Use of Stainless-Steel, Balloon-Expandable Chimney Grafts Is Durable Though Caution Is Required When Lining Angulated Renal Arteries.

PURPOSE: To analyze the overall performance of flexible nitinol stents used to line chimney grafts (CGs) during chimney endovascular aneurysm repair (chEVAR) of pararenal pathologies. MATERIALS AND METHODS: A retrospective review was conducted of all 116 elective patients (mean age 74.3±7.2 years; 103 men) who underwent chEVAR with balloon-expandable Advanta V12/iCAST CGs in combination with the Endurant stent-graft between January 2009 and December 2017 at a single center. CG lining with a nitinol stent was electively performed in 43 target vessels of 32 patients. The Kaplan-Meier method was used to estimate the primary outcomes of CG patency and freedom from reintervention (FFR) at the patient level and according to the use of a stent to line the CG. Estimates are reported with the 95% confidence interval (CI). Adjusted odds ratios (ORs) were calculated to identify any confounding effect between the presence/absence of a stent lining or according to the number of CGs. RESULTS: The mean radiological follow-up was 27.3 months (range 22.1-32.6). During this time, 8 CGs (4.7%) became occluded, 6 of them were lined with stents. Restoration of patency was possible in 3 of the 4 occluded stents that were associated with symptoms. First-year primary patency estimates were 96.9% (95% CI 92.5% to 100%) for the unlined group vs 77.1% (95% CI 58% to 95.3%; p=0.001) for the lined group, while FFR was 87.6% (95% CI 79.9% to 95.2%) vs 83.4% (95% CI 68.1% to 98.6%; p=0.82), respectively. Lining represented an independent risk factor for CG occlusion (OR 9.9, p=0.006). CONCLUSION: CG lining performed mainly in angulated renal arteries during chEVAR was significantly associated with CG occlusion. These findings highlight the importance of not having the distal part of the CG impinge on the angulated segment of the target vessel.

Fluorescence Imaging of Breast Tumors and Gastrointestinal Cancer.

Optical imaging offers a high potential for noninvasive detection and therapy of cancer in humans. Recent advances in instrumentation for diffuse optical imaging have led to new capabilities for the detection of cancer in highly scattering tissue such as the female breast. In particular, fluorescence imaging was made applicable as a sensitive technique to image molecular probes in vivo. We review recent developments in the detection of breast cancer and fluorescence-guided surgery of the breast by contrast agents available for application on humans. Detection of cancer has been investigated with the unspecific contrast agents "indocyanine green" and "omocianine" so far. Hereby, indocyanine green was found to offer high potential for the differentiation of malignant and benign lesions by exploiting vessel permeability for macromolecules as a cancer-specific feature. Tumor-specific molecular targeting and activatable probes have been investigated in clinical trials for fluorescence-guided tumor margin detection. In this application, high spatial resolution can be achieved, since tumor regions are visualized mainly at the tissue surface. As another example of superficial tumor tissue, imaging of lesions in the gastrointestinal tract is discussed. Promising results have been obtained on high-risk patients with Barrett´s esophagus and with ulcerative colitis by administering 5-aminolevulinic acid which induces accumulation of protoporphyrin IX serving as a tumor-specific fluorescent marker. Time-gated fluorescence imaging and spectroscopy are effective ways to suppress underlying background from tissue autofluorescence. Furthermore, recently developed tumor-specific molecular probes have been demonstrated to be superior to white-light endoscopy offering new ways for early detection of malignancies in the gastrointestinal tract.

Target-Specific Imaging of Cathepsin and S100A8/A9 Reflects Specific Features of Malignancy and Enables Estimation of Tumor Malignancy.

