Lisa T. MacKenzie

In vivo magnetic resonance imaging of single cells in mouse brain with optical validation.

In the current work we demonstrate, for the first time, that single cells can be detected in mouse brain in vivo using magnetic resonance imaging (MRI). Cells were labeled with superparamagnetic iron oxide nanoparticles and injected into the circulation of mice. Individual cells trapped within the microcirculation of the brain could be visualized with high‐resolution MRI using optimized MR hardware and the fast imaging employing steady state acquisition (FIESTA) pulse sequence on a 1.5 T clinical MRI scanner. Single cells appear as discrete signal voids on MR images. Direct optical validation was provided by coregistering signal voids on MRI with single cells visualized using high‐resolution confocal microscopy. This work demonstrates the sensitivity of MRI for detecting single cells in small animals for a wide range of application from stem cell to cancer cell tracking. Magn Reson Med, 2006.

In vivo MRI of cancer cell fate at the single-cell level in a mouse model of breast cancer metastasis to the brain.

Metastasis (the spread of cancer from a primary tumor to secondary organs) is responsible for most cancer deaths. The ability to follow the fate of a population of tumor cells over time in an experimental animal would provide a powerful new way to monitor the metastatic process. Here we describe a magnetic resonance imaging (MRI) technique that permits the tracking of breast cancer cells in a mouse model of brain metastasis at the single‐cell level. Cancer cells that were injected into the left ventricle of the mouse heart and then delivered to the brain were detectable on MR images. This allowed the visualization of the initial delivery and distribution of cells, as well as the growth of tumors from a subset of these cells within the whole intact brain volume. The ability to follow the metastatic process from the single‐cell stage through metastatic growth, and to quantify and monitor the presence of solitary undivided cells will facilitate progress in understanding the mechanisms of brain metastasis and tumor dormancy, and the development of therapeutics to treat this disease. Magn Reson Med, 2006. Published 2006 Wiley‐Liss, Inc.

Three-dimensional High-Frequency Ultrasound Imaging for Longitudinal Evaluation of Liver Metastases in Preclinical Models

Liver metastasis is a clinically significant contributor to the mortality associated with melanoma, colon, and breast cancer. Preclinical mouse models are essential to the study of liver metastasis, yet their utility has been limited by the inability to study this dynamic process in a noninvasive and longitudinal manner. This study shows that three-dimensional high-frequency ultrasound can be used to noninvasively track the growth of liver metastases and evaluate potential chemotherapeutics in experimental liver metastasis models. Liver metastases produced by mesenteric vein injection of B16F1 (murine melanoma), PAP2 (murine H-ras-transformed fibroblast), HT-29 (human colon carcinoma), and MDA-MB-435/HAL (human breast carcinoma) cells were identified and tracked longitudinally. Tumor size and location were verified by histologic evaluation. Tumor volumes were calculated from the three-dimensional volumetric data, with individual liver metastases showing exponential growth. The importance of volumetric imaging to reduce uncertainty in tumor volume measurement was shown by comparing three-dimensional segmented volumes with volumes estimated from diameter measurements and the assumption of an ellipsoid shape. The utility of high-frequency ultrasound imaging in the evaluation of therapeutic interventions was established with a doxorubicin treatment trial. These results show that three-dimensional high-frequency ultrasound imaging may be particularly well suited for the quantitative assessment of metastatic progression and the evaluation of chemotherapeutics in preclinical liver metastasis models.

Noninvasive Quantification of Tumor Volume in Preclinical Liver Metastasis Models Using Contrast-enhanced X-ray Computed Tomography

Objectives:To determine a timepoint after contrast injection that yields equal liver parenchymal and vascular enhancement in micro-computed tomography images. To evaluate the utility of images acquired during this time period for the noninvasive measurement of liver-tumor volume. Materials and Methods:The imaging timepoint was determined by quantifying the enhancement kinetics of Fenestra VC (0.015 mL/g) in NIH III mice. In respiratory-gated images of tumor bearing mice, the ability to measure tumor volume was evaluated with a measurement variability study, and by comparing in vivo and histologically measured tumor volume. Results:Eight hours after contrast injection the liver parenchyma and vasculature were equally enhanced allowing for clear delineation of the unenhanced tumors. The smallest tumor detected in this study was 1.1 mm in diameter. The coefficient of variation for tumor-volume measurement ranged from 3.6% to 12.9% and from 6.3% to 25.8% for intra and interobserver variability, respectively. In vivo and histologic tumor-volume measurements were closely correlated (r = 0.98, P < 0.0001). Conclusions:Imaging at a time period of equal liver parenchyma and vascular enhancement after contrast injection allows for clear delineation of liver-tumor borders, thereby enabling quantitative tumor-volume monitoring.

