Deciphering Tumor Heterogeneity in Triple-Negative Breast Cancer: The Crucial Role of Dynamic Cell-Cell and Cell-Matrix Interactions*

Epithelial tissue development, wound healing, and tumor progression are intricate processes influenced by the dynamic interplay between cells and the surrounding extracellular matrix. These physical cell-cell and cell-matrix interactions are especially complex in triple-negative breast cancer (TNBC), which displays dramatic spatiotemporal heterogeneity over the course of cancer progression. Consequently, TNBC is highly aggressive and associated with poorer patient prognosis compared to other breast cancer subtypes, posing a significant clinical challenge due to the limited availability of targeted therapies. To address this issue, we have established a scalable method that captures the spatial heterogeneity of multicellular cluster-induced matrix deformations. We find that induction of the epithelial-mesenchymal transition dramatically alters the 3D mechanophenotype of mammary cell clusters through a progressive loss of protrusive and circumferential tractions to more localized contractile tractions. To reveal how individual cell differences may contribute to overall tumor progression, we used a microfluidic model to strat­egically engineer tumors with precise composition using noninvasive and invasive clonal TNBC cell subpopulations exhibiting epithelial and mesenchymal characteristics, respectively. Our findings revealed that the physical presence of invasive clonal cells in multiclonal tumors could dramatically enhance overall tumor growth, invasion, and escape to a nearby vessel, which could be delayed by selectively targeting the invasive subpopulation. Moreover, we used a reverse-engineering approach to locally pattern regions of noninvasive and invasive cell subpopulations, revealing how spatial heterogeneity at the cellular level affects the dynamics of tumor progression. Overall, our work enables the systematic interrogation of breast tumor heterogeneity in the context of dynamic cell-cell and cell-matrix interactions, providing insight into the physical basis of TNBC progression. These findings shed light on how selective targeting of tumor subpopulations with distinct mechanophenotype and invasion signatures may offer a promising therapeutic strategy for managing heterogeneous tumors.