PURPOSE: Tumor development and metastasis are dependent on tumor infiltrating immune cells which form a characteristic tumor microenvironment (TME). Activated monocytes secrete the protein heterodimer S100A8/A9 promoting TME formation. Monocyte-dependent proteases facilitate local tumor cell invasion by degradation of the extracellular matrix. We aimed for target specific in vivo imaging of S100A8 and proteases to provide differentiating biomarkers for local tumor growth and metastatic potential. PROCEDURES: Murine breast cancer cells of the 4T1 model with graduated metastatic potential (4T1 and 4T07: both hematogenous metastasis > 168FAR: lymph-node metastasis > 67NR: no metastasis) were orthotopically implanted into female BALB/c mice. At 4 mm size, tumors were investigated by injecting the protease-specific probe ProSense 750EX (PerkinElmer, 4T1 n = 7, 4T07 n = 10, 168FAR n = 16, 67NR n = 15) and anti-S100A8-Cy5.5 (n = 6 each) and performing fluorescence reflectance imaging at 0 and 24 h after injection. In vivo imaging was validated with immunohistochemistry. RESULTS: At 24 h, S100A8-specific signals in 4T1 and 4T07 were significantly higher (1714.05/1683.45 AU) as compared to 168FAR and 67NR (174.85/167.95 AU, p = 0.0012/p = 0.0003), reflecting the capability of hematogenous spread. Protease-specific signals were significantly higher in 4T1 and 4T07 (348.01/409.93 AU) as compared to 168FAR (214.91 AU) and 67NR (129.78 AU p < 0.0001 each), reflecting local vessel invasion and tumor cell shedding. Immunohistology supported the in vivo imaging results. CONCLUSIONS: Non-invasive in vivo imaging of S100A8 and monocytic proteases allows for differentiation of the tumors' local invasive and systemic metastatic potential in reflecting the TME formation. While proteases augment local tumor cell invasion, solid metastases seem to be dependent on a pro-tumoral microenvironment.

Gadofosveset-enhanced MRI as simple surrogate parameter for real-time evaluation of the initial tumour vessel infarction by retargeted tissue factor tTF-NGR.

Truncated tissue factor (tTF)-NGR consists of the extracellular domain of the human TF and the binding motif NGR. tTF-NGR activates blood coagulation within the tumour vasculature following binding to CD13, and is overexpressed in the endothelial cells of tumour vessels, resulting in tumour vessel infarction and subsequent retardation/regression of tumour growth. The aim of the present study was to investigate gadofosveset-based real-time dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in evaluating the initial therapeutic effects of the anti-vascular tTF-NGR approach. DCE-MRI (3.0 T) was performed in human U87-glioblastoma tumour-bearing nude mice. During a dynamic T1w GE-sequence, a gadolinium-based blood pool contrast agent (gadofosveset) was injected via a tail vein catheter. Following the maximum contrast intensity inside the tumour being obtained, tTF-NGR was injected (controls received NaCl) and the contrast behaviour of the tumour was monitored by ROI analysis. The slope difference of signal intensities between controls and the tTF-NGR group was investigated, as well as the differences between the average area under the curve (AUC) of the two groups. The association between intensity, group (control vs. tTF-NGR group) and time was analysed by fitting a linear mixed model. Following the injection of tTF-NGR, the signal intensity inside the tumours exhibited a statistically significantly stronger average slope decrease compared with the signal intensity of the tumours in the NaCl group. Furthermore, the initial average AUC values of mice treated with tTF-NGR were 5.7% lower than the average AUC of the control animals (P<0.05). Gadofosveset-enhanced MRI enables the visualization of the initial tumour response to anti-vascular treatment in real-time. Considering the clinical application of tTF-NGR, this method may provide a simple alternative parameter for monitoring the tumour response to vascular disrupting agents and certain vascular targeting agents in humans.

Structural cartilage damage attracts circulating rheumatoid arthritis synovial fibroblasts into affected joints.

BACKGROUND: Rheumatoid arthritis synovial fibroblasts (RASFs) are known to travel via the bloodstream from sites of cartilage destruction to new locations where they reinitiate the destructive processes at distant articular cartilage surfaces. In this study, we examined the role of interleukin (IL)-1-induced cartilage changes and their chemotactic effect on RASF transmigratory capacity. METHODS: To investigate synovial fibroblast (SF) transmigration through endothelial layers, we used a modified Boyden chamber with an endothelioma cell layer (bEnd.5) as a barrier and IL-1-treated murine cartilage explants as a chemotactic stimulus for SFs from human tumor necrosis factor-transgenic (hTNFtg) mice. We injected recombinant IL-1 or collagenase into knee joints of wild-type mice, followed by tail vein injection of fluorescence-labeled hTNFtg SFs. The distribution and intensity of transmigrating hTNFtg SFs were measured by fluorescence reflectance imaging with X-ray coregistration. Toluidine blue staining was performed to evaluate the amount of cartilage destruction. RESULTS: Histomorphometric analyses and in vivo imaging revealed a high degree of cartilage proteoglycan loss after intra-articular IL-1 and collagenase injection, accompanied by an enhanced in vivo extravasation of hTNFtg SFs into the respective knee joints, suggesting that structural cartilage damage contributes significantly to the attraction of hTNFtg SFs into these joints. In vitro results showed that degraded cartilage was directly responsible for the enhanced transmigratory capacity because stimulation with IL-1-treated cartilage, but not with IL-1 or cartilage alone, was required to increase hTNFtg SF migration. CONCLUSIONS: The present data indicate that structural cartilage damage facilitates the migration of arthritic SF into affected joints. The prevention of early inflammatory cartilage damage may therefore help prevent the progression of rheumatoid arthritis and its spread to previously unaffected joints.

