RAPID RARE CANCER CELL ISOLATION FOR MOLECULAR DIAGNOSIS

某些肿瘤在体内检测到原发肿瘤生长之前，会将肿瘤细胞释放到血液中。最近有研究表明，在血液中可以检测到低至每毫升血液中1个细胞的肿瘤上皮细胞，血液中罕见癌细胞的存在具有重要的诊断价值。目前的筛查方法受到荧光激活细胞扫描速度相对较慢的限制。(FACS)，以及预富集方法的复杂性。这项建议的总体目标是开发一种高通量、基于免疫磁学的细胞分离系统，以尽可能多地从人类血液中回收稀有癌细胞，用于进一步的分子分析(如聚合酶链式反应、生物检测和其他)。具体目标如下。首先：一次分离将使用我们实验室开发的新型连续流通式免疫磁性单元进行。连续单元在高吞吐量方面本质上更有效率。我们建议为下一代系统开发实验和理论基础，该系统将以10/7细胞/S的速度分选细胞，并允许在短时间(1小时)内对用于癌细胞治疗的整个体积的血液产品(通常为0.5至1.0升)进行非破坏性筛选。第二：与目前使用的间歇系统不同，连续细胞分离过程可以分阶段进行。我们建议为连续分段分离过程建立实验和理论基础，这将进一步提高细胞产品的纯度和体积，从而提高细胞分离步骤下游分析的成功率。第三：目前的细胞标记程序可能需要修改和优化，以便在靶向和分离罕见癌细胞方面获得最佳性能。我们建议使用我们实验室开发的独特的细胞跟踪测速仪来筛选具有最高灵敏度和特异度的针对罕见癌细胞的可用的单抗和胶体磁性标记。综上所述，这项建议侧重于癌症筛查的“前端”，即一种高通量输出设备，用于从大量细胞中快速分离和浓缩稀有癌细胞。这项建议是对PAR-98-067《癌症分子分析的创新技术》的响应，我们相信它解决了PAR的第二个目标，即“将允许高通量分析基因改变、基因组产品的表达和监测癌症信号转导途径的新技术”。

Certain tumors shed neoplastic cells into the blood before the primary tumor growth can be detected in the body. It has been shown recently that epithelial cells from the tumor can be detected in the blood at concentrations as low as 1 cell per ml of blood, and that the presence of rare cancer cells in blood has an important diagnostic value. The current screening methods are limited by relatively low speed of Fluorescent Activated Cell Scanning. (FACS),and complexity of preenrichment methods. The overall objective of this proposal is to develop a high-throughput, immunomagnetically based cell separating system to recover as many rare cancer cells in human blood as possible, for further molecular analysis (such as PCR, biological assays and other). The specific aims are as follows. First: the primary separation will be conduced using a novel continuos, flow-through immunomagnetic unit developed in our laboratories. Continuos units are intrinsically more efficient with respect to high throughput. We propose to develop experimental and theoretical basis for the next generation system which will sort cells at a rate of 10/7 cells/s, and allow a non- destructive screening of an entire volume of blood product used for cancer cell therapy ( typically 0.5 to 1.0 liters) in a short period of time (<1hour). Second: the continuos cell separation process can be staged, unlike the currently used batch systems. We propose to develop experimental and theoretical basis for the continuos staged separation process which will further increase the purity and decrease the volume of the cell product, and thus increase the rate of success of analysis downstream of the cell separation step. Third: the current cell labeling procedure may require modifications and optimization for the best performance in targeting and isolating rare cancer cells. We propose to screen available monoclonal antibodies and colloidal magnetic labels for the highest sensitivity and specificity in targeting rare cancer cells using unique Cell Tracking Velocimetry instrument developed in our laboratories. In summary, this proposal focuses on the "front-end" of cancer screening namely a high-throughoutput device to rapidly isolate and concentrate rare cancer cells form large numbers of cells. This proposal is responsive to PAR-98-067, Innovative Technologies for the Molecular analysis of Cancer, and we believe it addresses the second objective of the PAR, namely "novel technologies that will allow high- throughput analysis of genetic alterations, expression of genome products, and monitoring of signal transduction pathways to cancer."