Intracellular In vivo Imaging

细胞跟踪研究对开发基于细胞的新疗法至关重要。例如，我们正在使用PET标记的成人干细胞来跟踪可能的骨髓移植的骨内细胞注射。类似地，我们通过PET标记这些细胞并注射它们来探索NK细胞注射的动力学。我们正在进行研究，以确定是否可以诱导树突状细胞向它们敏感的肿瘤转移。在体内，分子成像剂专门针对细胞表面或微环境。然而，将癌细胞与正常细胞区分开来的大多数高度特异的变化发生在细胞内。因此，挑战是开发既能报告胞浆内变化又能在体内成像的试剂。实现这一目标的第一步是将显像剂靶向细胞表面，这需要与细胞表面标记的亲和力。然后，配体必须被内吞作用内化，然后结合到适当的位置，在那里它被激活。也可以使用内吞作用，而不是特定的细胞表面结合，而是内部结合。这些要求对合成化学提出了很大的要求，因为分子结构必须具有多种功能。我们正在开发可激活的光学结构，只有当它们内化到细胞质时才会发出荧光。利用一系列商业上可获得的染料，这些染料结合靶向化合物，然后经过修饰，在特定的细胞内条件下，如较低的pH和存在特定的酶活性的情况下，我们正在朝着细胞内体内成像的目标前进。这项工作是与东京大学化学系的乌拉诺教授合作进行的。通过证明基于BODIPY和罗丹明骨架的高活性光学显像剂是可能的，我们在这一领域取得了长足的进步。我们还在率先努力创造多模式成像代理；这种代理既可以在光学相机上看到，也可以在PET、MR或放射性核素相机上看到。正在设计的试剂具有高度的生物兼容性，这些元素已经在人类身上使用。例如，我们用罗丹明绿(GSA-RHG)标记的半乳糖基血清白蛋白(GSA)在癌细胞内迅速内化，可能是一种可行的人类使用的试剂。我们正在开发活性荧光分子显像剂，并在过去一年中取得了一些成功。然而，我们继续寻求一种仅在癌细胞内激活而不在其他正常细胞内激活的解决方案。除了GSA作为靶向配体外，我们还使用了商业上可用的抗体，如曲妥珠单抗和西妥昔单抗，它们将实现靶向、结合和内化。具体地说，我们将曲妥珠单抗与吲哚青绿(ICG)联合使用，ICG是FDA批准的一种近红外(NIR)染料，用于靶向原位肿瘤。当抗体与其同源受体结合时，它被内化释放ICG，然后ICG开始发出荧光。这一结构的两个组成部分都是FDA批准的，因此从理论上讲，这项技术可以相当容易地转化为临床。同时成像多个目标的能力引导我们探索多激发和多发射相机。我们曾希望单个激发光能够激活不同波长的多个荧光团，但事实证明这是不现实的。相反，我们使用了多波长激发光，使用了新的Maestro相机。这使我们能够同时在近红外成像多达4个目标，非常有希望在临床应用。此外，我们正在开发带有荧光接收器的光纤显微镜，以允许使用基于光纤的小型显微镜对非常小的区域进行经皮检查。最近，我们已经证明，通过使用高度调谐的实时摄像机可以对未麻醉的活体小鼠进行成像。在不久的将来，开发多靶点多彩色成像以更好地表征肿瘤是可能的。最近，我们已经使用Halotag技术进行体内成像。目前，如果研究人员想要用荧光标记物(如绿色荧光蛋白(GFP))标记细胞，他们必须将合适的基因导入细胞。如果他们对彩色成像的需求发生变化(即，他们需要将细胞标记为红色，以避免与发出绿色荧光的药物竞争)，他们必须重新获得表达红色荧光蛋白(RFP)的细胞系。Halotag是一种基因结构，导致细胞表面表达一种特殊的、非自然的酶。然后，一个Halo配体可以强烈地结合这种酶。通过将不同颜色的荧光团连接到每个卤素配体上，可以改变细胞的颜色，而不需要重新衍生细胞系，这在多色实验中是一个潜在的优势。此外，我们实验室的最新结果表明，通过简单地将靶向的Halo配体与另一个荧光团交换，可以获得良好的成像结果。这种方法应该会增加类似实验的灵活性。最近，我们开发了基于H-二聚体形成的高度特异的可激活探针。例如，当罗丹明与抗体的Fc部分结合时，罗丹明会二聚化，导致荧光猝灭。PH值可激活的探测器以及上转换纳米晶体已经被开发出来，这些纳米晶体在比它们发射的波长更高的波长下被激发。最后，开发了具有独特靶向性的近红外探测器，这些探测器似乎具有特定的细胞膜毒性，适合于高度靶向性的光免疫疗法。相关癌症部位：卵巢癌、胃癌、结直肠癌、胃肠道、胰腺癌。相关研究领域：生物工程、纳米技术、生物材料研究、生物技术、临床研究。

Cell tracking studies are vital to the development of new cell based therapies. For instance we are using PET labeled adult stem cells to track the intrabone injection of cells for possible bone marrow transplants. Similarly we exploring the kinetics of NK cell injections by PET labeling these cells and injecting them. We are conducting studies to see whether dendritic cells can be induced to traffic to tumors to which they are sensitized.In vivo molecular imaging agents specifically target the cell surface or microenvironment. However, most of the highly specific changes of cancer cells that differentiate cancers cells from normal cells occur intracellularly. The challenge, therefore, is to develop agents that report intracytoplasmic changes yet still are capable of being imaged in vivo. The first step in achieving this goal is to target the imaging agent to the cell surface which requires affinity for a cell surface marker. The ligand must then be internalized by endocytosis and then bind to the appropriate site whereupon it activates. Endocytosis can also be used without specific cell surface binding, but internal binding. These requirements place large demands on synthetic chemistry since the molecular construct must have multiple functionalities. We are developing activatable optical constructs which only fluoresce when they are internalized to the cytoplasm. Using a series of commercially available dyes that are bound to targeting compounds and then modified to fluoresce under specific intracellular conditions such as lower pH and in the presence of specific enzymatic activity we are making progress toward the goal of intracellular in vivo imaging. This work is being performed in collaboration with Prof. Urano from the University of Tokyo Chemistry Department. We have made considerable advances in this area by proving that it is possible to create highly activatable optical imaging agents based on the BODIPY and Rhodamine backbones. We are also pioneering efforts to create multimodal imaging agents; agents that can be seen