Background:In cancer research, the multicellular tumour spheroids (MCTSs) model has attracted great attention as a transitional stage betweenin vitrotwo-dimensional (2D) cultures andin vivostudies, contributing to a better understanding of tumour biology. The objective of this study was to construct three-dimensional (3D) homotypic bone and cancer spheroids together with heterotypic spheroids resembling breast cancer metastasis to the bone.Methods:To construct spheroid models, cells were seeded in ultra-low attachment (ULA) plates with varying concentrations of Matrigel followed by forced aggregation via centrifugation. The models were validated by growth kinetics, cell viability and quantitative real-time polymerase chain reaction (RT-qPCR).Results:The addition of Matrigel allowed the formation and growth of the spheroids. Matrigel enhanced the circularity of the spheroids for homotypic cancer spheroids but significantly reduced their viability. Matrigel had the opposite effect on homotypic bone spheroids, significantly improving viability and negatively affecting circularity. The formation of heterotypic spheroids with 1 % Matrigel represented the optimum condition with high viability and circularity index. The model was subsequently validated to accurately represent bone metastasis via the upregulation of the pro-metastatic genes.Conclusions:Incorporating Matrigel as an extracellular matrix (ECM) protein mixture into spheroid models is crucial as it enhances cellular interactions and is the key to simulatingin vivoconditions. Consequently, we constructed a 3D heterotypic tumour spheroid model using 1 % Matrigel as a robust and versatile model to study different aspects of breast cancer metastasis to the bone and for further drug testing.