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，癌症复发和治疗效果的早期标志，并且检测转移细胞的检测目前仅在体外技术不足以完成这些任务，因此我们的建议将其作为其最终目标，光声（PA）流式细胞术（FC）在体内的发展，这是一种超灵敏的实时，血液循环中黑色素瘤细胞的无标记无创定量检测。我们将通过实现以下具体目标来实现这一目标。研制一种先进的PA流式细胞仪（PAFC）并在体外验证其参数。一种新的，先进的PA流式细胞仪将开发，使用近红外（IR）激光具有高脉冲重复率。其主要特点，特别是其敏感性阈值，将在体外使用静态和流动黑色素瘤细胞进行评估。目标2。估计PAFC在动物模型中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将通过PA光谱在不同激光波长的小鼠模型体内测定。PAFC实时、无标记检测黑色素瘤细胞的能力将通过将细胞注射到小鼠尾静脉中来验证。目标3。在动物模型中确定PAFC在肿瘤发展的不同阶段监测循环转移性黑色素瘤细胞的能力。转移性肿瘤细胞将通过PA技术在肿瘤发展不同阶段的动物模型血液循环中实时检测，现有的常规检测作为独立对照。目标4。将PAFC转化为人类应用，用于黑色素瘤发展不同阶段ctc的定量监测。无痛、无创、无标签、基于纤维的PAFC定量检测人体循环黑色素瘤细胞的能力将分四个阶段进行评估：(1)对健康供体血液样本进行体外PA研究，其中加入了黑色素瘤人类细胞系；(2)黑色素瘤患者血液样本的体外PA研究，经常规检测验证；(3)以不同皮肤色素沉着的健康个体为对照组进行体内研究；4)处于不同疾病阶段的黑色素瘤患者。在本研究过程中，我们将获得具有统计学意义的数据，这些数据将证明这种创新技术具有前所未有的能力，可以在不需要标记的情况下定量监测体内循环的黑色素瘤细胞。实现这一目标对公共卫生的益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物，以及评估治疗效果。公共卫生相关性：一种无痛、无创、无标签、基于纤维的光声流式细胞术（PAFC）选择性地、时间分辨地检测来自不同血管的PA信号的能力将在(1)白种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进行评估，在这些患者中循环转移性黑色素瘤细胞将被定量测定。在本研究过程中，我们将获得具有统计学意义的数据，这些数据将证明这种创新技术具有前所未有的能力，可以在不需要标记的情况下定量监测体内循环的黑色素瘤细胞。实现这一目标对公共卫生的好处延伸到对循环细胞的常规监测，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。

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.