on both optical cameras as well as PET, MR or radionuclide cameras. The agents being designed are highly biocompatible and elements have already been used in humans. For instance, the agent Galactosylserum Albumin (GSA) which we have labeled with Rhodamine Green (GSA-RhG) is internalized rapidly within cancer cells and may be viable as an agent for human use. We are developing activated fluorescent molecular imaging agents and have a number of successes over the year. However, we continue to pursue a solution that will activate only within cancer cells and not within other, normal cells. In addition to GSA as a targeting ligand we are employing commercially available antibodies such as trastuzumab and cetuximab that will enable targeting, binding and internalization. Specifically, we have used trastuzumab in combination with a self quenched indocyanine green, ICG, an FDA approved Near InfraRed (NIR) dye to target in situ tumors. When the antibody binds its cognate receptor, it is internalized releasing the ICG which then begins to fluoresce. Both components of this construct are FDA-approved so in theory this technique could be translated clinically fairly easily.The ability to image multiple targets simulanteously led us to explore multiexcitation and multiemission cameras. We had hoped that a single excitation light would be able to activate multiple fluorophores at differing wavelengths but this proved to be unrealistic. Instead, we use multiple wavelength excitation light using a new Maestro camera. This has allowed us to simultaneously image up to 4 targets in the near infrared and is very promising for clinical application. Additionally, we are developing fiberoptic scopes with fluorescence receptors to allow very small areas to be examined percutaneously using small fiber-based scopes. Recently, we have demonstrated that is possible to image live unanesthetized mice by using a highly tuned real time camera. It may be possible in the near future to develop multi-targeted multi color imaging to better characterize tumors.Recently, we have used the Halotag technology to perform in vivo imaging. Currently, if researchers want to label cells with a fluorescent marker (such as Green Fluorescent Protein (GFP)), they must transfect the cell with a suitable gene. If their needs for color imaging change (i.e., they need to label the cells red so as not to compete with a drug that fluoresces in the green) they must rederive a cell line that expresses Red Fluorescent Protein (RFP). Halotag is a genetic construct that leads to a special, non-natural enzyme expression on the cell surface. A Halo ligand can then bind this enzyme avidly. By attaching different color fluorophores to each Halo ligand it is possible to change the colors of the cells without rederiving the cell line, a potential advantage in multicolor experiments. Moreover, recent results in our lab demonstrate that is possible to get excellent imaging results by simply exchanging the targeting Halo ligand with another fluorophore. This method should add flexibility to similar experiments.Most recently, we have developed highly specific activatable probes based on H dimer formation. For instance, Rhodamine dimerizes when bound to the Fc portion of an antibody leading to a quenching of fluorescence. pH activatable probes have been developed as well as upconverting nanocrystals that are excited at higher wavelengths than they emit at. Finally, uniquely targeted near infrared probes have been developed that appear to have specific cell membrane toxicity suitable for photoimmunotherapy therapy that is highly targeted.Relevant cancer sites: Ovarian Cancer, Stomach Cancer, Colorectal Cancer, Gastrointestinal Tract, Pancreatic Cancer. Relevant Research Areas: Bioengineering, Nanotechnology, Biomaterials Research, Biotechnology, Clinical Research.