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A High-Throughput Molecular Imaging of Renal Tissue Responses using NIR-based Probes

Molecular imaging is an emerging technology in medical science that facilitates improved understanding of many diseased states, as well as response to novel therapies. Optical imaging techniques, including fluorescence and bioluminescence, are widely used in small animal imaging due in part to their sensitivity, cost effectiveness, speed, and ease of use. For imaging in small animals, the deep tissue propagation of near-infrared (NIR) light between 700-900 nm offers considerably improved probe detection in non-superficial tissues and organs such as the kidney. As illustrated below, NIR-based fluorescent imaging probes that specifically target various diseased states can be prepared and have utility for assessing renal function and damage (1). In this section, we will apply this method for the assessments of “molecular” events in murine renal disease. 

VUIIS and VUMC have devoted considerable resources and infrastructure towards the development of a world-class molecular imaging facility. Our molecular imaging capability includes nuclear (PET and SPECT), high-field MR (up to 9.4T), and optical methods. Furthermore, within our VUIIS infrastructure we have established a significant imaging probe discovery and development program that includes high-throughput screening technology and modern chemical synthesis methods. Thus far, a considerable portion of our molecular imaging probe development has centered around optical imaging, particularly utilizing near-infrared (NIR)-based fluorophores. Optical imaging using NIR-based reagents is highly sensitive, non-invasive, and allows “large numbers” of small animals to be rapidly screened (≤ 60 per hr). Since the absorption of water and hemoglobin are minimized between 700nm-900nm, most tissues are quite transparent to NIR light, enabling the detection of NIR-based probes in vivo at tissue depths approaching centimeters. Furthermore, reduced tissue autofluorescence and scatter of excitation and emission light in the NIR contribute to the sensitivity of NIR-based probes. To support VKDC investigators, we will develop and employ NIR-based probes that will be prepared using unique, and spectroscopically complementary, fluorophores (700nm and 800nm emission, e.g. NIR700-Annexin-V, NIR800-VEGFR2). In doing so, we anticipate that as part of ongoing and future investigations it will be possible to utilize multi-spectral optical imaging to simultaneously profile the uptake of multiple probes in vivo.

Preparation of Optical Imaging Probe (e.g. NIR700-Annexin-V): Phosphatidyl serine can be found in the plasma membrane of healthy cells, but its antigenic sites are only exposed externally to the cell upon triggering of cellular apoptosis. For this reason, Annexin V, which binds phosphatidyl serine, has become a useful probe for the assessment of apoptosis in cell culture, in preserved tissues, and more recently in live animals and humans (2, 3). In order to develop an optical agent for imaging apoptosis, we labeled Annexin V from human placenta (Sigma Aldrich, St. Louis, MO) with LI-COR 700DX (LI-COR Biosciences, Lincoln, NE). The spectroscopic properties of NIR-Annexin-V were evaluated in PBS at room temperature. A 0.3mM aqueous solution gave an absorbance maximum centered at 680nm, and fluorescence emission centered at 698nm (Figure M1-A). To determine whether NIR700-Annexin-V would signal therapy-induced apoptosis in vitro, we treated DiFi cells with an EGFR blocking monoclonal antibody, mAb-C225 (cetuximab), at four concentrations (0 (control), 0.3mg/mL, 3ƒnmg/mL, and 30ƒnmg/mL). We then labeled control and drug treated cells with NIR700-Annexin-V and measured the resultant probe binding using an Odyssey NIR plate reader. In parallel, we independently evaluated apoptosis in identically treated populations of control and drug-treated cells using a commercially available apoptosis kit measuring caspase 3/7 activity (Caspase-glow, Promega). As shown in Figure M1-B, the raw NIR700-Annexin-V fluorescence signal for each condition is plotted as a percent of the control (no drug) binding and appears to indicate a dose-dependant apoptosis response. These data are in good agreement with results obtained using the commercial apoptosis kit (Figure M1).

