Janine Ring

Detection of Asymptomatic Cerebral Microbleeds: A Comparative Study at 1.5 and 3.0 T

RATIONALE AND OBJECTIVES The magnitude of iron-induced susceptibility changes in gradient echo T2*-weighted magnet resonance imaging (T2* MRI) increases with the field strength and should increase the sensitivity for detection of cerebral microbleeds (CMBs) at 3.0 T. To test these hypotheses, we prospectively examined individuals with documented CMBs at 1.5 and 3.0 T. MATERIALS AND METHODS Five hundred fifty elderly individuals, who participated in an interdisciplinary study of healthy aging, were examined at 3.0 T using T2* MRI sequences (repetition time [TR]/echo time [TE]/flip angle [FA] = 573 ms/16 ms/18 degrees ). Individuals positive for CMBs were asked to undergo an additional examination at 1.5 T (TR/TE/FA = 663 ms/23 ms/18 degrees ). Images were analyzed independently by two observers. CMBs were counted throughout the brain and were qualitatively analyzed comparing the degree of visible hypointensity on a 5-point scale from 1 (complete signal loss) to 5 (no detection) for both field strengths. Contrast-to-noise ratio of CMBs to surrounding brain tissue was calculated. RESULTS At 3.0 T, CMBs were detected in 45 of 550 individuals; 25 agreed to an additional examination at 1.5 T. In this group (n = 25), a total of 53 CMBs were detected at 3.0 T, compared to 41 CMBs at 1.5 T. The mean contrast-to-noise ratio of CMBs was significantly increased at 3.0 T compared to 1.5 T (27.4 +/- 8.2 vs. 17.4 +/- 8.0; p < .001). On qualitative analysis, visibility of CMBs was ranked significantly higher at 3.0 T (1.3 +/- 0.4 vs. 2.9 +/- 1.1; p < .001). CONCLUSION Evidence of past microbleeds may even be found in neurologically normal elderly individuals by MRI. Detection rate and visibility of CMBs benefit from the higher field strength, resulting in a significantly improved depiction of iron-containing brain structures (CMBs) at 3.0 T with potential clinical relevance.

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.

Tumor Blood Volume Determination by Using Susceptibility-corrected ΔR2* Multiecho MR

PURPOSE To evaluate a susceptibility-corrected multiecho magnetic resonance (MR) relaxometry technique for an accurate and robust determination of DeltaR2* as a noninvasive surrogate parameter of the perfused tumor blood volume. MATERIALS AND METHODS All experiments were approved by the institutional animal care committee. In a glass tube phantom with different superparamagnetic iron oxide (SPIO) particle concentrations and at tumor mice xenografts with DU-4475, HT-1080, and MDA-MB-435 tumors (n = 15 total, n = 5 per model) with different degrees of neovascularization after injection of different ultrasmall SPIO (USPIO) doses changes of the transverse relaxation rate (DeltaR2*) were determined by using a fixed echo time (TE) of 22 msec and a susceptibility-corrected multigradient-echo technique. The mean DeltaR2* value and the vascular volume fraction (VVF) of each tumor was determined and compared with independent in vivo fluorescent tumor perfusion measurements and histologic analysis helped determine microvessel density (MVD). Statistical differences were tested by using analysis of variance and linear correlations. RESULTS For the phantom study, DeltaR2* maps calculated with a fixed TE of 22 msec showed a higher standard deviation of the noise index compared with the susceptibility-corrected multiecho technique. For the xenograft model, mean tumor DeltaR2* values (+/- standard error of the mean) showed significant differences between the various tumors (eg, DU-4475: 12.3 sec(-1) +/- 2.67, HT-1080: 36.47 sec(-1) +/- 5.84, and MDA-MB-435: 64.01 sec(-1) +/- 8.87 at 80 mumol of iron per kilogram; P < .05). DeltaR2* values increased dose dependently and in a linear fashion, resulting in reproducibly stable VVF measurements. Fluorescent tumor perfusion measurements and MVD counts corroborated the MR results. CONCLUSION Susceptibility-corrected multiecho MR relaxometry allows a highly accurate and robust determination of DeltaR2* and VVF with an excellent dynamic range for tumor characterization at clinically relevant doses of USPIO.

