Wael Yared

Dual In Vivo Quantification of Integrin-targeted and Protease-activated Agents in Cancer Using Fluorescence Molecular Tomography (FMT)

PurposeIntegrins, especially αvβ3 and αvβ5, are upregulated in tumor cells and activated endothelial cells and as such, serve as cancer biomarkers. We developed a novel near-infrared-labeled optical agent for the in vivo detection and quantification of αvβ3/αvβ5.ProceduresA small peptidomimetic αvβ3 antagonist was synthesized, coupled to a near-infrared fluorescent (NIRF) dye, and tested for binding specificity using integrin-overexpressing cells, inhibition of vitronectin-mediated cell attachment, binding to tumor and endothelial cells in vitro, and competition studies. Pharmacokinetics, biodistribution, specificity of tumor targeting, and the effect of an antiangiogenic treatment were assessed in vivo.ResultsThe integrin NIRF agent showed strong selectivity towards αvβ3/αvβ5in vitro and predominant tumor distribution in vivo, allowing noninvasive and real-time quantification of integrin signal in tumors. Antiangiogenic treatment significantly inhibited integrin signal in vivo but had no effect on a cathepsin-cleavable NIR agent. Simultaneous imaging revealed different patterns of distribution reflecting the underlying differences in integrin and cathepsin biology during tumor progression.ConclusionsNIRF-labeled integrin antagonists allow noninvasive molecular fluorescent imaging and quantification of tumors in vivo, improving and providing more refined approaches for cancer detection and treatment monitoring.

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.

Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors.

A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.

Noninvasive In Vivo Quantification of Neutrophil Elastase Activity in Acute Experimental Mouse Lung Injury

We developed a neutrophil elastase-specific near-infrared fluorescence imaging agent, which, combined with fluorescence molecular tomographic imaging, allowed us to detect and quantify neutrophil elastase activity in vivo, in real time, and noninvasively in an acute model of lung injury (ALI). Significantly higher fluorescent signal was quantified in mice with LPS/fMLP-induced ALI as compared to healthy controls, correlating with increases in the number of bronchoalveolar lavage cells, neutrophils, and elastase activity. The agent was significantly activated ex vivo in lung sections from ALI but not from control mice, and this activation was ablated by the specific inhibitor sivelestat. Treatment with the specific inhibitor sivelestat significantly reduced lung signal in mice with ALI. These results underscore the unique ability of fluorescence molecular imaging to quantify specific molecular processes in vivo, crucial for understanding the mechanisms underlying disease progression and for assessing and monitoring novel pharmacological interventions.

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments.

Detection and quantification of enzymatically active prostate-specific antigen in vivo

Abstract. Assays for blood levels of prostate-specific antigen (PSA), performed in prostate cancer detection, measure mostly inactive/complexed PSA and do not provide information regarding enzymatically active PSA, which is biologically more relevant. Thus, we designed and synthesized an enzymatically cleavable peptide sequence labeled with near-infrared (NIR) fluorophores (ex/em 740/770  nm) and coupled it to a pharmacokinetic modifier designed to improve its plasma kinetics. In its native state, the agent, PSA750 FAST™ (PSA750), is optically quenched (>95%) and only becomes fluorescent upon cleavage by active PSA, yielding a significant increase in signal. This activation is highly selective for PSA relative to a large panel of disease-relevant enzymes. Active PSA was detected in tumor frozen sections using PSA750 and this activity was abolished in the presence of the inhibitor, alpha-1 anti-chymotrypsin. In vivo imaging of tumor-bearing mice using fluorescence molecular tomography demonstrated a significantly higher fluorescent signal in PSA+ LNCaP tumors as compared to PSA− prostate cancer 3 tumors (13.0±3.7 versus 2.8±0.8  pmol, p=0.023). Ex vivo imaging of tumor sections confirms PSA750-derived NIR signal localization in nonvascular tissue. This is the first report that demonstrates the feasibility and effectiveness of noninvasive, real time, fluorescence molecular imaging of PSA enzymatic activity in prostate cancer.

