Significance: Metastasis accounts for most cancer-associated death. To metastasize, cells can move collectively where cells travel together as cohorts to invade surrounding tissues. However, the mechanisms by which cells move collectively are unclear. Utilizing a combination of in vitro, ex vivo, and in silico approaches, we demonstrated that cancer cell collective invasion is regulated by the energetic states of leader–follower cells. Leader cells require more energy than follower cells, and forward invasion of the leader cell consumes and depletes its available energy. A follower cell then takes over the leader position to sustain invasion. Clinically, there is significant interest in metabolic inhibitors to treat cancer, and our results suggest that metastatic migration might also be inhibited by targeting metabolic pathways.

Cancer-associated fibroblasts (CAFs) have distinct roles within the tumor microenvironment, which can impact the mode and efficacy of tumor cell migration. CAFs are known to increase invasion of less-aggressive breast cancer cells through matrix remodeling and leader–follower dynamics. Here, we demonstrate that CAFs communicate with breast cancer cells through the formation of contact-dependent tunneling nanotubes (TNTs), which allow for the exchange of cargo between cell types. CAF mitochondria are an integral cargo component and are sufficient to increase the 3D migration of cancer cells. This cargo transfer results in an increase in mitochondrial ATP production in cancer cells, whereas it has a negligible impact on glycolytic ATP production. Manually increasing mitochondrial oxidative phosphorylation (OXPHOS) by providing extra substrates for OXPHOS fails to enhance cancer cell migration unless glycolysis is maintained at a constant level. Together, these data indicate that tumor–stromal cell crosstalk via TNTs and the associated metabolic symbiosis is a finely controlled mechanism by which tumor cells co-opt their microenvironment to promote cancer progression and may become a potential therapeutic target.

Significance: We discovered that cells on micropatterned strips change from highly persistent migration into striking oscillations upon the disassembly of microtubules. The oscillation phenomenon then allowed us to apply computer modeling to understand how the positive feedback in the local-excitation–global-inhibition (LEGI) mechanism, responsible for the persistence of migration, might be converted into negative feedback to drive oscillations upon the disassembly of microtubules. Our analyses led to the conclusion that microtubules facilitate the transport of inhibitory signals and their global distribution. Depending on the relative position of excitation and inhibitory signals, the resulting feedback in the integrated control circuit may be either positive or negative. Our finding therefore provides important insights into the role of microtubules in the control circuit of cell migration.

The importance of centrosome in directional cell migration has long been recognized. However, the conventional view that centrosome determines cell’s front, based on its often-observed position in front of the nucleus, has been challenged by contradictory observations. Here we show that centrosome defines the rear instead of the front, using cells plated on micropatterned adhesive strips to facilitate directional migration. We found that centrosome is always located proximal to the future rear before polarity is established through symmetry breaking or reversed as the cell reaches a dead end. In addition, using microsurgery to alter the distance of centrosomes from cells’ ends, we show that centrosomal proximity is predictive of the placement of the rear. Removal of centrosome impairs directional cell migration, whereas the removal of nucleus alone makes no difference in most cells. Computer modeling under the framework of a local-enhancement/global-inhibition mechanism further demonstrates that positioning of rear retraction, mediated by signals concentrated near the centrosome, recapitulates all the experimental observations. Our results resolve a long-standing controversy and explain how cells use centrosome and microtubules to maintain directional migration.

Extracellular vesicles released by cancer cells have recently been implicated in the differentiation of stromal cells to their activated, cancer-supporting states. Microvesicles, a subset of extracellular vesicles released from the plasma membrane of cancer cells, contain biologically active cargo, including DNA, mRNA, and miRNA, which are transferred to recipient cells and induce a phenotypic change in behavior. While it is known that microvesicles can alter recipient cell phenotype, little is known about how the physical properties of the tumor microenvironment affect fibroblast response to microvesicles. Here, we utilized cancer cell-derived microvesicles and synthetic substrates designed to mimic the stiffness of the tumor and tumor stroma to investigate the effects of microvesicles on fibroblast phenotype as a function of the mechanical properties of the microenvironment. We show that microvesicles released by highly malignant breast cancer cells cause an increase in fibroblast spreading, α-smooth muscle actin expression, proliferation, cell-generated traction force, and collagen gel compaction. Notably, our data indicate that these phenotypic changes occur only on stiff matrices mimicking the stiffness of the tumor periphery and are dependent on the cell type from which the microvesicles are shed. Overall, these results show that the effects of cancer cell-derived microvesicles on fibroblast activation are regulated by the physical properties of the microenvironment, and these data suggest that microvesicles may have a more robust effect on fibroblasts located at the tumor periphery to influence cancer progression.