Tissue-factor fusion proteins induce occlusion of tumor vessels

A variety of fusion proteins consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) fused to the peptides GRGDSP (abbr. RGD), GNGRAHA (abbr. NGR) or derivates of these peptides, have been synthesized. These binding motif peptides target av-integrins or aminopeptidase N (CD13), respectively, on tumor endothelial cells. After expression and deposition as inclusion bodies in Escherichia coli BL21 (DE3), the tTF-fusion proteins were refolded and purified in a multi-step chromatography process. The upscaling process of fusion protein synthesis in order to produce amounts needed for clinical studies is presented. The proteins retained their specific proteolytic ability to activate FX by FVIIa and were able to bind to endothelial cells in vitro. Western blot analysis, analytic chromatography, FX coagulation assay and in vivo experiments have been performed to test for the in vitro stability of the tTF-NGR protein after long-term incubation at 5 degrees C or 25 degrees C, respectively. In vivo xenograft studies in nude mice bearing different malignant human tumors (mammary carcinoma SKBR3, adenocarcinoma of the lung A549) revealed that intravenous or subcutaneous administration of tTF-NGR or -RGD fusion proteins, but not the tTF protein without binding motif, induced thrombosis of tumor vessels which led to significant tumor growth retardation or regression. The anti-vascular mechanism of the tTF fusion proteins was verified by the molecular imaging methods such as magnetic resonance imaging (MRI) and fluorescence reflectance imaging (FRI); MRI showed a reduction of the relative tumor blood volume (BV) and FRI the formation of fibrin in the tTF-fusion protein treated tumors.

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 mm; 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.

Concurrent MR blood volume and vessel size estimation in tumors by robust and simultaneous ΔR2 and ΔR2* quantification

This work presents a novel method for concurrent estimation of the fractional blood volume and the mean vessel size of tumors based on a multi‐gradient‐echo‐multi‐spin‐echo sequence and the injection of a super‐paramagnetic blood‐pool agent. The approach further comprises a post‐processing technique for simultaneous estimation of changes in the transverse relaxation rates R2 and R  2* , which is robust against global B0 and B1 field inhomogeneities and slice imperfections. The accuracy of the simultaneous ΔR2 and ΔR  2* quantification approach is evaluated in a phantom. The simultaneous blood volume and vessel size estimates, obtained with MR, compare well to the immunohistological findings in a preclinical experiment (HT1080 cells, implanted in nude mice). Clinical translation is achieved in a patient with a pleomorphic sarcoma in the left pubic bone. The latter demonstrates the robustness of the technique against changes in the contrast agent concentration in blood during washout. Magn Reson Med, 2011.

NG2 proteoglycan as a pericyte target for anticancer therapy by tumor vessel infarction with retargeted tissue factor

tTF-TAA and tTF-LTL are fusion proteins consisting of the extracellular domain of tissue factor (TF) and the peptides TAASGVRSMH and LTLRWVGLMS, respectively. These peptides represent ligands of NG2, a surface proteoglycan expressed on angiogenic pericytes and some tumor cells. Here we have expressed the model compound tTF-NGR, tTF-TAA, and tTF-LTL with different lengths in the TF domain in E. coli and used these fusion proteins for functional studies in anticancer therapy. We aimed to retarget TF to tumor vessels leading to tumor vessel infarction with two barriers of selectivity, a) the leaky endothelial lining in tumor vessels with the target NG2 being expressed on pericytes on the abluminal side of the endothelial cell barrier and b) the preferential expression of NG2 on angiogenic vessels such as in tumors. Chromatography-purified tTF-TAA showed identical Factor X (FX)-activating procoagulatory activity as the model compound tTF-NGR with Km values of approx. 0.15 nM in Michaelis-Menten kinetics. The procoagulatory activity of tTF-LTL varied with the chosen length of the TF part of the fusion protein. Flow cytometry revealed specific binding of tTF-TAA to NG2-expressing pericytes and tumor cells with low affinity and dissociation KD in the high nM range. In vivo and ex vivo fluorescence imaging of tumor xenograft-carrying animals and of the explanted tumors showed reduction of tumor blood flow upon tTF-TAA application. Therapeutic experiments showed a reproducible antitumor activity of tTF-TAA against NG2-expressing A549-tumor xenografts, however, with a rather small therapeutic window (active/toxic dose in mg/kg body weight).

Monitoring of bevacizumab-induced antiangiogenic treatment effects by "steady state" ultrasmall superparamagnetic iron oxide particles magnetic resonance imaging using robust multiecho ΔR2* relaxometry.

