Time Domian Electron Paramagnetic Resonance Imaging

pO2成像的动态范围扩展：在之前的研究中，我们检查并确认了自旋探针Ox071 （Ox063的氘化版本）在R1和R2*的EPR血氧测定中的实用性。由于线宽窄，信号衰减速度较慢，预计使用Ox071进行pO2估计不仅适用于缺氧组织，而且适用于缺氧较少的组织，如实体器官。在这项工作中，我们提出了我们的第一个3D体内EPR血氧测定研究，使用Ox071与Ox063血氧测定进行比较。R2*与[Ox071]和pO2的变化分别使用标准模体溶液在1、2、5、10 mM和0、2、5、10、21%处进行校准。用Ox071或Ox063连续几天对携带MIA Paca-2肿瘤的小鼠进行体内EPR成像。Ox063和Ox071在MRI共配准标记的肿瘤区域的自旋密度、pO2图和pO2直方图相似。使用Ox071和Ox063对胸腺小鼠进行健康肾脏成像。Ox071血氧测定结果显示，与Ox063相比，Ox071在肾脏中pO2分布更为均匀，表明Ox071适用于更高pO2范围的组织血氧测定。肿瘤微环境对依福环磷酰胺疗效的影响：胰腺导管腺癌（Pancreatic ductal adencarcinoma, PDACs）形成低血管和缺氧肿瘤，目前的化疗方案难以治疗。吉西他滨（GEM）通常被用作pdac的一线治疗，但存在化疗耐药和穿透肿瘤内部的问题。环磷酰胺是一种缺氧激活的前药，已被证明与GEM联合使用有效，尽管每种药物相互作用的机制尚未确定。我们使用来自不同肿瘤微环境特征的两种细胞系（MIA Paca-2和SU 86.86）的小鼠异种移植物来探讨每种药物对另一种药物的作用。GEM处理提高了SU.86.86异种移植物小鼠的存活时间（HR =0.35, 95% CI=0.13 ~ 0.90 p=0.03），但对MIA Paca-2小鼠无影响（HR =0.91, 95% CI=0.37 ~ 2.25, p=0.84）。相反，进化环磷酰胺对SU86.86小鼠没有影响，也没有显著提高生存时间（HR=0.57, 95% CI=0.23 ~ 1.42, p=0.22）。在最初灌注不良的MIA Paca-2肿瘤中，电子顺磁共振（EPR）成像显示GEM治疗后氧合恶化，这为GEM激活evofosfamide以及evofosfamide和GEM联合使用的有效性提供了直接机制。亚致死量的两种处理均增强了其他处理在体外对Su86.86的毒性，但对MIAPaca-2没有增强作用。双链DNA损伤的修复在Su86.86联合治疗中得到增强，而在MIA Paca-2中没有。有人提出了进化环磷酰胺和GEM之间协同作用的可能机制。GEM与进化环磷酰胺之间的协同作用似乎源于GEM对肿瘤血管的作用和GEM对同源重组DNA修复过程的抑制的双重作用。各通路的相对重要性取决于肿瘤微环境，值得进一步研究。缺氧激活的前药依福环磷酰胺改善肿瘤氧合。肿瘤有低密度的区域。氧气水平被称为缺氧区，这些区域对放射治疗和化疗有抵抗力。缺氧激活的前药，如环氧环磷酰胺，被开发用于特异性杀死缺氧区域的细胞。在低氧肿瘤微环境中，强还原氧化还原状态将环氧环磷酰胺（TH-302）转化为还原形式，并释放细胞毒性溴异磷酰胺（Br-IPM）片段。因此，这种药物优先攻击肿瘤的缺氧区域，而其他标准的抗癌治疗，如化疗和放疗，往往无效。已提出并在临床前和临床环境中测试了各种与进化环磷酰胺的联合疗法。然而，evofosfamide单药治疗肿瘤缺氧的效果尚未完全了解，部分原因是缺乏定量方法来评估肿瘤体内pO2。在这里，我们使用EPR定量pO2成像来评估肿瘤缺氧在两个胰腺导管腺癌异种移植模型中对evofosfamide治疗的反应；MIA Paca-2肿瘤对进化环磷酰胺有反应，而Su.86.86肿瘤对进化环磷酰胺无反应。EPR成像显示，在缺氧MIA Paca-2肿瘤中，evofosfamide治疗后氧合全面改善，与吡莫硝唑缺氧染色的离体结果一致，与DCE MRI观察到的Ktrans减少明显相反。evolofosfamide不仅可以杀死肿瘤的缺氧区域，还可以改善残留肿瘤区域的氧合，这一观察结果为在evolofosfamide后使用放射和抗增殖药物联合治疗以改善预后提供了理论依据。

Dynamic range extension of pO2 imaging: In the preceding research, we examined and confirmed the utility of spin probe Ox071, deuterated version of Ox063, for both R1 and R2* based EPR oximetry. Due to the narrow line width and slower signal decay, it was expected that pO2 estimation using Ox071 is suitable not only in hypoxic tissue but also in less hypoxic tissue such as solid organs. In this work, we present our first 3D in vivo EPR oximetry study using Ox071 in comparison with Ox063 oximetry. The R2* change with [Ox071] and pO2 was calibrated using standard phantom solutions at 1,2,5,10 mM and 0, 2, 5, 10, 21%, respectively. In vivo EPR imaging of a mouse bearing MIA Paca-2 tumor was performed on successive days by using either Ox071 or Ox063. The spin density, pO2 maps, and pO2 histograms in the tumor regions marked by co-registration with MRI were similar between Ox063 and Ox071. Healthy kidney imaging was also performed on athymic mice using both Ox071 and Ox063. Ox071 oximetry showed more homogeneous pO2 profile in kidney compared with Ox063, suggesting that Ox071 is suitable for oximetry in tissue at higher pO2 range. Tumor microenvironment determinants in evofosfamide efficacy: Pancreatic ductal adenocarcinomas (PDACs) form hypovascular and hypoxic tumors which are difficult to treat with current chemotherapy regimens. Gemcitabine (GEM) is often used as a first line treatment for PDACs, but has issues with chemoresistance and penetration in the interior of the tumor. Evofosfamide, a hypoxia activated prodrug, has