Fluorescence Molecular In Vivo Liquid Biopsy of Circulating Tumor Cells

项目摘要 这个项目的目标是开发新的技术来直接检测循环中的肿瘤细胞(CTCs)。 无需抽取血液样本即可检测血液流动。转移是大多数与癌症有关的原因 死亡，通常是由CTCs通过血管系统传播引起的。因此，CTC非常令人感兴趣 在临床和基础癌症研究方面。几乎所有研究CTCS的方法都依赖于绘图和分析 少量(7.5毫升)血液样本，也就是广为人知的“液体活检”。然而，由于CTC的稀缺性 从少量血液样本中估计CTC数量是非常不准确的，稀有细胞可能逃脱检测 完全是。液体活检对短期内CTC数值的自然变化也不敏感。 我们的团队最近开发了一种新技术，体内扩散流式细胞术(DIFC)，以检测罕见的， 荧光标记的CTCs直接在小动物体内。DIFC使用漫反射光对大循环进行采样 大块组织中的血量。因此，DIFC的主要优势是敏感性：我们证明了DIFC可以 准确检测每毫升PB少于1个四氯化碳。因为它不需要抽血，DIFC还允许 对单个动物进行的纵向研究。DIFC还可以很容易地扩展到更大的肢体、物种和潜在的 即使对人类来说也是如此。 然而，在人类中使用DIFC需要对靶CTCs进行明亮和特异的荧光标记。 活着。幸运的是，可注射分子示踪剂技术在技术和监管方面取得了重大进展。 用于荧光引导的癌症手术。特别值得注意的是，OTL38是一种近红外(NIR) 小分子叶酸受体(FR)靶向探针正在进行卵巢癌和肝癌的III期临床试验。 OTL38对血液中的CTCs具有很高的特异性和亲和力，其他血细胞或 血管壁。此外，由于近红外光在生物组织中的光衰减最小， OTL38适用于较深部位的血管检测。 该项目的目标是开发和验证体内荧光分子的使能技术 CTCs的液体活检。在目标1中，我们将建立一个用于人类手腕或前臂的H-DIFC系统，其中 动脉流速为每分钟100s毫升。在目标2中，我们将使用OTL38验证FR+CTCs的标记，并 H-DIFC在体外模拟手臂流动模型中的可探测性。我们将量化具体情况， 亮度和外部可探测性在组织中最深可达4毫米。在目标3中，我们将测试OTL38和H-DIFC 小鼠转移模型和体内无毛豚鼠(具有与人皮肤相似的光学特性)。 如果成功，H-DIFC将允许对四氯化碳进行敏感和准确的连续计数，而无需绘制 血样。这将是一种用于分级、管理和研究的全新诊断工具 广泛的恶性肿瘤的转移。此外，由于OTL38(和其他荧光示踪剂) 已经在进行高级临床试验，将会有一条快速的途径进行第一次人体试验。

Project Summary The goal of this project is to develop new technology to detect circulating tumor cells (CTCs) directly in the bloodstream without having to draw blood samples. Metastasis is responsible for the majority of cancer-related deaths, and is often mediated by dissemination of CTCs via the vasculature. CTCs are therefore of great interest clinically and in basic cancer research. Nearly all methods for the study of CTCs rely on drawing and analyzing small (7.5 mL) blood samples, which is broadly known as ‘liquid biopsy’. However, due to the rarity of CTCs estimation of CTC numbers from small blood samples is extremely inaccurate, and rare cells may escape detection entirely. Liquid biopsy is also insensitive to natural changes in CTC numbers that occur over short time periods. Our team recently developed new technology, Diffuse in vivo Flow Cytometry (DiFC), to detect rare, fluorescently-labeled CTCs directly in vivo in small animals. DiFC uses diffuse light to sample large circulating blood volumes in bulk tissue. The main advantage of DiFC is therefore sensitivity: we showed that DiFC can accurately detect fewer than 1 CTC per mL of PB. Because it does not require blood draws, DiFC also allows longitudinal studies in individual animals. DiFC is also readily scalable to larger limbs, species, and potentially even to humans. However, use of DiFC in humans would require bright and specific fluorescent labeling of target CTCs in vivo. Fortunately, there has been major technical and regulatory progress in injectable molecular tracer technology for fluorescence guided surgery of cancer. Of particular interest for this proposal, OTL38 is a near-infrared (NIR) small-molecule folate-receptor (FR)-targeted probe that is in phase-III clinical trials for ovarian and liver cancer. OTL38 has high specificity and affinity for CTCs in blood, with low non-specific uptake by other blood cells or vessel walls. In addition, because NIR light experiences minimal light attenuation through biological tissue, OTL38 is suitable for detection from deeper-seated blood vessels. The goal of this project is to develop and validate the enabling technology for ‘fluorescence molecular in vivo liquid biopsy of CTCs’. In Aim 1, we will build an H-DiFC system for use in the human wrist or forearm, where arterial flow rates are 100s of mL per minute. In Aim 2, we will validate labeling of FR+ CTCs with OTL38, and detectability with H-DiFC in an arm-mimicking flow phantom model in vitro. We will quantify specificity, brightness, and external detectability up to 4 mm deep in tissue. In Aim 3, we will test OTL38 and H-DiFC in a mouse metastasis model and in hairless guinea pigs (which have similar optical properties to human skin) in vivo. If successful, H-DiFC would allow sensitive and accurate enumeration of CTCs continuously without drawing blood samples. This would represent a completely new diagnostic tool for staging, managing, and studying metastasis for a broad range of malignancies. Moreover, because OTL38 (and other fluorescent tracers) are already in advanced clinical trials, there would be a rapid pathway to first-in-human testing.