Rationale and Objectives:The purpose of this study was to evaluate whether “steady state” magnetic resonance imaging (MRI) using a robust multiecho &Dgr;R2* MR relaxometry technique is suitable for the early assessment of a clinically approved antiangiogenic treatment regimen using bevacizumab (Avastin). Methods:A673 rhabdomyosarcoma-bearing mice were treated with bevacizumab (n = 6) or saline as control, respectively (n = 6). MRI using a multigradient echo sequence was performed before and after 2 doses of 100 &mgr;g bevacizumab at baseline and day 7. Ultrasmall superparamagnetic iron oxide particles (SH U 555 C) induced changes of the transverse relaxation rate R2* (&Dgr;R2*) were measured in regions of interest. From these results, the vascular volume fraction was estimated, providing a surrogate marker for the microvessel density (MVD). The actual MVD was determined by immunohistochemistry and correlated with the MRI results. Results:Bevacizumab treatment resulted in a significant reduction of the &Dgr;R2* values compared with the control group (bevacizumab: 10.47 ± 0.78 seconds−1 vs. control: 17.91 ± 2.63 seconds−1; P = 0.01), reflecting the significant decrease of the vascular volume fraction by 33% (bevacizumab: 2.21% ± 0.15% vs. control: 3.31% ± 0.22%; P = 0.001). Immunohistochemistry confirmed the MR results showing an approximately 25% reduction of the MVD after treatment (bevacizumab: 7.11 ± 0.3 vs. control: 9.45 ± 0.38; P = 0.001). Conclusion:Multiecho &Dgr;R2* MR relaxometry allows an early and quantitative assessment of tumor vascularization changes in response to an antiangiogenic treatment with a clinically approved vascular endothelial growth factor inhibitor. With the availability of long circulating ultrasmall superparamagnetic iron oxide particles s for clinical use, this imaging technique could be instantly translated to antiangiogenic treatment monitoring in clinical studies.

Abstract 2629: Site-directed and random PEGylation of retargeted tissue factor can improve the activity/toxicity profile of the molecule

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Retargeted tissue factor can induce tumor vessel infarction as a new approach for tumor therapy via vascular targeting. To optimize this anti-vascular approach with retargeted tissue factor (tTF) and to simplify the characterization and batch-to-batch reproducibility, single polyethylene glycol (PEG) units were site-specifically linked to tTF proteins such as tTF-NGR and compared to randomly PEGylated tTF derivatives. Experimental procedures Site-directed (SD) coupling of PEG units (4 and 20 kDa, respectively) to the N-terminus of recombinant tTF-fusion proteins was performed by reductive alkylation according to the PEGylation of granulocyte-colony stimulating factor G-CSF (Kinstler et al., 1996 & 2002). Random PEGylation was accomplished by nucleophilic substitution of short, branched PEG units (2.4 kDa each) to primary amines such as lysine residues. Successful coupling and purification steps were verified by HPLC, SDS-PAGE, Western blotting and mass spectrometry. The biological ability of PEGylated tTF derivatives to induce coagulation was assessed by FX-activation assay according to Ruf et al. (1991). Pharmacokinetic analyses were performed with blood probes from CD-1 mice after intravenous application of the PEG-tTF proteins, using a tissue factor ELISA kit. For in vivo evaluation of tumor growth, immunodeficient CD-1 nude mice were xenotransplanted with human tumor cells; tolerability studies were carried out with non-tumor-bearing mice. Histological analyses of tumor tissues and normal organs were performed according to standard protocols using an anti-PEG antibody. The anti-vascular mechanism was further verified by molecular imaging methods such as MRI. Results SD-PEGylation revealed mono-PEGylated tTF proteins, clearly separable from non-PEGylated tTF by using cation-exchange HPLC, leading to a homogeneous protein solution with a high batch-to-batch reproducibility. The ability of the SD-PEGylated tTF-NGR to induce coagulation within the FX-activation assay was barely affected in comparison to the non-PEGylated tTF-NGR protein, while the pharmacokinetic profile of the mono-PEGylated tTF-NGR resembles the profile of the randomly PEGylated protein (area under the curve was increased more than 1-log step). In vivo studies of mono-PEGylated tTF-NGR revealed a markedly reduced effective dose for tumor growth inhibition compared to the non-PEGylated protein (0.2 vs. 1 mg/kg bw). Tolerability and molecular imaging studies are ongoing to study in vivo activity/safety of mono-PEGylated tTF-NGR. Conclusion Promising results have been achieved to 1. simplify the characterization and batch-to-batch reproducibility and 2. to optimize the activity/toxicity profile of tumor-vessel infarction by retargeted tTF. The therapeutic range of tTF-NGR fusion protein can be improved by using SD- and random-PEGylation techniques, respectively. Citation Format: Caroline Zerbst, Janine Ring, Max Frohlich, Christoph Schliemann, Rolf M. Mesters, Wolfgang E. Berdel, Christian Schwoppe. Site-directed and random PEGylation of retargeted tissue factor can improve the activity/toxicity profile of the molecule. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2629. doi:10.1158/1538-7445.AM2015-2629