The magnetic resonance shutter speed discriminates vascular properties of malignant and benign breast tumors in vivo

Wei Huang, Xin Li, Elizabeth A. Morris, Luminita A. Tudorica, Venkatraman E. Seshan, William D. Rooney, Ian Tagge, Ya Wang, Jingang Xu and Charles S. Springer

The pharmacokinetic analysis of dynamic-contrast-enhanced (DCE) MRI data yields Ktrans and kep, two parameters independently measuring the capillary wall contrast reagent transfer rate. The almost universally used standard model (SM) embeds the implicit assumption that equilibrium transcytolemmal water exchange is effectively infinitely fast. In analyses of routine DCE-MRI data from 22 patients with suspicious breast lesions initially ruled positive by institutional screening protocols, the SM Ktrans values for benign and malignant lesions exhibit considerable overlap. A form of the shutter-speed model (SSM), which allows for finite exchange kinetics, agrees with the SM Ktrans value for each of the 15 benign lesions. However, it reveals that the SM underestimates Ktrans for each of the seven malignant tumors in this population. The fact that this phenomenon is unique to malignant tumors allows their complete discrimination from the benign lesions, as validated by comparison with gold-standard pathology analyses of subsequent biopsy tissue samples. Likewise, the SM overestimates kep, particularly for the benign tumors. Thus, incorporation of the SSM into the screening protocols would have precluded all 68% of the biopsy/pathology procedures that yielded benign findings. The SM/SSM difference is well understood from molecular first principles.

Quantitative Molecular Magnetic Resonance Imaging of Tumor Angiogenesis Using cNGR-Labeled Paramagnetic Quantum Dots

Marlies Oostendorp, Kim Douma, Tilman M. Hackeng, Anouk Dirksen, Mark J. Post, Marc A.M.J. van Zandvoort and Walter H. Backes

The objective of this study was to develop and apply cyclic Asn-Gly-Arg (cNGR)-labeled paramagnetic quantum dots (cNGR-pQDs) for the noninvasive assessment of tumor angiogenic activity using quantitative in vivo molecular magnetic resonance imaging (MRI). cNGR was previously shown to colocalize with CD13, an aminopeptidase that is highly overexpressed on angiogenic tumor endothelium. Because angiogenesis is important for tumor growth and metastatization, its in vivo detection and quantification may allow objective diagnosis of tumor status and evaluation of treatment response. I.v. injection of cNGR-pQDs in tumor-bearing mice resulted in increased quantitative contrast, comprising increased longitudinal relaxation rate and decreased proton visibility, in the tumor rim but not in tumor core or muscle tissue. This showed that cNGR-pQDs allow in vivo quantification and accurate localization of angiogenic activity. MRI results were validated using ex vivo two-photon laser scanning microscopy (TPLSM), which showed that cNGR-pQDs were primarily located on the surface of tumor endothelial cells and to a lesser extent in the vessel lumen. In contrast, unlabeled pQDs were not or only sparsely detected with both MRI and TPLSM, supporting a high specificity of cNGR-pQDs for angiogenic tumor vasculature.

Vessel Fractions in Tumor Xenografts Depicted by Flow- or Contrast-Sensitive Three-Dimensional High-Frequency Doppler Ultrasound Respond Differently to Antiangiogenic Treatment

Moritz Palmowski, Jochen Huppert, Peter Hauff, Michael Reinhardt, Karin Schreiner, Michaela A. Socher, Peter Hallscheidt, Guenter W. Kauffmann, Wolfhard Semmler and Fabian Kiessling

High-frequency volumetric Power Doppler ultrasound (HF-VPDU) captures flow-dependent signals in blood vessels and can be used to assess antiangiogenic therapy effects in rodent tumors. However, the sensitivity is limited to vessels larger than capillaries. Contrast-enhanced HF-VPDU reveals all perfused vessels by assessing stimulated acoustic emissions from disintegrating microbubbles. Thus, we investigated whether flow-sensitive and contrast-enhanced HF-VPDU can depict different vessel fractions and assess their early response to antiangiogenic therapy. Mice with A431 tumors were scanned before and after administration of polybutylcyanoacrylate microbubbles by HF-VPDU. Animals received either antiangiogenic treatment (SU11248) or a control substance and were imaged repeatedly over 9 days. At each time point, tumors were removed for immunohistochemical analysis. During growth of untreated tumors, vascularization decreased correspondingly on flow-sensitive and contrast-enhanced scans. Treated tumors showed a significantly (P < 0.05) stronger decline in vascularization than controls, which was more pronounced in contrast-enhanced scans. Surprisingly, whereas vascularization remained low in contrast-enhanced scans, flow-sensitive ultrasound indicated a reincrease after day 6 with a higher vascularization than the controls at day 9. Histologic evaluation indicated that immature vessels degraded markedly on therapy, whereas large mature vessels on the tumor periphery were more therapy resistant and drew closer due to tumor shrinkage. In conclusion, contrast-enhanced HF-VPDU and flow-sensitive HF-VPDU are both capable of assessing the effects of antiangiogenic therapy. Because contrast-sensitive ultrasound is more sensitive for small immature vessels and flow-sensitive ultrasound mostly captures large vessels at the tumor periphery, the combination of both methods can provide evidence of vascular maturity in tumors.

