Ultra-Rare Cell In Vivo Flow Cytometry

 描述：在生物医学研究的许多领域，稀有循环细胞的研究非常重要。例子包括癌症转移，血液恶性肿瘤，器官移植生物学，免疫学，生殖医学和干细胞疗法。在这个项目中，我们将开发一种新的高通量光学扫描仪，具有前所未有的研究小动物体内稀有循环细胞的能力。 循环细胞通常通过抽取少量血液样品来定量，所述血液样品经纯化并用血细胞计数法或流式细胞术分析。然而，众所周知，处理和纯化血液样品会影响细胞活力，并且由于采样体积小，稀有细胞可能无法检测到。最近，开发了“体内流式细胞术”（IVFC）方法，可以在不抽取样本的情况下对细胞进行计数。虽然非常有用，但这些技术通常依赖于对流速较小的微观血管的询问，因此无法检测到稀有细胞。用于研究循环细胞的新型高灵敏度和高精度工具 因此，研究界非常需要。 在这个提议中，我们将开发一种小型化的光学扫描仪，它将使用漫射光子来询问小鼠肢体中的循环血液。该技术-被称为“超稀有细胞IVFC”（UR-IVFC）-将采用许多独特的设计元素，包括：i）多个具有光纤耦合激光器和荧光检测器的断层光学环，ii）有效的几何光收集，iii）频率 编码激光器和检测器通道，以及，iv）用于精确计数和跟踪细胞的高级信号处理算法。结合使用，UR-IVFC将允许单细胞灵敏度 在10分钟的扫描中，并且错误警报率小于每分钟0.001。 我们预计UR-IVFC的独特功能将在许多研究领域产生直接影响。我们将首先使用UR-IVFC研究多发性骨髓瘤（MM）的治疗，MM是一种目前无法治愈的血液恶性肿瘤。我们将研究“细胞动员疗法”，这是一种新兴的MM治疗策略。MM细胞被化学迫使从保护性骨髓龛进入循环，在那里它们容易受到化疗的影响。我们将使用UR-IVFC来研究MM细胞的特定克隆亚群是否对动员有抵抗力，我们将测试 动员剂对微小残留病（MRD）的疗效。除了阐明MM生物学和治疗耐药性的分子机制外，这些研究还可以为MM患者提供新的治疗策略。此外，使用现有技术进行这些研究非常困难（或完全不可行）。

 DESCRIPTION: There are many areas of biomedical research where the study of rare circulating cells is important. Examples include cancer metastasis, hematological malignancies, organ transplant biology, immunology, and reproductive medicine and stem-cell therapies. In this project we will develop a new high-throughput optical scanner with unprecedented capabilities for studying rare circulating cells in small animals in vivo. Circulating cells are normally quantified by drawing small blood samples which are purified and analyzed with hemocytometry or flow cytometry. However, it is known that handling and purifying blood samples can affect cell viability, and that rare cells can escape detection due to the small sampling volume. More recently, `in vivo flow cytometry' (IVFC) methods have been developed that allow enumeration of cells without drawing samples. While extremely useful, these generally rely on interrogation of microscopic blood vessels with small flow rates, so that rare cells are undetectable. New high-sensitivity and high-accuracy tools for studying circulating cells are therefore greatly needed by the research community. In this proposal we will develop a miniaturized optical scanner that will use diffuse photons to interrogate circulating blood in the limb of a mouse. The technology - termed "ultra-rare cell IVFC" (UR-IVFC) - will employ a number of unique design elements including, i) multiple tomographic optical rings with fiber-coupled lasers and fluorescence detectors, ii) efficient geometric light collection, iii) frequency encoded lasers and detector channels, and, iv) advanced signal processing algorithms for accurate counting and tracking of cells. In combination, UR-IVFC will allow single-cell sensitivity in a 10 minute scan and with a false alarm rate less than 0.001 per minute. We anticipate that the unique capabilities of UR-IVFC will have immediate impact in many research fields. We will first use UR-IVFC to study treatment of multiple myeloma (MM), a hematological malignancy for which there is currently no cure. We will study "cell mobilization therapy", which is an emerging treatment strategy for MM. MM cells are chemically forced from the protective bone marrow niche into circulation where they are vulnerable to chemotherapy. We will use UR-IVFC to study whether specific clonal sub-populations of MM cells are resistant to mobilization, and we will test the efficacy of mobilizing agents for minimal residual disease (MRD). In addition to illuminating molecular mechanisms of MM biology and treatment resistance, these studies could yield new therapeutic strategies for patients suffering from MM. Moreover they are extremely difficult (or outright infeasible) to perform with existing technology.