Comparison of Optical and Power Doppler Ultrasound Imaging for Non-Invasive Evaluation of Arsenic Trioxide as a Vascular Disrupting Agent in Tumors

Small animal imaging provides diverse methods for evaluating tumor growth and acute response to therapy. This study compared the utility of non-invasive optical and ultrasound imaging to monitor growth of three diverse human tumor xenografts (brain U87-luc-mCherry, mammary MCF7-luc-mCherry, and prostate PC3-luc) growing in nude mice. Bioluminescence imaging (BLI), fluorescence imaging (FLI), and Power Doppler ultrasound (PD US) were then applied to examine acute vascular disruption following administration of arsenic trioxide (ATO).During initial tumor growth, strong correlations were found between manual caliper measured tumor volume and FLI intensity, BLI intensity following luciferin injection, and traditional B-mode US. Administration of ATO to established U87 tumors caused significant vascular shutdown within 2 hrs at all doses in the range 5 to 10 mg/kg in a dose dependant manner, as revealed by depressed bioluminescent light emission. At lower doses substantial recovery was seen within 4 hrs. At 8 mg/kg there was >85% reduction in tumor vascular perfusion, which remained depressed after 6 hrs, but showed some recovery after 24 hrs. Similar response was observed in MCF7 and PC3 tumors. Dynamic BLI and PD US each showed similar duration and percent reductions in tumor blood flow, but FLI showed no significant changes during the first 24 hrs.The results provide further evidence for comparable utility of optical and ultrasound imaging for monitoring tumor growth, More specifically, they confirm the utility of BLI and ultrasound imaging as facile assays of the vascular disruption in solid tumors based on ATO as a model agent.     

Multimodality Imaging of Tumor and Bone Response in a Mouse Model of Bony Metastasis

Cancer drug development generally performs in vivo evaluation of treatment effects that have traditionally relied on detection of morphologic changes. The emergence of new targeted therapies, which may not result in gross morphologic changes, has spurred investigation into more specific imaging methods to quantify response, such as targeted fluorescent probes and bioluminescent cells. The present study investigated tissue response to docetaxel or zoledronic acid (ZA) in a mouse model of bony metastasis. Intratibial implantations of breast cancer cells (MDA-MB-231) were monitored throughout this study using several modalities: molecular resonance imaging (MRI) tumor volume and apparent diffusion coefficient (ADC), micro-computed tomography (µCT) bone volume, bioluminescence imaging (BLI) reporting cancer cell apoptosis, and fluorescence using Osteosense 800 and CatK 680-FAST. Docetaxel treatment resulted in tumor cell kill reflected by ADC and BLI increases and tumor volume reduction, with delayed bone recovery seen in µCT prefaced by increased osteoblastic activity (Osteosense 800). In contrast, the ZA treatment group produced similar values in MRI, BLI, and Osteosense 800 fluorescence imaging readouts when compared to controls. However, µCT bone volume increased significantly by the first week post-treatment and the CatK 680-FAST signal was slightly diminished by 4 weeks following ZA treatment. Multimodality imaging provides a more comprehensive tool for new drug evaluation and efficacy screening through identification of morphology as well as function and apoptotic signaling.     

Preparation of a cathepsin D sensitive near-infrared fluorescence probe for imaging.

A variety of proteases are overexpressed or activated during pathogenesis and represent important targets for therapeutic drugs. We have previously shown that optical imaging probes sensitive in the near-infrared fluorescence (NIRF) spectrum can be used for in vivo imaging of enzyme activity. In the current study, we show that these probes can be designed with specificity for specific enzymes, for example, cathepsin D which is known to be overexpressed in many tumors. A NIR cyanine fluorochrome served as the optical reporter and was attached to the amino terminal of an 11 amino acid peptide sequence with specificity for cathepsin D. The peptides were subsequently attached to a synthetic graft copolymer for efficient tumoral delivery. The close spatial proximity of the multiple fluorochromes resulted in quenching of fluorescence in the bound state. A 350-fold signal amplification was observed post cleavage during in vitro testing. Cell culture experiments using a rodent tumor cell line stably transfected with human cathepsin D confirmed enzyme specific activation within cells. This sequence but not a scrambled control sequence showed enzyme specificity in vitro. We conclude that activatable NIRF optical probes can be synthesized to potentially probe for specific enzymes in living organisms.     