Three-dimensional MR mapping of angiogenesis with 51(3)-targeted theranostic nanoparticles in the MDA-MB-435 xenograft mouse model

Anne H. Schmieder, Shelton D. Caruthers, Huiying Zhang, Todd A. Williams, J. David Robertson, Samuel A. Wickline, and Gregory M. Lanza

Our objectives were 1) to characterize angiogenesis in the MDA-MB-435 xenograft mouse model with three-dimensional (3D) MR molecular imaging using 51(RGD)- or irrelevant RGS-targeted paramagnetic nanoparticles and 2) to use MR molecular imaging to assess the antiangiogenic effectiveness of 51(3)- vs. 3-targeted fumagillin (50 µg/kg) nanoparticles. Tumor-bearing mice were imaged with MR before and after administration of either 51(RGD) or irrelevant RGS-paramagnetic nanoparticles. In experiment 2, mice received saline or 51(3)- or 3-targeted fumagillin nanoparticles on days 7, 11, 15, and 19 posttumor implant. On day 22, MRI was performed using 51(3)-targeted paramagnetic nanoparticles to monitor the antiangiogenic response. 3D reconstructions of 51(RGD)-signal enhancement revealed a sparse, asymmetrical pattern of angiogenesis along the tumor periphery, which occupied <2.0% tumor surface area. 51-targeted rhodamine nanoparticles colocalized with FITC-lectin corroborated the peripheral neovascular signal. 51(3)-fumagillin nanoparticles decreased neovasculature to negligible levels relative to control; 3-targeted fumagillin nanoparticles were less effective (P>0.05). Reduction of angiogenesis in MDA-MB-435 tumors from low to negligible levels did not decrease tumor volume. MR molecular imaging may be useful for characterizing tumors with sparse neovasculature that are unlikely to have a reduced growth response to targeted antiangiogenic therapy.

Antivascular effects of combretastatin A4 phosphate in breast cancer xenograft assessed using dynamic bioluminescence imaging and confirmed by MRI

Dawen Zhao, Edmond Richer, Peter P. Antich and Ralph P. Mason

Bioluminescence imaging (BLI) has found significant use in evaluating long-term cancer therapy in small animals. We have now tested the feasibility of using BLI to assess acute effects of the vascular disrupting agent combretastatin A4 phosphate (CA4P) on luciferase-expressing MDA-MB-231 human breast tumor cells growing as xenografts in mice. Following administration of luciferin substrate, there is a rapid increase in light emission reaching a maximum after about 6 min, which gradually decreases over the following 20 min. The kinetics of light emission are highly reproducible; however, following i.p. administration of CA4P (120 mg/kg), the detected light emission was decreased between 50 and 90%, and time to maximum was significantly delayed. Twenty-four hours later, there was some recovery of light emission following further administration of luciferin substrate. Comparison with dynamic contrast-enhanced MRI based on the paramagnetic contrast agent Omniscan showed comparable changes in the tumors consistent with the previous literature. Histology also confirmed shutdown of tumor vascular perfusion. We believe this finding provides an important novel application for BLI that could have widespread application in screening novel therapeutics expected to cause acute vascular changes in tumors.

In vivo Optical Molecular Imaging of Vascular Endothelial Growth Factor for Monitoring Cancer Treatment

Sung K. Chang, Imran Rizvi, Nicolas Solban and Tayyaba Hasan

Purpose: Vascular endothelial growth factor (VEGF) expression is a critical component in tumor growth and metastasis. Capabilities to monitor VEGF expression in vivo can potentially serve as a useful tool for diagnosis, prognosis, treatment planning, monitoring, and research. Here, we present the first report of in vivo hyperspectral molecular imaging strategy capable of monitoring treatment-induced changes in VEGF expression.

Experimental Design: VEGF was targeted with an anti-VEGF antibody conjugated with a fluorescent dye and was imaged in vivo using a hyperspectral imaging system. The strategy was validated by quantitatively monitoring VEGF levels in three different tumors as well as following photodynamic treatment. Specificity of the molecular imaging strategy was tested using in vivo competition experiments and mathematically using a quantitative pharmacokinetic model.

Results: The molecular imaging strategy successfully imaged VEGF levels quantitatively in three different tumors and showed concordance with results from standard ELISA. Changes in tumoral VEGF concentration following photodynamic treatment and Avastin treatment were shown. Immunohistochemistry shows that (a) the VEGF-specific contrast agent labels both proteoglycan-bound and unbound VEGF in the extracellular space and (b) the bound VEGF is released from the extracellular matrix in response to photodynamic therapy. In vivo competition experiments and quantitative pharmacokinetic model-based analysis confirmed the high specificity of the imaging strategy.

Conclusion: This first report of in vivo quantitative optical molecular imaging-based monitoring of a secreted cytokine in tumors may have implications in providing tools for mechanistic investigations as well as for improved treatment design and merits further investigation.