Legumain is activated in macrophages during pancreatitis.

Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68(+) macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.

Dual-Modality Activity-Based Probes as Molecular Imaging Agents for Vascular Inflammation.

Macrophages are cellular mediators of vascular inflammation and are involved in the formation of atherosclerotic plaques. These immune cells secrete proteases such as matrix metalloproteinases and cathepsins that contribute to disease formation and progression. Here, we demonstrate that activity-based probes (ABPs) targeting cysteine cathepsins can be used in murine models of atherosclerosis to noninvasively image activated macrophage populations using both optical and PET/CT methods. The probes can also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations. METHODS: Macrophage-rich carotid lesions were induced in FVB mice fed on a high-fat diet by streptozotocin injection followed by ligation of the left common carotid artery. Mice with carotid atherosclerotic plaques were injected with the optical or dual-modality probes BMV109 and BMV101, respectively, via the tail vein and noninvasively imaged by optical and small-animal PET/CT at different time points. After noninvasive imaging, the murine carotid arteries were imaged in situ and ex vivo, followed by immunofluorescence staining to confirm target labeling. Additionally, human carotid plaques were topically labeled with the probe and analyzed by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunofluorescence staining to confirm the primary targets of the probe. RESULTS: Quantitative analysis of the signal intensity from both optical and PET/CT imaging showed significantly higher levels of accumulation of BMV109 and BMV101 (P < 0.005 and P < 0.05, respectively) in the ligated left carotid arteries than the right carotid or healthy arteries. Immunofluorescence staining for macrophages in cross-sectional slices of the murine artery demonstrated substantial infiltration of macrophages in the neointima and adventitia of the ligated left carotid arteries compared with the right. Analysis of the human plaque tissues by sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that the primary targets of the probe were cathepsins X, B, S, and L. Immunofluorescence labeling of the human tissue with the probe demonstrated colocalization of the probe with CD68, elastin, and cathepsin S, similar to that observed in the experimental carotid inflammation murine model. CONCLUSION: We demonstrate that ABPs targeting the cysteine cathepsins can be used in murine models of atherosclerosis to noninvasively image activated macrophage populations using both optical and PET/CT methods. The probes could also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations. Therefore, ABPs targeting the cysteine cathepsins are potentially valuable new reagents for rapid and noninvasive imaging of atherosclerotic disease progression and plaque vulnerability.

Non-invasive Imaging of Idiopathic Pulmonary Fibrosis Using Cathepsin Protease Probes.

Idiopathic pulmonary fibrosis (IPF) is a lethal, chronic, progressive disease characterized by formation of scar tissue within the lungs. Because it is a disease of unknown etiology, it is difficult to diagnose, to predict disease course and to devise treatment strategies. Recent evidence suggests that activated macrophages play key roles in the pathology of IPF. Therefore, imaging probes that specifically recognize these pools of activated immune cells could provide valuable information about how these cells contribute to the pathobiology of the disease. Here we demonstrate that cysteine cathepsin-targeted imaging probes can be used to monitor the contribution of macrophages to fibrotic disease progression in the bleomycin-induced murine model of pulmonary fibrosis. Furthermore, we show that the probes highlight regions of macrophage involvement in fibrosis in human biopsy tissues from IPF patients. Finally, we present first-in-human results demonstrating non-invasive imaging of active cathepsins in fibrotic lesions of patients with IPF. Together, our findings validate small molecule cysteine cathepsin probes for clinical PET imaging and suggest that they have the potential to be used to generate mechanistically-informative molecular information regarding cellular drivers of IPF disease severity and progression.

Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes.

Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4-8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis.

Design of Protease Activated Optical Contrast Agents That Exploit a Latent Lysosomotropic Effect for Use in Fluorescence-Guided Surgery.

There is a need for new molecular-guided contrast agents to enhance surgical procedures such as tumor resection that require a high degree of precision. Cysteine cathepsins are highly up-regulated in a wide variety of cancers, both in tumor cells and in the tumor-supporting cells of the surrounding stroma. Therefore, tools that can be used to dynamically monitor their activity in vivo could be used as imaging contrast agents for intraoperative fluorescence image guided surgery (FGS). Although multiple classes of cathepsin-targeted substrate probes have been reported, most suffer from overall fast clearance from sites of protease activation, leading to reduced signal intensity and duration in vivo. Here we describe the design and synthesis of a series of near-infrared fluorogenic probes that exploit a latent cationic lysosomotropic effect (LLE) to promote cellular retention upon protease activation. These probes show tumor-specific retention, fast activation kinetics, and rapid systemic distribution. We demonstrate that they are suitable for detection of diverse cancer types including breast, colon and lung tumors. Most importantly, the agents are compatible with the existing, FDA approved, da Vinci surgical system for fluorescence guided tumor resection. Therefore, our data suggest that the probes reported here can be used with existing clinical instrumentation to detect tumors and potentially other types of inflammatory lesions to guide surgical decision making in real time.

Improved quenched fluorescent probe for imaging of cysteine cathepsin activity.

