In vivo real-time detection of circulating melanoma cells

描述(申请人提供)：死于癌症，尤其是黑色素瘤，通常与转移有关。由于血液循环中罕见癌细胞的定量检测似乎是转移发展、癌症复发和治疗效果的早期标志，而且目前检测转移细胞的方法仅限于体外技术，不能满足这些任务，因此我们的建议将体内光声(PA)流式细胞术(FC)的发展作为其最终目标，这是一种超灵敏的实时、无标记的血流中循环黑色素瘤细胞的新方法。我们将通过实现以下具体目标来实现这一目标：目的1.研制一种先进的PA流式细胞仪，并在体外验证其参数。一种新的、先进的PA流式细胞仪将被开发出来，它使用具有高脉冲重复频率的近红外(IR)激光。它的主要特征，特别是其敏感性阈值，将在体外使用静态和流动的黑色素瘤细胞进行评估。目的2.在动物模型中评价PAFC实时检测单个黑色素瘤细胞的能力。黑色素瘤细胞相对于血液和皮肤组织的吸收对比度将通过体内不同激光波长下的小鼠模型的PA光谱来确定。PAFC实时、无标记检测黑色素瘤细胞的能力将通过向小鼠尾静脉注射细胞来验证。目的3.在动物模型中确定PAFC在肿瘤发展不同阶段监测循环转移黑色素瘤细胞的能力。在肿瘤发展的不同阶段，将利用PA技术在动物模型的血液循环中实时检测转移的肿瘤细胞，可用的常规检测方法作为独立的对照。目的4.将PAFC转化为人类应用，用于对黑色素瘤不同发展阶段的CTCs进行定量监测。无痛、无创、无标签、基于纤维的PAFC在人体内定量检测循环黑色素瘤细胞的能力将被分四个阶段进行评估：(1)含有黑色素瘤人类细胞系的健康捐赠者血液样本的体外PA研究；(2)经常规检测方法证实的黑色素瘤患者血液样本的体外PA研究；(3)以具有不同皮肤色素沉着的健康人为对照的体内研究；以及4)不同疾病阶段的黑色素瘤患者的体外PA研究。在这项研究的过程中，我们将获得具有统计学意义的数据，这些数据将证明这项创新技术在不需要标记的情况下，在体内定量监测循环黑色素瘤细胞的前所未有的能力。实现这一目标对公众健康的好处延伸到常规监测循环肿瘤细胞作为黑色素瘤患者体内微转移发展和癌症复发的早期标志，以及评估治疗效果。

DESCRIPTION (provided by applicant): Death from cancer, especially melanoma, is most often related to metastasis. As the quantitative detection of rare cancer cells in the blood circulation appears to be an early marker of metastatic development, cancer recurrence, and therapeutic efficacy, and as assays for detecting metastatic cells are currently only in vitro techniques that are inadequate to these tasks, our proposal has as its ultimate goal the development of photoacoustic (PA) flow cytometry (FC) in vivo, a new method for ultrasensitive real-time, label-free noninvasive quantitative detection of circulating melanoma cells in blood flow. We will pursue this goal through accomplishment of the following Specific Aims: Aim 1. Develop an advanced PA flow cytometer (PAFC) and verify its parameters in vitro. A new, advanced PA flow cytometer will be developed that uses a near- infrared (IR) laser with a high pulse repetition rate. Its main characteristics, especially its sensitivity threshold, will be evaluated in vitro with the use of static and flowing melanoma cells. Aim 2. Estimate the capability of PAFC to detect single melanoma cells in real time in vivo in an animal model. The absorption contrast of melanoma cells in relation to blood and skin tissues will be determined by PA spectroscopy in vivo in a mouse model at different laser wavelengths. The capability of PAFC for real-time, label-free detection of melanoma cells will be verified by the injection of cells into the tail veins of mice. Aim 3. Ascertain the capability of PAFC to monitor circulating metastatic melanoma cells at different stages of tumor development in an animal model. Metastatic tumor cells will be detected in real time with the PA technique in blood circulation in an animal model at different stages of tumor development, with available conventional assays serving as independent controls. Aim 4. Translate PAFC to human application for quantitative monitoring of CTCs at different stages of melanoma development. The capability of a painless, non-invasive, label-free, fiber- based PAFC for quantitative detection of circulating melanoma cells in vivo in humans will be assessed in four stages: (1) ex vivo PA study of blood samples from healthy donors spiked with melanoma human cell lines; (2) ex vivo PA study of blood samples from melanoma patients verified with conventional assays; (3) in vivo study of healthy individuals as a control group with different skin pigmentation; and 4) melanoma patients at different stages of disease. In the course of this study, we will obtain statistically significant data that will demonstrate this innovative technique's unprecedented capability for quantitatively monitoring circulating melanoma cells in vivo without the need for labeling. The benefits to the public health of achieving this goal extend to routinely monitoring circulating tumor cells as early marker for the micrometastasis development and cancer recurrence in vivo in melanoma patients, as well as to evaluating the efficacy of therapy.