Molecular Imaging of Cancer Using X‑ray Computed Tomography with Protease Targeted Iodinated Activity-Based Probes

X-ray computed tomography (CT) is a robust, precise, fast, and reliable imaging method that enables excellent spatial resolution and quantification of contrast agents throughout the body. However, CT is largely inadequate for molecular imaging applications due mainly to its low contrast sensitivity that forces the use of large concentrations of contrast agents for detection. To overcome this limitation, we generated a new class of iodinated nanoscale activity-based probes (IN-ABPs) that sufficiently accumulates at the target site by covalently binding cysteine cathepsins that are exceptionally highly expressed in cancer. The IN-ABPs are comprised of a short targeting peptide selective to specific cathepsins, an electrophilic moiety that allows activity-dependent covalent binding, and tags containing dendrimers with up to 48 iodine atoms. IN-ABPs selectively bind and inhibit activity of recombinant and intracellular cathepsin B, L, and S. We compared the in vivo kinetics, biodistribution, and tumor accumulation of IN-ABPs bearing 18 and 48 iodine atoms each, and their control counterparts lacking the targeting moiety. Here we show that although both IN-ABPs bind specifically to cathepsins within the tumor and produce detectable CT contrast, the 48-iodine bearing IN-ABP was found to be optimal with signals over 2.1-fold higher than its nontargeted counterpart. In conclusion, this study shows the synthetic feasibility and potential utility of IN-ABPs as potent contrast agents that enable molecular imaging of tumors using CT

Cathepsin Activity-Based Probes and Inhibitor for Preclinical Atherosclerosis Imaging and Macrophage Depletion

<div><p>Background and Purpose</p><p>Cardiovascular disease is the leading cause of death worldwide, mainly due to an increasing prevalence of atherosclerosis characterized by inflammatory plaques. Plaques with high levels of macrophage infiltration are considered “vulnerable” while those that do not have significant inflammation are considered stable; cathepsin protease activity is highly elevated in macrophages of vulnerable plaques and contributes to plaque instability. Establishing novel tools for non-invasive molecular imaging of macrophages in plaques could aid in preclinical studies and evaluation of therapeutics. Furthermore, compounds that reduce the macrophage content within plaques should ultimately impact care for this disease.</p><p>Methods</p><p>We have applied quenched fluorescent cathepsin activity-based probes (ABPs) to a murine atherosclerosis model and evaluated their use for <i>in vivo</i> imaging using fluorescent molecular tomography (FMT), as well as <i>ex vivo</i> fluorescence imaging and fluorescent microscopy. Additionally, freshly dissected human carotid plaques were treated with our potent cathepsin inhibitor and macrophage apoptosis was evaluated by fluorescent microscopy.</p><p>Results</p><p>We demonstrate that our ABPs accurately detect murine atherosclerotic plaques non-invasively, identifying cathepsin activity within plaque macrophages. In addition, our cathepsin inhibitor selectively induced cell apoptosis of 55%±10% of the macrophage within excised human atherosclerotic plaques.</p><p>Conclusions</p><p>Cathepsin ABPs present a rapid diagnostic tool for macrophage detection in atherosclerotic plaque. Our inhibitor confirms cathepsin-targeting as a promising approach to treat atherosclerotic plaque inflammation.</p></div

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

Non-invasive imaging of plaques in murine atherosclerosis.

<p>Diabetic, fat-fed mice with a ligated carotid artery were injected with non-quenched probe GB123 or quenched probe GB137 as indicated. Fluorescent molecular tomography (FMT) was used to monitor and follow the pharmacokinetics and signal accumulation in plaques. <b>(a, b)</b> Left images: front overlay of fluorescence and bright field. Middle images: side view of fluorescence alone. These images show strong fluorescence signal (arrows) (GB123 at 4 hours and GB137 at 2 hours post probe injection) around the ligated left carotid artery. Right images show <i>ex vivo</i> fluorescent image of excised heart and carotid arteries (ligated artery is marked).</p

Macrophage labeling with fluorescent activity based probe.

<p>Ligated and control carotid arteries from mice treated with GB123 (<b>a</b>) or GB137 (<b>b</b>) (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160522#pone.0160522.g001" target="_blank">Fig 1</a>) were embedded in OCT and serial sectioned. Samples were stained for F4/80, a macrophage marker, and scanned by a confocal microscope: DAPI (blue), Cy5 labeled by probe (red), F4/80 (green), yellow color is overlay of red and green fluorescence. Cathepsin probes were found to co-localize with F4/80 macrophages.</p

Cathepsin inhibitor induces specific macrophage apoptosis.

<p>Freshly excised human atherosclerotic tissue samples were treated with the cathepsin inhibitor GB111-NH₂ for 24 hours. Serial frozen sections were stained for CD68 and cleaved caspase-3 and visualized by a confocal microscope: DAPI (blue), cleaved caspase-3 (green), CD68 (red), yellow color is overlay of red and green fluorescence. GB111-NH₂ was found to induce specific macrophage cell death (a). Co-localization analysis of CD68 and cleaved Caspase 3 positive cells. Bar graphs present the fraction of apoptotic macrophages out of total CD68 population (b) and the fraction of macrophages out of total apoptotic cells is shown in (c). Data is mean ± SEM (n = 3).</p

GB119 specifically labels cells <i>in vitro</i> and tumor tissue <i>ex vivo</i>.