The cysteine cathepsins are a family of proteases that play important roles in both normal cellular physiology and many human diseases. In cancer, the activity of many of the cysteine cathepsins is upregulated and can be exploited for tumor imaging. Here we present the design and synthesis of a new class of quenched fluorescent activity-based probes (qABPs) containing a phenoxymethyl ketone (PMK) electrophile. These reagents show enhanced in vivo properties and broad reactivity resulting in dramatically improved labeling and tumor imaging properties compared to those of previously reported ABPs.

Functional imaging of legumain in cancer using a new quenched activity-based probe.

Legumain is a lysosomal cysteine protease whose biological function remains poorly defined. Legumain activity is up-regulated in most human cancers and inflammatory diseases most likely as the result of high expression in populations of activated macrophages. Within the tumor microenvironment, legumain activity is thought to promote tumorigenesis. To obtain a greater understanding of the role of legumain activity during cancer progression and inflammation, we developed an activity-based probe that becomes fluorescent only upon binding active legumain. This probe is highly selective for legumain, even in the context of whole cells and tissues, and is also a more effective label of legumain than previously reported probes. Here we present the synthesis and application of our probe to the analysis of legumain activity in primary macrophages and in two mouse models of cancer. We find that legumain activity is highly correlated with macrophage activation and furthermore that it is an ideal marker for primary tumor inflammation and early stage metastatic lesions.

Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape.

Breast cancer metastasis is a key determinant of long-term patient survival. By comparing the transcriptomes of primary and metastatic tumor cells in a mouse model of spontaneous bone metastasis, we found that a substantial number of genes suppressed in bone metastases are targets of the interferon regulatory factor Irf7. Restoration of Irf7 in tumor cells or administration of interferon led to reduced bone metastases and prolonged survival time. In mice deficient in the interferon (IFN) receptor or in natural killer (NK) and CD8(+) T cell responses, metastasis was accelerated, indicating that Irf7-driven suppression of metastasis was reliant on IFN signaling to host immune cells. We confirmed the clinical relevance of these findings in over 800 patients in which high expression of Irf7-regulated genes in primary tumors was associated with prolonged bone metastasis-free survival. This gene signature may identify patients that could benefit from IFN-based therapies. Thus, we have identified an innate immune pathway intrinsic to breast cancer cells, the suppression of which restricts immunosurveillance to enable metastasis.

Cathepsin B inhibition limits bone metastasis in breast cancer.

Metastasis to bone is a major cause of morbidity in breast cancer patients, emphasizing the importance of identifying molecular drivers of bone metastasis for new therapeutic targets. The endogenous cysteine cathepsin inhibitor stefin A is a suppressor of breast cancer metastasis to bone that is coexpressed with cathepsin B in bone metastases. In this study, we used the immunocompetent 4T1.2 model of breast cancer which exhibits spontaneous bone metastasis to evaluate the function and therapeutic targeting potential of cathepsin B in this setting of advanced disease. Cathepsin B abundancy in the model mimicked human disease, both at the level of primary tumors and matched spinal metastases. RNA interference-mediated knockdown of cathepsin B in tumor cells reduced collagen I degradation in vitro and bone metastasis in vivo. Similarly, intraperitoneal administration of the highly selective cathepsin B inhibitor CA-074 reduced metastasis in tumor-bearing animals, a reduction that was not reproduced by the broad spectrum cysteine cathepsin inhibitor JPM-OEt. Notably, metastasis suppression by CA-074 was maintained in a late treatment setting, pointing to a role in metastatic outgrowth. Together, our findings established a prometastatic role for cathepsin B in distant metastasis and illustrated the therapeutic benefits of its selective inhibition in vivo.

Telomere length in lymphoblast cell lines derived from clinically radiosensitive cancer patients.

Approximately 1-5 percent of cancer patients suffer from significant side effects in normal tissue after radiotherapy (RT). Although RT is an effective cancer therapy, treatment dose intensities are restricted to minimize the incidence of such normal tissue reactions. Therefore, most patients receive lower dose intensities than can be tolerated in normal tissue. A primary aim for radiation oncology is to identify radiosensitive (RS) individuals prior to treatment. Such predictive ability should result in an improvement in tumor control rates and/or a reduction in the incidence of RT side effects. Recent evidence suggests a link between RS and telomere length. A positive correlation between cellular RS and telomere length in a cohort of breast cancer patients has been reported. Furthermore,individuals with cancer-prone recessive RS syndromes, such as ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome(NBS), have shortened telomeres. To determine whether the association between telomere length and RS could be used as a predictive assay to prospectively identify RS cancer patients, we utilized a bank of lymphoblastoid cell lines (LCLs) derived from 33 RS patients, along with 18 LCL samples from RT patients who did not have severe reactions, to assess the link between RS and telomere length. We found a subset of RS patient LCLs had abnormally long telomere lengths, so these data suggest that RS could potentially be predicted for a subset of RS patients based on telomere length in LCLs, and contribute to therapy individualization.