Breast tumor cells must survive during transit through the bloodstream or lymphatics to form the metastatic tumors that threaten the health of breast cancer patients. Recent studies have shown that circulating tumor cells (CTCs), which are softer and more flexible, are able to survive better in the bloodstream and pose a greater metastatic risk. However, the genetic alterations that affect the flexibility of circulating breast tumor cells are not known, which creates a barrier to developing therapies that can target these characteristics of metastatic tumor cells. In this project, we will bring together an international consortium of physicists, engineers, tumor cell biologists, and breast cancer clinicians to define a molecular framework that governs the mechanical properties of circulating tumor cells. Since large epithelial tumor cells often fragment in narrow capillaries, a better understanding of the molecular determinants of tumor cell mechanics could reveal opportunities to destroy them in the circulation. Our investigations of microtentacles on breast tumor cells have revealed that the surface of circulating tumor cells is regulated by a balance between expansion of tubulin filaments from the cell center and contraction in the actin filaments that lie beneath the plasma membrane. Breast tumor cells can improve their metastatic potential by either over-stabilizing tubulin or weakening the containment provided by actin. In order to directly measure the mechanical alterations in circulating tumor cells, we will use an innovative technology, known as the optical cell stretcher, which uses laser illumination to deform and measure the dynamic physical responses of the circulating tumor cell cytoskeleton. The optical cell stretcher can analyze tumor cells in a high-throughput and non-contact manner that preserves the cells for follow-up studies. We will use two genetic models of tubulin stabilization (Tau and EMT) and two genetic models of actin disruption (PTEN loss and Src) to develop a molecular framework for the regulation of CTC cytoskeletal physics. The metastatic efficiency of CTCs with different mechanical signatures will be tested with whole-animal imaging of CTCs in living mice. The ability of targeted therapies to alter the mechanical properties of CTCs will also be examined. Finally, freshly isolated CTCs from human breast cancer patients will be analyzed on the optical cell stretcher using the molecular framework established with the experimental models to clarify the metastatic potential of patient-derived CTCs as well as the responses of these cells to experimental therapies. The completion of this project will improve the molecular understanding of mechanical changes that alter metastatic efficiency and further develop a novel technology to gauge patient prognosis and therapeutic response from circulating tumor cells.