Isolation of Tumor Initiating Cells (TICs) using Contactless Dielectrophoresis

描述(申请人提供)：肿瘤起始细胞(Tic)，也称为癌症干细胞，是具有移植新肿瘤能力的有限数量的癌细胞。 从现有的一个。据推测，它们也是肿瘤转移特性的原因。分离抽动是了解这些细胞在癌症发病机制和进展中的作用的第一步，也是开发改进的癌症特异性治疗方法的关键。TIC是最相关的治疗靶点。然而，由于缺乏合适的高通量和快速分离这些细胞的方法，目前对TIC的分离和鉴定工作受到了阻碍。无接触介电泳法(CDEP)是一种新的、非侵入性的技术，可以根据悬浮在介质中的稀有细胞的物理和电学性质来检测和丰富它们，而不依赖于它们的基因。介质电泳法(DEP)依赖于粒子在非均匀电场作用下的极化运动。传统的DEP微流控系统容易受到电极结垢的影响，并且需要复杂的制造程序，因为电极与样品直接接触，这限制了它们的寿命和成本效益。CDEP通过使用放置在二次、高导电性通道中的外部电极来缓解这些限制，这些二次通道由一层薄的绝缘层与细胞样品隔开。由于该设备不需要复杂的制造技术，因此很容易实现大规模生产，因此，该技术可以被大量生物学家和研究人员用于细胞分离。在过去的二十年里，DEP在分离靶细胞方面的应用得到了广泛的研究，结果表明稀有细胞可以与细胞混合物中的其他细胞分离。我们的中心假设是CDEP能够区分肿瘤模拟细胞(TICS)和非TICS。我们将利用包括实验和计算建模的多方面方法来设计新的CDEP设备，以评估和优化CDEP技术的有效性，以物理地从肿瘤中发现的其他细胞群中分离和丰富肿瘤起始细胞。这一多方面的方法将包括1)与典型肿瘤细胞相比，表征肿瘤启动细胞的双向电泳反应，2)评估CDEP在分离和选择性浓缩肿瘤启动细胞方面的性能，3)通过使用CDEP微设备分选细胞来研究可能的TIC亚群，以及4)使用原代人类前列腺组织，并对原代人类前列腺细胞重复目标1、2和3。成功分离抽搐将导致对消除这一细胞群的最适当治疗策略的了解增加。 公共卫生相关性：非接触介电泳法(CDEP)是一种从细胞混合物或生物液中分离和浓缩稀有细胞的新技术。该项目的目标是开发CDEP作为医生从非TICs中分离患者特异性肿瘤起始细胞(TIC)的工具。这将有助于了解TICS在癌症发病机制和进展中的作用，并将导致对消除这一细胞群体的最适当治疗策略的更多了解。

DESCRIPTION (provided by applicant): Tumor initiating cells (TICs), also known as cancer stem cells, are a finite population of cancer cells that have the ability to transplant a new tumor from an existing one. They are also putatively responsible for the metastatic properties of tumors. Isolation of TICs is the first step towards understanding the role of these cells in the pathogenesis and progression of cancer and is critical towards the development of improved specific therapies for cancer. The TIC is the most relevant therapeutic target. However, current efforts in isolation and characterization of the TIC are hampered by the lack of suitable high throughput and rapid methods to isolate these cells. Contactless-dielectrophoresis (cDEP) is a new, non-invasive technique to detect and enrich rare cells suspended in a medium based on their physical and electrical properties, independent of their genotype. Dielectrophoresis (DEP) relies upon the motion of a particle due to its polarization induced by a non-uniform electric fiel. Conventional DEP microfluidic systems are susceptible to electrode fouling and require complicated fabrication procedures, because the electrodes are in direct contact with the sample, which limits their lifetime and cost effectiveness. cDEP alleviates these limitations by using external electrodes placed in secondary, highly conductive, channels that are separated from the cell sample by a thin insulating layer. Since the devices do not require complicated fabrication techniques, mass production is readily achievable and, therefore, this technique can be used by a large population of biologists and researchers for cell isolation. The application of DEP to separate target cells has been studied extensively in the last two decades, and the results indicate that rare cells can be separated from other cells in a cell mixture. Our central hypothesis is that cDEP is capable of discriminating between tumor imitating cells (TICs) and non-TICs. We will utilize a multifaceted approach involving experimentation and computational modeling to design new cDEP devices to assess and optimize the effectiveness of cDEP technology to physically separate and enrich tumor initiating cells from other cell populations found in a tumor. This multi-faceted approach will include 1) characterize the dielectrophoretic response of tumor initiating cells compared to typical tumor cells, 2) assess the performance of cDEP at isolating and selectively concentrating tumor initiating cells, 3) investigate possible subpopulation of TICs by sorting cells using cDEP microdevices, and 4) use primary human prostate tissue and repeat Aims 1, 2, and 3 with primary human prostate cells. Success in isolating TICs will lead to increased understanding of the most appropriate therapeutic strategies to ablate this cell population. PUBLIC HEALTH RELEVANCE: Contactless Dielectrophoresis (cDEP) is a new technique to isolate and enrich rare cells from a cell mixture or from biological fluids. The goal of this projet is to develop cDEP as a tool for physicians to isolate patient specific tumor initiating cells (TIC) from non-TICs. This will facilitate understanding of TICs role in the pathogenesis, progression of cancer, and will lead to increased understanding of the most appropriate therapeutic strategies to ablate this cell population.