In vivo imaging of tumors with protease-activated near-infrared fluorescent probes

We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.     

A near-infrared cell tracker reagent for multiscopic in vivo imaging and quantification of leukocyte immune responses

The complexity of the tumor microenvironment necessitates that cell behavior is studied in a broad, multi-scale context. Although tomographic and microscopy-based far and near infrared fluorescence (NIRF, >650 nm) imaging methods offer high resolution, sensitivity, and depth penetration, there has been a lack of optimized NIRF agents to label and track cells in their native environments at different scales. In this study we labeled mammalian leukocytes with VivoTag 680 (VT680), an amine reactive N-hydroxysuccinimide (NHS) ester of a (benz) indolium-derived far red fluorescent probe. We show that VT680 diffuses into leukocytes within minutes, covalently binds to cellular components, remains internalized for days in vitro and in vivo, and does not transfer fluorescence to adjacent cells. It is biocompatible, keeps cells fully functional, and fluoresces at high intensities. In a tumor model of cytotoxic T lymphocyte (CTL) immunotherapy, we track and quantify VT680-labeled cells longitudinally at the whole-body level with fluorescence-mediated molecular tomography (FMT), within tissues at single cell resolutions by multiphoton and confocal intravital microscopy, and ex vivo by flow cytometry. Thus, this approach is suitable to monitor cells at multiple resolutions in real time in their native environments by NIR-based fluorescence imaging.     

Non-Invasive Detection of a Small Number of Bioluminescent Cancer Cells In Vivo

Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice.     

In vivo bioluminescence imaging of bone marrow-derived cells in brain inflammation

It has been accepted that bone marrow cells infiltrate the brain and play important roles in neuroinflammation. However, there is no good tool for the visualization of these cells in living animals. In this study, we generated mice that were transplanted with GFP- or luciferase-expressing bone marrow cells, and performed in vivo fluorescence imaging (FLI) and in vivo bioluminescence imaging (BLI) to visualize the infiltrated cells. Brain inflammation was induced by intrahippocampal injection of lipopolysaccharide (LPS). Immunohistochemical investigation demonstrated an increase in the infiltration of bone marrow cells into the hippocampus because of the LPS injection and differentiation of the infiltrated cells into microglia, but not into neurons or astrocytes. BLI, but not FLI, successfully detected an increase in signal intensity with the LPS injection, and the increase of BLI coincided with that of luciferase activity in hippocampus. BLI could quantitatively and continuously monitor bone marrow-derived cells in vivo.     

Secreted Gaussia Luciferase as a Biomarker for Monitoring Tumor Progression and Treatment Response of Systemic Metastases

Background

Currently, only few techniques are available for quantifying systemic metastases in preclinical model. Thus techniques that can sensitively detect metastatic colonization and assess treatment response in real-time are urgently needed. To this end, we engineered tumor cells to express a naturally secreted Gaussia luciferase (Gluc), and investigated its use as a circulating biomarker for monitoring viable metastatic or primary tumor growth and their treatment responses.

Methodology/Principal Findings

We first developed orthotopic primary and metastatic breast tumors with derivative of MDA-MB-231 cells expressing Gluc. We then correlated tumor burden with Gluc activity in the blood and urine along with bioluminescent imaging (BLI). Second, we utilized blood Gluc assay to monitor treatment response to lapatinib in an experimental model of systemic metastasis. We observed good correlation between the primary tumor volume and Gluc concentration in blood (R² = 0.84) and urine (R² = 0.55) in the breast tumor model. The correlation deviated as a primary tumor grew due to a reduction in viable tumor fraction. This was also supported by our mathematical models for tumor growth to compare the total and viable tumor burden in our model. In the experimental metastasis model, we found numerous brain metastases as well as systemic metastases including bone and lungs. Importantly, blood Gluc assay revealed early growth of metastatic tumors before BLI could visualize their presence. Using secreted Gluc, we localized systemic metastases by BLI and quantitatively monitored the total viable metastatic tumor burden by blood Gluc assay during the course of treatment with lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER2.

