Amelie M. Lutz

US Imaging of Tumor Angiogenesis with Microbubbles Targeted to Vascular Endothelial Growth Factor Receptor Type 2 in Mice

PURPOSE To prospectively evaluate contrast material-enhanced ultrasonography (US) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in two murine tumor models. MATERIALS AND METHODS Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. A US contrast agent, consisting of encapsulated gaseous microbubbles, was developed specifically to bind to VEGFR2 (by using anti-VEGFR2 antibodies and biotin-streptavidin interaction) which is up-regulated on endothelial cells of tumor blood vessels. VEGFR2-targeted microbubbles (MB(V)), control microbubbles (MB(C)), and nonlabeled microbubbles (MB(N)) were tested for binding specificity on cells expressing VEGFR2 (mouse angiosarcoma SVR cells) and control cells (mouse skeletal myoblast C2C12 cells). Expression of mouse VEGFR2 in culture cells was tested with immunocytochemical and Western blot analysis. Contrast-enhanced US imaging with MB(V) and MB(C) was performed in 28 tumor-bearing nude mice (mouse angiosarcoma, n = 18; rat malignant glioma, n = 10). Differences were calculated by using analysis of variance. RESULTS In cell culture, adherence of MB(V) on SVR cells (2.1 microbubbles per SVR cell) was significantly higher than adherence of control microbubbles (0.01-0.10 microbubble per SVR cell; P < .001) and significantly more MB(V) attached to SVR cells than to C2C12 cells (0.15 microbubble per C2C12 cell; P < .001). In vivo, contrast-enhanced US imaging showed significantly higher average video intensity when using MB(V) compared with MB(C) for angiosarcoma and malignant glioma tumors (P < .001). Results of immunohistochemical analysis confirmed VEGFR2 expression on vascular endothelial cells of both tumor types. CONCLUSION US imaging with contrast microbubbles targeted to VEGFR2 allows noninvasive visualization of VEGFR2 expression in tumor vessels in mice.

Dual-targeted contrast agent for US assessment of tumor angiogenesis in vivo.

PURPOSE To develop and validate a dual-targeted ultrasonographic (US) imaging agent with microbubbles (MBs) that attaches to both vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and alpha(v)beta(3) integrin and to compare the US imaging signal obtained from dual-targeted MBs (MB(D)) with that from single-targeted MBs (MB(S)) in a murine model of tumor angiogenesis. MATERIALS AND METHODS Animal protocols were approved by the institutional Administrative Panel on Laboratory Animal Care. Single- and dual-targeted US imaging agents were prepared by attaching anti-VEGFR2, anti-alpha(v)beta(3) integrin, or both antibodies to the shell of perfluorocarbon-filled MBs. Binding specificities of targeted MBs compared with isotype-matched immunoglobulin G-labeled control MBs (MB(C)) and nontargeted nonlabeled MBs (MB(N)) were tested with VEGFR2-positive and alpha(v)beta(3) integrin-positive cells (mouse SVR cells) and control cells (mouse 4T1 cells). In vivo imaging signals of contrast material-enhanced US by using anti-VEGFR2-targeted MBs (MB(V)), anti-alpha(v)beta(3) integrin-targeted MBs (MB(I)), MB(D), and MB(C) were quantified in 49 mice bearing SK-OV-3 tumors (human ovarian cancer). Tumor tissue was stained for VEGFR2, alpha(v)beta(3) integrin, and CD31. RESULTS Attachment of MB(D) to SVR cells (mean, 0.74 MBs per cell +/- 0.05 [standard deviation]) was significantly higher than attachment to 4T1 cells (mean, 0.04 +/- 0.03), and attachment to SVR cells was higher for MB(D) than for MB(V) (mean, 0.58 +/- 0.09), MB(I) (mean, 0.42 +/- 0.21), MB(C) (mean, 0.11 +/- 0.13), and MB(N) (mean, 0.01 +/- 0.01) (P < .05). Imaging signal in the murine tumor angiogenesis model was significantly higher (P < .001) for MB(D) (mean, 16.7 +/- 7.2) than for MB(V) (mean, 11.3 +/- 5.7), MB(I) (mean, 7.8 +/- 5.3), MB(C) (mean, 2.8 +/- 0.9), and MB(N) (mean, 1.1 +/- 0.4). Immunofluorescence confirmed expression of VEGFR2 and alpha(v)beta(3) integrin on tumor vasculature. CONCLUSION Dual-targeted contrast-enhanced US directed at both VEGFR2 and alpha(v)beta(3) integrin improves in vivo visualization of tumor angiogenesis in a human ovarian cancer xenograft tumor model in mice. SUPPLEMENTAL MATERIAL http://radiology.rsnajnls.org/cgi/content/full/248/3/936/DC1.

