Non-invasive Imaging of Endothelial Progenitor Cells in Tumor Neovascularization Using a Novel Dual-modality Paramagnetic/Near-Infrared Fluorescence Probe

Objective

Bone-marrow derived endothelial progenitor cells (EPCs) play an important role in tumor neovasculature. Due to their tumor homing property, EPCs are regarded as promising targeted vectors for delivering therapeutic agents in cancer treatment. Consequently, non-invasive confirmation of targeted delivery via imaging is urgently needed. This study shows the development and application of a novel dual-modality probe for in vivo non-invasively tracking of the migration, homing and differentiation of EPCs.

Methods

The paramagnetic/near-infrared fluorescence probe Conjugate 1 labeled EPCs were systemically transplanted into mice bearing human breast MDA-MB-231 tumor xenografts. Magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence optical imaging were performed at different stages of tumor development. The homing of EPCs and the tumor neovascularization were further evaluated by immunofluorescence.

Results

Conjugate 1 labeled EPCs can be monitored in vivo by MRI and NIR fluorescence optical imaging without altering tumor growth for up to three weeks after the systemic transplantation. Histopathological examination confirmed that EPCs were recruited into the tumor bed and then incorporated into new vessels two weeks after the transplantation. Tumor size and microvessel density was not influenced by EPCs transplantation in the first three weeks.

Conclusions

This preclinical study shows the feasibility of using a MRI and NIR fluorescence optical imaging detectable probe to non-invasively monitor transplanted EPCs and also provides strong evidence that EPCs are involved in the development of endothelial cells during the tumor neovascularization.     

Molecular Imaging of Pancreatic Duct Adenocarcinoma Using a Type 2 Cannabinoid Receptor-Targeted Near-Infrared Fluorescent Probe

Imaging probes targeting type 2 cannabinoid receptor (CB₂R) overexpressed in pancreatic duct adenocarcinoma (PDAC) tissue have the potential to improve early detection and surgical outcome of PDAC. The aim of our study was to evaluate the molecular imaging potential of a CB₂R-targeted near-infrared (NIR) fluorescent probe (NIR760-XLP6) for PDAC. CB₂R overexpression was observed in both PDAC patient tissues and various pancreatic cancer cell lines. In vitro fluorescence imaging indicated specific binding of NIR760-XLP6 to CB₂R in human PDAC PANC-1 cells. In a xenograft mouse tumor model, NIR760-XLP6 showed remarkable 50- (ex vivo) and 3.2-fold (in vivo) tumor to normal contrast enhancement with minimal liver and kidney uptake. In a PDAC lymph node metastasis model, significant signal contrast was observed in bilateral axillary lymph nodes with PDAC metastasis after injection of the probe. In conclusion, NIR760-XLP6 exhibits promising characteristics for imaging PDAC, and CB₂R appears to be an attractive target for PDAC imaging.     

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.     

Bioconjugated quantum dots for cancer research: Present status,prospects and remaining issues

Semiconductor quantum dots (QDs) are nanoparticles in which charge carriers are three dimensionally confined or quantum confined. The quantum confinement provides size-tunable absorption bands and emission color to QDs. Also, the photoluminescence (PL) of QDs is exceptionally bright and stable, making them potential candidates for biomedical imaging and therapeutic interventions. Although fluorescence imaging and photodynamic therapy (PDT) of cancer have many advantages over imaging using ionizing radiations and chemo and radiation therapies, advancement of PDT is limited due to the poor availability of photostable and NIR fluorophores and photosensitizing (PS) drugs. With the introduction of biocompatible and NIR QDs, fluorescence imaging and PDT of cancer have received new dimensions and drive. In this review, we summarize the prospects of QDs for imaging and PDT of cancer. Specifically, synthesis of visible and NIR QDs, targeting cancer cells with QDs, in vitro and in vivo cancer imaging, multimodality, preparation of QD-PS conjugates and their energy transfer, photosensitized production of reactive oxygen intermediates (ROI), and the prospects and remaining issues in the advancement of QD probes for imaging and PDT of cancer are summarized.     

