In vivo real time detection of circulating melanoma cells

描述(申请人提供)：大多数癌症患者死于转移性疾病，原因是循环中的肿瘤细胞(CTCs)从原发肿瘤通过血液扩散到远处的器官。临床研究已经证明，使用CTC计数作为转移发展和治疗效果的预后或预测标志物具有巨大的潜力。尽管在体外CTC检测方面取得了很大进展，但由于采集的血样较少，它们的灵敏度仍然较低。为了克服这些问题，我们发展了体内光声(PA)流式细胞术(PAFC)，并在黑色素瘤患者的第一个试点试验中证明了其可行性。然而，这些试验仅揭示了PAFC的主要局限性，而揭示了体内大多数光学诊断和治疗方法。这些问题包括对深层组织的非侵入性光辐射传输不佳，激光安全问题，以及在癌症早期诊断、预防转移和克服肿瘤耐药性方面进展甚微。同时，这些试验帮助确定了进一步显著改进体内光学技术，特别是PAFC的创新方法，包括将更高水平的LasR能量安全地传递到更深的组织，以及在复杂的生物背景下更高效地检测低色素CTCs。这项建议的目标是开发创新的方法，以显著提高体内光学方法的灵敏度，重点是PAFC平台，并在大量黑色素瘤患者中展示其能力，通过检测和识别CTCs以及潜在的CTP来实时控制治疗效果。特别是，我们提出了诊断激光能量的分步传输、人体内非线性纳米气泡相关信号放大、可切换波长激光的皮秒光谱、控制CTC尺寸、聚焦球形超声换能器阵列以及光学皮肤透明的概念，以减少人体皮肤中的光散射。为了实现这一目标，我们将追求以下具体目标：(1)测试非线性、分步传输激光能量的概念。(2)优化CTCs的非线性、分级PA检测和PT体外清除的参数。(3)探讨黑色素瘤患者常规治疗过程中和治疗后CTCs的体内非线性、分级PA诊断和PT治疗的临床能力。新的无标签PA-PT治疗平台的临床原型将在人体内进行三个阶段的测试：(1)健康人的体内研究，2)晚期黑色素瘤患者的体内PA-PT治疗，以及3)在手术和给药期间控制黑色素瘤患者的CTCs。在这项研究的过程中，我们将获得有统计学意义的数据，这些数据将证明这项创新技术前所未有的灵敏度阈值为1CTC/500毫升；我们还将尝试达到1CTC/1-3L的阈值。

DESCRIPTION (provided by applicant): Most cancer patients die from metastatic disease as the result of circulating tumor cells (CTCs) spreading from a primary tumor through the blood to distant organs. Clinical studies have demonstrated the tremendous potential of using CTC counts as prognostic or predictive markers of metastatic development and therapeutic efficacy. Despite significant progress in the development of in vitro CTC assays, they still have low sensitivity due to the small samples of blood taken. To overcome these problems, we developed in vivo photoacoustic (PA) flow cytometry (PAFC) and have demonstrated its feasibility in the first pilot trials in melanoma patients. However, these trials revealed the principal limitations nt only of PAFC but most optical diagnostic and therapeutic methods in vivo. These include poor delivery of noninvasive optical radiation to deep tissue, laser safety concerns, and little progres in early cancer diagnosis, prevention metastasis, and overcoming of tumor resistance. Simultaneously, these trials helped identify innovative ways to further significantly improve optical technology in vivo, particularly PAFC, including the safe delivery of a higher level of lasr energy to deeper tissue and more efficient detection of low pigmented CTCs in complex biological backgrounds. The goal of this proposal is to develop innovative approaches to significantly enhance the sensitivity of optical methods in vivo, with a focus on the PAFC platform, and to demonstrate its capabilities in a large cohort of melanoma patients for real-time control of therapeutic efficacy through detection and identification of CTCs, and potentially CTPs. In particular, we propose the concept of fractionated delivery of diagnostic laser energy, nonlinear nanobubble-associated signal amplification in humans, picosecond spectroscopy with switchable wavelength laser, controlling of CTC sizes, and a focused spherical ultrasound transducer array, together with optical skin clearing to reduce light scattering in human skin. To achieve this goal, we will pursue the following Specific Aims: (1) Test the concept of nonlinear, fractionated delivery of laser energy. (2) Optimize the parameters of the nonlinear, fractionated PA detection and PT eradication of CTCs in vitro. (3) Explore the clinical capabilities of in vivo nonlinear, fractionated PA diagnosis and PT therapy of CTCs in melanoma patients during and after conventional treatment. The new clinical prototype of label-free PA-PT theranostic platform will be tested in vivo in humans in three stages: (1) in vivo study of healthy individuals, 2) in vvo PA-PT theranostics in advanced-stage melanoma patients, and 3) controlling CTCs at surgery and during drug administration in melanoma patients. In the course of this study, we will obtain statistically significant data that will demonstrate this innovative technique's unprecedented sensitivity threshold of 1 CTC/500 mL; we will also attempt to achieve a threshold of 1 CTC/1-3 L.