Magnetophoretic Cell Sorting and Analysis

说明(由申请人提供)：这是一项竞争性的续展申请，其基础是在前几个资助期中发展起来的工具和专门知识。我们建议研究将当前的磁性细胞分离方法扩展到依赖于细胞自身的自然(固有)磁化作用的应用的可行性。这一应用建立在恶性细胞转化和细胞磁化率增加之间存在联系的假设之上。支持证据来自对组织磁性的其他研究的大量数据，特别是来自电子(自旋)顺磁共振、EPR和实体肿瘤研究的数据。其他支持证据来自我们自己的数据，即癌细胞系(HeLa和Hep 3B)的生理性电解质溶液中的磁场诱导运动(磁电泳性)，显示出与对照组(含氧红细胞)相比，统计上更快的速度。我们发现，在培养基中加入可溶性铁化合物后，细胞的磁电迁移增加，这表明细胞内的铁摄取对细胞的磁化有影响。建议的努力分为以下几个方面：具体目标：SA1：研究所观察到的选定癌细胞系与匹配的正常细胞相比磁电迁移(MM)增加的分子机制。与转包地点(俄亥俄州立大学)的细胞跟踪测速专家(Chalmers博士)和EPR光谱分析专家(Kuppusamy博士)合作，将在癌细胞系上研究介质中的铁对细胞磁电迁移和细胞内顺磁含量增加的影响。SA2：为了提高细胞磁电迁移率分析仪的灵敏度和分辨率，细胞跟踪测速仪(CTV)。通过增加设备的磁能密度和磁场梯度，通过改进细胞跟踪硬件和软件的成像能力，以及通过与场流分馏专家合作对流体动力学伪影进行校正，可以实现将磁致伸缩分辨率提高10倍(威廉姆斯博士)。SA3：以正常血细胞为基准，在单个细胞水平上测量选定的原发肿瘤的磁化率。我们将与一位肿瘤专家(波登博士)合作，对原发肿瘤细胞系与正常对照进行比较，以确定其磁电迁移能力的差异。SA4：在模型血细胞悬液上测试无标记、磁性癌细胞分离的可行性。拟议的策略是基于可溶性铁的运输过程，而不是颗粒铁的运输过程，因此可能比现在的做法更好地适应癌细胞的病理生物学。它可以对如何实践和利用磁胞分离产生深远的影响，并可以补充和扩展现有的磁胞分离方法的应用。 公共卫生相关性：人们对确定癌症生物标志物以改善疾病治疗中的诊断和预后有着浓厚的兴趣。一个特别有前景的应用是血液中循环肿瘤细胞(CTC)的检测、计数和分析。有证据表明，来自与这项赠款申请合作的实验室，对已经历恶性转化的细胞的磁化率有异常的顺磁贡献。我们建议通过使用我们最先进的磁性研究设施来系统地研究这种影响，这些设施包括主要(克利夫兰诊所)和分包(俄亥俄州立大学)站点提供的磁性细胞分离实验室和电子(自旋)顺磁共振(EPR)光谱设施。这项研究可能会对磁分离方法在生物医学研究和癌症治疗中的应用产生深远的影响。

DESCRIPTION (provided by applicant): This is a competing renewal application that builds on the instrumentation and expertise developed in the course of the previous funding periods. We propose to investigate the feasibility of extending the current magnetic cell separation methods to applications relying on the cell's own, natural (intrinsic) magnetization. The application rests on the hypothesis that there is a link between malignant cell transformation and an increase in the cell magnetic susceptibility. The supporting evidence comes from a large body of data from other studies on the magnetic properties of tissues, in particular, from electron (spin) paramagnetic resonance, EPR, and studies of solid tumors. Additional supporting evidence comes from our own data on the magnetic field-induced motion (magnetophoresis) in physiologic electrolyte solutions of cancer cell lines (HeLa and Hep 3B), that demonstrate statistically greater velocity compared to controls (oxygenated red blood cells). We have shown an additional increase in the cell magnetophoretic mobility following the addition of soluble iron compounds to the culture media, suggesting an effect of intracellular iron uptake on the cell magnetization. The proposed effort is divided between: Specific Aims: SA1: To investigate the molecular mechanism for the observed increase in magnetophoretic mobility (MM) in selected cancer cell lines as compared to matched normal cells. In collaboration with experts in cell tracking velocimetry (Dr. Chalmers) and EPR spectrometry (Dr. Kuppusamy) at the subcontract site (The Ohio State University), the effect of iron in the medium on the cell magnetophoretic mobility and the increased intracellular paramagnetic content will be investigated on cancer cell lines. SA2: To improve the sensitivity and resolution of the cell magnetophoretic mobility analyzer, the Cell Tracking Velocimetry (CTV). The increase of magnetophoretic mobility resolution by a factor up to 10 fold will be accomplished by increasing the magnetic energy density and field gradient of the apparatus, by improving the imaging capability of the cell tracking hardware and software, and by including corrections for fluid dynamics artifacts in collaboration with an expert in field-flow fractionation (Dr. Williams). SA3: To measure the magnetic susceptibility of selected primary tumors at a single cell level against a baseline of normal blood cells. Primary tumor cell lines will be tested against normal controls for differences in the magnetophoretic mobility, in collaboration with an expert oncologist (Dr. Borden). SA4: To test the feasibility of the label-less, magnetic cancer cell separation on model blood cell suspensions. The proposed strategy is based on the soluble - rather than particulate - iron transport processes, and may therefore be more finely tuned to the pathobiology of the cancer cell than is practiced today. It could have a profound impact on how the magnetic cell separation is practiced and utilized and could complement and extend the applications of the already existing magnetic cell separation methods. PUBLIC HEALTH RELEVANCE: There is an intense interest in identifying cancer biomarkers for improved diagnosis and prognosis in the treatment of the disease. One particularly promising application is the detection, enumeration and analysis of circulating tumor cells (CTC's) in blood. There is evidence of abnormal paramagnetic contributions to the magnetic susceptibility of cells that have undergone malignant transformation coming from the laboratories collaborating on this grant application. We propose to systematically investigate this effect by using our state-of-the-art magnetic research facilities that include the magnetic cell separation laboratory and electron (spin) paramagnetic resonance (EPR) spectrometry facility available at the primary (Cleveland Clinic) and the subcontract (The Ohio State University) sites. The proposed research could have a profound impact on the magnetic separation methods in application to biomedical research and the treatment of cancer.