Conclusion/Significance

We demonstrated secreted Gluc assay accurately reflects the amount of viable cancer cells in primary and metastatic tumors. Blood Gluc activity not only tracks metastatic tumor progression but also serves as a longitudinal biomarker for tumor response to treatments.     

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)}     

Novel α(2)β(1) integrin-targeted peptide probes for prostate cancer imaging

Accumulating experimental evidence indicates that overexpression of α(2)β(1) integrin may correlate with progression in human prostate cancer. The objective of this study was to design a novel imaging probe based on the Asp-Gly-Glu-Ala (DGEA) peptide for near-infrared-fluorescent (NIRF) imaging of α(2)β(1) integrin expression in prostate cancer. The peptides were conjugated with appropriate fluorescent dyes, and the binding affinity of these probes was evaluated by flow cytometry in three human prostate cell lines (PC-3, CWR-22, and LNCaP). In vivo NIRF imaging of the α(2)β(1)-positive PC-3 xenograft model was performed to evaluate the α(2)β(1) targeted probe. In vitro immunofluorescence staining was carried out to confirm the α(2)β(1) integrin expression level. Flow cytometry analysis showed that PC-3 had the highest probe uptake, followed by CWR-22 and LNCaP tumor cells. In the subcutaneous PC-3 model, the tumor demonstrated prominent uptake with good tumor to background contrast. Immunohistochemistry staining also supported the in vivo optical imaging results. DGEA-based optical agents have been developed for specific imaging of α(2)β(1) integrin expression. In vitro and in vivo localization demonstrated the potential of this agent to identify tumor subtypes amenable to anti-α(2)β(1) integrin treatment and potentially provide prognostic information regarding tumor progression.     

多模态分子影像对肝癌进展和血管生成的研究

通过对HCCLM3-fLuc-GFP肝癌的进展和血管生成的多模态成像分析,对肝癌细胞在体内生长的适应过程和级数生长特性,分别进行了生物自发荧光成像(bioluminescent imaging,BLI)研究、激发荧光成像(fluorescent imaging,FMI)研究和小动物计算机断层成像(micro-compu...     

Functional in vivo imaging of cysteine cathepsin activity in murine model of inflammation

Near-infrared fluorophore (NIRF)-labeled imaging probes are becoming increasingly important in bio-molecular imaging applications, that is, in animal models for tumor imaging or inflammation studies. In this study we showed that the previously introduced chemical concept of ‘Reverse Design’ represents an efficient strategy for the generation of selective probes for cysteine proteases from chemically optimized protease inhibitors for investigations in proteomic lysates as well as for in vivo molecular imaging studies. The newly developed activity-based probe AW-091 was demonstrated to be highly selective for cathepsin S in vitro and proved useful in monitoring cysteine cathepsin activity in vivo, that is, in zymosan-induced mouse model of inflammation. AW-091 showed higher signal-to-background ratios at earlier time points than the commercially available polymer-based ProSense680 (VisEn Medical) and thus represents an efficient new tool for studying early proteolytic processes leading to various diseases, including inflammation, cancer, and rheumatoid arthritis. In addition, the fluorescent signal originating from the cleaved AW-091 was shown to be reduced by the administration of an anti-inflammatory drug, dexamethasone and by the cathepsin inhibitor E-64, providing a valuable system for the evaluation of small-molecule inhibitors of cathepsins.     

