In Vivo Flow Cytometry to Measure Circulating Tumor Load

描述（由申请人提供）： 许多患有前列腺癌、卵巢癌和其他癌症的患者都有循环肿瘤细胞，即使是在疾病的早期。大多数研究显示循环计数与疾病分期、治疗反应和进展风险相关。这种计数目前难以测量，因为1-20个肿瘤细胞/cc血液在30 - 60亿个红细胞的海洋中丢失。流式细胞术-离体染色细胞流过固定的检测器-是一种已建立的技术，可以检测（和分选）溶液中的稀有细胞。在这里，我们使用毛细血管床中的细胞作为流动室的模拟物，以允许新的体内稀有细胞计数。 我们建议通过静脉注射靶向染料荧光标记毛细血管中的细胞，并使用毛细血管中细胞的物理流动产生的时间尖峰通过非侵入性光学检测器来定量靶细胞计数。在第一阶段，我们构建了一个基于纤维的非侵入性细胞检测器，可选地包括共聚焦成像/空间滤波，放置在浅表毛细血管床的组织与薄粘膜，如在嘴唇或舌下。我们测试了至少两种可注射的类别特异性染料：（1）PSMA靶向前列腺导管上皮来源的细胞，和（2）叶酸靶向叶酸受体过表达的癌症。因为染料和它们的内吞细胞表面靶标都是血管内的，所以在nM浓度下，递送是快速的（秒）并且结合是强的（100 K/细胞），产生高的细胞-血液比。我们在毛细血管模型和粗糙的老鼠中进行测试。相同激发/发射波长的血红蛋白允许校正检测到的血管体积的计数。如果成功，GMP染料合成和人体试验开始在第二阶段。 在这项修订的I期（R43）研究中，具体目标是：（a）生产基于纤维的探针，具有或不具有共聚焦光学器件，用于收集来自亚表面毛细血管床的光，（b）使用PSMA染料标记的LNCaP细胞和叶酸染料标记的KB细胞来证明毛细血管床模型中的操作，以及（B）在患有LNCaP和KB肿瘤的裸小鼠中证明，使用标记和原位造影剂注射。如果成功，II期（R44）的目标将是a）开发药物的GMP生产;（B）在临床前试验中证明该药物的安全性;（c）申请并获得IND、IDE和RB批准最新的至少一种药物，以及（d）在患有/不患有卵巢癌的女性（和患有/不患有前列腺癌的男性）中进行测试，以测试体内性能。 如果成功，这可能会导致改善分期，治疗监测，并可能早期诊断。

DESCRIPTION (provided by applicant): Many patients with prostate, ovarian, and other cancers have Circulating tumor cells, even in early-stage disease. Most studies show an association of circulating counts with disease stage, treatment response, and progression risk. Such counts are currently difficult to measure, as the 1-20 tumor cells/cc of blood are lost in a sea of 3-6 billion red blood cells. Flow cytometry -- ex vivo stained cells flowing past a fixed detector-- is an established technique which has allowed for detection (and sorting) of rare cells in solution. Here, we use cell low in the capillary bed to serve as an analogue of a flow chamber, to permit novel IN VIVO rare-cell counts. We propose to fluorescently label cells lowing in the capillaries, via i.v. injection of targeted dyes, and to use temporal spikes created by the physical flow of cells in the capillaries past a noninvasive optical detector to quantitate target cell count. In Phase I, we construct a fiber-based noninvasive cell detector, optionally including confocal imaging/spatial-filtering, to be placed over superficial capillary beds in tissues with thin mucosa, such as on the lip or sublingually. We test at least two injectable class-specific dyes: (1) PSMA-targeted to cells of prostate duct epithelial origin, and (2) folate-targeted to folate-receptor-overexpressing cancers. Because both the dyes and their endocytosing cell-surface targets are intravascular, delivery is rapid (secs) and binding is strong (100K/cell) at nM concentrations, yielding high cell-to-blood ratios. We test in a capillary model and rude mice. Hemoglobin at the same excite/emit wavelengths allows correction of counts for detected vascular volume. If successful, GMP dye synthesis and human testing begin in Phase II. In this revised Phase I (R43) study, specific aims are: (a) to produce a fiber-based probe, with or without confocal optics, for collecting light from subsurface capillary beds, (b) to demonstrate operation in a capillary bed model using PSMA-dye labeled LNCaP cells and folate-dye labeled KB cells, and (c) to demonstrate in nude mice with LNCaP and KB tumors, using both labeled and in-situ contrast injection. If successful, Phase II (R44) aims will be a) to develop GMP production of the agent; (b) to demonstrate safety of this agent in pre-clinical trials; (c) to apply for and obtain IND, IDE, and RB approval latest at least one agent, and (d) to test in women with/without ovarian cancer (and men with/without prostate cancer), to test performance in vivo. If successful, this may lead to improved staging, treatment monitoring, and possibly earlier diagnosis.