剪切力调控肿瘤细胞粘附与迁移新机制：整合素alpha(v)beta(3)和GPIIb/IIIa作用规律及其力学生物学机制研究

Carcinoma cells metastasis to distant organs through blood vessels is a mainly leading cause of death in cancer patients. Hematogenous metastasis is a highly regulated and dynamic process in which cancer cells separate from a primary tumor, migrate across blood vessel walls into the bloodstream, and disperse throughout the body to establish new colonies. Cancer cells will interact with many host cells in the blood or blood vessel, for instance, endothelial cells and platets, during the hemotogenous metastasis preocess. Our previous studies demonstrated that this interaction could be mediated by the integrins of alpha(v)beta(3) and GP IIb/IIIa, and the metastasis potential of cancer cells is positively related to the aggregation of platelets and the expression of alpha(v)beta(3) and GP IIb/IIIa. However, the the dynamic interaction and the mechanobiological mechanisms remain to be elucidated. In this project, we first construct the parallel flow chamber and co-culture flow chamber system, and aim to investigate how the activated states or active partterns of alpha(v)beta(3) and GP IIb/IIIa integrins affect the interaction of cancer cell-endothelial cell. We also further explore the mechanotransduction pathways for cancer cells under various shear flow conditions, and to elucidate the relationship between mechanotransduction pathways and the ability of cancer cell adhesion, migration and metastasis. The detailed interaction of cancer cell-endothelial cell, the contribution of alpha(v)beta(3) and GP IIb/IIIa integrins, and the mechnobiological mechanism will be illuminated through this poposed project investigation. These studies will promote our understanding of the roles of alpha(v)beta(3) and GPIIb/IIIa and integrins under shear flow conditions for cancer metastasis study, and may provide new strategy to cancer treatment in the future.

肿瘤细胞经血道的粘附与转移是肿瘤细胞扩散并导致患者死亡的重要因素之一，而肿瘤细胞转移与新病灶形成是一个十分复杂的生物学过程，其中涉及肿瘤细胞与很多宿主细胞间相互作用，如内皮细胞、血小板等，我们前期研究表明，这种相互作用可通过整合素alpha(v)beta(3)和GPIIb/IIIa来介导，且肿瘤细胞转移能力与上述两种整合素的表达及血小板聚集程度密切相关，但其在血流剪切力作用下相互作用的动力学规律及其力学生物学机制并不清楚。该项目拟通过平板流动腔技术和细胞共培养的流体力学加载平台，研究alpha(v)beta(3)和GPIIb/IIIa活化形式和活化状态对肿瘤细胞与内皮细胞粘附动力学、信号转导途径、以及肿瘤细胞粘附和侵袭能力的影响规律，深入阐明alpha(v)beta(3) 和GPIIb/IIIa在肿瘤细胞粘附与转移过程中作用规律、贡献大小及力学生物学机制，为探索肿瘤治疗新策略提供实验证据。

血源性转移是恶性肿瘤转移的一个重要方式，它是一个高度受控、多因子参与的动态级联病理过程，而血液流动产生的切应力在肿瘤转移过程中作用与调控机制尚未完全阐明，该项目以乳腺癌细胞系MDA-MB-231或MCF-7为研究对象，重点探讨整合素αvβ3和GP IIb/IIIa在低切应力作用下力学-生物学耦合机制与规律。主要研究结果总结如下：(1) 定性和定量分析了低切应力条件下血小板与肿瘤细胞间的相互作用(粘附规律)。采用平板流动腔技术研究了低切应力作用(1.84 dyn)后，血小板存在有无、活化与否、整合素αvβ3阻断与否等不同实验条件下MDA-MB-231细胞的粘附、迁移和运动规律，发现整合素αvβ3和GP IIb/IIIa显著参与调控肿瘤细胞的粘附、运动与转移行为，尤其是发现血小板的活化与聚集为肿瘤细胞在血管内皮细胞层上的稳固粘附提供了便利条件；(2) 发现低切应力作用可以活化肿瘤细胞表面整合素αvβ3，导致其在细胞膜脂筏部位聚集显著增多，进而引发了依赖于Src活化的PI3K/Akt这一信号转导途径，并与NF-kB信号通路存在明显的交叉对话机制，从而调控肿瘤细胞MMP-2/9、VEGF等细胞因子的表达与活性，来共同调控肿瘤细胞的恶性转移行为；(3) 阐明了低切应力作用对肿瘤细胞迁移板状伪足形成与细胞迁移的关系及其分子调控机制。低切应力作用可以明显影响MDA-MB-231细胞骨架的动态组装，促进了板状伪足的形成，发现mTOR/70S6K信号通路通过Cdc42 和Rac1 参与了细胞骨架和板状伪足形成的调控，进一步丰富了肿瘤血源性转移的内容与机制；(4) 揭示了肿瘤微环境理化特性(如炎性微环境)在癌细胞侵袭转移中的影响机制,发现微环境中的趋化因子MCP-1通过ERK/GSK-3β/Snail 信号转导途径促进了乳腺癌MCF-7细胞上表间质转换(EMT)和细胞骨架重排，对肿瘤微环境物理和生化因素如何协同影响其转移行为提供了初步的实验数据与参考信息。相关研究成果发表在Cancer Letters、Oncotarget、Cellular & Molecular Immunology、BBA-Molecular Cell Research、Scientific Reports、Medical Oncology等国际重要学术期刊上。通过该项目的研究，阐明了低切应力调控肿瘤细胞血源性转移