Bioluminescence and magnetic resonance imaging of macrophage homing to experimental abdominal aortic aneurysms

Macrophage infiltration is a prominent feature of abdominal aortic aneurysm (AAA) progression. We used a combined imaging approach with bioluminescence (BLI) and magnetic resonance imaging (MRI) to study macrophage homing and accumulation in experimental AAA disease. Murine AAAs were created via intra-aortic infusion of porcine pancreatic elastase. Mice were imaged over 14 days after injection of prepared peritoneal macrophages. For BLI, macrophages were from transgenic mice expressing luciferase. For MRI, macrophages were labeled with iron oxide particles. Macrophage accumulation during aneurysm progression was observed by in situ BLI and by in vivo 7T MRI. Mice were sacrificed after imaging for histologic analysis. In situ BLI (n = 32) demonstrated high signal in the AAA by days 7 and 14, which correlated significantly with macrophage number and aortic diameter. In vivo 7T MRI (n = 13) at day 14 demonstrated T?* signal loss in the AAA and not in sham mice. Immunohistochemistry and Prussian blue staining confirmed the presence of injected macrophages in the AAA. BLI and MRI provide complementary approaches to track macrophage homing and accumulation in experimental AAAs. Similar dual imaging strategies may aid the study of AAA biology and the evaluation of novel therapies.     

Activatable fluorescent probes in fluorescence-guided surgery: Practical considerations

Fluorescence-guided imaging during surgery is a promising technique that is increasingly used to aid surgeons in identifying sites of tumor and surgical margins. Of the two types of fluorescent probes, always-on and activatable, activatable probes are preferred because they produce higher target-to-background ratios, thus improving sensitivity compared with always-on probes that must contend with considerable background signal. There are two types of activatable probes: 1) enzyme-reactive probes that are normally quenched but can be activated after cleavage by cancer-specific enzymes (activity-based probes) and 2) molecular-binding probes which use cancer targeting moieties such as monoclonal antibodies to target receptors found in abundance on cancers and are activated after internalization and lysosomal processing (binding-based probes). For fluorescence-guided intraoperative surgery, enzyme-reactive probes are superior because they can react quickly, require smaller dosages especially for topical applications, have limited side effects, and have favorable pharmacokinetics. Enzyme-reactive probes are easier to use, fit better into existing work flows in the operating room and have minimal toxicity. Although difficult to prove, it is assumed that the guidance provided to surgeons by these probes results in more effective surgeries with better outcomes for patients. In this review, we compare these two types of activatable fluorescent probes for their ease of use and efficacy.     

Effects of a Non Thermal Plasma Treatment Alone or in Combination with Gemcitabine in a MIA PaCa2-luc Orthotopic Pancreatic Carcinoma Model

Pancreatic tumors are the gastrointestinal cancer with the worst prognosis in humans and with a survival rate of 5% at 5 years. Nowadays, no chemotherapy has demonstrated efficacy in terms of survival for this cancer. Previous study focused on the development of a new therapy by non thermal plasma showed significant effects on tumor growth for colorectal carcinoma and glioblastoma. To allow targeted treatment, a fibered plasma (Plasma Gun) was developed and its evaluation was performed on an orthotopic mouse model of human pancreatic carcinoma using a MIA PaCa2-luc bioluminescent cell line. The aim of this study was to characterize this pancreatic carcinoma model and to determine the effects of Plasma Gun alone or in combination with gemcitabine. During a 36 days period, quantitative BLI could be used to follow the tumor progression and we demonstrated that plasma gun induced an inhibition of MIA PaCa2-luc cells proliferation in vitro and in vivo and that this effect could be improved by association with gemcitabine possibly thanks to its radiosensitizing properties.     

Thermoacoustic molecular imaging of small animals

We have designed, constructed, and tested a thermoacoustic computed tomography (TCT) scanner for imaging optical absorption in small animals in three dimensions. The device utilizes pulsed laser irradiation (680-1064 nm) and a unique, 128-element transducer array. We quantified the isotropic spatial resolution of this scanner to be 0.35 mm. We describe a dual-wavelength subtraction technique for isolating optical dyes with TCT. Phantom experiments demonstrate that we can detect 5 fmol of a near-infrared dye (indocyanine green, ICG) in a 1-microL volume using dual-wavelength subtraction. Initial TCT imaging in phantoms and in two sacrificed mice suggests that three-dimensional, optical absorption patterns in small animals can be detected with an order of magnitude better spatial resolution and an order of magnitude better low-contrast detectability in small animals when compared to fluorescence imaging or diffusion optical tomography.     