Optical in vivo imaging of the alarmin S100A9 in tumor lesions allows for estimation of the individual malignant potential by evaluation of tumor-host cell interaction.

UNLABELLED: Tumors recruit and reprogram immune cells to support tumor development and spread, the most prominent among them being of monocytic origin such as tumor-associated macrophages (TAM) or myeloid-derived suppressor cells (MDSC). The alarmin S100A8/A9 has been implicated in the induction of TAM and MDSC. We assessed S100A9 as a molecular imaging marker for the activity of tumor-associated immune cells in a syngeneic murine breast cancer model. S100A9 could serve as a surrogate marker for tumor immune crosstalk as a function of malignancy, providing a tool with the potential for both basic research in tumor immunology and clinical stratification of patients. METHODS: BALB/c mice were inoculated with murine breast cancer cells of common origin but different metastatic capability. At different times during tumor development, optical imaging was performed using a S100A9-specific probe to visualize activated monocytes. To further explore the impact of tumor-educated monocytes, splenic myeloid cells were isolated from either healthy or tumor-bearing animals and injected into tumor-bearing mice. We analyzed the effect of the cell transfer on immune cell activity and tumor development. RESULTS: We could prove S100A9-driven imaging to sensitively and specifically reflect monocyte activity in primary tumor lesions. The imaging results were corroborated by histology and fluorescence-activated cell sorting analyses. In a prospective experiment, S100A9 imaging proved indicative of the individual tumor growth, with excellent correlation. Moreover, we could show that the monocyte activity as depicted by S100A9 activity in the primary tumor lesion mirrored the tumor's metastatic behavior. Treatment with tumor-primed splenic monocytes induced increased tumor growth, accompanied by an augmented infiltration of activated myeloid cells (MDSC and TAM) into the tumor. The consecutive S100A9 expression as depicted by in vivo imaging was significantly increased. CONCLUSION: S100A9 proved to be a sensitive and specific marker for the activity of tumor-associated immune cells. To our knowledge, S100A9 imaging represents a first in vivo imaging approach for the estimation of recruitment and activity of tumor-associated myeloid immune cells. We demonstrated the potential value of this imaging approach for prediction of local and systemic tumor development.

In vivo monitoring of angiogenesis during tendon repair: a novel MRI-based technique in a rat patellar tendon model.

PURPOSE: Recent in vivo studies were able to show the impairing effect of neoangiogenesis in degenerative tendon diseases. Clinical in vivo monitoring of angiogenesis in injured tendons therefore seems to be crucial for an accurate therapeutic approach. The aim of this study was to develop a novel magnetic resonance imaging (MRI)-based technique for observing angiogenesis during tendon healing in vivo. METHODS: Tendinopathy was induced by an in situ freezing model of rat patellar tendon and monitored after 7, 14, and 28 days. Animals were randomly divided into an imaging and immunohistochemical group. MRI with a 'blood pool' contrast agent was used to determine neoangiogenesis during tendon healing. MRI was compared to histochemical staining and quantification of blood vessels in injured and native tendons. RESULTS: MRI data revealed a peak in changes in the transverse relaxation rate (ΔR 2*), which is proportional to relative blood volume, 7 days after surgery and decrease until day 28. Histological microvessel density and vascular endothelial growth factor synthesis were also most evident at day 7 and decreased over time. CONCLUSIONS: The current results are demonstrating a time-dependent correlation between microvessel density and ΔR 2*. Thus, MRI-based evaluation of angiogenesis in the tendon might be a new promising technique for in vivo monitoring of angiogenesis and therapy response in the future.