In summary, we have successfully prepared and demonstrated the feasibility of employing a novel molecular imaging agent (NIR700-Annexin-V) to non-invasively assess treatment response in cultured cells and in vivo in small animal models of cancer. We wish to emphasize that we have intentionally prepared NIR700-Annexin-V using a NIR-dye that is spectrally distinct from the other proposed NIR-based imaging probes which bear 800nm-based labels (such as NIR800-αVEGFR2). Thus, we envision capitalizing upon the spectroscopically distinct nature of the 700/800nm chemistry to simultaneously profile multiple biological readouts in a single animal using multispectral optical imaging. In addition to NIR-Annexin-V, we have labeled, purified, and prepared a panel of complementary NIR-imaging probes, including NIR-VEGFR2 antibody, NIR-VEGF-E and NIR-PlGF (angiogenesis), NIR-EGF (EGFR expression), NIR-d-thymidine (DNA replication), and NIR-GLu (Glucose metabolism). These NIR probes have been successfully applied to imaging murine tumors.

Preliminary Studies of Optical Kidney Imaging: We have considerable experience utilizing NIR-based imaging probes targeting cell apoptosis and EGFR that we have developed. We therefore tested and characterized these probes, Annexin-V (both NIR700-Annexin-V and 99mTc-Annexin-V) and NIR800-EGF, as an imaging carrier to assess renal tissue injury or response. As expected, NIR800-EGF strongly binds to the kidneys of normal mice (shown in Figure M2), in consistent with abundant EGFR expression in kidney (4). This probe can be used to assess renal EGFR-ligand interactions non-invasively in vivo. The Figure M3 demonstrates SPECT/CT imaging of 99mTc-Annexin-V. As shown in this Figure, 99mTc-Annexin-V illustrates considerable specific binding of Annexin-V in the normal cortex of the mouse kidney. There is no binding in the medulla, nor unrinary excretion of the probe, indicating that NIR700-Annexin-V binds to renal cortex with high affinity. This agrees with a recent report showing that Annexin-V probe is prominently trapped in renal cortex (5). The observed level of specific binding in the normal kidney precludes the use of Annexin-V-based probes for assessment of apoptosis in this organ, however, as shown in Figure M4, we have illustrated with the optical tracer (NIR700-Annexin-V) that renal retention of the probe appears to be an indicator of kidney health. As shown by the clearance profile analysis of the imaging probe, kidneys subjected to HgCl2 renal tubular injury show less imaging probe retention when compared to healthy controls. Thus, the probe may be useful for assessing renal health. Since Annexin-V strongly binds to renal cortex without apoptosis, we will consider another apoptosis imaging probe, Aposense (6, 7), which has successfully been applied to renal apoptosis imaging.

Some NIR probes are cleared from the animals mainly through kidney. Also, the uptake and clearance may be altered in pathological conditions (8). We will therefore inject non-targeting NIR probe as well as targeting probe to the mouse simultaneously, image the kidneys on time course, compare the retention of these two probes, and determine the renal NIR-light signals of the specific probe. The binding of the targeting probe to kidney will be determined by subtracting non-targeting probe signals from targeting probe signals. The Figure M5 demonstrates the capability to use non-targeted NIR-based imaging probes for this purpose (imaged using the Cambridge Research Instruments “Maestro” optical systems). As illustrated in Figure M5, we administered a non-targeted NIR-based probe that is normally cleared through the kidney. Following ischemic injury, affected mice exhibit impaired clearance of the non-targeted probe, particularly at longer imaging time points as shown. It is noteworthy that non-targeting probe may be useful for assessing renal perfusion as described recently (8).

