Single cell determination of ROS and calcium roles in breast cancer apoptotic resistance

分离的乳腺上皮细胞中的微管蛋白微触手。 乳腺肿瘤细胞通过非粘附性微环境转移到远处器官，如 血液或血液循环。然而，很少有人知道的动态行为和药物反应， 非粘附性肿瘤细胞，这是由于在不因细胞漂移而模糊的情况下对非粘附性细胞进行成像的挑战。 PI的实验室在非粘附肿瘤细胞表面发现了独特的微触角（McTN）， 促进循环肿瘤细胞（CTC）在活小鼠肺毛细血管中的聚集和滞留。 这个项目将测试肌动蛋白皮层收缩调节分子机制的假设， McTN可以通过独立的途径靶向，以减少CTC的聚集和再附着 在转移过程中。这一假设的预测将在以下具体目标中得到检验。 具体目标1：抑制调节肌动蛋白皮质收缩的激酶以影响McTN。 A）定义通路抑制剂对微触手的影响和支持分子机制。 B）分析抑制剂对肿瘤细胞机械性质的影响（布里渊显微术，AFM）。 C）在斑马鱼（CTC再附着）、小鼠（原位，PDX）和活的患者肿瘤细胞中测试优先药物。 具体目标2：测试X-ROS机械转导对McTN机制和功能的作用。 A）化学抑制TRPM 8和钙信号传导以影响McTN机制。 B）途径抑制剂对MCAM和遗传调节MCAM或TRPM 8（CRISPR）的计量效应。 C）在斑马鱼、小鼠和患者肿瘤细胞中测试优先的机械转导基因和药物。 具体目标3：McTN介导的肿瘤聚集的靶向机制。 A）定义用洋地黄毒苷或哇巴因抑制同型/异型聚类的McTN机制。 B）下调桥粒蛋白DSG 3以减少McTN介导的肿瘤细胞聚集。 C）在斑马鱼、小鼠和活的患者肿瘤细胞中测试优先的抗聚集机制。 该项目将使用创新的生物工程技术（TetherChip，Brillouin显微镜）， 高度保守的机械转导原理（X-ROS），最近由PI的实验室在上皮肿瘤中鉴定 细胞在当前的临床试验中纳入FDA批准的疗法和药物将增加快速治疗的潜力。 将该项目的成果转化为影响转移性乳腺癌的临床治疗。

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.