In Vivo image Analysis Using iRFP Transgenic Mice

Fluorescent proteins with light wavelengths within the optical window are one of the improvements in in vivo imaging techniques. Near-infrared (NIR) fluorescent protein (iRFP) is a stable, nontoxic protein that emits fluorescence within the NIR optical window without the addition of exogenous substrate. However, studies utilizing an in vivo iRFP model have not yet been published. Here, we report the generation of transgenic iRFP mice with ubiquitous NIR fluorescence expression. iRFP expression was observed in approximately 50% of the offspring from a matings between iRFP transgenic and WT mice. The serum and blood cell indices and body weights of iRFP mice were similar to those of WT mice. Red fluorescence with an excitation wavelength of 690 nm and an emission wavelength of 713 nm was detected in both newborn and adult iRFP mice. We also detected fluorescence emission in whole organs of the iRFP mice, including the brain, heart, liver, kidney, spleen, lung, pancreas, bone, testis, thymus, and adipose tissue. Therefore, iRFP transgenic mice may therefore be a useful tool for various types of in vivo imaging.     

Photoimmunotherapy of Gastric Cancer Peritoneal Carcinomatosis in a Mouse Model

Photoimmunotherapy (PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with the toxicity induced by photosensitizers after exposure to near infrared (NIR) light. We performed PIT in a model of disseminated gastric cancer peritoneal carcinomatosis and monitored efficacy with in vivo GFP fluorescence imaging. In vitro and in vivo experiments were conducted with a HER2-expressing, GFP-expressing, gastric cancer cell line (N87-GFP). A conjugate comprised of a photosensitizer, IR-700, conjugated to trastuzumab (tra-IR700), followed by NIR light was used for PIT. In vitro PIT was evaluated by measuring cytotoxicity with dead staining and a decrease in GFP fluorescence. In vivo PIT was evaluated in a disseminated peritoneal carcinomatosis model and a flank xenograft using tumor volume measurements and GFP fluorescence intensity. In vivo anti-tumor effects of PIT were confirmed by significant reductions in tumor volume (at day 15, p<0.0001 vs. control) and GFP fluorescence intensity (flank model: at day 3, PIT treated vs. control p<0.01 and peritoneal disseminated model: at day 3 PIT treated vs. control, p<0.05). Cytotoxic effects in vitro were shown to be dependent on the light dose and caused necrotic cell rupture leading to GFP release and a decrease in fluorescence intensity in vitro. Thus, loss of GFP fluorescence served as a useful biomarker of cell necrosis after PIT.     

Two-dimensional Fast Surface Imaging Using a Handheld Optical Device: In Vitro and In Vivo Fluorescence Studies

Near-infrared (NIR) optical imaging is a noninvasive and nonionizing modality that is emerging as a diagnostic tool for breast cancer. The handheld optical devices developed to date using the NIR technology are predominantly developed for spectroscopic applications. A novel handheld probe-based optical imaging device has been recently developed toward area imaging and tomography applications. The three-dimensional (3D) tomographic imaging capabilities of the device have been demonstrated from previous fluorescence studies on tissue phantoms. In the current work, fluorescence imaging studies are performed on tissue phantoms, in vitro, and in vivo tissue models to demonstrate the fast two-dimensional (2D) surface imaging capabilities of this flexible handheld-based optical imaging device, toward clinical breast imaging studies. Preliminary experiments were performed using target(s) of varying volume (0.23 and 0.45 cm³) and depth (1–2 cm), using indocyanine green as the fluorescence contrast agent in liquid phantom, in vitro, and in vivo tissue models. The feasibility of fast 2D surface imaging (∼5 seconds) over large surface areas of 36 cm² was demonstrated from various tissue models. The surface images could differentiate the target(s) from the background, allowing a rough estimate of the target''s location before extensive 3D tomographic analysis (future studies).     

Magnetic resonance and near-infrared imaging using a novel dual-modality nano-probe for dendritic cell tracking in vivo