Future directions: We will assess a correlation between renal NIR-EGF light signals and EGFR expression by Western blot and immunohistochemistry. To do a better control study, we will also prepare the NIR-EGF mutant probe which does not bind to EGFR by mutating the EGFR receptor-binding domain in EGF. A potential
problem in this method will be that renal pathological changes may limit the accessibility of NIR-probe to the target molecule, including reduced renal blood flow, decreased glomerular filtration or postglomerular circulation resulted from glomerulosclerosis, renal vascular regression, and impaired urine flow. It might be difficult to assess molecular events in advanced renal lesions by this method. However, this method would be effectively used for testing and screening pharmacological agents as well as for elucidating molecular events at the early stage of renal disease. Once we established the method and the protocol, this imaging assay and the probes will be available to the investigators through the Core. Furthermore, we will continue the efforts to develop and test new NIR-based imaging reagents. As an example, we will prepare and test NIR-Lycopersion esculentum lectin which labels vascular endothelium in vivo to assess renal vascularization. PET with a radio-label such as 18F is emerging as the leading modality for molecular imaging, enabling non-invasive, sensitive, and quantitative and “high and 3D resolution” imaging of biological processes. The method can also be clinically translated. Once NIR-probe is validated, we will develop the probe of PET version.

Publications for Molecular imaging (8)

Abulrob A, Brunette E, Slinn J, Baumann E, Stanimirovic D. In vivo time domain optical imaging of renal ischemia-reperfusion injury: discrimination based on fluorescence lifetime. Mol Imaging (2007) 6:304-14
View abstract View in PubMed

Fluorescence lifetime is an intrinsic parameter of the fluorescent probe, independent of the probe concentration but sensitive to changes in the surrounding microenvironment. Therefore, fluorescence lifetime imaging could potentially be applied to in vivo diagnostic assessment of changes in the tissue microenvironment caused by disease, such as ischemia. The aim of this study was to evaluate the utility of noninvasive fluorescence lifetime imaging in distinguishing between normal and ischemic kidney tissue in vivo. Mice were subjected to 60-minute unilateral kidney ischemia followed by 6-hour reperfusion. Animals were then injected with the near-infrared fluorescence probe Cy5.5 or saline and imaged using a time-domain small-animal optical imaging system. Both fluorescence intensity and lifetime were acquired. The fluorescence intensity of Cy5.5 was clearly reduced in the ischemic compared with the contralateral kidney, and the fluorescence lifetime of Cy5.5 was not detected in the ischemic kidney, suggesting reduced kidney clearance. Interestingly, the two-component lifetime analysis of endogenous fluorescence at 700 nm distinguished renal ischemia in vivo without the need for Cy5.5 injection for contrast enhancement. The average fluorescence lifetime of endogenous tissue fluorophores was a sensitive indicator of kidney ischemia ex vivo. The study suggests that fluorescence lifetime analysis of endogenous tissue fluorophores could be used to discriminate ischemic or necrotic tissues by noninvasive in vivo or ex vivo organ imaging.

Kietselaer BL, Reutelingsperger CP, Boersma HH, Heidendal GA, Liem IH, Crijns HJ, Narula J, Hofstra L. Noninvasive detection of programmed cell loss with 99mTc-labeled annexin A5 in heart failure. J Nucl Med (2007) 48:562-7
View abstract View in PubMed

Apoptosis, or programmed cell death (PCD), contributes to the decline in ventricular function in heart failure. Because apoptosis comprises a programmed cascade of events, it is potentially reversible, and timely intervention should delay the development of cardiomyopathy. (99m)Tc-Labeled annexin A5 has successfully been used for the noninvasive detection of PCD in myocardial infarction and heart transplant rejection. The present study evaluated the role of annexin A5 imaging for detection of PCD in heart failure patients. METHODS: Annexin A5 imaging was performed on 9 consecutive heart failure patients with advanced nonischemic cardiomyopathy (dilated, n = 8; hypertrophic, n = 1) and in 2 relatives having the same genetic background as the hypertrophic cardiomyopathy patient but no heart failure. RESULTS: Four of the patients with dilated cardiomyopathy and the 1 with hypertrophic cardiomyopathy and heart failure showed focal, multifocal, or global left ventricular uptake of annexin A5. No uptake was visualized in the remaining 4 patients or in the 2 controls. All cases showing annexin A5 uptake within the left ventricle experienced significant reduction in left ventricular function or functional class. In cases with no annexin A5 uptake, left ventricular function and clinical status remained stable. CONCLUSION: These data indicate the feasibility of noninvasive PCD detection with annexin imaging in heart failure patients. Annexin A5 uptake is associated with deterioration in left ventricular function, and this association may lend itself to the development of novel management strategies.