Targeted microbubbles for imaging tumor angiogenesis: assessment of whole-body biodistribution with dynamic micro-PET in mice.

PURPOSE To evaluate in vivo whole-body biodistribution of microbubbles (MBs) targeted to tumor angiogenesis-related vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by using dynamic micro-positron emission tomography (PET) in living mice. MATERIALS AND METHODS Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Lipid-shell perfluorocarbon-filled MBs, targeted to VEGFR2 via anti-VEGFR2 antibodies, were radiolabeled by conjugating the radiofluorination agent N-succinimidyl-4-[(18)F]fluorobenzoate (SFB) to the anti-VEGFR2 antibodies. These MBs were then injected intravenously into nude mice (n = 4) bearing angiosarcomas, and the whole-body biodistribution of these probes was assessed for 60 minutes by using dynamic micro-PET. Results were compared with ex vivo gamma counting (n = 6) and immunofluorescence staining (n = 6). Control studies in angiosarcoma-bearing mice were performed with injection of the radiolabeled antibodies alone (n = 3) or free SFB (n = 3). A mixed-effects regression of MB accumulation on fixed effects of time and tissue type (tumor or muscle) and random effect of animal was performed. RESULTS VEGFR2-targeted MBs rapidly cleared from the blood circulation (50% blood clearance after approximately 3.5 minutes) and accumulated in the liver (mean, 33.4% injected dose [ID]/g +/- 13.7 [standard deviation] at 60 minutes) and spleen (mean, 9.3% ID/g +/- 6.5 at 60 minutes) on the basis of micro-PET imaging. These findings were confirmed with ex vivo gamma counting. Uptake of targeted MBs was significantly higher (P < .0001) in tumor than in adjacent skeletal muscle tissue. Immunofluorescence staining demonstrated accumulation of the targeted MBs within hepatic Kupffer cells and splenic macrophages. Biodistribution of the radiolabeled antibodies and free SFB differed from the distribution of the targeted MBs. CONCLUSION Dynamic micro-PET allows assessment of in vivo biodistribution of VEGFR2-targeted MBs.

Targeted Contrast-Enhanced Ultrasound Imaging of Tumor Angiogenesis with Contrast Microbubbles Conjugated to Integrin-Binding Knottin Peptides

