A Three-Dimensional Model-on-a-Chip of Multifocal and Multicentric Breast Cancer for Investigating Multitumor Aggressiveness

Multifocal and multicentric breast cancer (MMBC) has been increasingly recognized due to its aggressiveness, heterogeneity, and association with poorer clinical outcomes compared to those of its unifocal counterparts. However, the underlying mechanisms of MMBC development and progression remain unclear. Traditional two-dimensional cell cultures and animal models are limited for replicating the physiological features of breast tissue and multitumor circumstances. Here, we present a three-dimensional (3D) model-on-a-chip that mimics the tumor microenvironment of MMBC to investigate multitumor aggressiveness. Our model integrated polydimethylsiloxane chips with chessboard-like patterned surfaces, 3D tumor-like spheroids from MDA-MB-231 and MCF-7 cells, and a collagen I hydrogel to mimic the breast extracellular matrix. Compact spheroids of 500-600 μm in diameter were consistently fabricated and 3D positioned on the chip as single and double spheroids, representing unifocal and MMBC, respectively. Using this on-chip model, we examined differences in cell sprouting and spheroid movement between single and double setups. MCF-7 showed limited sprouting, whereas MDA-MB-231 cells sprouted vigorously from the spheroid core to the extracellular matrix (ECM) area in both setups. MDA-MB-231 in the double setup particularly exhibited unique directional collective cell migration. The spheroids could move through the ECM and merge with each other in the double setup. The merging distance exhibited a strong linear relationship over time in both cell lines, with a larger slope observed in MDA-MB-231. These data suggest intertumor communication and a possible mechanism for MMBC progression, while the degree varied across different breast cancer molecular subtypes. This on-chip model, therefore, provides a potential <i>in vitro</i> platform for gaining insights into the malignant transformation of MMBC and advancing our knowledge of other multiple synchronous tumor diseases.