Fluorescent non-peptidic RGD mimetics with high selectivity for αVß3 vs αIIbß3 integrin receptor: novel probes for in vivo optical imaging.

Integrins are heterodimeric transmembrane protein receptors consisting of different α and ß subunits. α(v)ß(3) integrins are overexpressed on many tumor cells and tumor-associated angiogenic vessels, whereas α(IIb)ß(3) is a receptor for, e.g., fibrinogen and mediates platelet aggregation. In this study, a near-infrared fluorescent imaging probe has been designed and synthesized by conjugating fluorescent dyes to a non-peptidic, pharmacophore-based ligand, based on a molecular modeling design approach. Affinity values were determined, and in vitro cell binding assays and preliminary in vivo xenograft studies in nude mice were performed to evaluate target binding. Competition assays revealed excellent binding and selectivity to α(v)ß(3) compared to that for α(IIb)ß(3). In vitro, the probe showed high target binding on α(v)ß(3)-positive M-21 cells and negligible binding to α(v)ß(3)-negative MCF-7 cells. In vivo, the tracer is able to image target expression in U-87 xenografts with a maximum signal-to-noise ratio (SNR) of 2.5:1 at 24 h after injection.

Alarmin S100A8/S100A9 as a biomarker for molecular imaging of local inflammatory activity.

Inflammation has a key role in the pathogenesis of various human diseases. The early detection, localization and monitoring of inflammation are crucial for tailoring individual therapies. However, reliable biomarkers to detect local inflammatory activities and to predict disease outcome are still missing. Alarmins, which are locally released during cellular stress, are early amplifiers of inflammation. Here, using optical molecular imaging, we demonstrate that the alarmin S100A8/S100A9 serves as a sensitive local and systemic marker for the detection of even sub-clinical disease activity in inflammatory and immunological processes like irritative and allergic contact dermatitis. In a model of collagen-induced arthritis, we use S100A8/S100A9 imaging to predict the development of disease activity. Furthermore, S100A8/S100A9 can act as a very early and sensitive biomarker in experimental leishmaniasis for phagocyte activation linked to an effective Th1-response. In conclusion, the alarmin S100A8/S100A9 is a valuable and sensitive molecular target for novel imaging approaches to monitor clinically relevant inflammatory disorders on a molecular level.

Optimizing the bioavailability of small molecular optical imaging probes by conjugation to an albumin affinity tag.

Small molecular imaging probes are often found to be rapidly cleared from the circulation. In order to improve signal to noise ratio (SNR) by high probe accumulation in the target tissue we intended to prolong the presence of the probes in the circulation by exploiting inherent transport mechanisms. Human serum albumin (HSA) is playing an increasingly important role as a drug carrier in clinical settings and drugs directly bound to albumin or attached to albumin binding moieties have been successfully developed for treatment approaches. To optimize the bioavailability of existing fluorescent probes, a hydrophobic affinity tag is installed, which enhances albumin binding. In a first experiment an endothelin-A receptor (ETAR) probe is modified by inserting a trivalent linker, attaching an albumin affinity tag and labeling the conjugate with the fluorescent dye Cy 5.5. The spectroscopic properties of the conjugate are examined by photometer- and fluorometer measurements in comparison to a probe without albumin binding tag. Albumin binding was proven by agarose gel electrophoresis. The affinity towards ETAR was confirmed in vitro by cell binding assays on human fibrosarcoma cells (HT-1080) and in vivo by murine xenograft imaging studies. In vitro, the modified probe retains high target binding in the absence and presence of albumin. Binding could be blocked by predosing with ETAR antagonist atrasentan, proving specificity. The in vivo examinations in comparison to the established probe showed a reduced renal elimination and a prolonged circulation of the tracer resulting in significantly higher signal intensity (SI) at the target and a higher signal-to-noise ratio (SNR) between 3h and 96 h after injection. In summary, we designed a small molecular, non-peptidic fluorescent probe which targets ETAR and reversibly binds to serum albumins. The reversible binding to albumin enhances the biological half-life of the probe substantially and enables near infrared optical imaging of subcutaneous tumors for several days. This approach of reversibly attaching probes to serum albumin may serve as a tool to optimize tracer distribution for more precise target characterization in molecular imaging experiments.