Targeted contrast-enhanced ultrasound imaging is increasingly being recognized as a powerful imaging tool for the detection and quantification of tumor angiogenesis at the molecular level. The purpose of this study was to develop and test a new class of targeting ligands for targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with small, conformationally constrained peptides that can be coupled to the surface of ultrasound contrast agents. Methods: Directed evolution was used to engineer a small, disulfide-constrained cystine knot (knottin) peptide that bound to αvβ3 integrins with a low nanomolar affinity (KnottinIntegrin). A targeted contrast-enhanced ultrasound imaging contrast agent was created by attaching KnottinIntegrin to the shell of perfluorocarbon-filled microbubbles (MB-KnottinIntegrin). A knottin peptide with a scrambled sequence was used to create control microbubbles (MB-KnottinScrambled). The binding of MB-KnottinIntegrin and MB-KnottinScrambled to αvβ3 integrin-positive cells and control cells was assessed in cell culture binding experiments and compared with that of microbubbles coupled to an anti-αvβ3 integrin monoclonal antibody (MBαvβ3) and microbubbles coupled to the peptidomimetic agent c(RGDfK) (MBcRGD). The in vivo imaging signals of contrast-enhanced ultrasound with the different types of microbubbles were quantified in 42 mice bearing human ovarian adenocarcinoma xenograft tumors by use of a high-resolution 40-MHz ultrasound system. Results: MB-KnottinIntegrin attached significantly more to αvβ3 integrin-positive cells (1.76 ± 0.49 [mean ± SD] microbubbles per cell) than to control cells (0.07 ± 0.006). Control MB-KnottinScrambled adhered less to αvβ3 integrin-positive cells (0.15 ± 0.12) than MB-KnottinIntegrin. After blocking of integrins, the attachment of MB-KnottinIntegrin to αvβ3 integrin-positive cells decreased significantly. The in vivo ultrasound imaging signal was significantly higher after the administration of MB-KnottinIntegrin than after the administration of MBαvβ3 or control MB-KnottinScrambled. After in vivo blocking of integrin receptors, the imaging signal after the administration of MB-KnottinIntegrin decreased significantly (by 64%). The imaging signals after the administration of MB-KnottinIntegrin were not significantly different in the groups of tumor-bearing mice imaged with MB-KnottinIntegrin and with MBcRGD. Ex vivo immunofluorescence confirmed integrin expression on endothelial cells of human ovarian adenocarcinoma xenograft tumors. Conclusion: Integrin-binding knottin peptides can be conjugated to the surface of microbubbles and used for in vivo targeted contrast-enhanced ultrasound imaging of tumor angiogenesis. Our results demonstrate that microbubbles conjugated to small peptide-targeting ligands provide imaging signals higher than those provided by a large antibody molecule.

Early Diagnosis of Ovarian Carcinoma: Is a Solution in Sight?

UNLABELLED Ovarian cancer is the most lethal of the gynecologic malignancies. Because ovarian cancer symptoms are subtle and nonspecific, the diagnosis is often delayed until the disease is well advanced. Overall 5-year survival is a rather dismal 50% but can be improved to greater than 90% if the disease is confined to the ovary at the time of diagnosis (generally in fewer than 25% of patients). Effective screening tools are currently not available. Owing to the rather low incidence of the disease in the general population, potential screening tests must provide very high specificity to avoid unnecessary interventions in false-positive cases. This article reviews currently available serum biomarkers and imaging tests for the early detection of ovarian cancer and provides an outlook on the potential improvements in these noninvasive diagnostic tools that may lead to successful implementation in a screening program. SUPPLEMENTAL MATERIAL http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.11090563/-/DC1.

Earlier Detection of Breast Cancer with Ultrasound Molecular Imaging in a Transgenic Mouse Model

While there is an increasing role of ultrasound for breast cancer screening in patients with dense breast, conventional anatomical ultrasound lacks sensitivity and specificity for early breast cancer detection. In this study, we assessed the potential of ultrasound molecular imaging using clinically translatable vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted microbubbles (MB(VEGFR2)) to improve the diagnostic accuracy of ultrasound in earlier detection of breast cancer and ductal carcinoma in situ (DCIS) in a transgenic mouse model [FVB/N-Tg(MMTV-PyMT)634Mul]. In vivo binding specificity studies (n = 26 tumors) showed that ultrasound imaging signal was significantly higher (P < 0.001) using MB(VEGFR2) than nontargeted microbubbles and imaging signal significantly decreased (P < 0.001) by blocking antibodies. Ultrasound molecular imaging signal significantly increased (P < 0.001) when breast tissue (n = 315 glands) progressed from normal [1.65 ± 0.17 arbitrary units (a.u.)] to hyperplasia (4.21 ± 1.16), DCIS (15.95 ± 1.31), and invasive cancer (78.1 ± 6.31) and highly correlated with ex vivo VEGFR2 expression [R(2) = 0.84; 95% confidence interval (CI), 0.72-0.91; P < 0.001]. At an imaging signal threshold of 4.6 a.u., ultrasound molecular imaging differentiated benign from malignant entities with a sensitivity of 84% (95% CI, 78-88) and specificity of 89% (95% CI, 81-94). In a prospective screening trail (n = 63 glands), diagnostic performance of detecting DCIS and breast cancer was assessed and two independent readers correctly diagnosed malignant disease in more than 95% of cases and highly agreed between each other [intraclass correlation coefficient (ICC) = 0.98; 95% CI, 97-99]. These results suggest that VEGFR2-targeted ultrasound molecular imaging allows highly accurate detection of DCIS and breast cancer in transgenic mice and may be a promising approach for early breast cancer detection in women.

