignant breast cancer cells.