Background aimsThe effect of cellular-based immunotherapy is highly correlated with the success of dendritic cells (DCs) homing to the draining lymph nodes (LNs) and interacting with antigen-specific CD4⁺ T cells. In this study, a novel magneto-fluorescent nano-probe was used to track the in vivo migration of DCs to the draining LNs.MethodsA dual-modality nano-probe composed of superparamagnetic iron oxide (SPIO) and near-infrared fluorescent (NIRF) dye (NIR797) was developed, and its magnetic and optical contrasting properties were characterized. DCs generated from mouse bone marrow were co-cultured with the probe at a lower concentration of 10 μg/mL. The cell phenotype and function of DCs were also investigated by fluorescence-activated cell sorting analysis and mixed leukocyte reactivity assay. Labeled DCs were injected into the footpad of C57BL/6 mice. Afterward, magnetic resonance imaging, NIRF imaging, Perls staining and CD11c immunofluorescence were used to observe the migration of the labeled DCs into draining LNs.ResultsThe synthetic SPIO-NIR797 nano-probe had a desirable superparamagnetic and near-infrared behavior. Perls staining showed perfect labeling efficiency. The cell phenotypes, including CD11c, CD80, CD86 and major histocompatibility complex class II, as well as the T-cell activation potential of the mature DCs were insignificantly affected after incubation (P > 0.05). Labeled DCs migrating into LNs could be detected by both magnetic resonance imaging and NIRF imaging simultaneously, which was further confirmed by Perls staining and immunofluorescence.ConclusionsThe novel dual-modality SPIO-NIR797 nano-probe has highly biocompatible characteristics for labeling and tracking DCs, which can be used to evaluate cancer immunotherapy in clinical applications.     

Minimally Invasive Electro-Magnetic Navigational Bronchoscopy-Integrated Near-Infrared-Guided Sentinel Lymph Node Mapping in the Porcine Lung

BackgroundThe use of near-infrared (NIR) fluorescence imaging with indocyanine green (ICG) for sentinel lymph node (SN) mapping has been investigated in lung cancer; however, this has not been fully adapted for minimally invasive surgery (MIS). The aim of our study was to develop a minimally invasive SN mapping integrating pre-operative electro-magnetic navigational bronchoscopy (ENB)-guided transbronchial ICG injection and intraoperative NIR thoracoscopic imaging.MethodsA NIR thoracoscope was used to visualize ICG fluorescence. ICG solutions in a 96-well plate and ex vivo porcine lungs were examined to optimize ICG concentrations and injection volumes. Transbronchial ICG injection (n=4) was assessed in comparison to a traditional transpleural approach (n=3), where after thoracotomy an ICG solution (100μL at 100μg/mL) was injected into the porcine right upper lobe for SN identification. For further translation into clinical use, transbronchial ICG injection prior to thoracotomy followed by NIR thoracoscopic imaging was validated (n=3). ENB was used for accurate targeting in two pigs with a pseudo-tumor.ResultsThe ICG fluorescence at 10 μg/mL was the brightest among various concentrations, unchanged by the distance between the thoracoscope and ICG solutions. Injected ICG of no more than 500μL showed a localized fluorescence area. All 7 pigs showed a bright paratracheal lymph node within 15 minutes post-injection, with persistent fluorescence for 60 minutes. The antecedent transbronchial ICG injection succeeded in SN identification in all 3 cases at the first thoracoscopic inspection within 20 minutes post-injection. The ENB system allowed accurate ICG injection surrounding the pseudo-tumors.ConclusionsENB-guided ICG injection followed by NIR thoracoscopy was technically feasible for SN mapping in the porcine lung. This promising platform may be translated into human clinical trials and is suited for MIS.     

A Versatile Technique for the In Vivo Imaging of Human Tumor Xenografts Using Near-Infrared Fluorochrome-Conjugated Macromolecule Probes

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins—an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG_2a—were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging.     

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque,a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.¹Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.⁴ While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.² The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR ''window'' can substantially improve the potential for in vivo imaging.^2,5Inflammatory cysteine proteases have been well studied using activatable NIRF probes¹⁰, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis⁸. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.^3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology⁶, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.¹⁰ Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque. In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,⁶ is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.^11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.Download video file.^{(61M, mov)}     

In Vivo Detection of Macrophage Recruitment in Hind-Limb Ischemia Using a Targeted Near-Infrared Fluorophore