MR imaging of the knee: position related changes of the menisci in asymptomatic volunteers.

Rationale and Objectives:To evaluate position related changes of the menisci in asymptomatic volunteers based on MR imaging of the knee in different positions. Methods:Twenty-two knees from 22 asymptomatic volunteers with no history of knee injury and no evidence of meniscal tears were examined with a 0.5-T open-configuration MR system. Sagittal and coronal images were obtained with the knee supine in neutral, supine in 90° flexion with external and internal rotation, as well as in upright weight-bearing positions. The position of the menisci from the outer inferior edge of the meniscus to the outermost edge of the articular cartilage of the tibial plateau was measured, and meniscal movement was calculated. The Wilcoxon signed-rank test was used for statistical analysis. Results:Meniscal movement in the sagittal plane was greatest in the anterior horn of the medial meniscus upon position change from supine neutral to supine in 90° flexion with external rotation (mean, 10.5 millimeters). The least meniscal movement was observed in the anterior horn of the lateral meniscus when changing from the supine neutral to the upright knee position (mean, 0.6 millimeters). Meniscal protrusion (ie, protrusion of any part of the meniscus beyond the tibial plateau) was noted most frequently for the anterior horn of the medial meniscus (14/22 instances; 63.6%) in the sagittal plane with the knee in neutral position (mean, 2.6 millimeters, range, 1.8–2.8 millimeters). In the coronal plane, medial meniscal protrusion was most frequently present in the upright weight-bearing position (11/22 instances (50%; mean, 2 millimeters; range, 1.2–2.6 millimeters). Conclusions:Meniscal movement is most prominent in the anterior horn of the medial meniscus with the knee in the supine position in 90° flexion with external rotation. Meniscal protrusion is more frequently present in the medial meniscus and averaged less than 3 millimeters in normal volunteers in either the sagittal or coronal MR imaging plane.

Quantification and Monitoring of Inflammation in Murine Inflammatory Bowel Disease with Targeted Contrast-enhanced US

PURPOSE To evaluate ultrasonography (US) by using contrast agent microbubbles (MBs) targeted to P-selectin (MB(P-selectin)) to quantify P-selectin expression levels in inflamed tissue and to monitor response to therapy in a murine model of chemically induced inflammatory bowel disease (IBD). MATERIALS AND METHODS All procedures in which laboratory animals were used were approved by the institutional administrative panel on laboratory animal care. Binding affinity and specificity of MB(P-selectin) were tested in cell culture experiments under flow shear stress conditions and compared with control MBs (MB(Control)). In vivo binding specificity of MB(P-selectin) to P-selectin was tested in mice with trinitrobenzenesulfonic acid-induced colitis (n = 22) and control mice (n = 10). Monitoring of anti-tumor necrosis factor α antibody therapy was performed over 5 days in an additional 30 mice with colitis by using P-selectin-targeted US imaging, by measuring bowel wall thickness and perfusion, and by using a clinical disease activity index score. In vivo targeted contrast material-enhanced US signal was quantitatively correlated with ex vivo expression levels of P-selectin as assessed by quantitative immunofluorescence. RESULTS Attachment of MB(P-selectin) to endothelial cells was significantly (P = .0001) higher than attachment of MB(Control) and significantly (ρ = 0.83, P = .04) correlated with expression levels of P-selectin on endothelial cells. In vivo US signal in mice with colitis was significantly higher (P = .0001) with MB(P-selectin) than with MB(Control). In treated mice, in vivo US signal decreased significantly (P = .0001) compared with that in nontreated mice and correlated well with ex vivo P-selectin expression levels (ρ = 0.69; P = .04). Colonic wall thickness (P ≥ .06), bowel wall perfusion (P ≥ .85), and clinical disease activity scoring (P ≥ .06) were not significantly different between treated and nontreated mice at any time. CONCLUSION Targeted contrast-enhanced US imaging enables noninvasive in vivo quantification and monitoring of P-selectin expression in inflammation in murine IBD.

