纳米薄膜基板结合量子点多分子成像对循环肿瘤细胞和循环肿瘤干细胞的研究

It has been well demonstrated that detection of circulating tumor cell (CTCs) contribute to early predict recurrence and metastasis, assess prognosis, and monitor curative effect. However, current CTCs capture systems fail to efficiently capture due to the scarcity, diverse shape and heterogeneity of CTCs. And, the current staining method of CTCs is lack of sensibility and fail to achieve information of multi-biomarker at one time. Capture and identification of CTCs from complex blood system is still a major challenge. Our previous studies introduced a CTCs capture system that was composed of hydroxyapatite-chitosan (HA-CTS) nanofilm and three-color immunofluorescence. This system capture and identified epithelial phenotype CTCs successfully. With the development of epithelial-mesenchymal transition studies, this system is difficult to achieve the information of heterogeneity CTCs (including EpCAM-negative cancer cells, mesenchymal phenotype cells, circulating tumor stem cells, etc.). This study attempts to change and modify the structure and fabrication parameters of HA-CTS nanofilm substrate to improve the capture efficiency of tumor cells, especially improve the capture efficiency of heterogeneous CTCs. At the same time, new pattern quantum dots-based imaging technology is introduced to label multiple targets information of epithelial, mesenchymal, and circulating tumor stem cells phenotypes at real-time and in situ. Further, the significance of heterogeneity CTCs in tumor invasion and metastasis will be explored here.

检测循环肿瘤细胞（CTCs）有助于早期预测肿瘤复发转移、评估预后、监测治疗效果。但CTCs数目稀少、形态多样、异质性较大，尚不能被目前检测手段高效、敏感的捕获；传统鉴定方法敏感性差，无法实现多分子标记物的同时成像，CTCs的捕获和鉴定仍存在巨大挑战。我们前期制备了羟基磷灰石-壳聚糖（HA-CTS）纳米薄膜基板，结合传统三色免疫荧光染色成功捕获并鉴定了肿瘤患者上皮来源的CTCs。随着上皮间质转化研究的深入，该系统难以实现对异质性CTCs（EpCAM阴性、间质表型、肿瘤干细胞）的高效捕获和分离。本研究试图通过改进HA-CTS纳米薄膜基板的结构和制作参数来提高异质性肿瘤细胞的捕获效率。同时，引入新型量子点多分子成像技术，同时显示上皮、间质及干细胞多个靶标信息，解决传统染色无法实时、原位观测异质性CTCs这一问题，并探索这类异质性CTCs在肿瘤侵袭转移中的意义。

检测循环肿瘤细胞（CTCs）有助于早期预测肿瘤复发转移、评估预后、监测治疗效果。但CTCs数目稀少、形态多样、异质性较大，尚不能被目前检测手段高效、敏感的捕获；传统鉴定方法敏感性差，无法实现多分子标记物的同时成像，CTCs的捕获和鉴定仍存在巨大挑战。本课题组前期研究中，开发了一种由HA-CTS复合材料构成的CTCs捕获装置。本项目是在前期基础上，通过进一步优化该捕获装置的制作方法和捕获过程，同时修饰上皮和干细胞捕获抗体EpCAM和ALDH1，充分利用纳米薄膜与CTCs表面超微结构的相互作用和抗原抗体间的免疫亲和反应，实现了对肿瘤细胞的高效捕获。同时，引入量子点多分子成像技术，实时、原位、共成像白细胞标志物CD45、上皮标志物CK、EMT标记物Snail。结果发现该装置可同时捕获和鉴定上皮和间质表型的CTCs，同时，上皮间质转化可能与肿瘤侵袭转移密切相关。这为深入了解CTCs提供了新的方向，有助于更好地了解肿瘤转移的生物学特性，鉴定出那些有着较高转移复发风险的肿瘤患者。.在本项目支持下，申请人作为第7参与者获得湖北省科技进步一等奖1项。