University of Rochester Medical Center, Department of Imaging Sciences, Rochester, New York 14642, USA.
J Biomed Opt. 2013 Oct;18(10):101314. doi: 10.1117/1.JBO.18.10.101314.
We demonstrate the use of an enzyme-activatable fluorogenic probe, Neutrophil Elastase 680 FAST (NE680), for in vivo imaging of neutrophil elastase (NE) activity in tumors subjected to photodynamic therapy (PDT). NE protease activity was assayed in SCC VII and EMT6 tumors established in C3H and BALB/c mice, respectively. Four nanomoles of NE680 was injected intravenously immediately following PDT irradiation. 5 h following administration of NE680, whole-mouse fluorescence imaging was performed. At this time point, levels of NE680 fluorescence were at least threefold greater in irradiated versus unirradiated SCC VII and EMT6 tumors sensitized with Photofrin. To compare possible photosensitizer-specific differences in therapy-induced elastase activity, EMT6 tumors were also subjected to 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-PDT. NE levels measured in HPPH-PDT-treated tumors were twofold higher than in unirradiated controls. Ex vivo labeling of host cells using fluorophore-conjugated antibodies and confocal imaging were used to visualize Gr1+ cells in Photofrin-PDT-treated EMT6 tumors. These data were compared with recently reported analysis of Gr1+ cell accumulation in EMT6 tumors subjected to HPPH-PDT. The population density of infiltrating Gr1+ cells in treated versus unirradiated drug-only control tumors suggests that the differential in NE680 fold enhancement observed in Photofrin versus HPPH treatment may be attributed to the significantly increased inflammatory response induced by Photofrin-PDT. The in vivo imaging of NE680, which is a fluorescent reporter of NE extracellular release caused by neutrophil activation, demonstrates that PDT results in increased NE levels in treated tumors, and the accumulation of the cleaved probe tracks qualitatively with the intratumor Gr1+ cell population.
我们展示了一种酶激活的荧光探针，中性粒细胞弹性蛋白酶 680 FAST（NE680），用于在接受光动力疗法（PDT）的肿瘤中体内成像中性粒细胞弹性蛋白酶（NE）的活性。NE 蛋白酶活性在分别在 C3H 和 BALB/c 小鼠中建立的 SCCVII 和 EMT6 肿瘤中进行了测定。在 PDT 照射后立即静脉注射 4 纳米摩尔的 NE680。在 NE680 给药后 5 小时，进行全鼠荧光成像。此时，用 Photofrin 敏化的照射和未照射的 SCCVII 和 EMT6 肿瘤中，NE680 荧光的水平至少高出三倍。为了比较治疗诱导的弹性蛋白酶活性中可能存在的光敏剂特异性差异，还对 EMT6 肿瘤进行了 2-（1-己氧基乙基）-2-去乙烯基焦脱镁叶绿酸-a（HPPH）-PDT。在 HPPH-PDT 处理的肿瘤中测量的 NE 水平比未照射的对照高两倍。使用荧光染料缀合的抗体对宿主细胞进行离体标记，并使用共聚焦成像来可视化 Photofrin-PDT 处理的 EMT6 肿瘤中的 Gr1+细胞。将这些数据与最近报道的在 EMT6 肿瘤中进行 HPPH-PDT 后 Gr1+细胞积累的分析进行了比较。与未照射的药物对照肿瘤相比，治疗后浸润性 Gr1+细胞的种群密度表明，在 Photofrin 与 HPPH 治疗中观察到的 NE680 倍数增强的差异可能归因于 Photofrin-PDT 诱导的炎症反应显著增加。NE680 的体内成像，它是由中性粒细胞激活引起的 NE 细胞外释放的荧光报告物，表明 PDT 导致处理肿瘤中的 NE 水平升高，并且切割探针的积累与肿瘤内的 Gr1+细胞群体定性地相关。
