Exploiting the MUC5AC Antigen for Noninvasive Identification of Pancreatic Cancer.

Pancreatic cancer (PC) remains the fourth leading cause of cancer death; therefore, there is a clinically unmet need for novel therapeutics and diagnostic markers to treat this devastating disease. Physicians often rely on biopsy or CT for diagnosis, but more specific protein biomarkers are highly desired to assess the stage and severity of PC in a noninvasive manner. Serum biomarkers such as carbohydrate antigen 19-9 are of particular interest as they are commonly elevated in PC but have exhibited suboptimal performance in the clinic. MUC5AC has emerged as a useful serum biomarker that is specific for PC versus inflammation. We developed RA96, an anti-MUC5AC antibody, to gauge its utility in PC diagnosis through immunohistochemical analysis and whole-body PET in PC. Methods: In this study, extensive biochemical characterization determined MUC5AC as the antigen for RA96. We then determined the utility of RA96 for MUC5AC immunohistochemistry on clinical PC and preclinical PC. Finally, we radiolabeled RA96 with 89Zr to assess its application as a whole-body PET radiotracer for MUC5AC quantification in PC. Results: Immunohistochemical staining with RA96 distinguished chronic pancreatitis, pancreatic intraepithelial neoplasia, and varying grades of pancreatic ductal adenocarcinoma in clinical samples. 89Zr-desferrioxamine-RA96 was able to detect MUC5AC with high specificity in mice bearing capan-2 xenografts. Conclusion: Our study demonstrated that RA96 can differentiate between inflammation and PC, improving the fidelity of PC diagnosis. Our immuno-PET tracer 89Zr-desferrioxamine-RA96 shows specific detection of MUC5AC-positive tumors in vivo, highlighting the utility of MUC5AC targeting for diagnosis of PC.

CD13 as target for tissue factor induced tumor vascular infarction in small cell lung cancer.

OBJECTIVES: Zinc-binding protease aminopeptidase N (CD13) is expressed on tumor vascular cells and tumor cells. It represents a potential candidate for molecular targeted therapy, e.g. employing truncated tissue factor (tTF)-NGR, which can bind CD13 and thereby induce tumor vascular infarction. We performed a comprehensive analysis of CD13 expression in a clinically well characterized cohort of patients with small cell lung cancer (SCLC) to evaluate its potential use for targeted therapies in this disease. MATERIAL AND METHODS: CD13 expression was analyzed immunohistochemically in 27 SCLC patients and correlated with clinical course and outcome. In CD-1 nude mice bearing human HTB119 SCLC xenotransplants, the systemic effects of the CD13-targeting fusion protein tTF-NGR on tumor growth were tested. RESULTS AND CONCLUSION: In 52% of the investigated SCLC tissue samples, CD13 was expressed in tumor stroma cells, while the tumor cells were negative for CD13. No prognostic effect was found in the investigated SCLC study collective with regard to overall survival (p>0.05). In CD-1 nude mice, xenografts of CD13 negative HTB119 SCLC cells showed CD13 expression in the intratumoral vascular and perivascular cells, and the systemic application of CD13-targeted tissue factor tTF-NGR led to a significant reduction of tumor growth. We here present first data on the expression of CD13 in SCLC tumor samples. Our results strongly recommend the further investigation of tTF-NGR and other molecules targeted by NGR-peptides in SCLC patients. Considering the differential expression of CD13 in SCLC samples pre-therapeutic CD13 analysis is proposed for testing as investigational predictive biomarker for patient selection.

Correction: Potential therapeutic impact of CD13 expression in non-small cell lung cancer.

[This corrects the article DOI: 10.1371/journal.pone.0177146.].

Potential therapeutic impact of CD13 expression in non-small cell lung cancer.

BACKGROUND: Aminopeptidase N (CD13) is a zinc-binding protease that has functional effects on both cancerogenesis and tumor angiogenesis. Since CD13 is an antigen suitable for molecular targeted therapies (e.g. tTF-NGR induced tumor vascular infarction), we evaluated its impact in NSCLC patients, and tested the effects of the CD13-targeted fusion protein tTF-NGR (truncated tissue factor (tTF) containing the NGR motif: asparagine-glycine-arginine) in vivo in nude mice. METHODS: Expression of both CD13 and CD31 was studied in 270 NSCLC patients by immunohistochemistry. Clinical correlations and prognostic effects of the expression profiles were analyzed using univariate and multivariate analyses. In addition, a microarray-based analysis on the basis of the KM plotter database was performed. The in vivo effects of the CD13-targeted fusion protein tTF-NGR on tumor growth were tested in CD1 nude mice carrying A549 lung carcinoma xenotransplants. RESULTS: CD13 expression in tumor endothelial and vessel associated stromal cells was found in 15% of the investigated samples, while expression in tumor cells was observed in 7%. Although no significant prognostic impact was observed in the full NSCLC study cohort, both univariate and multivariate models identified vascular CD13 protein expression to correlate with poor overall survival in stage III and pN2+ NSCLC patients. Microarray-based mRNA analysis for either adenocarcinomas or squamous cell carcinomas did not reveal any significant effect. However, the analysis of CD13 mRNA expression for all lung cancer histologies demonstrated a positive prognostic effect. In vivo, systemic application of CD13-targeted tissue factor tTF-NGR significantly reduced CD13+ A549 tumor growth in nude mice. CONCLUSIONS: Our results contribute a data basis for prioritizing clinical testing of tTF-NGR and other antitumor molecules targeted by NGR-peptides in NSCLC. Because CD13 expression in NSCLC tissues was found only in a specific subset of NSCLC patients, rigorous pre-therapeutic testing will help to select patients for these studies.

