不同刚度三维脱细胞肿瘤工程支架构建及其对肿瘤细胞耐药性的影响

Three-dimensional mechanical microenvironment is an important and nonnegligible factor in tumor development. It is a hot topic and difficulty to build a tumor engineering scaffold that can simulate the mechanical properties of tumor at different stages. Our previous study funded by the National Natural Science Foundation of China found that suspension mechanical state (which can be seen as an extreme form of soft matrix) can significantly enhance the expression of adenosine triphosphate binding cassette subfamily C member 3 (ABCC3) and protein accumulation of Lamin A/C in breast tumor cells MDA-MB-231. The aim of this project is to prepare a novel kind of three-dimensional tumor engineering scaffold with different stiffness through decellularing cells from tumor tissue, which is developed by subcutaneously tumorigenesis in the armpits of nude mice by injecting MDA-MB-231 cells with different expression of lysine oxidase (LOX) gene. In this project, drug resistance of breast tumor cells to methotrexate will further be proposed as the finite research object. The novel kind of scaffold will be used to investigate the effects of three-dimensional matrix stiffness on the drug resistance of tumor cells. The regulation rules of ABCC3 and Lamin A/C in this process will be explored. In addition, the difference of gene expression caused by matrix stiffness and drug treatment will be detected, among them, the key genes related to drug resistance of tumor cells will be verified. Applicants aim to investigate the regulation of three-dimensional matrix stiffness on the drug resistance of breast tumor cells and its mechanobiological mechanism. The finding of this project is hoped to provide new ideas for seeking novel treatment methods to breast tumor.

三维力学微环境是调控肿瘤发生发展不可忽视的重要因素，构建可模拟不同发展阶段肿瘤力学特性的肿瘤工程支架是本领域的研究热点和难点。申请人前一个国家自然基金项目研究发现悬浮力学状态(可看做基质变软的一种极端形式)可显著提高乳腺肿瘤细胞MDA-MB-231三磷酸腺苷结合盒亚家族C成员3 (ABCC3)的表达和核纤层蛋白A/C的蛋白累积。本项目拟使赖氨酰氧化酶表达不同的MDA-MB-231细胞在裸鼠腋窝皮下成瘤，经脱细胞制备一种新型具有不同刚度的三维肿瘤工程支架。进一步以乳腺肿瘤细胞对甲氨蝶呤药物的耐药性为有限研究目标，利用该肿瘤工程支架考察三维基质刚度对肿瘤细胞耐药性的影响，探索ABCC3和核纤层蛋白A/C在其中的调控规律；检测基质刚度和药物处理引起的基因表达差异，验证与肿瘤细胞耐药性相关的关键基因；以期解析三维基质刚度调控肿瘤细胞耐药性的力学生物学机制，为探寻治疗乳腺肿瘤新方法开拓新思路。

三维力学微环境是调控肿瘤发生发展不可忽视的重要因素，构建可模拟不同发展阶段肿瘤力学特性的肿瘤工程支架是本领域的研究热点和难点。本项目使赖氨酰氧化酶(LOX)表达不同的MDA-MB-231细胞在裸鼠腋窝皮下成瘤，经脱细胞制备了一种新型具有不同刚度的三维肿瘤工程支架。进一步以乳腺肿瘤细胞对顺铂(DDP)药物的耐药性为有限研究目标，利用该肿瘤工程支架考察了三维基质刚度对肿瘤细胞耐药性的影响，探索三磷酸腺苷结合盒转运子B亚家族成员1 (ABCB1)在其中的调控规律，检测基质刚度和药物处理引起的基因表达差异，验证与肿瘤细胞耐药性相关的关键基因。研究表明，利用差异表达LOX基因的MDA-MB-231细胞成功获得肿瘤组织，经脱细胞后，各组肿瘤脱细胞基质(DECM)具有明显的孔且分布均匀，中刚度组孔隙率为48.47 ± 5.27%，低刚度组为68.12 ± 5.74%，高刚度组为32.68 ± 4.30%。低刚度组基质刚度为0.74 ± 0.1 kPa，显著小于中刚度组的1.60 ± 0.14 kPa和高刚度组的1.99 ± 0.19 kPa。低刚度组DECM中细胞活性高于其他两支架组，且浸润程度同样高于其他两支架组。三维基质刚度的增加能够显著提高MDA-MB-231细胞对DDP的耐药性，随着基质刚度的增加，肿瘤细胞Yes相关蛋白(YAP)、ABCB1等基因表达上调，抑制ABCB1蛋白表达能够降低刚度依赖的耐药性，细胞增殖能力显著下降。本项目研究结果解析了三维基质刚度调控肿瘤细胞耐药性的力学生物学机制，有望为探寻治疗乳腺肿瘤新方法开拓新思路。