Comprehensive Evaluation of the Anti-Angiogenic and Anti-Neoplastic Effects of Endostar on Liver Cancer through Optical Molecular Imaging

Molecular imaging enables non-invasive monitoring of tumor growth, progression, and drug treatment response, and it has become an important tool to promote biological studies in recent years. In this study, we comprehensively evaluated the in vivo anti-angiogenic and anti-neoplastic effects of Endostar on liver cancer based on the optical molecular imaging systems including micro-computer tomography (Micro-CT), bioluminescence molecular imaging (BLI) and fluorescence molecular tomography (FMT). Firefly luciferase (fLuc) and green fluorescent protein (GFP) dual labeled human hepatocellular carcinoma cells (HCC-LM3-fLuc-GFP cells) were used to establish the subcutaneous and orthotopic liver tumor model. After the tumor cells were implanted 14∼18 days, Endostar (5 mg/kg/day) was administered through an intravenous tail vein injection for continuous 14 days. The computer tomography angiography (CTA) and BLI were carried out for the subcutaneous tumor model. FMT was executed for the orthotopic tumor model. The CTA data showed that tumor vessel formation and the peritumoral vasculature of subcutaneous tumor in the Endostar treatment group was significantly inhibited compared to the control group. The BLI data exhibited the obvious tumor inhibition day 8 post-treatment. The FMT detected the tumor suppression effects of Endostar as early as day 4 post-treatment and measured the tumor location. The above data confirmed the effects of Endostar on anti-angiogenesis and tumor suppression on liver cancer. Our system combined CTA, BLI, and FMT to offer more comprehensive information about the effects of Endostar on the suppression of vessel and tumor formation. Optical molecular imaging system enabled the non-invasive and reliable assessment of anti-tumor drug efficacy on liver cancer.     

Purification method directly influences effectiveness of an epidermal growth factor-coupled targeting agent for noninvasive tumor detection in mice

Receptor targeting is an effective method of enhancing fluorescence signal in tumors for optical imaging. We previously used epidermal growth factor (EGF) conjugated to IRDye 800CW to detect and track orthotopic prostate tumors in mice. In this study, our goal was to identify a reliable assay for targeting agent integrity in vitro that correlated with signal strength in vivo. Binding of IRDye 800CW EGF to intact A431 human epidermoid carcinoma cells was quantified in a microplate assay. Specificity was confirmed by competition with unlabeled EGF or monoclonal antibody blocking. Biological activity of intact and damaged targeting agents relative to unlabeled EGF was determined by binding and stimulation of extracellular signal-regulated kinase (ERK) phosphorylation. Both assays indicated a reduction of up to 60% of the fluorescence intensity with damaged agents. Using a research prototype imaging system optimized for IRDye 800CW detection, we compared the efficacy of intact and damaged targeting agents for imaging subcutaneous tumors in mice. In live animal images and in sections of the excised tumors, damaged targeting agents consistently yielded diminished fluorescence signals corresponding to the reduction observed in microplate assays. This is the first study to directly correlate targeting agent signal strength in whole cell binding, In-Cell Western, and in vivo near-infrared imaging.     

In vivo imaging of siRNA delivery and silencing in tumors

With the increased potential of RNA interference (RNAi) as a therapeutic strategy, new noninvasive methods for detection of siRNA delivery and silencing are urgently needed. Here we describe the development of dual-purpose probes for in vivo transfer of siRNA and the simultaneous imaging of its accumulation in tumors by high-resolution magnetic resonance imaging (MRI) and near-infrared in vivo optical imaging (NIRF). These probes consisted of magnetic nanoparticles labeled with a near-infrared dye and covalently linked to siRNA molecules specific for model or therapeutic targets. Additionally, these nanoparticles were modified with a membrane translocation peptide for intracellular delivery. We show the feasibility of in vivo tracking of tumor uptake of these probes by MRI and optical imaging in two separate tumor models. We also used proof-of-principle optical imaging to corroborate the efficiency of the silencing process. These studies represent the first step toward the advancement of siRNA delivery and imaging strategies, essential for cancer therapeutic product development and optimization.     

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.