Combinatorial effects of doxorubicin and retargeted tissue factor by intratumoral entrapment of doxorubicin and proapoptotic increase of tumor vascular infarction.

Truncated tissue factor (tTF), retargeted to tumor vasculature by GNGRAHA peptide (tTF-NGR), and doxorubicin have therapeutic activity against a variety of tumors. We report on combination experiments of both drugs using different schedules. We have tested fluorescence- and HPLC-based intratumoral pharmacokinetics of doxorubicin, flow cytometry for cellular phosphatidylserine (PS) expression, and tumor xenograft studies for showing in vivo apoptosis, proliferation decrease, and tumor shrinkage upon combination therapy with doxorubicin and induced tumor vascular infarction. tTF-NGR given before doxorubicin inhibits the uptake of the drug into human fibrosarcoma xenografts in vivo. Reverse sequence does not influence the uptake of doxorubicin into tumor, but significantly inhibits the late wash-out phase, thus entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with doxorubicin in vitro increases PS concentrations in the outer layer of the cell membrane as a sign of early apoptosis. Cells expressing increased PS concentrations show comparatively higher procoagulatory efficacy on the basis of equimolar tTF-NGR present in the Factor X assay. Experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in athymic nude mice indeed show a combinatorial tumor growth inhibition applying doxorubicin and tTF-NGR in sequence over single drug treatment. Combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by new mechanisms, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in tumor endothelial and tumor cells.

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).

Tumor Growth Inhibition via Occlusion of Tumor Vasculature Induced by N-Terminally PEGylated Retargeted Tissue Factor tTF-NGR.

tTF-NGR retargets the extracellular domain of tissue factor via a C-terminal peptide GNGRAHA, a ligand of the surface protein aminopeptidase N (CD13) and upon deamidation of integrin αvß3, to tumor vasculature. tTF-NGR induces tumor vascular infarction with consecutive antitumor activity against xenografts and selectively inhibits tumor blood flow in cancer patients. Since random PEGylation resulted in favorable pharmacodynamics of tTF-NGR, we performed site-directed PEGylation of PEG units to the N-terminus of tTF-NGR to further improve the antitumor profile of the molecule. Mono-PEGylation to the N-terminus did not change the procoagulatory activity of the tTF-NGR molecule as measured by Factor X activation. Experiments to characterize pharmacokinetics in mice showed a more than 1 log step higher mean area under the curve of PEG20k-tTF-NGR over tTF-NGR. Acute (24 h) tolerability upon intravenous application for the mono-PEGylated versus non-PEGylated tTF-NGR compounds was comparable. PEG20k-tTF-NGR showed clear antitumor efficacy in vivo against human tumor xenografts when systemically applied. However, site-directed mono-PEGylation to the N-terminus does not unequivocally improve the therapeutic profile of tTF-NGR.

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.

MRI visualization of Staphyloccocus aureus-induced infective endocarditis in mice.

Infective endocarditis (IE) is a severe and often fatal disease, lacking a fast and reliable diagnostic procedure. The purpose of this study was to establish a mouse model of Staphylococcus aureus-induced IE and to develop a MRI technology to characterize and diagnose IE. To establish the mouse model of hematogenous IE, aortic valve damage was induced by placing a permanent catheter into right carotid artery. 24 h after surgery, mice were injected intravenously with either iron particle-labeled or unlabeled S. aureus (strain 6850). To distinguish the effect of IE from mere tissue injury or recruited macrophages, subgroups of mice received sham surgery prior to infection (n = 17), received surgery without infection (n = 8), or obtained additionally injection of free iron particles to label macrophages (n = 17). Cardiac MRI was performed 48 h after surgery using a self-gated ultra-short echo time (UTE) sequence (TR/TE, 5/0.31 ms; in-plane/slice, 0.125/1 mm; duration, 12∶08 min) to obtain high-resolution, artifact-free cinematographic images of the valves. After MRI, valves were either homogenized and plated on blood agar plates for determination of bacterial titers, or sectioned and stained for histology. In the animal model, both severity of the disease and mortality increased with bacterial numbers. Infection with 105 S. aureus bacteria reliably caused endocarditis with vegetations on the valves. Cinematographic UTE MRI visualised the aortic valve over the cardiac cycle and allowed for detection of bacterial vegetations, while mere tissue trauma or labeled macrophages were not detected. Iron labeling of S. aureus was not required for detection. MRI results were consistent with histology and microbial assessment. These data showed that S. aureus-induced IE in mice can be detected by MRI. The established mouse model allows for investigation of the pathophysiology of IE, testing of novel drugs and may serve for the development of a clinical diagnostic strategy.

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.

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.

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 ΔR2* 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 µg bevacizumab at baseline and day 7. Ultrasmall superparamagnetic iron oxide particles (SH U 555 C) induced changes of the transverse relaxation rate R2* (ΔR2*) 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 ΔR2* 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 ΔR2* 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.

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 R(2) and R(2)*, which is robust against global B(0) and B(1) field inhomogeneities and slice imperfections. The accuracy of the simultaneous ΔR(2) 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.

Tumor blood volume determination by using susceptibility-corrected DeltaR2* 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.

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.