Enzyme-activatable imaging probe reveals enhanced neutrophil elastase activity in tumors following photodynamic therapy.

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 Gr1+ cells in irradiated versus control EMT6 mouse mammary carcinoma tumors.6,7 Several animal studies have also investigated the importance of intratumor neutrophil accumulation to the long-term curative outcome of PDT using selective depletion or inactivation of neutrophils and have demonstrated that the depletion of these effector cells results in a decrease of the PDT-mediated tumor cure rate.3,8,9 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. In this context, NE has received particular attention.10,11 Although NE has been implicated in a variety of diseases due to its capacity to degrade the extracellular matrix, new findings provide compelling evidence that NE contributes considerably to the regulation of host functions.12 A recent study reported that NE is involved in polymorphonuclear leukocyte-mediated host protection in a mouse model of Pseudomonas aeruginosa-induced pneumonia.13 The study also demonstrated that extracellularly active NE has the capacity to induce mRNA expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α), macrophage inflammatory protein-2, and interleukin-6 (IL-6). The growing recognition of the role of proteinases and proteolytic cascades in both the growth and metastasis of tumors has led to the development not only of therapeutic strategies using proteinase inhibitors but also of methods to detect and image tumors in vivo via tumor-associated proteolytic activities.14–16 These selective protease probes belong to a family of imaging agents that are optically invisible or at least display reduced fluorescence when administered intravenously until activated by enzymatic cleavage at the disease site. This allows localization of fluorescence signal in areas of enzyme activity with low background in tissue regions that do not activate the probes. Thus, increased signal levels report enhanced proteolytic activity. Recently, several of these activatable imaging probes have been developed. For example, 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.17–19 In light of recent findings that NE mediates the upregulation of proinflammatory cytokines13 and based on the knowledge that the local inflammatory response to PDT is also 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 evaluated if PDT conditions that induce significant neutrophil accumulation in treated tumor sites alter local NE protease activity. We report that in vivo imaging results are consistent with significantly increased NE activity in PDT-treated tumors, and the extent of probe accumulation correlates with the magnitude of intratumor neutrophil influx associated with different treatment protocols.