Macrophages are an essential component of the immune system and have protective and pathogenic functions in various diseases. Imaging of macrophages in vivo could furnish new tools to advance evaluation of disease and therapies. Critical limb ischemia is a disease in which macrophages have considerable pathogenic roles, and are potential targets for cell-based immunotherapy. We sought to develop a new near-infrared fluorescence (NIRF) imaging probe to target macrophages specifically in vivo in various pathological states, including hind-limb ischemia. We rapidly screened the photostable cyanine-based NIRF library against different blood cell lines. The identified monocyte/macrophage-selective hit was tested in vitro in live-cell labeling assay. Non-invasive NIRF imaging was performed with murine models of paw inflammation by lipopolysaccharide challenge and hind-limb ischemia with femoral artery ligation. in vivo macrophage targeting was further evaluated using intravital microscopy with Csf1r-EGFP transgenic mice and immunofluorescent staining with macrophage-specific markers. We discovered MF800, a Macrophage-specific near-infrared Fluorophore, which showed selective live-cell imaging performance in a panel of cell lines and primary human blood samples. MF800 outperforms the clinically-available NIRF contrast agent ICG for in vivo specificity in paw inflammation and hind-limb ischemia models. We observed a marked overlap of MF800-labeled cells and EGFP-expressing macrophages in intravital imaging of Csf1r-EGFP transgenic mice. In the histologic analysis, MF800-positive cells also expressed the macrophage markers CD68 and CD169. NIRF imaging showcased the potential of using MF800 to understand macrophage behavior in vivo, characterize macrophage-associated diseases, and may help in assessing therapeutic responses in the clinic.     

Structure–activity relationships of succinimidyl-Cys-C(O)-Glu derivatives with different near-infrared fluorophores as optical imaging probes for prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA), which is overexpressed in malignant prostate cancer (PCa), is an ideal target for imaging and therapy of PCa. We previously reported a PSMA imaging probe, 800CW-SCE, based on succinimidyl-Cys-C(O)-Glu (SCE) for optical imaging of PCa. In this study, we investigated the structure–activity relationships of novel SCE derivatives with five different near-infrared (NIR) fluorophores (IRDye 680LT, IRDye 750, Indocyanine Green, Cyanine 5.5, and Cyanine 7) as optical imaging probes targeting PSMA. An in vitro binding assay revealed that 800CW-SCE, 680LT-SCE, and 750-SCE exhibited higher binding affinity than 2-PMPA, which is known as a PSMA inhibitor. These three SCE derivatives were internalized into PSMA-positive cells (LNCaP cells) but not into PSMA-negative cells (PC-3 cells). In the in vivo imaging study, 800CW-SCE and 750-SCE were highly accumulated in LNCaP tumors but not in PC-3 tumors, and the ratio of LNCaP/PC-3 accumulation of 800CW-SCE was higher than that of 750-SCE. The present study may provide valuable molecular design information for the future development of new PSMA imaging probes based on the SCE scaffold.     

Synthesis and characterization of new porphyrazine-Gd(III) conjugates as multimodal MR contrast agents

Magnetic resonance imaging (MRI) has long been used clinically and experimentally as a diagnostic tool to obtain three-dimensional, high-resolution images of deep tissues. These images are enhanced by the administration of contrast agents such as paramagnetic Gd(III) complexes. Herein, we describe the preparation of a series of multimodal imaging agents in which paramagnetic Gd(III) complexes are conjugated to a fluorescent tetrapyrrole, namely, a porphyrazine (pz). Zinc metalated pzs conjugated to one, four, or eight paramagnetic Gd(III) complexes are reported. Among these conjugates, Zn-Pz-8Gd(III) exhibits an ionic relaxivity four times that of the monomeric Gd(III) agent, presumably because of increased molecular weight and a molecular relaxivity that is approximately thirty times larger, while retaining the intense electronic absorption and emission of the unmodified pz. Unlike current clinical MR agents, Zn-Pz-1Gd(III) is taken up by cells. This probe demonstrates intracellular fluorescence by confocal microscopy and provides significant contrast enhancement in MR images, as well as marked phototoxicity in assays of cellular viability. These results suggest that pz agents possess a new potential for use in cancer imaging by both MRI and near-infrared (NIR) fluorescence, while acting as a platform for photodynamic therapy.     

Detection of Cancer Metastases with a Dual-labeled Near-Infrared/Positron Emission Tomography Imaging Agent

