Research Project
10298137
Tubulin microtentacles in detached mammary epithelial cells. Breast tumor cells metastasize to distant organs through non-adherent microenvironments, such as the bloodstream or lymphatics. However, very little is known about the dynamic behavior and drug responses of non-adherent tumor cells, due to the challenges of imaging non-adherent cells without blurring from cell drift. The PI?s lab discovered unique microtentacles (McTNs) on the surface of non-adherent tumor cells that promote the aggregation and retention of circulating tumor cells (CTCs) in the lung capillaries of living mice. This project will test the hypothesis that actin cortical contraction regulates molecular mechanisms underlying McTNs and can be targeted through independent pathways to reduce the clustering and reattachment of CTCs during metastasis. Predictions of this hypothesis will be tested in the following specific aims. Specific Aim 1: Inhibit kinases regulating actin cortical contraction to impact McTNs. A) Define impact of pathway inhibitors on microtentacles and supporting molecular mechanisms. B) Analyze inhibitor impacts on tumor cell mechanical properties (Brillouin microscopy, AFM). C) Test prioritized drugs in zebrafish (CTC reattachment), mice (orthotopic, PDX) and live patient tumor cells. Specific Aim 2: Test role of X-ROS mechanotransduction on McTN mechanisms and function. A) Chemically inhibit TRPM8 and calcium signaling to influence McTN mechanisms. B) Gauge effects of pathway inhibitors on MCAM and genetically regulating MCAM or TRPM8 (CRISPR). C) Test prioritized mechanotransduction genes and drugs in zebrafish, mice and patient tumor cells. Specific Aim 3: Target mechanisms of McTN-mediated tumor clustering. A) Define McTN mechanisms that inhibit homotypic/heterotypic clustering with Digitoxin or Ouabain. B) Downregulate desmosomal protein DSG3 to reduce McTN-mediated tumor cell clustering. C) Test prioritized anti-clustering mechanisms in zebrafish, mice and live patient tumor cells. This project will use innovative bioengineering techniques (TetherChip, Brillouin microscopy) and examine highly-conserved mechanotransduction principles (X-ROS), recently identified by the PI?s lab in epithelial tumor cells. Inclusion of FDA-approved therapies and drugs in current clinical trials will increase the potential to rapidly translate the outcomes of this project to impact the clinical treatment of metastatic breast cancer.
