Continuous, Non-Invasive Optical Monitoring of Circulating Tumor Cell-Mediated Metastasis in Awake Mice

项目摘要 血行转移是造成绝大多数癌症相关死亡的原因，其中循环肿瘤转移是导致癌症死亡的主要原因。 细胞（CTC）从原发性肿瘤脱落到外周血（PB）中。少数CTC可能形成 继发部位，临床上极难控制。研究CTC的大多数方法依赖于 抽取和分析分数小的PB血液样品（“液体活组织检查”）。虽然CTC和多细胞 CTC簇（CTCCs）已经研究了几十年，但对其在体内的“动力学”（瞬时）知之甚少 PB中它们的数量变化），以及这些可能如何影响转移发展和对抗癌药物的反应 治疗例如，已知放射疗法可促进癌症的转移性扩散，然而， 对此的机制仍然知之甚少。 我们的团队最近开发了一种新的方法，用于在小动物中体内计数CTC，称为“扩散计数”。 体内流式细胞术”（DiFC）。DiFC使用漫射光来检测大的表达荧光蛋白的CTC， 深血管DiFC每分钟可以采集大约100微升的血液样本， 每毫升PB检测不到1个CTC，并在数分钟内对整个外周血容量进行采样。我们 先前使用DiFC研究小鼠异种移植模型中罕见的CTC和CTCC传播。DiFC透露 CTC数量是高度动态的，并且可以在24小时周期内以数量级或更大的数量级变化。 这些变化在很大程度上被CTC计数方法遗漏，因为这些方法不经常抽血。 该项目的目标是建立一个“可穿戴”拴w-DiFC仪器，将允许连续，非- 在小鼠中长时间侵入性监测CTC数量。w-DiFC光学探头和信号 处理设计将允许在环境照明条件下在自由移动的小鼠中收集数据。我们将首先 使用w-DiFC研究原位异种移植和转基因小鼠疾病发展期间的CTC动力学 转移模型。 我们还将使用w-DiFC来测量髓母细胞瘤放射治疗后的连续CTC动力学 (MB)小鼠模型MB是一种常见的儿童脑癌， 脑和脊柱的软脑膜表面。有证据表明，辐射可能 通过触发CTC向血液中的动员来加剧转移。我们希望w-DiFC能够 在短期、中期和长期时间尺度上衡量CTC将为这一过程提供独特的见解。我们 还将使用w-DiFC来研究使用抗炎药物来阻断促转移作用。所以 这里提出的研究可能最终导致更好地理解转移和改善治疗 儿童MB的治疗方案我们预计，将在这里开发的独特技术将具有 在未来广泛应用于其他癌症和抗癌治疗。

Project Summary Hematogenous metastasis is responsible for a large majority of cancer-related deaths, where circulating tumor cells (CTCs) shed from the primary tumor into the peripheral blood (PB). A small number of CTCs may form secondary sites, which are extremely difficult to control clinically. Most methods for studying CTCs rely on drawing and analyzing fractionally small PB blood samples (“liquid biopsy”). Although CTCs and multicellular CTC clusters (CTCCs) have been studied for decades, little is known about their “dynamics” in vivo (transient changes in their numbers in PB), and how these may affect metastasis development and response to anti-cancer treatment. For example, it is know that radiation therapy may encourage metastatic dissemination of cancer, yet the mechanisms for this are still poorly understood. Our team recently developed a new method for in vivo enumeration of CTCs in small animals called “diffuse in vivo flow cytometry” (DiFC). DiFC uses diffuse light to detect fluorescent-protein expressing CTCs in large, deeply-seated blood vessels. DiFC can sample approximately 100 microliters of blood per minute, permitting detection of fewer than 1 CTC per mL of PB, and sampling of the entire peripheral blood volume in minutes. We previously used DiFC to study rare CTC and CTCC dissemination in mouse xenograft models. DiFC revealed that CTC numbers are highly dynamic and may change by an order-of-magnitude or more over 24 hour periods. These changes are largely missed by CTC enumeration methods that involve infrequent blood draws. The goal of this project is to build a “wearable” tethered w-DiFC instrument that will allow continuous, non- invasive monitoring of CTC numbers over extended periods in mice. The w-DiFC optical probe and signal processing design will permit data collection in freely-moving mice in ambient lighting conditions. We will first use w-DiFC to study CTC dynamics during disease development in an orthotopic xenograft and transgenic mouse model of metastasis. We will use also w-DiFC to measure continuous CTC dynamics after radiation therapy in a medulloblastoma (MB) mouse model. MB is a common form of childhood brain cancer that aggressively metastasizes to the leptomeningeal surfaces of the brain and spine via the PB. There is significant evidence that radiation may exacerbate metastasis by triggering mobilization of CTCs into the blood. We expect that the ability of w-DiFC to measure CTCs over short-, medium-, and long-term timescales will provide unique insights into this process. We will also use w-DiFC to study the use of anti-inflammatory drugs to block the pro-metastatic effect. Hence, the studies proposed here could ultimately lead to better understanding of metastasis and improved treatment protocols for childhood MB. We anticipate that the unique technologies that will be developed here will have broad application to other cancers and anti-cancer therapies in the future.