Magnetophoretic Cell Sorting and Analysis

描述（由申请人提供）：这是一个竞争性的续期申请，建立在之前资助期间开发的仪器和专业知识的基础上。我们建议研究将目前的磁性电池分离方法扩展到依赖于电池自身自然（固有）磁化的应用的可行性。该应用基于恶性细胞转化和细胞磁化率增加之间存在联系的假设。支持性证据来自于其他组织磁性研究的大量数据，特别是电子（自旋）顺磁共振（EPR）和实体肿瘤研究。额外的支持证据来自我们自己的数据，即癌细胞系（HeLa和Hep 3B）生理电解质溶液中的磁场诱导运动（磁电泳），与对照组（氧合红细胞）相比，统计上显示速度更快。我们已经证明，在培养基中添加可溶性铁化合物后，细胞磁泳流动性进一步增加，这表明细胞内铁摄取对细胞磁化有影响。具体目的：SA1：研究与匹配的正常细胞相比，在选定的癌细胞系中观察到的磁电泳迁移率（MM）增加的分子机制。在与分包站点（俄亥俄州立大学）的细胞跟踪速度测定（Chalmers博士）和EPR光谱测定（Kuppusamy博士）的合作中，将在癌细胞系上研究培养基中铁对细胞磁电泳迁移率和细胞内顺磁性含量增加的影响。为了提高细胞磁泳迁移率分析仪的灵敏度和分辨率，采用了细胞跟踪测速仪（CTV）。通过增加仪器的磁能密度和场梯度，通过提高细胞跟踪硬件和软件的成像能力，以及通过与场流分馏法专家（Williams博士）合作，包括对流体动力学伪影的校正，可以将磁电泳迁移率分辨率提高10倍。SA3：以正常血细胞为基准，在单细胞水平上测量选定原发肿瘤的磁化率。与肿瘤学专家（Borden博士）合作，将原发肿瘤细胞系与正常对照进行磁电泳流动性差异测试。SA4：测试在模型血细胞悬浮液上无标记、磁性癌细胞分离的可行性。提出的策略是基于可溶性铁转运过程，而不是颗粒铁转运过程，因此可能比目前的做法更能适应癌细胞的病理生物学。它将对磁细胞分离的实践和利用产生深远的影响，并可以补充和扩展现有磁细胞分离方法的应用。

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.