Breast cancer is the most commonly diagnosed malignancy worldwide, with molecular subtypes following distinct clinical trajectories. While Luminal A breast cancers are typically indolent, a subset enriched in α-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs) exhibits aggressive behavior, facilitating tumor invasion. However, the biophysical mechanisms by which CAFs drive invasion and extracellular matrix (ECM) remodeling remain unclear. In addition, the temporal and spatial dynamics of CAF interactions with the collagen matrix and cancer cell spheroids remain unknown, raising the question of whether these processes follow a deterministic sequence or occur stochastically. To address this, we conducted histological analysis of Luminal A tumors, which revealed variation in CAF, cancer cell, and ECM organization at tumor boundaries. To assess the impact of CAF on cancer cell invasion, we use a 3D in-vitro model co-embedding 19TT breast CAF and MCF7 luminal breast cancer spheroids within a three-dimensional (3D) collagen-I hydrogel and performed time-lapse imaging. We demonstrate that inter-spheroid distance critically determines 19TT CAF-induced MCF7 spheroid behavior. Moreover, we showed that CAF-mediated collagen matrix remodeling and degradation precede the observed MCF7 spheroid disruption and are critical in promoting cancer cell spheroid expansion and cell dissemination. While broad-spectrum matrix metalloproteinase inhibition suppressed CAF-driven collagen degradation and MCF7 spheroid expansion, it did not prevent ECM remodeling, CAF migration, or single-cell dissemination of cancer cell spheroids. Furthermore, a complementary heterospheroid model revealed similar ECM remodeling and invasion dynamics despite the altered cellular arrangement of cancer cells and CAFs. Our findings enhance our understanding of the relationship between CAF activity and collagen matrix remodeling processes that promote cancer cell invasion, providing insights into the potential therapeutic benefits of targeting CAFs in breast cancer treatment. Statement of Significance This research provides key insights into breast cancer-associated fibroblasts (CAFs) mediated remodeling of the extracellular matrix (ECM) and subsequent breast cancer cell dissemination and invasion. Herein, we demonstrated that CAFs remodel collagen fibres before migration and matrix metalloproteinase (MMP)-mediated degradation. Using a 3D in-vitro model, we showed that distinct mechanisms govern cancer cell spheroid expansion and single-cell dissemination: while expansion depends on collagen matrix integrity, dissemination relies on CAF-driven collagen remodeling. These findings advance our understanding of the relationship between CAF activity and collagen matrix remodeling processes that promote cancer cell invasion, providing insights into the potential therapeutic benefits of targeting CAFs in breast cancer treatment.

Tissue surface tension influences cell sorting and tissue fusion. Earlier mechanical studies suggest that multicellular spheroids actively reinforce their surface tension with applied force. Here we study this open question through high-throughput microfluidic micropipette aspiration measurements on cell spheroids to identify the role of force duration and spheroid deformability. In particular, we aspirate spheroid protrusions of mice fibroblast NIH3T3 and human embryonic HEK293T homogeneous cell spheroids into micron-sized capillaries for different pressures and monitor their viscoelastic creep behavior. We find that larger spheroid deformations lead to faster cellular retraction once the pressure is released, regardless of the applied force. Additionally, less deformable NIH3T3 cell spheroids with an increased expression level of alpha-smooth muscle actin, a cytoskeletal protein upregulating cellular contractility, also demonstrate slower cellular retraction after pressure release for smaller spheroid deformations. Moreover, HEK293T cell spheroids only display cellular retraction at larger pressures with larger spheroid deformations, despite an additional increase in viscosity at these larger pressures. These new insights demonstrate that spheroid viscoelasticity is deformation-dependent and challenge whether surface tension truly reinforces at larger aspiration pressures.

An early step of metastasis requires a complex and coordinated migration of invasive tumor cells into the surrounding tumor microenvironment (TME), which contains extracellular matrix (ECM). It is being appreciated that 3D matrix-based microfluidic models have an advantage over conventional in vitro and animal models to study tumor progression events. Recent microfluidic models have enabled recapitulation of key mechanobiological features present within the TME to investigate collective cancer cell migration and invasion. Microfluidics also allows for functional interrogation and therapeutic manipulation of specific steps to study the dynamic aspects of tumor progression. In this review, we focus on recent developments in cancer cell migration and how microfluidic strategies have evolved to address the physiological complexities of the TME to visualize migration modes adapted by various tumor cells.

Epithelial to mesenchymal transition (EMT) is crucial during embryonic development, tissue fibrosis, and cancer progression. Epithelial cells that display a cobblestone-like morphology can undergo a switch to mesenchymal-like phenotype, displaying an elongated spindle shape or a fibroblast-like morphology. EMT is characterized by timely and reversible alterations of molecular and cellular processes. The changes include loss of epithelial and gain of mesenchymal marker expression, loss of polarity, increased cell migratory and invasive properties. Epithelial cells can progress unevenly during this transition and attain hybrid E/M states or metastable EMT states, referred to as epithelial cell plasticity. To gain a deeper insight into the mechanism of EMT, understanding the dynamic aspects of this process is essential. One of the most prominent factors to induce EMT is the cytokine transforming growth factor-β (TGF-β). This chapter discusses molecular and cellular techniques to monitor TGF-β-induced signaling and EMT changes in normal and cancer cell lines. These methods include measuring the TGF-β-induced activation of its intracellular SMAD effectors proteins and changes in epithelial/mesenchymal marker expression and localization. Moreover, we describe assays of cell migration and dynamic reorganization of the actin cytoskeleton and stress filaments that are frequently part of the TGF-β-induced EMT cellular response.