Abstract 4246: Multispectral open-air fluorescence-guided imaging and detection of tumors using a hands-free translational platform with liquid crystal tunable filters (LCTF)

Intraoperative identification and resection of tumors currently relies on the ability of the surgeon to visually detect or palpate the tumors and residual malignant tissue. As such, minuscule tumor nodules can go undetected or be inadequately removed, with such cases often resulting in the need for secondary treatment or additional surgical intervention. The Solaris™ platform is an open-air fluorescent imaging instrument designed for large animal fluorescence-guided surgery, with the advantage of real-time acquisition of fluorescence images/video under surgical light conditions. Solaris supports four fixed fluorescent channels ranging from visible to near infrared (NIR), and a multispectral channel where a liquid crystal tunable filter (LCTF) is used to acquire spectral data by sweeping across the green-to-red portion of the visible spectrum. This range of imaging channels allows for single-wavelength and multispectral imaging of widely used reagents (e.g. indocyanine green [ICG] and Fluorescein isothiocyanate [FITC]) and unique NIR fluorescent dyes used for detecting and labeling tumors. While fluorescent imaging using NIR imaging agents (680, 750, 800 nm) offered effective tumor detection, identification of tumors implanted in nude mice or rats using visible (400-650 nm) reagents such as FITC presented challenges considering the presence of auto-fluorescence originating from tissue and food (alfalfa). For these reagents, Solaris acquired multispectral images using the LCTF under ambient light conditions, and an automated spectral unmixing algorithm was applied to the multispectral data, after background correction and ambient light removal, to separate tissue and food auto-fluorescence from the reagent fluorescent signal. The algorithm used vertex component analysis to automatically extract the primary pure spectra present in the multispectral images and unmix the reagent fluorescent signal by non-negative least squares fitting. To test the spectral unmixing capabilities of Solaris, in vivo experiments were performed using small amounts of locally injected FITC in mice and rats. In the absence of unmixing, it was not possible to accurately detect sites of FITC signal, but with unmixing the labeled regions were well defined. Additional studies in tumor-bearing mice and rats substantiated the ability to spectrally unmix FITC agent signal in deep tumor masses imaged under ambient light, enhancing the ability to surgically resect them. To further validate this concept, bioluminescent tumor cell lines were implanted in mice. After image-guided tumor resection, both the residual tumor bed and the resected tumors were imaged to confirm complete removal. These data demonstrate that intraoperative image-guided resection of fluorescent-labeled tumors can be achieved using LCTF-based open-air multispectral imaging on the Solaris. Citation Format: Ali Behrooz, Kristine Vasquez, Peter Waterman, Jeff Meganck, Jeffrey Peterson, Peter Miller, Joshua Kempner, Wael Yared. Multispectral open-air fluorescence-guided imaging and detection of tumors using a hands-free translational platform with liquid crystal tunable filters (LCTF). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4246.

Abstract 4939: Detection and quantification of enzymatically active tissue prostate-specific antigen in vivo

