Intracellular In vivo Imaging

细胞跟踪研究对于开发新的基于细胞的疗法至关重要。例如，我们正在使用PET标记的成体干细胞来跟踪可能的骨髓移植的细胞的骨内注射。同样，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，看看是否可以诱导树突状细胞运输到它们敏感的肿瘤。体内分子成像剂特异性靶向细胞表面或微环境。然而，大多数癌细胞的高度特异性变化发生在细胞内，这些变化将癌细胞与正常细胞区分开来。因此，挑战是开发报告胞浆内变化但仍能够在体内成像的试剂。实现这一目标的第一步是将成像剂靶向细胞表面，这需要对细胞表面标记物的亲和力。然后配体必须通过内吞作用内化，然后结合到适当的位点，从而激活。内吞作用也可以在没有特异性细胞表面结合但内部结合的情况下使用。这些要求对合成化学提出了很高的要求，因为分子构建体必须具有多种功能。我们正在开发可激活的光学结构，只有当它们被内化到细胞质中时才会发出荧光。使用一系列商业上可获得的染料，这些染料与靶向化合物结合，然后在特定的细胞内条件下（如较低的pH值）和特定酶活性的存在下被修饰为荧光，我们正在朝着细胞内体内成像的目标取得进展。这项工作是与东京大学化学系的Urano教授合作进行的。我们已经在这一领域取得了相当大的进展，证明它是可能的，以创建基于BODIPY和罗丹明主链的高度可活化的光学成像剂。我们还开创性地努力创造多模态成像剂;可以在光学相机以及PET，MR或放射性核素相机上看到的试剂。正在设计的药剂具有高度生物相容性，并且已经在人体中使用。例如，我们用罗丹明绿色（GSA-RhG）标记的试剂半乳糖基血清白蛋白（GSA）在癌细胞内迅速内化，并且可以作为人类使用的试剂。我们正在开发激活的荧光分子成像剂，并在过去一年中取得了一些成功。然而，我们继续寻求一种只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作为靶向配体之外，我们还采用了市售的抗体，如曲妥珠单抗和西妥昔单抗，这些抗体将能够实现靶向、结合和内化。具体地，我们已经使用曲妥珠单抗与自猝灭吲哚菁绿色ICG（FDA批准的近红外（NIR）染料）组合以靶向原位肿瘤。当抗体结合其同源受体时，它被内化，释放ICG，然后开始发荧光。这种结构的两个组件都是FDA批准的，因此理论上这种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使我们探索多激发和多发射相机。我们曾希望单一激发光能够激活不同波长的多个荧光团，但这被证明是不现实的。相反，我们使用多波长激发光，使用新的Maestro相机。这使我们能够同时成像多达4个目标在近红外，是非常有前途的临床应用。此外，我们正在开发带有荧光接收器的光纤内窥镜，以便使用小型光纤内窥镜对非常小的区域进行连续检查。最近，我们已经证明，是可能的图像活未麻醉小鼠使用高度调谐的真实的时间相机。在不久的将来，有可能开发多靶点多色成像，以更好地表征肿瘤。最近，我们已经使用Halotag技术来进行体内成像。目前，如果研究人员想用荧光标记（如绿色荧光蛋白（GFP））标记细胞，他们必须用合适的基因标记细胞。如果他们对彩色成像的需求改变（即，他们需要将细胞标记为红色，以便不与发绿色荧光的药物竞争），他们必须重新衍生表达红色荧光蛋白（RFP）的细胞系。Halotag是一种基因构建体，可在细胞表面上导致特殊的非天然酶表达。然后，Halo配体可以贪婪地结合这种酶。通过将不同颜色的荧光团连接到每个Halo配体上，可以改变细胞的颜色，而无需重新衍生细胞系，这是生物学实验中的一个潜在优势。此外，我们实验室最近的结果表明，通过简单地将靶向Halo配体与另一种荧光团交换，可以获得优异的成像结果。这种方法应该为类似的实验增加灵活性。最近，我们已经开发了高度特异性的可激活的探针的基础上H二聚体的形成。例如，若丹明在与抗体的Fc部分结合时二聚化，导致荧光淬灭。已经开发了pH可激活的探针以及在比它们发射的波长更高的波长下激发的上转换纳米晶体。最后，已经开发出独特的靶向近红外探针，其似乎具有适合于高度靶向的光免疫疗法的特异性细胞膜毒性。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.