By dual labeling a targeting moiety with both nuclear and optical probes, the ability for noninvasive imaging and intraoperative guidance may be possible. Herein, the ability to detect metastasis in an immunocompetent animal model of human epidermal growth factor receptor 2 (HER-2)-positive cancer metastases using positron emission tomography (PET) and near-infrared (NIR) fluorescence imaging is demonstrated. METHODS: (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m was synthesized, characterized, and administered to female Balb/c mice subcutaneously inoculated with highly metastatic 4T1.2neu/R breast cancer cells. (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m (150 µg, 150 µCi, m = 2, n = 2) was administered through the tail vein at weeks 2 and 6 after implantation, and PET/computed tomography and NIR fluorescence imaging were performed 24 hours later. Results were compared with the detection capabilities of F-18 fluorodeoxyglucose (¹⁸FDG-PET). RESULTS: Primary tumors were visualized with ¹⁸FDG and (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m, but resulting metastases were identified only with the dual-labeled imaging agent. ⁶⁴Cu-PET imaging detected lung metastases, whereas ex vivo NIR fluorescence showed uptake in regions of lung, skin, skeletal muscle, and lymph nodes, which corresponded with the presence of cancer cells as confirmed by histologic hematoxylin and eosin stains. In addition to detecting the agent in lymph nodes, the high signal-to-noise ratio from NIR fluorescence imaging enabled visualization of channels between the primary tumor and the axillary lymph nodes, suggesting a lymphatic route for trafficking cancer cells. Because antibody clearance occurs through the liver, we could not distinguish between nonspecific uptake and liver metastases. CONCLUSION: (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m may be an effective diagnostic imaging agent for staging HER-2-positive breast cancer patients and intraoperative resection.     

In vivo detection of phospholipase C by enzyme-activated near-infrared probes

In this article, the characterization of the first near-infrared (NIR) phospholipase-activated molecular beacon is reported, and its utility for in vivo cancer imaging is demonstrated. The probe consists of three elements: a phospholipid (PL) backbone to which the NIR fluorophore, pyropheophorbide a (Pyro), and the NIR Black Hole Quencher 3 (BHQ) were conjugated. Because of the close proximity of BHQ to Pyro, the Pyro-PtdEtn-BHQ probe is self-quenched until enzyme hydrolysis releases the fluorophore. The Pyro-PtdEtn-BHQ probe is highly specific to one isoform of phospholipase C, phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine. Incubation of Pyro-PtdEtn-BHQ in vitro with PC-PLC demonstrated a 150-fold increase in fluorescence that could be inhibited by the specific PC-PLC inhibitor tricyclodecan-9-yl xanthogenate (D609) with an IC(50) of 34 ± 8 μM. Since elevations in phosphocholine have been consistently observed by magnetic resonance spectroscopy in a wide array of cancer cells and solid tumors, we assessed the utility of Pyro-PtdEtn-BHQ as a probe for targeted tumor imaging. Injection of Pyro-PtdEtn-BHQ into mice bearing DU145 human prostate tumor xenografts followed by in vivo NIR imaging resulted in a 4-fold increase in tumor radiance over background and a 2 fold increase in the tumor/muscle ratio. Tumor fluorescence enhancement was inhibited with the administration of D609. The ability to image PC-PLC activity in vivo provides a unique and sensitive method of monitoring one of the critical phospholipase signaling pathways activated in cancer, as well as the phospholipase activities that are altered in response to cancer treatment.     

Non-Invasive Imaging of Tumors by Monitoring Autotaxin Activity Using an Enzyme-Activated Near-Infrared Fluorogenic Substrate

Autotaxin (ATX), an autocrine motility factor that is highly upregulated in metastatic cancer, is a lysophospholipase D enzyme that produces the lipid second messenger lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (AR-2) that enables visualization of ATX activity in vivo. AR-2 exhibits minimal fluorescence until it is activated by ATX, which substantially increases fluorescence in the near-infrared (NIR) region, the optimal spectral window for in vivo imaging. In mice with orthotopic ATX-expressing breast cancer tumors, ATX activated AR-2 fluorescence. Administration of AR-2 to tumor-bearing mice showed high fluorescence in the tumor and low fluorescence in most healthy tissues with tumor fluorescence correlated with ATX levels. Pretreatment of mice with an ATX inhibitor selectively decreased fluorescence in the tumor. Together these data suggest that fluorescence directly correlates with ATX activity and its tissue expression. The data show that AR-2 is a non-invasive and selective tool that enables visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo.     

