Intracellular oxygen sensing using 19F MRI

描述(申请人提供)：使用纳米颗粒探针进行MRI细胞跟踪的一个公认的局限性是，它们无法报告体内的细胞活动或细胞存活情况。非侵入性细胞跟踪方法可以监测细胞存活和激活的变化，将在癌症治疗、细胞治疗和抗炎药物的开发中具有重要价值。为了实现这些目标，我们将探索使用全氟化碳(PFC)乳胶成像探针实时监测活体细胞内血氧含量的用途。这项建议建立在我们之前的工作基础上，开发了用于体外和PFC成像试剂的细胞原位标记的MRI细胞跟踪方法。对于体外细胞标记，分离的感兴趣的细胞(例如，白细胞、干细胞或癌细胞)在培养中用PFC乳剂标记，并在转移到受试者后，使用氟-19(19F)MRI在体内跟踪细胞。氟信号产生没有背景的特定于细胞的图像，可以用来量化聚集部位的表观细胞数量。为了原位标记，PFC乳剂被静脉注射并被巨噬细胞吸收，巨噬细胞是炎症部位的家园，可以通过19F MRI进行可视化。在这项计划中，我们将利用细胞内PFC分子的氧气感应特性。氧与PFC的结合导致了19F自旋-晶格弛豫时间(T1)的减少，其中T1与氧分压(PO2)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，以特定于细胞的方式监测细胞内的PO2。我们实验室在癌症模型中的结果表明，测量肿瘤细胞内绝对PO2和抗癌治疗的反应是可行的。总体而言，该提案有两个具体目标。目的1：细胞内PO2能否在体内检测细胞活性？我们将检验这一假设，即pO2的可测量增加是细胞凋亡过程的间接结果。在肿瘤细胞中，我们将描述化疗、自杀基因治疗和效应性T细胞免疫治疗后的细胞PO2反应，所有这些都推动细胞走向凋亡，但使用不同的触发因素。建立细胞活性和细胞内PO2之间的关系可用于新出现的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，这些血氧仪技术可能会被用于细胞治疗的临床试验，以确定细胞移植物在交付给患者后是否可以存活。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能检测到免疫活性吗？在进一步的扩展中，我们将测试这一假设，即定位于炎症部位的巨噬细胞将通过改变细胞内PO2水平来对药物免疫抑制做出反应。将使用炎症性肠病的小鼠模型，并对巨噬细胞进行原位PFC标记。我们将测试巨噬细胞内PO2是否可以作为非侵入性评估抗炎药物的工具。总体而言，拟议的实验将为使用细胞内PFC细胞跟踪剂获取以下信息的广泛领域的调查奠定基础 体内细胞代谢的实时监测。

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.