been shown to be effective in combination with GEM, although the mechanism of each drug on the other has not been established. We used two mouse xenografts from two cell lines (MIA Paca-2 and SU 86.86) with different tumor microenvironmetal characteristics to probe the action of each drug on the other. GEM treatment enhanced survival times in mice with SU.86.86 xenografts (HR =0.35, 95% CI=0.13 to 0.90 p=0.03) but had no effect on MIA Paca-2 mice (HR =0.91, 95% CI=0.37 to 2.25, p=0.84). Conversely, evofosfamide had no effect on SU86.86 mice and did not improve survival times to a statistically significant degree (HR=0.57, 95% CI=0.23 to 1.42, p=0.22). In MIA Paca-2 tumors, which were initially poorly perfused, electron paramagnetic resonance (EPR) imaging showed that oxygenation worsened when treated with GEM, providing a direct mechanism for the activation of evofosfamide by GEM and the effectiveness of evofosfamide and GEM combinations. Sublethal amounts of either treatment enhanced the toxicity of other treatment in vitro in Su86.86 but not in MIAPaca-2. Repair of double stranded DNA lesions was enhanced in the combination treatment in Su86.86 but not MIA Paca-2. A possible mechanism for the synergy between evofosfamide and GEM has been proposed. The synergy between GEM and evofosfamide appears to stem from the dual action of GEM's effect on tumor vasculature and the GEM inhibition of the homologous recombination DNA repair process. The relative importance of each pathway is dependent on the tumor microenvironment and merits further study. The hypoxia activated prodrug Evofosfamide improves tumor oxygenation. Tumors have regions with low. Levels of oxygen called hypoxic zones, These regions are resistant to radiation therapy and chemotherapy. Hypoxia activated prodrugs such as Evofosfamide are developed to specifically kill cells in hypoxic regions. In hypoxic tumor microenvironments, the strongly reducing redox state converts evofosfamide (TH-302) to a reduced form and releases a cytotoxic bromo-isophosphoramide (Br-IPM) moiety. This drug therefore preferentially attacks hypoxic regions in tumors where other standard anti-cancer treatments such as chemotherapy and radiation therapy are often ineffective. Various combination therapies with evofosfamide have been proposed and tested in preclinical and clinical settings. However, the treatment effect of evofosfamide monotherapy on tumor hypoxia has not been fully understood, partly due to the lack of quantitative methods to assess tumor pO2 in vivo. Here, we use quantitative pO2 imaging by EPR to evaluate the change in tumor hypoxia in response to evofosfamide treatment using two pancreatic ductal adenocarcinoma xenograft models; MIA Paca-2 tumors responding to evofosfamide and Su.86.86 tumors which do not respond. EPR imaging showed oxygenation improved globally after evofosfamide treatment in hypoxic MIA Paca-2 tumors, in agreement with the ex vivo results obtained from hypoxia staining by pimonidazole and in apparent contrast to the decrease in Ktrans observed in DCE MRI. The observation that evofosfamide not only kills the hypoxic region of the tumor but also improves oxygenation in the residual tumor regions provides a rationale for combination therapies using radiation and anti-proliferatives post evofosfamide for improved outcomes.