三维模型调控EMT/MET表型转化探讨肿瘤微环境在乳腺癌侵袭转移中的作用机制

EMT/MET switch is an important dynamic process during the invasion and metastasis of tumor cells which intimately correlated with the tumor microenvironment. It is still not clear how breast cancer cells undergo EMT/MET switch and remodel their microenvironment to suit for their colonization. Our research has revealed that by mimicking the EMT/MET switch of breast cancer cells in three-dimensional (3D) culture model in vitro, the expression of extracellular matrix (ECM) constituent Col IV, inter-cellular adhesion molecule 1 (ICAM-1) and surface receptor β1 integrin has changed significantly. We propose that cells with differential expression level of ECM constituents have advantages in choosing the metastasis site microenvironment. By summarizing the research results of EMT, MET, tumor microenvironment and “seed and soil” theory, we propose to study the correlation of EMT/MET switch and the ECM of cells themselves (seed), the targeted metastasis microenvironment (soil), and their roles in the metastasis process by 3D culture model in vitro. Our project focuses on the phenotype switch of EMT/MET, study the changes of cells themselves and the direct interactions between breast cancer cells and other host cells in tumor microenvironment by use of ECM screening and co-culture in 3D culture model. Furthermore, we investigate the pathways involved in the regulation of EMT/MET switch. Our research will provide theoretical and practical basis for blockage of the EMT/MET switch and then the inhibition of invasion and metastasis of breast cancer.

EMT/MET表型转化是肿瘤侵袭转移中重要的动态进程，并与肿瘤微环境有着密切联系。但目前细胞如何进行EMT/MET转化、重塑微环境进而适于其转移的作用机制尚不明确。我们前期已利用3D体外模型构建出乳腺癌细胞的EMT/MET表型转化模型，揭示表型转化伴随着胞外基质Col IV、粘附因子ICAM-1和表面受体β1 integrin等的显著变化，我们推测差异表达胞外基质的细胞在转移微环境上具有选择性优势。结合EMT、MET、肿瘤微环境和“seed与soil”研究成果，我们提出以3D模型研究EMT/MET与转移细胞本身（seed）胞外成分，转移肿瘤微环境（soil）之间的联系。本课题以EMT/MET为切入点，通过基质成分筛选及细胞共培养模式研究转移细胞自身变化，转移细胞与肿瘤微环境各成分及细胞之间的直接作用，分析调控表型转化的信号通路，为阻断乳腺癌EMT/MET进而遏制侵袭转移奠定理论和实验依据。

复发转移是导致乳腺癌患者死亡的最主要原因，乳腺癌远处转移机制的研究是乳腺肿瘤研究的核心问题，对是否能有效控制乳腺癌、提高患者的生存期至关重要。EMT/MET表型转化是肿瘤侵袭转移中重要的动态进程，与肿瘤微环境关系密切。如何调控微环境及EMT/MET可塑性，进而有效抑制乳腺癌侵袭转移是亟待解决的关键科学问题。本课题以EMT/MET为切入点，利用3D体外模型构建出乳腺癌细胞的EMT/MET表型转化模型，研究表型可塑性与胞外基质及侵袭转移的相关性，探讨EMT/MET表型转化的乳腺癌细胞与肿瘤微环境之间的相互作用，并分析调控表型转化的信号通路及关键调控子，为阻断乳腺癌EMT/MET进而遏制侵袭转移奠定理论和实验依据。研究结果揭示，细胞EMT/MET表型转化伴随着胞外微环境，细胞粘附分子，上皮与间质特征蛋白，细胞干性及侵袭转移能力等的显著变化。同时关键整合素分子integrinβ1的糖基化状态发生变化，影响了细胞与细胞间，细胞与胞外基质间的作用。研究发现特定微环境下EMT化乳腺癌细胞可形成血管拟态表型。内质网应激，TGF-β1/Smad2/3及β-catenin通路参与了表型可塑性的调控。研究通过对EMT/MET差异表型乳腺癌细胞行转录组学分析，获取了细胞mRNA水平的整体变化情况，差异基因列表及其相关功能。课题组借此进一步发掘了EMT/MET表型转化动态过程中E/M表型双向调控子keratin5的作用，通过keratin5的敲降或过表达建立了稳定的混合E/M表型细胞模型，并在细胞水平、动物水平和临床数据上进行多方面验证。项目研究结果有望带来新的肿瘤模型技术的进步，并为临床抗转移研究提供新思路、新方法及新策略，具有重要的理论意义和应用前景。