A Nonpeptidic Cathepsin S Activity-Based Probe for Noninvasive Optical Imaging of Tumor-Associated Macrophages

Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Chemistry & biology (Impact Factor: 6.65). 05/2012; 19(5):619-28. DOI: 10.1016/j.chembiol.2012.03.012
Source: PubMed


Macrophage infiltration into tumors has been correlated with poor clinical outcome in multiple cancer types. Therefore, tools to image tumor-associated macrophages could be valuable for diagnosis and prognosis of cancer. Herein, we describe the synthesis and characterization of a cathepsin S-directed, quenched activity-based probe (qABP), BMV083. This probe makes use of an optimized nonpeptidic scaffold leading to enhanced in vivo properties relative to previously reported peptide-based probes. In a syngeneic breast cancer model, BMV083 provides high tumor-specific fluorescence that can be visualized using noninvasive optical imaging methods. Furthermore, analysis of probe-labeled cells demonstrates that the probe primarily targets macrophages with an M2 phenotype. Thus, BMV083 is a potential valuable in vivo reporter for tumor-associated macrophages that could greatly facilitate the future studies of macrophage function in the process of tumorigenesis.

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Available from: Laura E Edgington-Mitchell
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    • "A major advance in this regard is the use of activity-based probes that covalently interact with activated proteases, allowing their localization by whole animal or cellular imaging and identification by proteomic approaches (99). This approach has been used to detect activated cathepsin S in macrophages of tumors and the inflamed colon, as well as in spinal microglial cells during colitis (67, 147). However, the use of probes is likely to reveal that multiple proteases become activated during physiological and pathological events, many of which could activate or disarm PARs. "
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    ABSTRACT: In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain, and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an emerging therapeutic target for major diseases. Most information about PAR activation and function derives from studies of a few proteases, for example thrombin in the case of PAR1, PAR3, and PAR4, and trypsin in the case of PAR2 and PAR4. These proteases cleave PARs at established sites with the extracellular N-terminal domains, and expose tethered ligands that stabilize conformations of the cleaved receptors that activate the canonical pathways of G protein- and/or β-arrestin-dependent signaling. However, a growing number of proteases have been identified that cleave PARs at divergent sites to activate distinct patterns of receptor signaling and trafficking. The capacity of these proteases to trigger distinct signaling pathways is referred to as biased signaling, and can lead to unique patho-physiological outcomes. Given that a different repertoire of proteases are activated in various patho-physiological conditions that may activate PARs by different mechanisms, signaling bias may account for the divergent actions of proteases and PARs. Moreover, therapies that target disease-relevant biased signaling pathways may be more effective and selective approaches for the treatment of protease- and PAR-driven diseases. Thus, rather than mediating the actions of a few proteases, PARs may integrate the biological actions of a wide spectrum of proteases in different patho-physiological conditions.
    Full-text · Article · May 2014 · Frontiers in Endocrinology
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    • "In addition to the production of Cat S by the tumor cells, further analysis confirmed that TAMs produced the protease as previously shown in the mouse models for pancreatic cancer and breast cancer [23,30,45,46]. Several studies have shown that expression of Cat S by tumour-infiltrating macrophages, could be an important contributor during prostate cancer progression [41]. "
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    ABSTRACT: Tumor-associated macrophages (TAMs) are the major component of tumor-infiltrating leukocytes. TAMs are heterogeneous, with distinct phenotypes influenced by the microenvironment surrounding tumor tissues, but relatively little is known about the key molecular in these cells that contribute to malignant phenotypes. Autophagic activity is a critical factor in tumor development that contributes to enhancing cellular fitness and survival in the hostile tumor microenvironment. However, the molecular basis and relations between autophagy and TAMs polarization remain unclear. Cathepsin S (Cat S) expression was analyzed in human colon carcinoma and normal colon tissues. In vivo effects were evaluated using PancO2 subcutaneous tumor model and SL4 hepatic metastasis model. Immunofluorescence staining, flow cytometry and real-time PCR were done to examine TAMs polarization. Western blotting assay, transmission electron microscopy, mCherry-GFP-LC3 transfection and DQ-BSA degradation assays were carried out to determine its role in regulating autophagy. In the present study, we showed that the enhanced expression of Cat S correlated with the severity of histologic grade as well as clinical stage, metastasis, and recurrence, which are known indicators of a relatively poor prognosis of human colon carcinoma. Cat S knockout led to decreased tumor growth and metastasis. Moreover, Cat S knockout inhibited M2 macrophage polarization during tumor development. We further demonstrated that Cat S was required for not only autophagic flux but also the fusion processes of autophagosomes and lysosomes in TAMs. Importantly, we found that Cat S contributed to tumor development by regulating the M2 phenotype of TAMs through the activation of autophagy. These results indicated that Cat S-mediated autophagic flux is an important mechanism for inducing M2-type polarization of TAMs, which leads to tumor development. These data provide strong evidence for a tumor-promoting role of autophagy in TAMs and suggest Cat S could be a potential target for cancer therapy.
    Full-text · Article · Mar 2014 · Molecular Cancer
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    • "Fluorescent ABPs have been developed most broadly for cysteine proteases, including caspases (Edgington et al., 2009, 2012; Li et al., 2012), cathepsins (Joyce et al., 2004; Blum et al., 2005, 2007; Gocheva et al., 2010; Verdoes et al., 2012), and legumain (Lee and Bogyo, 2010; Edgington et al., 2013). ABPs have also been developed for the threonine-dependent catalytic subunits of the proteasome (Verdoes et al., 2006) and for serine proteases (Liu et al., 1999). "
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    ABSTRACT: Activity-based probes (ABPs) are reactive small molecules that covalently bind to active enzymes. When tagged with a fluorophore, ABPs serve as powerful tools to investigate enzymatic activity across a wide variety of applications. In this article, detailed protocols are provided for using fluorescent ABPs to biochemically characterize the activity of proteases in vitro. Furthermore, descriptions are provided of how these probes can be applied to image protease activity in live animals and tissues along with subsequent analysis by histology, flow cytometry, and SDS-PAGE. Curr. Protoc. Chem. Biol. 5:25-44 © 2013 by John Wiley & Sons, Inc.
    Full-text · Article · Mar 2013
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