Visualizing Immune Responses in vivo: a systems biology approach

描述（由申请人提供）：本研究的目的是建立一种结合纳米晶体免疫构建物和荧光多光子激光扫描显微镜的多路活体细胞术来研究肿瘤中的免疫细胞。在针对肿瘤的细胞免疫应答研究中，原位肿瘤模型中细胞间相互作用、细胞表型和肿瘤内细胞分布的检测对于解剖免疫治疗成功或失败的机制至关重要。不幸的是，对多种细胞类型与传统荧光团的时空相互作用的观察受到其广谱特性的限制。我们建议通过开发和优化使用纳米晶体抗体复合物的小鼠输注方案来解决这个问题，这应该允许我们通过多光子激光扫描显微镜同时区分五种或更多特定的探针（目的1）。目标是在免疫治疗期间建立和利用靶向纳米晶体免疫构建物对肿瘤免疫细胞进行体内细胞测定和功能分析（目的2）。利用纳米晶体免疫构建物建立这种多路复用体内成像技术，将为进一步了解与活肿瘤或其他病变组织基质中免疫反应相关的分子、细胞和生理事件创造独特的机会，有可能转化为发现和验证新的治疗靶点。此外，这里采用的实验系统有可能加强对一种预后不佳的疾病（肝癌）的癌症研究。

DESCRIPTION (provided by applicant): The objective of this study is to establish a multiplexed intravital cytometry technique by combining nanocrystal-immunoconstructs and fluorescence multiphoton laser-scanning microscopy to study immune cells in tumors. In studies of the cellular immune response against tumors, the detection of cell-cell interactions, cell phenotype and intra-tumor cell distribution in orthotopic tumor models are critical to dissect the mechanisms of successful or failed immunotherapies. Unfortunately, observation of the temporal and spatial interactions of multiple cell types with traditional fluorophores is limited by their broad spectral characteristics. We propose to solve this problem by developing and optimizing infusion protocols in mice using nanocrystal-antibody complexes, which should allow us to distinguish simultaneously five or more specific probes by multiphoton laser-scanning microscopy (Aim 1). The goal is to establish and utilize targeted nanocrystal-immunoconstructs for in vivo cytometric and functional analyses of immune cells in tumors during immunotherapy (Aims 2). Establishing this multiplexed in vivo imaging technique with nanocrystal-immunoconstructs will create unique opportunities for further understanding the molecular, cellular and physiological events associated with immune responses in the stroma of living tumors or other diseased tissues, potentially translating into the discovery and validation of new therapeutic targets. In addition, the experimental system employed here has the potential to enhance cancer research in a disease - liver cancer - with dismal prognosis.) PUBLIC HEALTH RELEVANCE: Currently available intravital imaging techniques for observation of temporal and spatial interactions of multiple immune cell types with tumors using traditional fluorophores are limited by difficulties in concomitantly imaging multiple colors. We propose to solve this problem by developing and optimizing infusion protocols in mice using novel nanocrystal-antibody complexes, which should allow us to distinguish simultaneously five or more specific probes by three-dimensional intravital multiphoton laser-scanning microscopy. Information from this grant may elevate the clinical utility of immunotherapy approaches for cancer.