In Vivo Imaging of Neoplasia

描述（由申请人提供）：本研究计划的广泛、长期目标是开发一种集成的光学分子成像策略，该策略使用荧光肽作为探针来靶向体内癌前（发育不良）组织的存在。宏观和微观荧光仪器被开发为肽检测的补充成像方法。该方法可用于监测小动物模型中的生物标志物表达，以及在接受筛查的人类患者中早期发现癌症。在这个应用中，结肠腺瘤被用作不典型增生的模型。具体目标是：1)利用噬菌体展示技术对培养细胞和新鲜切除的人结肠粘膜标本进行生物筛选，鉴定5种候选肽；2)利用双轴共聚焦结构开发新的光学成像仪器，以可视化与肽结合相关的组织微结构。3)在体内通过体细胞激活cre -重组酶在远端结肠形成息肉的基因工程小鼠和接受常规结肠镜筛查的人类受试者中验证肽与发育不良的优先结合。之所以选择多肽作为探针，是因为多肽的多样性和小尺寸可以提供高亲和力结合，深入组织，并且其低风险的免疫原性和毒性有利于翻译用于临床。噬菌体展示是一种强大的组合技术，它提供了一种公正的方法来识别与发育不良粘膜具有亲和力结合的独特肽，非常适合适应与体内微环境相关的细微变化。双轴共焦结构提供了一种仪器，可以实现亚细胞分辨率与长工作距离，克服组织散射使用离轴荧光收集的影响。此外，这种设计可以缩小尺寸以适应内窥镜。在治疗内窥镜中，直径为5mm的仪器包足够小，可以通过直径为6mm的仪器通道。高速扫描是用一个微小的MEMS（微机电系统）反射镜进行的，该反射镜以足够的帧速率收集光学部分，以生成3D体积图像。候选肽将局部应用于结肠腺瘤和周围正常粘膜，然后进行宏观荧光成像以定位肽结合增加的区域，随后进行显微镜荧光成像以评估发育不良隐窝微结构中肽结合的空间分布。

DESCRIPTION (provided by applicant): The broad, long-term objectives of this research plan are to develop an integrated optical molecular imaging strategy that uses fluorescence peptides as probes to target the presence of pre-malignant (dysplastic) tissue in viva Macroscopic and microscopic fluorescence instruments are developed as complementary imaging methods for peptide detection. This methodology can be used for monitoring biomarker expression in small animal models and for the early detection of cancer in human patients undergoing screening. In this application, the colonic adenoma is used as a model for dysplasia. The specific aims are to 1) identify 5 candidate peptides using techniques of phage display for biopanning against cultured cells and freshly excised specimens of human colonic mucosa, 2) develop new optical imaging instrumentation using the dual axes confocal architecture to visualize the tissue micro architecture relevant to peptide binding, and 3) validate preferential peptide binding to dysplasia in vivo in a genetically engineered mouse that forms polyps in the distal colon by somatic activation of Cre-recombinase and in human subjects undergoing routine screening colonoscopy. Peptides are chosen for development as probes because their high diversity and small size can provide high affinity binding with deep tissue penetration, and their low risk of immunogenicity and toxicity facilitate translation for use in the clinic. Phage display is a powerful combinatorial technique that provides an unbiased approach for identifying unique peptides that exhibit affinity binding to dysplastic mucosa as is well suited to accommodate subtle changes associated with the in vivo microenvironment. The dual axes confocal architecture provides an instrument that can achieve sub-cellular resolution with long working distance and overcomes the effects of tissue scattering using off-axis fluorescence collection. Moreover, this design can be scaled down in size for endoscope compatibility. A 5 mm diameter instrument package is sufficiently small to pass through a 6 mm diameter instrument channel in a therapeutic endoscope. High speed scanning is performed with a tiny MEMS (micro-electro-mechanical systems) mirror that collects optical sections at a sufficient frame rate for generating 3D volumetric images. The candidate peptides will be topically applied to colonic adenomas and surrounding normal appearing mucosa followed by macroscopic fluorescence imaging to localize regions of increased peptide binding for subsequent microscopic fluorescence imaging to assess the spatial distribution of peptide binding within the dysplastic crypt micro architecture. Public Health: The proposed studies will result in the development of novel optical imaging probes and instruments that can be evaluated in pre-clinical models as well as translated to the clinic as practical screening tool for the early detection of cancer in hollow organs.