Tubulin microtentacles in detached mammary epithelial cells

脱落的乳腺上皮细胞内的微管蛋白微触须。 乳腺肿瘤细胞通过非黏附的微环境转移到远处的器官，例如 血液或淋巴管。然而，对其动态行为和药物反应知之甚少。 非粘附性肿瘤细胞，这是由于成像非粘附性细胞而不会因细胞漂移而模糊的挑战。 PI的实验室在非粘附性肿瘤细胞表面发现了独特的微触手(McTns)， 促进活体小鼠肺毛细血管内循环肿瘤细胞(CTCs)的聚集和滞留。 该项目将检验肌动蛋白皮质收缩调节潜在的分子机制的假设。 并可通过独立的途径作为靶点，以减少四氯化碳的聚集和重新附着 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。 具体目的1：抑制调节肌动蛋白皮质收缩的激酶影响McTns。 A)确定通路抑制剂对微触须的影响和支持的分子机制。 B)分析抑制剂对肿瘤细胞机械性能的影响(布里渊显微镜，AFM)。 C)在斑马鱼(CTC再附着)、小鼠(原位，PDX)和活患者肿瘤细胞中测试优先药物。 具体目标2：检测X-ROS机械转导在McTN机制和功能中的作用。 A)化学抑制TRPM8和钙信号以影响McTN机制。 B)测量途径抑制剂对MCAM和基因调控MCAM或TRPM8的影响(CRISPR)。 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)确定用洋地黄毒素或哇巴因抑制同型/异型聚集的McTN机制。 B)下调桥粒蛋白DSG3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，布里渊显微镜)并检查 高度保守的机械转导原理(X-ROS)，最近由Pi‘s实验室在上皮性肿瘤中发现 细胞。将FDA批准的疗法和药物纳入当前的临床试验将增加迅速 翻译这个项目的结果，以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.