<p>(<b>a</b>) Gli36Δ5EGFR cells were incubated with JPM-OEt, a general papain-family inhibitor (+, lanes 2,4 and 6) or 0.1% DMSO vehicle control (−, lanes 1,3 and 5) before the addition of the indicated probe GB119, GB123, or GB125 (the control probe, GB125, contains the Cy5 fluorescence label with a non-reactive amide in place of the acyloxymethylketone (AOMK) “warhead”(22)). (<b>b</b>) <i>In vitro</i> cell labeling with NIRF-APBs. C2C12/ras cells were incubated with JPM-OEt, a general papain-family inhibitor (+) or 0.1% DMSO as a vehicle control (−) before the addition of the indicated probe. Samples were analyzed by SDS-PAGE and Cy5 fluorescence was measured by scanning the gels with a Typhoon scanner. Specific bands are indicated as follows: cathepsin L single-chain form (Cat L sc); cathepsin L heavy-chain (Cat L hc), and cathepsin B (Cat B). (<b>c</b>) Gli36D5EGFR flank tumor tissue was immersed in GB119 for the indicated time or DMSO vehicle control (D) for 30 minutes. Specific cathepsin bands are indicated as follows: cathepsin L single-chain form (Cat L sc,); cathepsin L heavy-chain (Cat L hc); and cathepsin B (Cat B).</p

Topical application of GB119 to a brain tumor on the dorsal surface labels the tumor's edge more robustly than the tumor's center.

<p>(<b>a</b>) Monochromatic image of a whole brain with a tumor growing near the dorsal surface (arrow) showing the treated areas (outlines). (<b>b</b>) Unmixed false colored map of pixel intensity representing activated GB119 at 35 minutes post application. (<b>c</b>) Horizontal H&E stained section showing higher magnification of square area in panel b demonstrating tumor mass and normal surrounding brain. (<b>d</b>) Adjacent sections revealing human vimentin positive cells labeling tumor edge and center (false colored green), (<b>e</b>) activated GB119 (Cy5, false colored red) revealing infiltrating cells, the tumor margin and to a lesser degree the interior of the tumor and (<b>f</b>) merged imaged demonstrating co-registration (yellow) of GB119-labeled cells and vimentin positive cells within the tumor mass (arrows) and outside of the main tumor mass, arrow head. (<b>g</b>) GB119 labeled cells at the tumor's edge more robustly than at the tumor's center, although not significantly, p = 0.124. (<b>h</b> and <b>i</b>) reveal that GB119 is associated with CD11b-positive cells, but only when they are tumor associated, (<b>h</b>) is a typical image observed from within the tumor interior and (<b>i</b>) shows the images typical of the tumor – brain interface. Note that only CD11b positive cells associated with normal brain do not activate the probe. Section thickness 25 µm (<b>d–f</b>) and 10 µm (<b>h</b>,<b>i</b>). Scale bar, 5 µm (<b>a</b>,<b>b</b>), and 100 µm (<b>c–f, h,i</b>).</p

<i>Ex vivo</i> activation of GB119 in orthotopic brain tumors.

<p>(<b>a</b>) Representative 2-mm sections from normal and tumor-bearing brains (see arrow for main tumor mass). Monochromatic images are shown on left and unmixed false colored maps of pixel intensity representing activated GB119 at 35 minutes are shown on the right. (<b>b</b>) The averages of total signal/area/volume from the slices for each animal is plotted over time for normal (n = 3) and tumor (n = 6) groups. The groups did not differ at time 0 (p = 0.65). Using Bonferroni correction with overall significance level 0.05, the groups differed significantly at times 5, 15, 30 and 35 minutes. (<b>c</b>) Inhibition of GB119 activation in an orthotopic brain tumor model using opposing brain slices (opened like a book) containing tumor (arrow, n = 11). (<b>d</b>) Percent inhibition of GB119 activity was determined for each mouse and is presented as mean +/− standard deviation at 35 minutes. Using Bonferroni correction for multiple testing at times the groups did not differ at time 0 (p = 0.86) but differed significantly at times 1, 15, 30 and 35 minutes (p<0.0003). Scale bar, 5 mm.</p

Comparison of tumor cell labeling between topical application of GB119 and systemic administration of GB123.

<p>(<b>a–d</b>) Representative unmixed false colored maps of pixel intensity of coronal 2 mm sections (<b>a</b>,<b>b</b>) and dorsal surface tumors (<b>c</b>,<b>d</b>) comparing the pattern of activation of topical GB119 (left side) and systemic GB123 (right side). (<b>e,f</b>) H&E coronal sections demonstrating tumor mass. (<b>g–j</b>) Merged fluorescent microscopy of adjacent sections showing labeling in the center (<b>g,h</b>) and edge (<b>I,j</b>) of the tumor. Sections were immunostained with anti-human vimentin antibody to reveal the brain tumor xenograft (false colored green), covalently bound Cy5 (false colored red). Yellow represents co-localized staining. (<b>k & i</b>) H&E sections demonstrating tumor cells that migrated away from the main tumor mass (outlined) via the ventricular system (<b>k</b>) or along the cortical meninges (<b>i</b>). Boxed regions indicate invading cells (<b>m</b> & <b>n</b>). Merged images of boxed regions from panels k & i showing labeling of invading tumor cells. Vimentin positive tumor cells (false colored green) and Cy5 cells (false colored red) reveals that invading cells were identified by topical GB119 (yellow cells) (<b>m</b>) but not by systemic GB123 (<b>n</b>). DAPI staining for cellular orientation was also included in panels <b>m,n</b>. Scale bar, 1 mm (<b>e,f</b>), 500 µm (<b>k,l</b>), and 50 µm (<b>g–j, m, n</b>).</p