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Prostate-Specific Antigen (PSA) is a 237 amino acid, 33 kDa, extracellular serine protease expressed in prostate epithelial tissue. In the normal prostate, high concentrations of PSA are stored in the prostatic ductal network. However, cancer leads to the disruption of normal tissue architecture and subsequent leakage of PSA into the tissue interstitium and circulation. Enzymatically active PSA is only present in prostate tissue and at other sites of prostate cancer growth. In circulation, active PSA immediately forms complexes with the serum protease inhibitor alpha-1-antichymotrypsin (ACT), while the inactive forms remains “free”. Traditional assays relied on testing for the presence of total PSA (mostly inactive and complexed) and do not provide information regarding the amount of enzymatically active PSA, a more biologically relevant tumor biomarker. To fill this gap, we developed a novel near infrared (NIR) fluorescent agent, designed to detect active PSA with no detection of inactive or complexed PSA. This agent, PSA750 FAST, contains a PSA-cleavable peptide sequence labeled with NIR fluorophores (ex/em 750/770 nm) and coupled to a pharmacokinetic modifier designed to improve its plasma availability. In its native state, the agent is nearly completely optically quenched (> 95%); and only becomes fluorescent upon enzymatic cleavage with active PSA, yielding a 300-1800 fold increase in signal as compared to the signal obtained using inactive or complexed PSA. Proteolytic cleavage was also selective for PSA over a large panel of enzymes, including Kallikrein 1, Cathepsin B, MMP-9, MMP-12, MMP-13, uPA, chymotrypsin and thrombin. We hypothesized that the enzymatic activity of PSA could be non-invasively and quantitatively monitored in 3D using Fluorescence Molecular Tomography (FMT), a powerful near-infrared imaging modality that enables 3D quantitative determination of fluorochrome distribution in tissues of live small animals. LNCaP (PSA positive) and PC3 (PSA negative) cells were implanted in the chest area of male nude mice. Real time imaging was performed after systemic administration of PSA750 FAST when tumors reached the desired size. Our results demonstrated a significantly higher fluorescent signal in LNCaP tumors as compared the adjoining muscle (34.1 +/- 3.8 nM versus 7.2 +/- 0.6 nM, p=0.0043). Likewise, fluorescence concentration in tumors was significantly higher in LNCaP as compared to PC3 tumors (34.1 +/- 3.8 nM vs 17.14 +/- 3.48 nM, p=0.0191; 12.99 +/- 3.7 vs 2.8 +/- 0.8 pmoles, p=0.0233). This is the first report that demonstrates the feasibility of non-invasive, real-time, molecular imaging of active PSA in vivo in a relevant tumor model. These findings demonstrate the effectiveness of this agent in conjunction with optical imaging as a functional platform for molecular imaging in the field of prostate cancer. Citation Format: Jeffrey D. Peterson, Guojie Ho, Jeffrey Morin, Jeannine Delaney, Wael Yared, Milind Rajopadhye, Sylvie C. Kossodo. Detection and quantification of enzymatically active tissue prostate-specific antigen in vivo . [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4939. doi:10.1158/1538-7445.AM2014-4939

Abstract 4934: Imaging and quantification of bombesin receptor expression in vivo using a NIR-labeled bombesin peptide