Highly shifted proton MR imaging: cell tracking by using direct detection of paramagnetic compounds.

PURPOSE: To explore the feasibility of tracking thulium (Tm)-1,4,7,10-tetraazacyclododecane-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraacetic acid (DOTMA)-labeled cells in vivo by means of highly shifted proton magnetic resonance (MR) imaging as a potential alternative to established cell-tracking methods. MATERIALS AND METHODS: All animal experiments were approved by the local ethics committee for animal experiments. Highly shifted proton MR imaging is based on the principle that the shifted resonances on Tm and dysprosium (Dy)-DOTMA can be detected separately from the tissue water signal at MR imaging with very short echo time and radial center-out readout (UTE, or "ultrashort echo time"). MR imaging of aqueous solutions and in mice in vivo was performed at 9.4 T. Human fibrosarcoma cells (HT-1080) and murine macrophages were labeled with different amounts of Tm-DOTMA. Labeled fibrosarcoma cells were injected subcutaneously into three mice. For cell tracking, labeled macrophages were administered intravenously into eight mice bearing local granulomatous inflammation. Three-dimensional UTE MR imaging was performed during 1 week. Macrophage viability and activity and fibrosarcoma cell viability were statistically analyzed by performing an unpaired two-tailed t test for labeled versus unlabeled cells by using data of at least six independent experiments. RESULTS: The strongly shifted MR lines of Tm- and Dy-DOTMA can be separated from the tissue water signal and from each other. A detection limit of about 25 µmol/L of Tm-DOTMA was calculated from in vitro MR measurements. A mean ± standard error of the mean intracellular uptake of (4.19 ± 0.88) × 10(9) (HT-1080) and (10.1 ± 3.0) × 10(10) (macrophages) of Tm-DOTMA molecules per cell was achieved. In vivo, Tm-DOTMA signal was detectable for 1 week in both tumors and macrophages, with a detection limit of approximately 10(4) HT-1080 and 600 macrophages. Histologic examination results and elemental bioimaging confirmed labeled cells as source of MR signal. CONCLUSION: Strongly shifted proton three-dimensional UTE MR imaging of Tm-DOTMA-labeled cells is a highly specific and sensitive tool for in vivo cell tracking.

Non-invasive monitoring of tumor-vessel infarction by retargeted truncated tissue factor tTF-NGR using multi-modal imaging.

The fusion protein tTF-NGR consists of the extracellular domain of the thrombogenic human tissue factor (truncated tissue factor, tTF) and the peptide GNGRAHA (NGR), a ligand of the surface protein CD13 (aminopeptidase N), upregulated on endothelial cells of tumor vessels. tTF-NGR preferentially activates blood coagulation within tumor vasculature, resulting in tumor vessel infarction and subsequent tumor growth retardation/regression. The anti-vascular mechanism of the tTF-NGR therapy approach was verified by quantifying the reduced tumor blood-perfusion with contrast-enhanced ultrasound, the reduced relative tumor blood volume by ultrasmall superparamagnetic iron oxide-enhanced magnetic resonance imaging, and by in vivo-evaluation of hemorrhagic bleeding with fluorescent biomarkers (AngioSense(680)) in fluorescence reflectance imaging. The accumulation of tTF-NGR within the tumor was proven by visualizing the distribution of the iodine-123-labelled protein by single-photon emission computed tomography. Use of these multi-modal vascular and molecular imaging tools helped to assess the therapeutic effect even at real time and to detect non-responding tumors directly after the first tTF-NGR treatment. This emphasizes the importance of imaging within clinical studies with tTF-NGR. The imaging techniques as used here have applicability within a wider scope of therapeutic regimes interfering with tumor vasculature. Some even are useful to obtain predictive biosignals in personalized cancer treatment.

Vessel size imaging (VSI) by robust magnetic resonance (MR) relaxometry: MR-VSI of solid tumors in correlation with immunohistology and intravital microscopy.

