Cellular 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骨内注射的细胞，以用于可能的骨髓移植。同样，我们通过PET标记NK细胞并注射NK细胞来探索NK细胞注射的动力学。我们正在进行研究，看看树突状细胞是否可以被诱导到它们敏感的肿瘤。体内分子显像剂专门针对细胞表面或微环境。然而，癌细胞分化为癌细胞与正常细胞的高度特异性变化大多发生在细胞内。因此，挑战在于开发能够报告胞浆内变化但仍能在体内成像的药物。实现这一目标的第一步是将显像剂靶向到细胞表面，这需要对细胞表面标记物具有亲和力。然后配体必须通过内吞作用内化，然后结合到适当的位置，然后它被激活。内吞作用也可以不通过特定的细胞表面结合，而是通过细胞内部结合。这些要求对合成化学提出了很高的要求，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中时才会发出荧光。利用一系列商业上可用的染料，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值和存在特定的酶活性）进行修饰，发出荧光，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，证明了基于BODIPY和罗丹明骨架可以制造出高度可激活的光学显像剂。我们也在努力开创多模式显像剂；可以在光学相机、PET、MR或放射性核素相机上看到的试剂。正在设计的药物具有高度的生物相容性，其中一些成分已经在人体中使用。例如，我们用罗丹明绿（GSA- rhg）标记的半乳糖血清白蛋白（GSA）在癌细胞内迅速内化，可能作为人类使用的药物是可行的。我们正在开发活化荧光分子显像剂，并在过去一年中取得了一些成功。然而，我们将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作为靶向配体外，我们还使用市售抗体，如曲妥珠单抗和西妥昔单抗，这些抗体将实现靶向、结合和内化。具体来说，我们已经将曲妥珠单抗与自猝灭吲哚菁绿（ICG）联合使用，ICG是一种FDA批准的近红外（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，释放出ICG，然后开始发出荧光。这个结构的两个组成部分都是fda批准的，所以理论上这项技术可以很容易地转化为临床。同时对多个目标成像的能力促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，但事实证明这是不现实的。取而代之的是，我们使用多波长的激发光，使用新的Maestro相机。这使我们能够同时在近红外成像多达4个目标，在临床应用中非常有前景。此外，我们正在开发带有荧光受体的光纤示波器，使用小型光纤示波器可以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来更好地表征肿瘤是可能的。最近，我们开发了基于H二聚体形成的高特异性激活探针。例如，罗丹明与抗体的Fc部分结合时会发生二聚，导致荧光猝灭。pH活化探针和上转换纳米晶体已经被开发出来，这些纳米晶体被激发的波长比它们发射的波长要高。最后，独特的靶向近红外探针已经开发出来，似乎具有特定的细胞膜毒性，适合于光免疫治疗，这是高度靶向的治疗。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。最近，MIP一直在开发使用Zr89氧标记跟踪细胞的新方法。所有类型的造血细胞都被标记和跟踪。我们打算尝试使用PET成像在体内跟踪细胞，并已在小鼠和灵长类动物的各种细胞类型和条件下证明了可行性，作为人体测试之前的初步数据。正在开发报告基因策略，以便在更长的时间内监测细胞。结合治疗和细胞追踪的细胞工程目前也在研究中。

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. 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. More recently, MIP has been developing new methods of tracking cells using Zr89 oxine labeling. Hematopoetic cells of all types have been labeled and tracked. We intend to try tracking cells in vivo using PET imaging and have demonstrated feasibility in a variety of cell types and conditions both in mice and primates as preliminary data prior to human testing. Reporter gene strategies are being developed to monitor cells over longer duration. Cellular engineering that combines therapies and cell tracking is also currently undergoing study.