In vivo real-time detection of circulating melanoma cells

描述（由申请人提供）：癌症，尤其是黑色素瘤，导致的死亡通常与转移有关。由于血液循环中罕见癌细胞的定量检测似乎是转移发展、癌症复发和治疗功效的早期标志，并且由于用于检测转移细胞的测定目前仅是不足以完成这些任务的体外技术，因此我们的提议将体内光声（PA）流式细胞术（FC）的发展作为其最终目标，一种新的方法，用于血流中循环黑色素瘤细胞的超灵敏实时，无标记非侵入性定量检测。我们将通过实现以下具体目标来实现这一目标：目标1。研制了先进的PA流式细胞仪（PAFC），并对其参数进行了体外验证。将开发一种新的、先进的PA流式细胞仪，其使用具有高脉冲重复率的近红外（IR）激光。其主要特征，特别是其敏感性阈值，将在体外评估与静态和流动的黑色素瘤细胞的使用。目标2.评估PAFC在动物模型中体内真实的检测单个黑素瘤细胞的能力。将通过PA光谱法在小鼠模型中在不同激光波长下体内测定黑素瘤细胞相对于血液和皮肤组织的吸收对比度。将通过将细胞注射到小鼠尾静脉中来验证PAFC用于实时、无标记检测黑素瘤细胞的能力。目标3。确定PAFC在动物模型中监测肿瘤发展不同阶段循环转移性黑色素瘤细胞的能力。在肿瘤发展的不同阶段，将在动物模型的血液循环中用PA技术真实的实时检测转移性肿瘤细胞，可用的常规测定法作为独立对照。目标4。将PAFC转化为人类应用，用于在黑色素瘤发展的不同阶段定量监测CTC。将在四个阶段中评估无痛、非侵入性、无标记、基于纤维的PAFC用于定量检测人体内循环黑素瘤细胞的能力：（1）来自掺有黑素瘤人细胞系的健康供体的血液样品的离体PA研究;（2）用常规测定验证的来自黑素瘤患者的血液样品的离体PA研究;（3）作为具有不同皮肤色素沉着的对照组的健康个体的体内研究;以及4）处于疾病的不同阶段的黑素瘤患者。在本研究的过程中，我们将获得统计学上显著的数据，这些数据将证明这种创新技术在体内定量监测循环黑色素瘤细胞而无需标记的前所未有的能力。实现这一目标对公众健康的益处扩展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物，以及评估治疗效果。公共卫生相关性：将在（1）白人和非裔美国人健康志愿者和（2）处于不同疾病阶段的黑色素瘤患者中评估无痛、无创、无标记、基于光纤的光声流式细胞术（PAFC）对来自不同血管的PA信号进行选择性、时间分辨检测的能力，将对这些患者的循环转移性黑色素瘤细胞进行定量测定。在本研究的过程中，我们将获得统计学上显著的数据，这些数据将证明这种创新技术在体内定量监测循环黑色素瘤细胞而无需标记的前所未有的能力。实现这一目标对公共卫生的益处扩展到常规监测循环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物，以及评估治疗的疗效。

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. PUBLIC HEALTH RELEVANCE: The capability of a painless, noninvasive, label-free, fiber-based photoacoustic flow cytometry (PAFC) for selective, time-resolved detection of PA signals from different vessels will be assessed in (1) Caucasian and African American healthy volunteers and (2) melanoma patients at different stages of disease, in whom circulating metastatic melanoma cells will be quantitatively determined. 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 the routine monitoring of circulating cells as early markers of micrometastatic development and cancer recurrence in vivo in melanoma patients, as well as to evaluating the efficacy of therapy.