悬浮力学状态对乳腺肿瘤细胞存活和转移的调控及其力学生物学机制

The mechanical microenvironment of tumor is an important and nonnegligible factor in regulating tumor development. However, little attention has been paid on the influences of the suspension mechanical state of tumor cells after spreading from primary site on the survival and metastasis of tumor cells. According to the preliminary experimental results, we found that the migration ability of breast tumor cells was significantly improved by suspension culture and the expression of epithelial/mesenchymal cells and cancer stem cells associated markers of breast tumor cells could be regulated by this method as well. In this project, mechanical change induced by suspension state of circulating tumor cells will be proposed as the finite research object. Breast tumor cells MDA-MB-231 and MCF-7 will be chosen as the research objects and the techniques of suspension culture, shRNA, micropipette aspiration technique for measuring cellular mechanics, high throughput transcriptome sequencing analysis and so on will be combined to study the effects of suspension mechanical state on the survival, migration, epithelial-mesenchymal transition, cancer stem cells property and mechanical property of tumor cells. The regulation rules of Ca2+/calcineurin/NFAT/COX-2 pathway and Lamin A/C relevant mechanical characteristic of tumor cells will be further analyzed. In addition, the difference of gene expression caused by suspension mechanical state will be detected, among them, the key genes relating to tumor survival and metastasis will be verified. Applicants aim to investigate the regulation of suspension mechanical state on the survival and metastasis of breast tumor cells and its mechanobiological mechanism. The finding of this project is hoped to provide new ideas for seeking novel methods to prevent and treat breast tumor metastasis.

力学微环境是调控肿瘤发生发展不可忽视的重要因素，但鲜有研究关注肿瘤细胞失巢后的悬浮状态对肿瘤细胞存活和转移的影响。申请人预实验发现悬浮培养能显著提高乳腺肿瘤细胞的迁移能力，可调控其上皮/间质和肿瘤干细胞相关标志物的表达。本项目拟以循环肿瘤细胞悬浮状态引起的力学变化为有限研究目标，以乳腺肿瘤细胞MDA-MB-231和MCF-7为研究对象，结合悬浮培养、shRNA、微吸管细胞力学测量和高通量转录组测序等技术，重点考察悬浮力学状态对肿瘤细胞存活、迁移、上皮间质转化、肿瘤干细胞特性和细胞力学特性的影响；分析Ca2+/calcineurin/NFAT/COX-2信号通路以及与核纤层蛋白A/C相关细胞力学特性的调控规律；检测悬浮力学状态引起的基因表达差异，并验证与肿瘤存活和转移相关的关键基因；以期解析悬浮力学状态调控乳腺肿瘤细胞存活和转移的力学生物学机制，为探寻预防和治疗乳腺肿瘤转移新方法开拓新思路

力学微环境是调控肿瘤发生发展不可忽视的重要因素，但鲜有研究关注肿瘤细胞失巢后的悬浮状态对肿瘤细胞存活和转移的影响。本项目以循环肿瘤细胞悬浮状态引起的力学变化为有限研究目标，结合悬浮培养、shRNA、微吸管细胞力学测量和高通量转录组测序等技术，研究了悬浮力学状态对乳腺肿瘤细胞存活、迁移、上皮间质转化、肿瘤干细胞特性和细胞力学特性的影响。研究表明，悬浮状态改变肌动蛋白骨架的聚合和细胞收缩状态，快速且可逆地提高核纤层蛋白A/C (Lamin A/C)的蛋白水平，促进再贴壁乳腺肿瘤细胞的粘附、铺展、应力纤维丝和粘着斑的形成，提高细胞核圆度和细胞刚度，通过上调整合素β1表达促进乳腺肿瘤细胞的粘附能力。悬浮状态下，乳腺肿瘤细胞增殖活性和体外的克隆形成能力降低，并出现一定程度的凋亡和再贴壁后增殖滞后，但其迁移和侵袭能力增强。与贴壁组相比，悬浮状态上调254个基因，下调190个基因(|log2 fold change| > 2, 错误发现率 < 0.05)，差异基因主要富集于细胞周期、细胞粘附、细胞迁移等生物过程，悬浮状态时间依赖性地上调促转移相关基因的表达。悬浮力学状态诱导环氧合酶-2 (cyclooxygenase-2, COX-2)上调的力学生物学机制及其在乳腺肿瘤细胞转移过程的作用的研究表明，悬浮状态提高乳腺肿瘤细胞内Ca2+含量、活化T细胞核因子2 (nuclear factor of activated T cells 2, NFAT2)核内转移和COX-2的表达，促进肿瘤细胞的存活，迁移和侵袭能力，并通过提高肿瘤细胞在肺的截留和存活促进肿瘤转移。本研究证明了悬浮力学状态促进乳腺肿瘤细胞迁移的力学生物学机制为,证实悬浮状态可逆地通过提高乳腺肿瘤细胞的再粘附、迁移、侵袭和存活能力从而促进其转移，研究结果有望为靶向Lamin A/C和COX-2的抗转移治疗策略提供理论支持。