Biological characterization of breast cancer spheroid formed by fast fabrication method.

Engineered three-dimensional (3D) tissue culture platforms are useful for reproducing and elucidating complex in vivo biological phenomena. Spheroids, 3D aggregates of living cells, are produced based on physicochemical or microfabrication technologies and are commonly used even in cancer pathology research. However, conventional methods have difficulties in constructing 3D structures depending on the cell types, and require specialized techniques/lab know-how to reproducibly control the spheroid size and shape. To overcome these issues, we have developed a fabrication method, which enables anyone to make and mature cancer spheroids using a superhydrophobic microwell made of the monolithic porous materials. Here, we characterize the biological behaviors of the breast cancer spheroids fabricated by our method under normoxic and hypoxic conditions. We found that the fabricated spheroid contracted to a certain size via activation of the actomyosin system. Cell proliferation induced a hypoxic state inside the spheroid (elevated expression of the hypoxia-inducible factor HIF-1α), followed by the formation of a necrotic core and cell escape from the spheroid. In addition, we observed a decrease in cancer spheroid contractility and cell escape from spheroids under hypoxic conditions compared to normoxic conditions, which were related to oxygen concentration-dependent cell motility. The fabricated spheroids perform as 3D tumor tissues in a highly reproducible manner and within a short culture period. Our findings indicate that this fabrication method has a wide range of applications in cancer research, such as elucidating the mechanisms of tumor invasion and metastasis and screening anticancer drugs, as with previous methods. Supplementary Information: The online version contains supplementary material available at 10.1007/s44164-024-00066-3.