The aim of this study was to evaluate a robust magnetic resonance (MR) vessel size imaging (VSI) method for the noninvasive assessment of mean vessel size in solid tumors in a clinical dose range of ultrasmall superparamagnetic particles of iron oxide (USPIO). Therefore, USPIO-enhanced MR-VSI was performed on DU-4475, MDA-MB-435, and EOMA tumor-bearing mice xenografts with known differences in angiogenic activity and vessel morphology. MR results were compared to vessel sizes determined by immunohistochemistry (anti-CD31) and by intravital microscopy (IVM). MR-VSI revealed significantly different mean vessel sizes between the xenograft models at both USPIO doses (DU-4475: 20.6 ± 4.9 µm; MDA-MB-435: 37.4 ± 8.8 µm; and EOMA: 60.3 ±9.6 µm at 80 µmol/kg; p < .05). Immunohistochemistry revealed lower values for all tumor entities, whereas the size distribution was in line with MR-measurements. IVM corroborated the MR results for DU-4475 and MDA-MB435, but showed similar vessel sizes for MDA-MB-435 and EOMA. Our MR-VSI method allowed a noninvasive estimation of the mean vessel size in mice xenograft solid tumors with variable vascularity using a clinically relevant USPIO dose range.

Design and synthesis of small-molecule fluorescent photoprobes targeted to aminopeptdase N (APN/CD13) for optical imaging of angiogenesis.

We report here the synthesis of a nonpeptide, small-molecule fluorescent imaging agent with high affinity to aminopeptidase N (APN/CD13), a key player in a variety of pathophysiological angiogenic processes. On the basis of a recently described lead structure, we synthesized three putative precursor compounds by introducing polyethylene glycol (PEG) spacers comprising amino groups for dye labeling. Different attachment sites resulted in substantial differences in target affinity, cell toxicity, and target imaging performance. In comparison to bestatin, a natural inhibitor of many aminopeptidases, two of our compounds (22, 23) exhibit comparable inhibition potency, while a third (21) does not show any inhibiting effect. Cell binding assays with APN-positive BT-549 and APN-negative BT-20 cells and the final fluorescent probes Cy 5.5-21 and Cy 5.5-23 confirm these findings. The favorable characteristics of Cy 5.5-23 will now be proven in in vivo experiments with murine models of high APN expression and may serve as a tool to better understand APN pathophysiology.

In vivo fluorescence-mediated tomography imaging demonstrates atorvastatin-mediated reduction of lesion macrophages in ApoE-/- mice.

BACKGROUND: Macrophage recruitment into atherosclerotic plaques drives lesion progression, destabilization, and rupture. Chronic statin treatment reduces macrophage plaque content. Information on dynamics of macrophage recruitment would help assessing plaque vulnerability and guiding therapy. Techniques to image macrophage homing to vulnerable plaques in vivo are scarcely available. The authors tested if noninvasive fluorescence-mediated tomography (FMT) can assess plaque-stabilizing effects of short-term high-dosage atorvastatin. METHODS: Macrophages from green-fluorescent-protein-transgenic mice were labeled with a near-infrared fluorescent dye and were injected IV in apolipoprotein E-deficient mice (n=9) on Western diet 7 days after guidewire-injury of the carotid artery. FMT-scans, 2 and 7 days thereafter, quantified macrophage recruitment into carotid artery plaques. Atorvastatin was tested for macrophage adhesion, proliferation, and viability (n=5 to 6) in vitro. Fourteen mice received atorvastatin or vehicle for 4 days after 16 weeks on Western diet. FMT assessed macrophage recruitment into aortic and innominate artery lesions. Means (±SD)% are reported. RESULTS: Double-labeled macrophages were recruited into carotid artery lesions. FMT resolved fluorescence projecting on the injured carotid artery and detected a signal increase to 300% (±191) after guidewire injury. Atorvastatin reduced macrophage adhesion to activated endothelial cells by 36% (±19). In a clinically relevant proof-of-concept intervention, FMT-imaging detected that 4 days atorvastatin treatment reduced macrophage recruitment by 57% (±8) indicating plaque stabilization. Immunohistochemistry confirmed reduced macrophage infiltration. CONCLUSIONS: FMT optical imaging proved its high potential for clinical applicability for tracking recruitment of near-infrared fluorescent-labeled macrophages to vulnerable plaques in vivo. FMT-based quantification of macrophage recruitment demonstrated rapid plaque stabilization by 4-day atorvastatin treatment in apolipoprotein E-deficient mice.

Claudin-4-targeted optical imaging detects pancreatic cancer and its precursor lesions.

