MICA: Whole body 3-D imaging of cancer and inflammation in live zebrafish using optical tomography and fluorescence lifetime readouts of signalling

我们的首要目标是开发一种新的光学技术平台，直接成像整个生物体的疾病机制，随着时间的推移，以期提高我们对疾病进展和特定治疗的“整体”反应的理解，从而开发新的治疗方法。要使内部过程可视化，就需要透明的有机体。斑马鱼的胚胎特别有趣，因为它们本身是透明的，而且是脊椎动物，因此比其他透明的生物更能代表人类生物学。它们越来越多地用于生物医学研究，使用显微镜研究细胞过程，并且已经制作了许多斑马鱼疾病模型，包括癌症和炎症。不幸的是，胚胎的使用阻碍了对肿瘤发展或慢性炎症等疾病进展的全面研究，如果能够在更成熟的鱼类中对这些过程进行成像，将是有用的。然而，由于光的吸收和散射，以及缺乏通过厘米尺度样品进行高分辨率3-D成像的仪器，对较大的鱼进行成像是一个挑战。我们将研究斑马鱼的非色素突变，并开发新的技术来对抗光散射，我们将在一种新的断层成像系统中实现，以实现对整个幼鱼和成年活斑马鱼的组织结构、细胞迁移和信号过程变化的详细三维成像。这种“全局”三维成像对癌症很重要，因为肿瘤细胞通常会从原发部位扩散到其他器官。这被称为转移，很难治疗，经常导致死亡。使用哺乳动物疾病模型（如经常用于研究癌症生物学的小鼠）来观察肿瘤细胞在整个生物体中的生长和扩散，以及它们如何在新的部位生长，这是不可能的，因为它们无法通过光学方法获得。全局三维成像对于研究对污染物的反应如何在整个生物体中扩散，导致慢性炎症，以及身体的防御系统如何招募免疫细胞也很重要。然而，即使是非色素斑马鱼，现有的光学成像技术也仅限于表面附近的图像，并且受到光学散射的严重影响。我们的方法将建立在一种叫做“光学投影断层扫描”的方法上，这种方法类似于x射线断层扫描，但在可见光下工作。我们将把它与新型图像重建软件结合起来，该软件建立在光传播的基本物理和先进技术的基础上，以利用散射光的特性。我们将把这种新的成像平台应用于利用转基因成年斑马鱼研究癌症和炎症。为了研究癌症，我们会培育斑马鱼，我们可以用化学试剂诱导肿瘤，或者移植斑马鱼衍生的肿瘤。这些癌症模型类似于人类的疾病状态。为了研究炎症，我们将使用与人类具有类似免疫反应的成年斑马鱼模型，我们将研究由化学污染物（包括香烟烟雾）引起的炎症的进展。各种斑马鱼模型也将经过基因工程改造，这样在肿瘤细胞、免疫细胞或血管中发现的特定蛋白质将被荧光分子标记，以促进与疾病相关的结构变化和细胞迁移的成像。我们还将使用荧光标记的“生物传感器”，当特定的分子相互作用作为决定正常细胞功能的信号过程的一部分发生时，它们会改变其荧光特性，这些信号过程在癌症和炎症中变得失调。通过观察信号过程何时何地被激活或以其他方式被激活，我们可以研究诸如抗癌或消炎药等潜在疗法的效果。

Our overarching aim is to develop a new optical technology platform to directly image mechanisms of disease throughout whole live organisms as they progress over time with a view to improving our understanding of" global" responses to disease progression, and to specific treatments, in order to develop new therapies. To visualise internal processes requires transparent organisms. Zebrafish embryos are particularly interesting because they are inherently transparent and are vertebrates - and so represent better human biology than other transparent organisms. They are increasingly used for biomedical research, with cellular processes being studied using microscopes, and many zebrafish disease models have been produced, including for cancer and inflammation. Unfortunately the use of embryos precludes full studies of disease progression such as tumour development or chronic inflammation and it would be useful to be able to image such processes in more mature fish. Imaging larger fish, however, is challenged by the onset of absorption and scattering of light and the lack of instrumentation for high resolution 3-D imaging through cm scale samples. We would work with non-pigmented mutations of zebrafish and develop new techniques to counter light scattering that we would implement in a novel tomographic imaging system to enable detailed 3-D imaging of changes in tissue structures, cell migration and signalling processes throughout whole juvenile and adult live zebrafish. Such "global" 3-D imaging is important for cancer because tumour cells typically spread from their original site to other organs. This is called metastasis, which is very difficult to treat and often leads to death. Observing the growth and spread of tumour cells throughout an organism and how they grow at new sites is not possible using the mammalian disease models such as mice that are often used to study cancer biology because they are not optically accessible. Global 3-D imaging is also important for inflammation to study how the response to pollutants spreads throughout an organism, leading to chronic inflammation, and how the body's defences recruit immune cells. However, even with non-pigmented zebrafish, the established optical imaging techniques are restricted to image near the surface and are strongly compromised by optical scattering. Our approach would build on a method called "optical projection tomography" that is similar to x-ray tomography but works with visible light. We would combine it with novel image reconstruction software that builds on the basic physics of light propagation and advanced techniques to exploit characteristic properties of the scattered light. We will apply this novel imaging platform to the study of cancer and inflammation using genetically modified adult zebrafish. To study cancer, we would breed zebrafish in which we can either induce tumours with a chemical reagent or in which we can transplant zebrafish-derived tumours. These cancer models resemble the disease state in humans. To study inflammation, we would use adult zebrafish models that have an analogous immune response to humans and we would study the progression of inflammation resulting from chemical pollutants including cigarette smoke. The various zebrafish models would also be genetically engineered such that specific proteins found in tumour cells, immune cells or blood vessels would be labelled with fluorescent molecules to facilitate imaging of the disease-related structural changes and cell migration. We would also use fluorescently labelled "biosensors" that change their fluorescence properties when specific molecular interactions take place as part of the signalling processes that determine normal cellular function and which become dysregulated in cancer and inflammation. By observing when and where signalling processes are activated or otherwise, we can study the effects of potential therapies such as anti-cancer or anti-inflammatory drugs.