Molecular Imaging of Inflammation in Inflammatory Bowel Disease with a Clinically Translatable Dual-Selectin–targeted US Contrast Agent: Comparison with FDG PET/CT in a Mouse Model

PURPOSE To develop and test a molecular imaging approach that uses ultrasonography (US) and a clinically translatable dual-targeted (P- and E-selectin) contrast agent (MBSelectin) in the quantification of inflammation at the molecular level and to quantitatively correlate selectin-targeted US with fluorodeoxyglucose (FDG) combined positron emission tomography (PET) and computed tomography (CT) in terms of visualization and quantification of different levels of inflammation in a murine acute colitis model. MATERIALS AND METHODS Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care at Stanford University. MBSelectin was developed by covalently binding an analog of the naturally occurring binding ligand P-selectin glycoprotein ligand 1 fused to a human fragment crystallizable(or Fc) domain onto the lipid shell of perfluorobutane and nitrogen-containing MBs. Binding specificity of MBSelectin was assessed in vitro with a flow chamber assay and in vivo with a chemically induced acute colitis murine model. US signal was quantitatively correlated with FDG uptake at PET/CT and histologic grade. Statistical analysis was performed with the Student t test, analysis of variance, and Pearson correlation analysis. RESULTS MBSelectin showed strong attachment to both human and mouse P- and E-selectin compared with MBControl in vitro (P ≤ .002). In vivo, US signal was significantly increased (P < .001) in mice with acute colitis (173.8 arbitrary units [au] ± 134.8 [standard deviation]) compared with control mice (5.0 au ± 4.5). US imaging signal strongly correlated with FDG uptake on PET/CT images (ρ = 0.89, P < .001). Ex vivo analysis enabled confirmation of inflammation in mice with acute colitis and high expression levels of P- and E-selectin in mucosal capillaries (P = .014). CONCLUSION US with MBSelectin specifically enables detection and quantification of inflammation in a murine acute colitis model, leveraging the natural pathway of leukocyte recruitment in inflammatory tissue. US imaging with MBSelectin correlates well with FDG uptake at PET/CT imaging.

Detection of Pancreatic Ductal Adenocarcinoma in Mice by Ultrasound Imaging of Thymocyte Differentiation Antigen 1

BACKGROUND & AIMS Early detection of pancreatic ductal adenocarcinoma (PDAC) allows for surgical resection and increases patient survival times. Imaging agents that bind and amplify the signal of neovascular proteins in neoplasms can be detected by ultrasound, enabling accurate detection of small lesions. We searched for new markers of neovasculature in PDAC and assessed their potential for tumor detection by ultrasound molecular imaging. METHODS Thymocyte differentiation antigen 1 (Thy1) was identified as a specific biomarker of PDAC neovasculature by proteomic analysis. Up-regulation in PDAC was validated by immunohistochemical analysis of pancreatic tissue samples from 28 healthy individuals, 15 with primary chronic pancreatitis tissues, and 196 with PDAC. Binding of Thy1-targeted contrast microbubbles was assessed in cultured cells, in mice with orthotopic PDAC xenograft tumors expressing human Thy1 on the neovasculature, and on the neovasculature of a genetic mouse model of PDAC. RESULTS Based on immunohistochemical analyses, levels of Thy1 were significantly higher in the vascular of human PDAC than chronic pancreatitis (P = .007) or normal tissue samples (P < .0001). In mice, ultrasound imaging accurately detected human Thy1-positive PDAC xenografts, as well as PDACs that express endogenous Thy1 in genetic mouse models of PDAC. CONCLUSIONS We have identified and validated Thy1 as a marker of PDAC that can be detected by ultrasound molecular imaging in mice. The development of a specific imaging agent and identification of Thy1 as a new biomarker could aid in the diagnosis of this cancer and management of patients.