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Abstract
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.
Highlights
The inflammatory response elicited by photodynamic therapy (PDT) represents a complex and dynamic process characterized by elements of the innate immune system.[1,2] A hallmark of this host response is the massive recruitment of leukocytes, a significant fraction of which are neutrophils, to the treated tumor site.[3,4] This phenomenon has been observed with several different photosensitizers, including the US FDA approved Photofrin.[5]
In our own work using in vivo imaging, we have recently demonstrated that PDT with the phthalocyanine photosensitizer, Pc4, or with 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH), induces a significant increase in Gr1þ cells in irradiated versus control EMT6 mouse mammary carcinoma tumors.[6,7]
We report that in vivo imaging results are consistent with significantly increased Neutrophil elastase (NE) activity in PDTtreated tumors, and the extent of probe accumulation correlates with the magnitude of intratumor neutrophil influx associated with different treatment protocols
Summary
The inflammatory response elicited by photodynamic therapy (PDT) represents a complex and dynamic process characterized by elements of the innate immune system.[1,2] A hallmark of this host response is the massive recruitment of leukocytes, a significant fraction of which are neutrophils, to the treated tumor site.[3,4] This phenomenon has been observed with several different photosensitizers, including the US FDA approved Photofrin.[5]. Neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G are three serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules These proteases are externalized in an active form during neutrophil activation at inflammatory sites, and recent studies have started to elucidate their contribution to the immune response. Commercially available protease activatable probes targeting matrix metalloproteinases (MMPs) (MMPSense®, Perkin Elmer, Hopkinton, MA) have been widely used to illustrate enzyme activity in tumors and in other non-neoplastic diseases, such as stroke and arthritis.[17,18,19] In light of recent findings that NE mediates the upregulation of proinflammatory cytokines[13] and based on the knowledge that the local inflammatory response to PDT is characterized by increased expression of several cytokines, including IL-1β, TNF-α and IL-6,20 we performed an imaging study to characterize the NE enzyme activity in PDT-treated tumors using a newly developed NE-selective near infrared activatable optical probe, neutrophil elastase 680 FAST (NE680). We report that in vivo imaging results are consistent with significantly increased NE activity in PDTtreated tumors, and the extent of probe accumulation correlates with the magnitude of intratumor neutrophil influx associated with different treatment protocols
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