Intracellular oxygen sensing using 19F MRI

描述（由申请人提供）：使用纳米颗粒探针的MRI细胞跟踪的一个广泛认可的局限性是它们不能报告体内的细胞活性或细胞活力。非侵入性细胞跟踪方法可以监测细胞活力和激活的变化，这将在癌症治疗、细胞疗法和抗炎药物的发展中具有重要价值。为了实现这些目标，我们将探索使用全氟碳（PFC）乳剂成像探针实时监测体内细胞内氧饱和度的效用。该建议建立在我们之前的工作基础上，开发了MRI细胞跟踪方法，用于用PFC成像试剂对细胞进行离体和原位标记。对于离体细胞标记，用PFC乳剂在培养物中标记感兴趣的分离细胞（如白细胞、干细胞或癌细胞），然后转移到受试者身上，使用氟-19 (19F) MRI在体内跟踪细胞。氟信号产生无背景的细胞特异性图像，可用于量化积聚部位的表观细胞数量。对于原位标记，PFC乳剂被静脉注射，并被巨噬细胞吸收，巨噬细胞进入炎症部位，可以通过19F MRI可视化。在这个提议中，我们将利用细胞内PFC分子的氧传感特性。氧与PFC结合导致19F自旋晶格弛豫时间（T1）的减少，其中T1与氧的分压（pO2）呈线性变化。因此，我们建议将基于19F的细胞跟踪与19F T1测量相结合，以细胞特异性的方式监测细胞内pO2。我们实验室在癌症模型上的结果已经证明了测量肿瘤细胞内绝对pO2和对抗癌治疗反应的可行性。总的来说，该提案有两个具体目标。目的1：胞内pO2能否检测体内细胞活力？我们将检验可测量的pO2增加是凋亡过程的间接后果的假设。在肿瘤细胞中，我们将描述化疗、自杀基因治疗和效应T细胞免疫治疗后的细胞pO2反应，所有这些都驱动细胞凋亡，但使用不同的触发因素。建立细胞活力和细胞内pO2之间的关系可以用于新兴癌症治疗的临床前评估。此外，随着19F细胞跟踪的人类临床翻译的成熟，这些血氧测定技术可用于细胞治疗临床试验，以确定细胞移植物在交付给患者后是否可行。肿瘤模型的研究将有助于为未来各种治疗细胞类型的研究奠定基础。目的2：巨噬细胞胞内pO2能否检测免疫活性？在进一步的扩展中，我们将验证位于炎症部位的巨噬细胞通过改变细胞内pO2水平来响应药理学免疫抑制的假设。将使用炎症性肠病的小鼠模型，以及巨噬细胞的原位PFC标记。我们将测试巨噬细胞胞内pO2是否可以作为无创评估抗炎药物的工具。总的来说，提出的实验将为使用细胞内PFC细胞跟踪代理获取信息的广泛调查领域奠定基础

DESCRIPTION (provided by applicant): A widely recognized limitation of MRI cell tracking using nanoparticle probes is that they cannot report on cellular activity or cell viability in vivo Non-invasive cell tracking methods that can monitor changes in cell viability and activation would be a great value in the development of cancer treatments, cell therapies, and anti-inflammatory drugs. Towards these goals, we will explore the utility of real-time monitoring of intracellular oximetry in vivo using perfluorocarbon (PFC) emulsion imaging probes. This proposal builds on our prior work developing MRI cell tracking methods for ex vivo and in situ labeling of cells with PFC imaging reagents. For ex vivo cell labeling, isolated cells of interest (e.g., leukocytes, stem cells, or cancer cells) are labeled in culture with PFC emulsion, and following transfer to the subject, cells are tracked in vivo using fluorine-19 (19F) MRI. The fluorine signal yields cell-specific images, with no background, that can be used to quantify apparent cell numbers at sites of accumulation. For in situ labeling, PFC emulsion is injected intravenously and taken up by macrophages that home to sites of inflammation and can be visualized by 19F MRI. In this proposal, we will exploit the oxygen sensing properties of the intracellular PFC molecules. Oxygen binding to PFC results in a reduction in the 19F spin-lattice relaxation time (T1), where T1 varies linearly with the partial pressure of oxygen (pO2). Hence, we propose combining 19F-based cell tracking with 19F T1 measurements to monitor intracellular pO2 in a cell-specific manner. Results from our lab in cancer models have demonstrated the feasibility of measuring the absolute intracellular pO2 in tumor cells and response to anti-cancer treatments. Overall, the proposal has two Specific Aims. Aim 1: Can intracellular pO2 detect cell viability in vivo? We will test the hypothesis that a measureable increase in pO2 is an indirect consequence of apoptotic processes. In tumor cells, we will characterize the cellular pO2 response following chemotherapy, suicide gene therapy, and effector T cell immunotherapy, all of which drive the cell towards apoptosis, but employ different triggers. The establishment of a relationship between cell viability and intracellular pO2 can be exploited in the preclinical evaluation of emerging cancer therapies. Moreover, as human clinical translation of 19F cell tracking matures, these oximetry techniques may be used in cell therapy clinical trials to determine whether the cellular graft is viable after delivery to the patient. Stdies in tumor models will help set the foundation for future studies in a variety of therapeutic cell types. Aim 2: Can macrophage intracellular pO2 detect immunoactivity? In a further extension, we will test the hypothesis that macrophages localized at sites of inflammation will respond to pharmacological immunosuppression by altering intracellular pO2 levels. A murine model of inflammatory bowel disease will be used, along with in situ PFC labeling of macrophages. We will test whether macrophage intracellular pO2 can be used as a tool to assess anti-inflammatory drugs non-invasively. Overall, the proposed experiments will set the foundation for a broad field of inquiries using intracellular PFC cell tracking agents to garner information about real-time cell metabolism in vivo.