OBJECTIVES: Novel imaging methods based on specific molecular targets to detect both established neoplasms and their precursor lesions are highly desirable in cancer medicine. Previously, we identified claudin-4, an integral constituent of tight junctions, as highly expressed in various gastrointestinal tumours including pancreatic cancer. Here, we investigate the potential of targeting claudin-4 with a naturally occurring ligand to visualise pancreatic cancer and its precursor lesions in vitro and in vivo by near-infrared imaging approaches. DESIGN: A non-toxic C-terminal fragment of the claudin-4 ligand Clostridium perfringens enterotoxin (C-CPE) was labelled with a cyanine dye (Cy5.5). Binding of the optical tracer was analysed on claudin-4 positive and negative cells in vitro, and tumour xenografts in vivo. In addition, two genetically engineered mouse models for pancreatic intraepithelial neoplasia (PanIN) and pancreatic cancer were used for in vivo validation. Optical imaging studies were conducted using 2D planar fluorescence reflectance imaging (FRI) technology and 3D fluorescence-mediated tomography (FMT). RESULTS: In vitro, the peptide-dye conjugate showed high binding affinity to claudin-4 positive CAPAN1 cells, while claudin-4 negative HT1080 cells revealed little or no fluorescence. In vivo, claudin-4 positive tumour xenografts, endogenous pancreatic tumours, hepatic metastases, as well as preinvasive PanIN lesions, were visualised by FRI and FMT up to 48 h after injection showing a significantly higher average of fluorochrome concentration as compared with claudin-4 negative xenografts and normal pancreatic tissue. CONCLUSIONS: C-CPE-Cy5.5 combined with novel optical imaging methods enables non-invasive visualisation of claudin-4 positive murine pancreatic tumours and their precursor lesions, representing a promising modality for early diagnostic imaging.

Performance of a new fluorescence-labeled MMP inhibitor to image tumor MMP activity in vivo in comparison to an MMP-activatable probe.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade structural components of the extracellular matrix and participate in pathologies such as cancer and inflammatory disorders. The development of novel contrast agents for optical imaging of MMP activity in vivo is of great interest. The commonly used near-infrared fluorescence-compatible agents are dye-quenched probes that do not emit fluorescence until they interact with MMPs. In contrast, fluorescent synthetic low-molecular-weight MMP inhibitors have not been systematically employed. The aim of this study was to evaluate the performance of our recently developed Cy5.5-labeled MMP inhibitor to image MMP activity in tumors in vivo compared with activatable fluorescent MMP-sensing probes. The dynamic uptake of Cy5.5-AF489 into four different tumor entities was analyzed in xenografted mice by intravenous injection and subsequent fluorescence reflectance imaging. Tumors were characterized in regard to their MMP-2 and -9 mRNA expressions (qRT-PCR analysis), proteins (immunohistochemistry) and gelatinase/collagenase activities (in situ zymography). Cy5.5-AF489 was compared with MMPSense™ 680 and MMPSense™ 750 FAST, two commercially available MMP-activatable probes. Cy5.5-AF489 showed a specific tumor uptake, which was blocked by pre-injection of the unlabeled MMPI, and discriminated between tumors with high or low MMP-2/-9 expressions. Our optical probe facilitated faster visualization of MMP-active tumors accompanied by excellent tumor-to-background ratios when compared with activatable probes. The MMP inhibitor Cy5.5-AF489 permits fast in vivo imaging of MMP expression/activity in tumors. Given its small molecular weight and non-peptidic structure, translational imaging from a preclinical application to a diagnostic tool for MMP-related diseases seems feasible.

Labeling of anti-MUC-1 binding single chain Fv fragments to surface modified upconversion nanoparticles for an initial in vivo molecular imaging proof of principle approach.

In vivo optical Imaging is an inexpensive and highly sensitive modality to investigate and follow up diseases like breast cancer. However, fluorescence labels and specific tracers are still works in progress to bring this promising modality into the clinical day-to-day use. In this study an anti-MUC-1 binding single-chain antibody fragment was screened, produced and afterwards labeled with newly designed and surface modified NaYF(4):Yb,Er upconversion nanoparticles as fluorescence reporter constructs. The MUC-1 binding of the conjugate was examined in vitro and in vivo using modified state-of-the-art small animal Imaging equipment. Binding of the newly generated upconversion nanoparticle based probe to MUC-1 positive cells was clearly shown via laser scanning microscopy and in an initial proof of principal small animal optical imaging approach.

