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

项目摘要 血源性转移是绝大多数癌症相关死亡的原因，在这种情况下，循环中的肿瘤 细胞(CTCs)从原发肿瘤脱落到外周血(PB)。可能会形成少量的CTC 继发部位，临床上极难控制。大多数研究CTC的方法都依赖于 抽取和分析少量PB血样(“液体活检”)。虽然CTC和多细胞 CTC簇(CTCC)已经被研究了几十年，对它们在体内的动力学(瞬时)知之甚少 外周血中它们数量的变化)，以及这些变化如何影响转移的发展和抗癌反应 治疗。例如，众所周知，放射治疗可能会鼓励癌症的转移扩散，但 实现这一点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，用于在小动物体内计数CTCs，该方法被称为“弥散” 体内流式细胞术“(DIFC)。DIFC使用漫反射光检测大型CTCs中表达荧光蛋白的CTCs， 深埋的血管。在允许的情况下，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，并在几分钟内对整个外周血量进行采样。我们 以前使用DIFC研究罕见的CTC和CTCC在小鼠异种移植模型中的扩散。DIFC透露 CTC数字是高度动态的，可能在24小时内发生数量级或更大的变化。 CTC计数方法很大程度上忽略了这些变化，因为这种方法涉及到不频繁的抽血。 该项目的目标是建立一个“可穿戴的”捆绑的w-DIFC仪器，将允许连续，非 对小鼠体内较长时间内CTC数量的侵入性监测。W-DIFC光学探头和信号 处理设计将允许在环境照明条件下自由移动的老鼠收集数据。我们将首先 应用w-DIFC研究异种原位移植和转基因小鼠疾病发展过程中CTC的动态变化 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，可侵袭性地转移到 大脑和脊椎的软脑膜表面。有重要证据表明，辐射可能 通过触发CTCs的动员进入血液而加剧转移。我们预计w-DIFC的能力将 在短期、中期和长期时间尺度上测量CTC将为这一过程提供独特的见解。我们 还将利用w-DIFC研究使用抗炎药物阻断促转移的作用。因此， 这里提出的研究可能最终导致对转移的更好理解和改进治疗。 儿童甲基溴方案。我们预计将在这里开发的独特技术将具有 广泛应用于其他癌症和未来的抗癌治疗。

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.