Ultra-Rare Cell In Vivo Flow Cytometry

 描述：在生物医学研究的许多领域中，稀有循环细胞的研究非常重要。例子包括癌症转移、血液系统恶性肿瘤、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，具有前所未有的能力，用于研究小动物体内罕见的循环细胞。循环细胞通常通过抽取少量血液样本进行量化，这些样本经过纯化，并用血细胞分析仪或流式细胞仪进行分析。然而，众所周知，处理和纯化血液样本会影响细胞活力，而且由于采样量较小，稀有细胞可能逃脱检测。最近，已经开发出‘体内流式细胞术’(IVFC)方法，允许在不抽取样本的情况下对细胞进行计数。虽然非常有用，但它们通常依赖于对流速较小的微小血管的询问，因此罕见的细胞是无法检测到的。研究循环细胞的新型高灵敏度、高精度工具 因此，研究界非常需要。在这项提议中，我们将开发一种微型光学扫描仪，它将使用漫反射光子来询问小鼠肢体中循环的血液。这项名为“超稀有细胞IVFC”(UR-IVFC)的技术将采用许多独特的设计元素，包括：i)带有光纤耦合激光器和荧光探测器的多个层析光学环；ii)高效的几何光收集；iii)频率 编码激光器和探测器通道，以及(Iv)用于准确计数和跟踪细胞的高级信号处理算法。结合起来，UR-IVFC将允许单细胞灵敏度 在10分钟的扫描中，并且每分钟的误警率低于0.001。我们预计UR-IVFC的独特能力将在许多研究领域产生立竿见影的影响。我们将首先使用UR-IVFC来研究多发性骨髓瘤(MM)的治疗，MM是一种血液系统恶性肿瘤，目前还没有治愈方法。我们将研究“细胞动员疗法”，这是一种新兴的治疗MM的策略。MM细胞在化学上被迫从受保护的骨髓利基进入血液循环，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细胞的特定克隆性亚群是否抵抗动员，我们将测试 微小残留病(MRD)动员剂的疗效。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，利用现有技术执行这些策略极其困难(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.