Assessment of endothelin-A receptor expression in subcutaneous and orthotopic thyroid carcinoma xenografts in vivo employing optical imaging methods.

Endothelin (ET) receptor dysregulation has been described in a number of pathophysiological processes, including cardiovascular disorders, renal failure, and cancer. The aim of this study was to evaluate the expression of the ET-A receptor (ET(A)R) in murine models of thyroid carcinoma using optical imaging methods. A recently developed near-infrared fluorescent tracer was first assessed in isolated artery preparations for its functional performance in comparison with known ET(A)R antagonists BQ123 and PD156707. Before evaluation of the tracer in vivo, different thyroid carcinoma cell lines were characterized with respect to their ET receptor expression by RT-PCR and autoradiography. In vivo, sc and orthotopic papillary thyroid tumor xenografts were clearly visualized by fluorescence reflectance imaging and fluorescence-mediated tomography up to 48 h after injection of the tracer. Binding specificity of the probe was demonstrated by predosing with PD156707 as a competing inhibitor. In conclusion, optical imaging with a fluorescent ET(A)R tracer allows the noninvasive imaging of tumor-associated ET(A)R expression in vivo. In the future, this technique may help surgeons to evaluate lesion dimensions in intraoperative settings (e.g. thyroidectomy).

Near-infrared imaging of the breast using omocianine as a fluorescent dye: results of a placebo-controlled, clinical, multicenter trial.

OBJECTIVES: To evaluate the efficacy of the near-infrared (NIR) dye Omocianine in a placebo-controlled, dose-escalating multicenter trial for the detection of malignant breast lesions by using a NIR imaging system. MATERIALS AND METHODS: The study was approved by the ethical review board of Berlin and Münster,, and all participants provided written informed consent. Fifty-two consecutive patients were examined with NIR imaging before, during, and after intravenous injection of Omocianine. Three-dimensional absorption and fluorescence diffuse optical tomography scans were recorded simultaneously on a prototype NIR imaging unit (Computed Tomography Laser Mammography, Imaging Diagnostic Systems, Inc., Ft. Lauderdale, FL). Two readers assessed the images in consensus and assigned visibility scores to lesions seen on the absorption and absorption-corrected fluorescence diffuse optical tomography mammograms. Imaging results were compared with histopathologic findings. To analyze whether lesion detection rate for malignant lesions depended on the size of the lesion, lesions were dichotomized into those measuring less than 20 mm and those measuring 20 mm or more. Moreover, the shortest diameter between the center of the target lesions and the skin was measured on axial optical mammography data. RESULTS: There were a total of 53 target lesions. Histopathologically, 22 target lesions were diagnosed as benign and 31 target lesions as malignant. In the absorption mode, a detection rate of 11.8% for benign and 44.4% for malignant lesions across all dose groups was found. In the fluorescence mode, a detection rate of 17.6% was revealed for benign and 55.6% for malignant lesions across all dose groups. For dose group 0.1 mg/kg, a detection rate of 100% was found for malignant lesions in the fluorescence mode and 71.4% in the absorption mode. Across all dose groups in the fluorescence mode, detection rate for malignant target lesions in breasts smaller than the median axial breast diameter of 12.8 cm was higher with 69.2% than in median diameters ≥ 12.8 cm with 46.2%. Omocianine-enhanced fluorescence optical mammography allowed a better detection of more superficially located lesions, with detection rates for a lesion-skin distance <20 mm of 63.6%, for <30 mm of 47.4% and for ≥ 30 mm of 25%. Malignant target lesions with a diameter ≥ 20 mm were slightly better detected with 61.5% in contrast to suspicious lesions <20 mm with 53.8%. Optimal imaging time points varied strongly among the different target lesions and Omocianine dose groups, with a mean optimal time point for malignant lesions at 188 ± 385 minutes. CONCLUSION: Preliminary data suggest that fluorescence imaging after Omocianine administration has the potential to detect malignant breast lesions. As our study showed considerable variations in the detection of breast cancer at different fluorophore concentrations ranging from 20% to 100%, future work needs to be done to assess the suitable dose for NIR imaging.