Gastrin-releasing peptide (GRP) is one of the mammalian analogs of bombesin-like peptides that function as growth factors in normal and neoplastic tissues upon binding to a family of bombesin-binding G protein-coupled receptors. Bombesin receptors are overexpressed in a variety of cancers, particularly prostate, breast, and gastrointestinal stromal tumors, and as such have been used to develop radiolabeled imaging tracers. As a pre-clinical alternative to using radiolabeled ligands, we developed a novel near infrared (NIR) fluorescent agent, BombesinRSense 680 (BRS680), designed to target and quantify bombesin receptors in vivo. BRS680 was produced from a modified 7 amino-acid GRP analog peptide labeled with a NIR fluorophore (ex/em 660/680 nm) and a pharmacokinetic modifier designed to improve its plasma availability (plasma t1/2 = 1.5 hours). In vitro labeling of human colonic adenocarcinoma HT-29 cells, which express GRP receptors, and blocking the signal by addition of unlabeled native bombesin, demonstrated the specificity of the agent by both fluorescence microscopy and flow cytometry. In vivo receptor expression was quantified by fluorescence tomography after BRS680 (2 nmol/mouse) was injected intravenously into nude mice bearing HT-29 tumor xenografts. HT-29 tumors showed a high level of receptor expression with approximately 30 pmol (1.5% injected dose) of BRS680 quantified in the tumors at 24 hours, and lower fluorescence in other tissues except for pancreas, a tissue known for high receptor expression, and kidneys, indicating renal clearance. In contrast to the fast clearance from circulation, the tumor tissue half-life of BRS680 was shown to be approximately 42 hours. In vivo targeting specificity was confirmed by collecting tumor tissue from injected mice and co-localizing BRS680 fluorescent signal with an anti-GRP receptor antibody on frozen sections. More importantly, treatment of HT-29 tumor-bearing mice with a tumor growth-arresting chemotherapy regime decreased in vivo BRS680 signal. Six days after beginning treatment with 5-fluorouracil and oxaliplatin in mice with established tumors, BRS680 fluorescent signal was significantly decreased in treated mice as compared to control mice (21.55 + 4.89 versus 34.10 + 2.90 pmoles, p=0.043) paralleling the inhibition of tumor growth (74.25 + 7.65 versus 141 + 19.39 mm3, p=0.003). Interestingly, chemotherapy did not consistently affect the fluorescent signal associated with ProSense 750 FAST, an agent that is specifically activated by the cathepsin family of inflammatory proteases, co-injected in the same animals (6.99 +1.68 versus 10.91 + 1.73 pmoles, p=0.104). These studies demonstrate the utility of BRS680 in tracking in vivo expression of bombesin receptors and underscores its potential to serve as an in vivo real-time indicator of anti-tumor treatment efficacy. Citation Format: Jeffrey D. Peterson, Nara Narayanan, Jeannine Delaney, Jeffrey Morin, Milind Rajopadhye, Wael Yared, Sylvie Kossodo. Imaging and quantification of bombesin receptor expression in vivo using a NIR-labeled bombesin peptide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4934. doi:10.1158/1538-7445.AM2014-4934

Abstract 2448: Development of a red fluorescent labeled agent for assessing HER2 expression in vitro and in vivo

Upregulation of tumor HER2 occurs in approximately 25% women with breast cancer and is often associated with poor prognosis. We developed a red fluorescent imaging agent to non-invasively image and quantify tumor-associated HER2 expression in vivo. A red fluorescent dye (VivoTag 645; α=210,000 μ/cm; abs/em max 643/660 nm) was used to label trastuzumab, which is currently used to treat breast and stomach cancer. The red-labeled trastuzumab (VM4003) preferentially labeled HER2+ SKOV-3 human ovarian adenocarcinoma cells over HER2− human colorectal adenocarcinoma Colo-205 cells (10 fold), and the specificity of binding was confirmed by control experiments using free dye, labeled non-specific IgG, and competitive blockade with unlabeled excess trastuzumab. Fluorescence microscopy confirmed the expected membrane localization of fluorescence. In vivo and ex vivo imaging by fluorescence molecular tomography (FMT), showed significantly higher signal within the tumors, peaking at 6-72 hours following intravenous injection of 2 mg/kg VM4003, decreasing thereafter with a tissue half-life of 3 days. In vivo quantification of tumor signal in nude mice showed significantly higher tumor signal in HER2+ than in HER2− tumors (14.36 + 4 versus 2.39+ 0.74 pmol, at 6h imaging time, p=0.007; 18.77 + 4.45 versus 3.50+ 0.98 pmol, at 24h, p=0.001). Specificity of targeting was confirmed by competition with excess intravenous unlabeled trastuzumab, which achieved 70% signal inhibition in the tumors (tumor signal 16.12 + 3.03 versus 4.72 + 1.96 pmol, p=0.022 at 24h). Tumor volumes, as determined by direct measurements of tumor size, were comparable between both groups of mice (p=0.193). Fluorescence microscopy of ex vivo frozen tissue sections confirmed tumor fluorescence and signal localization associated to cell membranes and cytoplasm. In summary, red fluorescent-labeled trastuzumab selectively targets αER2, allowing both imaging in vitro and the non-invasive real-time tomographic imaging and quantification in vivo of HER-2 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2448. doi:1538-7445.AM2012-2448