Aloya R, Shirvan A, Grimberg H, Reshef A, Levin G, Kidron D, Cohen A, Ziv I. Molecular imaging of cell death in vivo by a novel small molecule probe. Apoptosis (2006) 11:2089-101
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Apoptosis has a role in many medical disorders, therefore assessment of apoptosis in vivo can be highly useful for diagnosis, follow-up and evaluation of treatment efficacy. ApoSense is a novel technology, comprising low molecular-weight probes, specifically designed for imaging of cell death in vivo. In the current study we present targeting and imaging of cell death both in vitro and in vivo, utilizing NST-732, a member of the ApoSense family, comprising a fluorophore and a fluorine atom, for both fluorescent and future positron emission tomography (PET) studies using an (18)F label, respectively. In vitro, NST-732 manifested selective and rapid accumulation within various cell types undergoing apoptosis. Its uptake was blocked by caspase inhibition, and occurred from the early stages of the apoptotic process, in parallel to binding of Annexin-V, caspase activation and alterations in mitochondrial membrane potential. In vivo, NST-732 manifested selective uptake into cells undergoing cell-death in several clinically-relevant models in rodents: (i) Cell-death induced in lymphoma by irradiation; (ii) Renal ischemia/reperfusion; (iii) Cerebral stroke. Uptake of NST-732 was well-correlated with histopathological assessment of cell-death. NST-732 therefore represents a novel class of small-molecule detectors of apoptosis, with potential useful applications in imaging of the cell death process both in vitro and in vivo.

Vanderheyden JL, Liu G, He J, Patel B, Tait JF, Hnatowich DJ. Evaluation of 99mTc-MAG3-annexin V: influence of the chelate on in vitro and in vivo properties in mice. Nucl Med Biol (2006) 33:135-44
View abstract View in PubMed

We conjugated mercaptoacetyltriglycine (MAG(3)) to rh-annexin V to permit radiolabeling with (99m)Tc in an effort to decrease the high kidney and liver accumulation observed for (99m)Tc-labeled Hynic-annexin V. The 36-kDa protein was conjugated at a 5:1 molar ratio with NHS-MAG(3) in HEPES buffer pH 7.8 at room temperature, then quenched with glycine and purified by dialysis. The biopotency of the resulting MAG(3)-annexin was similar to that of Hynic-annexin as determined by a sensitive red blood cell membrane affinity binding assay and a surface plasmon resonance (SPR) assay. The (99m)Tc radiolabeling of MAG(3)-annexin resulted in radiochemical yields of 90% under mildly basic pH conditions. Biodistribution data in normal mice clearly showed a significant decrease in kidney and liver uptake at 1 h postinjection for the (99m)Tc MAG(3)-annexin compared to the (99m)Tc Hynic-annexin (from 24% ID to 4% ID for the liver, and 45% ID to 15% ID for the kidneys, respectively). Autoradiography of the kidneys showed retention of radioactivity in the collecting tubules following administration of both labeled annexins. The (99m)Tc MAG(3)-annexin biodistribution was also characterized by a lower retention of radioactivity in the whole body, but with small intestine accumulation over fivefold higher than observed with (99m)Tc Hynic-annexin. These findings show a definite improvement in renal and hepatic clearance of the MAG(3) radioligand. However, due to the increased radioactivity uptake in the small intestines, the early in vivo detection of ongoing apoptosis in the lower abdomen might be more difficult with (99m)Tc MAG(3)-annexin. Nevertheless, (99m)Tc MAG(3)-annexin may be an attractive alternative to (99m)Tc Hynic-annexin for the in vivo imaging of phosphatidylserine receptors.

