利用功能化纸基芯片研究肺部炎症对前列腺癌转移至肺的影响及作用机理

Recurrence and metastasis is one of the biggest obstacles to further improve the anti-tumor effect. Lung inflammation is associated with the development of lung cancer, but whether inflammation also increases the risk of metastasis to the lungs of other tumors, it needs to be explored further. In vitro biomimetic tumor metastasis model is a new method of tumor research, which reproduces the tumor metastasis in vitro and clarifies the mechanism by replicating the physiological/pathological structure similar to the tumor. The function of tissues and organs relies upon their overall constituent and organization of cells. The function and dysfunction of tissues and organs are the result of intricate spatiotemporal cell-cell interactions and cell- extracellular microenvironment interactions. Microfluidic technology provides opportunities to create in vitro models with physiological microenvironment for cell study. This study aims constructing the in vitro model of lung metastasis of prostate cancer cells based on functional paper matrix. To this aim, a microfluidic platform re-constructing the physiological structures of lung is proposed to mimic the tumor lung metastasis process by co-culturing tumor cell, blood vessel cell and lung cell in a controllable fashion. To facility the on-chip tumor specific-gene testing, reverse transcription loop-mediated isothermal amplification (RT-LAMP) will be conducted on the paper-based cell analysis platform for the first time to investigated the molecular properties of the tumor cell with strong metastatic capability. In addition, target-responsive DNA hydrogel will be implanted on the functional paper-based in vitro tumor lung metastasis mold to trace the tumor cell secretion. The prostate cancer metastasis recaptured on paper-based in vitro lung metastasis mold will be analyzed along with the lung metastasis mouse mold to demonstrate the potential of this platform for tumor biology study. Finally, the impact of pre-existing lung inflammation on the risk of metastasis to the lungs of other tumors will be investigated on functional paper-based lung model. It is anticipated this project not only provides a novel and efficient platform for tumor biology studies, but also renders useful fundamental information for underline the impact of pathology condition of lung to metastasis of other tumors.

恶性肿瘤转移是提高抗肿瘤疗效的最大障碍之一。肺是肿瘤转移最高发的器官。目前尚不清楚是否肺部炎症增加了其他类型肿瘤转移至肺的风险，因而此问题亟待系统深入的研究。仿生芯片是肿瘤研究的新手段, 旨在芯片上通过细胞共育和诱导，构建芯片上的类器官模型。本项目以微流控技术为核心，构建一种新型具有原位生物分析功能的仿生肺芯片，研究肺部炎症对前列腺癌向肺组织侵袭的影响和机制。首先，制备具有生物活性的即用型气凝胶@纸基，为不同类型细胞提供三维生长环境；通过多层纸基的叠加-拆分，构建细胞共育仿生结构；在仿生芯片中嵌入环介导等温扩增功能区和分析物响应型凝胶功能区，实现原位肿瘤细胞基因分析和无标记的蛋白检测；最后，利用构建的仿生芯片研究肺部炎症病理条件对前列腺癌向肺部转移的影响，解析高侵袭力肿瘤细胞的基因特征，初步阐明侵袭机理。本项目的执行可拓展仿生芯片生物分析手段，为肿瘤生物学研究和抗肿瘤药物开发提供新的思路。

恶性肿瘤在治疗后的复发、转移，成了提高抗肿瘤疗效和提升患者存活率的最大障碍。仿生芯片是肿瘤研究的新手段，旨在芯片上通过细胞共育，模拟实肿瘤在体内的迁移，探讨致病机理,评价抗肿瘤疗效。纸质基底中存在大量类似组织间隙的微小孔隙,允许物质穿透并为细胞提供养分。同时，通过多层叠加可实现 3D 结构组织/器官构建,而通过拆解多层纸基可实现从 3D 至 2D 的结构转化,便于观测分析芯片内部细胞形态和功能。本项目通过改变类纸基材料的表面微纳结构、化学功能基团、亲水性等理化性质，调控类纸基-细胞生物表界面关系，支持细胞三维培养。结果显示通过冷冻干燥法可在类纸基纤维孔隙间填充具有10μm微孔的明胶气凝胶，促进肿瘤细胞在纤维间形成的微龛中形成肿瘤球，并提高肿瘤干细胞丰度；在丝素蛋白修饰的类纸基中，通过调节丝素蛋白螺旋蛋白构像比例，促进肿瘤细胞聚集成球；而琼脂修饰纸基可提高类纸基的透光性，有利于显微镜下实时观测非标记的肿瘤细胞成球与增殖。类纸基功能材料上生长的肿瘤球为仿生实体肿瘤提供了条件。利用类纸基材料良好的机械稳定性，通过多层纸基的叠加-拆分，构建细胞共育结构，研究实体肿瘤与血管内皮细胞的相互关系、肿瘤细胞对上皮的侵入等肿瘤迁移现象。结果显示，细胞因子VEGF促进肿瘤细胞在多层类纸基结构间的迁移；而共育体系中的血管内皮细胞会促进肿瘤球中的细胞离开原发球体，侵入到基质胶层生长。在此基础上，利用多层纸基的叠加-拆分特点，利用环介导等温扩增（LAMP）纸基芯片对迁移至不同基层的细胞进行分子生物学分析。结果显示，与未转移肿瘤细胞相比，高侵袭肿瘤细胞的干性相关基因表达增加，且具有更强的化疗药物耐受性。同时，高侵袭里肿瘤细胞的肿瘤相关抗原表达量明显高于未转移肿瘤细胞。研究结构提示，可针对高侵袭力肿瘤细胞的分子特征，设计靶向该类型细胞的抗肿瘤新策略。