Reducing Metastatic Potential by Targeting Tumor Metabolism and Understanding the Free-floating Tumor Microenvironments of Metastasis

The dynamic balance between microtubule extension and actin contraction regulates mammalian cell shape, division and motility, which has made the cytoskeleton an attractive and very successful target for cancer drugs. Circulating breast tumor cells use tubulin-based structures known as microtentacles (McTNs) to re-attach to endothelial cells and arrest in distant organs. McTN formation is dependent on the opposing cytoskeletal forces of stable microtubules and the actin network. Numerous compounds in clinical use to reduce tumor growth indiscriminately alter the assembly and dynamics of all microtubules, which causes significant dose-limiting toxicities on normal tissues. Therefore, novel molecular targets on microtubules are necessary to better exploit the cytoskeleton of circulating tumor cells (CTCs). AMP- activated protein kinase (AMPK) is a cellular metabolic regulator that can alter actin and microtubule organization in epithelial cells. We used drugs targeting AMPK to better understand the role of this pathway on the cytoskeleton of breast tumor cells. AMPK inhibition increased both microtubule stability and cofilin activation, which also resulted in higher McTN formation and re-attachment. Conversely, AMPK activation decreased microtubule stability and coflin activation with concurrent decreases in McTN formation and cell re-attachment. We also investigated a downstream substrate of AMPK, clip-170, which is a microtubule end-binding protein. We found that constitutive phosphorylation of clip-170 also reduces McTN frequency. These results support a model where AMPK activators may be used therapeutically to reduce the metastatic efficiency of CTCs. However, effective strategies to study the free-floating behavior of freshly isolated CTCs from patients remains a major barrier limiting our understanding of CTC biology. Therefore, we engineered a strategy to spatially immobilize detached tumor cells while maintaining their free-floating character. The goal was achieved using a microfluidic cell tethering device that allows capture of high-resolution images of McTNs on viable free-floating cells. In addition, we showed that tethering allows for real-time analysis of McTN dynamics on individual tumor cells and in response to tubulin-targeting drugs. The ability to image detached tumor cells and test existing drugs can enhance our understanding of CTCs, improve the development of more precise cytoskeletal cancer therapies, and advance personalized cancer treatment for patients.