Damianovich M, Ziv I, Heyman SN, Rosen S, Shina A, Kidron D, Aloya T, Grimberg H, Levin G, Reshef A, Bentolila A, Cohen A, Shirvan A. ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis. Eur J Nucl Med Mol Imaging (2006) 33:281-91
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PURPOSE: Acute renal tubular necrosis (ATN), a common cause of acute renal failure, is a dynamic, rapidly evolving clinical condition associated with apoptotic and necrotic tubular cell death. Its early identification is critical, but current detection methods relying upon clinical assessment, such as kidney biopsy and functional assays, are insufficient. We have developed a family of small molecule compounds, ApoSense, that is capable, upon systemic administration, of selectively targeting and accumulating within apoptotic/necrotic cells and is suitable for attachment of different markers for clinical imaging. The purpose of this study was to test the applicability of these molecules as a diagnostic imaging agent for the detection of renal tubular cell injury following renal ischemia. METHODS: Using both fluorescent and radiolabeled derivatives of one of the ApoSense compounds, didansyl cystine, we evaluated cell death in three experimental, clinically relevant animal models of ATN: renal ischemia/reperfusion, radiocontrast-induced distal tubular necrosis, and cecal ligature and perforation-induced sepsis. RESULTS: ApoSense showed high sensitivity and specificity in targeting injured renal tubular epithelial cells in vivo in all three models used. Uptake of ApoSense in the ischemic kidney was higher than in the non-ischemic one, and the specificity of ApoSense targeting was demonstrated by its localization to regions of apoptotic/necrotic cell death, detected morphologically and by TUNEL staining. CONCLUSION: ApoSense technology should have significant clinical utility for real-time, noninvasive detection of renal parenchymal damage of various types and evaluation of its distribution and magnitude; it may facilitate the assessment of efficacy of therapeutic interventions in a broad spectrum of disease states.

Belhocine TZ, Blankenberg FG. 99mTc-Annexin A5 uptake and imaging to monitor chemosensitivity. Methods Mol Med (2005) 111:363-80
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Most anticancer agents act by inducing apoptosis in sensitive tumor cells. Hence, in many types of cancers, significant increase of tumor apoptosis after chemotherapy correlates with tumor chemosensitivity. Theoretically, a reliable evaluation of apoptotic changes, postchemotherapy to baseline, may provide valuable insights into the apoptotic competence of cancers. Until now, assessment of chemosensitivity has usually relied upon histological evidence of tumor response (i.e., partial or complete disappearance of tumor cells) or demonstration of tumor shrinkage by means of morphological imaging (i.e., computed tomography or magnetic resonance imaging). In clinical practice, however, these conventional methods are proving ineffective for monitoring tumor chemosensitivity on a daily basis. Recent developments in molecular imaging have allowed the synthesis of a new radiolabeled agent, 99mTc-recombinant human Annexin A5, designed to the assessment of apoptotic response of cancers after a single course of chemotherapy. Such in vivo technique opens promising perspectives for evaluating, noninvasively and early, tumor response to anticancer therapies. Alternative methods for Annexin A5 labeling and imaging may improve the detection of drug-induced apoptosis to monitor chemosensitivity.

Sevick-Muraca EM, Houston JP, Gurfinkel M. Fluorescence-enhanced, near infrared diagnostic imaging with contrast agents. Curr Opin Chem Biol (2002) 6:642-50
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The deep tissue propagation of near-infrared (NIR) light between 700-900 nm offers new opportunities for diagnostic imaging when employing sensitive detection techniques and NIR excitable fluorescent agents that target and report disease and metabolism. Herein, we highlight approaches for illuminating tissues and monitoring the re-emitted fluorescence for tomographic reconstruction, strategies for developing fluorescent dye constructs, and clinical opportunities for fluorescence-enhanced NIR optical imaging.

Partanen AM, Thesleff I. Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages. Dev Biol (1987) 120:186-97
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Last updated on 2013-11-06 Moderated by Takamune Takahashi