基于纳米表面的循环肿瘤癌细胞俘获与分析

Circulating tumor cells (CTCs) are cancer cells that break away from either the primary tumor or metastatic site(s) and circulate in the peripheral blood. In addition to conventional diagnostic imaging and serum marker detection, enumeration and characterization of CTCs in patient blood provides valuable information for examining early-stage cancer metastases, predicting patient prognosis and monitoring therapeutic interventions and outcomes. Over the past decade, a variety of technologies capable of isolating and counting CTCs have been developed based on different strategies, e.g., immunomagnetic beads, flow cytometry, microfluidics, etc. Some of these technologies have been demonstrated in the clinical setting and allow reproducible detection of CTCs in the patient blood. However, challenges remain in improving CTC capture efficiency, reducing measurement costs and conducting sequential molecular analysis of these cells. The long term goal of this research proposal is to develop a new technology platform capable of isolating viable CTCs from whole blood with profound advantages of enhanced cell capture efficiency, low operation cost and ease of use. We will also test the feasibility of performing multiparametric molecular analysis of the isolated CTCs using an established quantitative immunocytochemistry (ICC) approach. .1. Challenges to Detect and Characterize CTCs in Whole Blood.(i).Due to extremely low abundance of CTCs compared to the number of hematologic cells in the blood, it is difficult to further improve the efficiency of the existing CTC capture technologies. .(ii).High operational costs of existing CTC capture technologies prohibit their dissemination for routine diagnosis in clinical settings.(iii).In addition to enumeration of CTCs in patient blood, it is essential to develop new methodologies capable of performing multiparametric molecular analysis to unveil the molecular properties and cellular heterogeneity of CTCs. .2. Our Approach to Overcome These Challenges.Based on a unique working mechanism, our group has demonstrated that a TiO2 nanowires-covered substrate, which is coated with anti-EpCAM (epithelial-cell-adhesion-molecule antibody), exhibits outstanding efficiency when employed to isolate viable CTCs from whole blood samples. With a simple stationary device setting and operation protocol, CTCs can be immobilized onto the TiO2 nanowires substrates because of the enhanced topographic interactions between the substrate-grated TiO2 nanowires and nanoscale cell surface components. Clinical studies of this TiO2 nanowires based CTC capture technology have been initiated with the help of Zhongnan hospital. These preliminary results constitute a solid foundation for realization of our proposed research.

循环肿瘤细胞（CTCs）检测技术作为一种具有高度可行性和可重复性的非侵入性新型癌症诊断技术，通常分为初步捕获富集和识别分析两个阶段。前者主要应用一些物理化学原理将这些细胞特异性地捕获富集起来，提高CTCs检测的敏感性。后者主要依靠肿瘤细胞上存在的一些肿瘤特异性或器官特异性标记以及肿瘤细胞本身的基因和形态学特征等进行识别分析。.本项目的研究目标是在透明材料衬底上制备可以进行实时光学观测的循环癌症细胞捕获微流系统，争取实现高效的循环肿瘤细胞捕获和在保持细胞活性的前提下对已捕获循环癌症细胞进行分离，以便进行后续生物医学分析和检测。从而为拓展循环癌症细胞研究在癌症早期诊断、癌症用药分析、放化疗疗效分析方和抗癌药物筛选等领域的应用创造条件。

循环肿瘤细胞（Circulating Tumor Cells, CTCs）是从原发肿瘤组织上脱离下来进入到肿瘤患者外周血中的肿瘤细胞，对于肿瘤的早期诊断和治疗评估有很重要的临床研究价值。然而患者外周血中 CTCs 的含量极其稀少，在对 CTCs 进行检测之前需要预先对其进行富集分选。近年来兴起的纳米技术和微流控技术因其在微纳米尺度范围的可控性，被逐渐发展成为比较成熟的 CTCs检测手段。.主要研究工作如下： .1. 通过自组装合成制备了 MnO2 纳米颗粒薄膜，将这种可透可见光的薄膜基 底应用在肿瘤细胞的静态捕获和释放当中。通过表面化学键合，基底上嫁接上了 上皮细胞粘附因子抗体（epithelial cell adhesion molecule antibody, anti-EpCAM）， 通过设定对照组实验，研究了经 anti-EpCAM 抗体修饰的薄膜基底对肿瘤细胞的捕 获具有特异性；研究了经抗体修饰的薄膜基底对肿瘤细胞的回收效率（从 DMEM II 和人造病人血中），以及研究了孵育时间对回收效率的影响；研究了不同的草酸 浓度对肿瘤细胞释放效率和细胞活性的影响；将释放下来的细胞进行培养，研究 了不同细胞的抗酸能力和分化能力。 .2. 我们使用表面包裹了明胶纳米颗粒的 SiO2 微球（SiO2@Gel MBs），联合 可进行尺寸分选工作的微流控芯片，开展了癌症患者外周血中 CTCs 的捕获与纯化 的研究工作。研究了 SiO2@Gel 微球表面通过嫁接上 anti-EpCAM 抗体对肿瘤细胞 捕获的特异性；研究了经抗体修饰的 SiO2@Gel 微球对肿瘤细胞的回收效率（从裂 红的血样和全血中），以及研究了所使用微球的量对肿瘤细胞回收效率的影响； 50µm 尺寸的 SiO2@Gel 微球在血样中捕获住肿瘤细胞后（cell-bead, 尺寸>50µm）， 通入芯片（过滤结构尺寸为 30µm）内，研究了不同芯片尺寸参数以及不同流速对 cell-beads 的纯化效率的影响；研究了基质金属蛋白酶-9（matrix metalloproteinases-9, MMP-9）的浓度对肿瘤细胞释放效率和细胞活性的影响；使用最优化实验条件进 一步研究了实验平台在临床癌症患者外周血中 CTCs 的捕获与鉴定的工作。 .3. 我们在包裹了明胶纳米颗粒的 SiO2 微球（SiO2@Gel