Enhanced tumor detection using a folate receptor-targeted near-infrared fluorochrome conjugate

Fluorescence optical imaging technologies are currently being developed to image specific molecular targets in vivo. Detection technologies range from those providing microscopic detail to whole body imaging systems with potential clinical use. A number of target-specific near-infrared imaging probes have recently been developed to image receptors, antigens, and enzymes. The goal of the current study was to evaluate a new near-infrared (NIR) folate receptor (FR)-targeted imaging probe for its ability to improve detection of FR-positive cancers. We hypothesized that modification of folate would retain receptor affinity in vivo, despite the bulkier NIR fluorochrome, NIR2 (em = 682 nm). Cellular uptake of the NIR conjugates was significantly higher in FR-positive nasopharyngeal epidermoid carcinoma, KB cells, compared to FR-negative human fibrosarcoma, HT1080 cells. When tumors were implanted in vivo, equal-sized KB tumors showed a 2.4-fold higher signal intensity compared to HT1080 tumors (24 h). The maximum signal-to-background ratio (3-fold) was observed at 24 h in KB tumor. Injection of the unmodified NIR2 fluorochrome did not result in persistent contrast increases under similar conditions. Furthermore, tumor enhancement with the NIR2-folate probe persisted over 48 h and was inhibitable in vivo by administration of unlabeled folate. These results indicate that folate-modified NIR fluorochrome conjugate can be used for improved detection of FR-positive tumors.     

Non-Invasive In Vivo Imaging of Near Infrared-labeled Transferrin in Breast Cancer Cells and Tumors Using Fluorescence Lifetime FRET

The conjugation of anti-cancer drugs to endogenous ligands has proven to be an effective strategy to enhance their pharmacological selectivity and delivery towards neoplasic tissues. Since cell proliferation has a strong requirement for iron, cancer cells express high levels of transferrin receptors (TfnR), making its ligand, transferrin (Tfn), of great interest as a delivery agent for therapeutics. However, a critical gap exists in the ability to non-invasively determine whether drugs conjugated to Tfn are internalized into target cells in vivo. Due to the enhanced permeability and retention (EPR) effect, it remains unknown whether these Tfn-conjugated drugs are specifically internalized into cancer cells or are localized non-specifically as a result of a generalized accumulation of macromolecules near tumors. By exploiting the dimeric nature of the TfnR that binds two molecules of Tfn in close proximity, we utilized a Förster Resonance Energy Transfer (FRET) based technique that can discriminate bound and internalized Tfn from free, soluble Tfn. In order to non-invasively visualize intracellular amounts of Tfn in tumors through live animal tissues, we developed a novel near infrared (NIR) fluorescence lifetime FRET imaging technique that uses an active wide-field time gated illumination platform. In summary, we report that the NIR fluorescence lifetime FRET technique is capable of non-invasively detecting bound and internalized forms of Tfn in cancer cells and tumors within a live small animal model, and that our results are quantitatively consistent when compared to well-established intensity-based FRET microscopy methods used in in vitro experiments.     

In vivo near-infrared imaging of fibrin deposition in thromboembolic stroke in mice

Objectives

Thrombus and secondary thrombosis plays a key role in stroke. Recent molecular imaging provides in vivo imaging of activated factor XIII (FXIIIa), an important mediator of thrombosis or fibrinolytic resistance. The present study was to investigate the fibrin deposition in a thromboembolic stroke mice model by FXIIIa–targeted near-infrared fluorescence (NIRF) imaging.

Materials and Methods

The experimental protocol was approved by our institutional animal use committee. Seventy-six C57B/6J mice were subjected to thromboembolic middle cerebral artery occlusion or sham operation. Mice were either intravenously injected with the FXIIIa-targeted probe or control probe. In vivo and ex vivo NIRF imaging were performed thereafter. Probe distribution was assessed with fluorescence microscopy by spectral imaging and quantification system. MR scans were performed to measure lesion volumes in vivo, which were correlated with histology after animal euthanasia.

Results

In vivo significant higher fluorescence intensity over the ischemia-affected hemisphere, compared to the contralateral side, was detected in mice that received FXIIIa-targeted probe, but not in the controlled mice. Significantly NIRF signals showed time-dependent processes from 8 to 96 hours after injection of FXIIIa-targeted probes. Ex vivo NIRF image showed an intense fluorescence within the ischemic territory only in mice injected with FXIIIa-targeted probe. The fluorescence microscopy demonstrated distribution of FXIIIa-targeted probe in the ischemic region and nearby micro-vessels, and FXIIIa-targeted probe signals showed good overlap with immune-fluorescent fibrin staining images. There was a significant correlation between total targeted signal from in vivo or ex vivo NIRF images and lesion volume.

Conclusion

Non-invasive detection of fibrin deposition in ischemic mouse brain using NIRF imaging is feasible and this technique may provide an in vivo experimental tool in studying the role of fibrin in stroke.