Effect of anti-fibrinolytic therapy on experimental melanoma metastasis

Anti-fibrinolytic agents such as aprotinin and ε-aminocaproic acid (EACA) are used clinically to decrease peri-operative bleeding. Use of these treatments during cancer-related surgeries has led to investigation of the effect of fibrinolysis inhibition on cancer cell spread. The ability of aprotinin to reduce proteolytic activity of proteases required for metastasis suggests that it could have an anti-metastatic effect in patients undergoing tumor resection. However, many metastatic cells in the vasculature of a secondary tissue are associated with a micro-thrombus. The association of tumor cells with thrombi has been shown to increase their survival; therefore inhibition of plasmin-mediated fibrinolysis might instead increase metastatic cell survival by enhancing the association between thrombi and tumor cells. The goal of this work was to determine the effect of anti-fibrinolytic treatment on experimental metastasis and to establish the role of coagulation factors in this effect. The metastatic ability of B16F10 melanoma cells was evaluated in vivo following cell or animal pre-treatment with aprotinin or EACA. Additionally, a novel in vivo technique was developed, to permit analysis of tumor cell association with thrombi in the lung microvasculature using confocal microscopy. Aprotinin and EACA treatment of mice resulted in a significant increase in lung metastasis. Aprotinin treatment increased the size of thrombi in association with cells arrested in lung capillaries. This study suggests that clinical use of anti-fibrinolytic agents for cancer-related surgeries could result in increased metastatic ability of those cells shed immediately prior to and during surgery, and that this approach thus requires further study.

Contrast-enhanced microcomputed tomography using intraperitoneal contrast injection for the assessment of tumor-burden in liver metastasis models.

Objectives:To determine if intraperitoneally (IP) administered contrast (iohexol), used in conjunction with a liver-specific agent (Fenestra), can improve measurement precision and accuracy when quantifying tumor volume from micro-CT images of a liver metastasis model. Materials and Methods:We compared images acquired with Fenestra alone to images acquired with the combination of Fenestra and IP iohexol. The variability in tumor volume and tumor-burden measurement was evaluated for both techniques. The tumor-burden measurement accuracy of both in vivo techniques was determined by comparison with tumor-burden quantified from ex vivo images. Results:The addition of IP iohexol decreased measurement variability for individual tumors and overall tumor-burden by 4–8 fold and 2–3 fold, respectively. IP iohexol significantly improved the accuracy of tumor-burden measurement for both low and high tumor-burdened animals. Conclusions:The combination of IP iohexol with Fenestra provides superior delineation of liver tumors, in comparison to Fenestra alone. The complete tumor delineation provided by this imaging strategy allows for noninvasive quantification of liver tumor-burden.

In vivo videomicroscopy reveals differential effects of the vascular-targeting agent ZD6126 and the anti-angiogenic agent ZD6474 on vascular function in a liver metastasis model

Metastases require a functional blood supply for progressive growth. Thus, therapies that target metastatic vasculature have potential clinical utility. The effects of the vascular-targeting agent (VTA), ZD6126, and the anti-angiogenic agent, ZD6474, on vascular development and function within metastases were compared in an experimental liver metastasis model. Ras-transformed PAP2 fibroblasts were injected into the mesenteric veins of SCID mice to produce a control liver metastasis burden of ∼40% at 14 days. Mice given a single dose of ZD6126 (200 mg/kg, i.p.) on day 13 were examined 24 h later. Histology revealed a significant reduction in metastatic burden, associated with extensive tumor necrosis, increased tumor cell apoptosis and a reduction in tumor-associated vasculature. In vivo videomicroscopy (IVVM) revealed disrupted, non-functional vascular channels within metastases, with no blood flow. Mice given ZD6474 on days 4 to 10 (50 mg/kg daily, oral gavage) were examined on day 11. Histology revealed a lower metastatic burden, significant reductions in metastasis size and vasculature, and a significant increase in tumor cell apoptosis. IVVM revealed extensive reductions in vascularity and blood flow within metastases. Neither ZD6126 nor ZD6474 treatment affected surrounding normal liver tissue. This study shows that both agents can reduce experimental liver metastasis with no apparent effect on normal vasculature. However, these reductions were attained through distinct effects on the metastatic vasculature. Understanding differences in the modes of action of VTAs and anti-angiogenic agents will be important in optimizing their clinical application and in developing appropriate combination strategies.

WE‐D‐I‐609‐07: Analysis of Growth Dynamics of Treated Murine Liver Metastases Using Volumetric Ultrasound Micro‐Imaging

Purpose: Two methods were compared for analyzingtherapeutic responses in an experimental murine livermetastasis model using longitudinal high‐frequency three‐dimensional (3D) ultrasound imaging.Method and Materials: B16F1 mouse melanoma cells were injected into the mesenteric vein of C57BL/6 mice to produce liver metastases. Treated mice received doxorubicin every second day starting seven days post injection and continuing until day 17. Untreated mice received saline injections on the same schedule. Imaging began eight days post injection and was performed every one to two days until day 19. Three‐dimensional images were acquired using a VisualSonics Vevo 660 ultrasound system with a 40 MHz transducer.Metastasis volumes were measured in 3D images by manual segmentation. Two growth curves were constructed for each metastasis by computing least‐squares fits of an exponential function and a Gompertz function, which is an exponential with a time‐varying rate parameter, to the volume data. Results: Eight untreated and 24 treated metastases were monitored. A significant (p < 0.05 in Wilcoxon rank‐sum tests) difference in untreated and treated tumor volumes was observed as early as day 12. The growth of untreated metastases was described equally well by exponential (mean ± standard deviation of r 2  = 0.963 ± 0.045 ) or Gompertz (r 2  = 0.974 ± 0.034) functions. However, Gompertz functions modeled the growth of treated metastases more precisely (r 2  = 0.985 ± 0.024) than exponential functions (r 2  = 0.970 ± 0.041) . The difference between exponential and Gompertz r 2 values for treated growth curves was significant (p = 0.040). Conclusion: High‐frequency 3D ultrasound imaging is sensitive to changes in murine livermetastasis growth rate produced by a chemotherapeutic agent, and may be capable of characterizing temporal variations in the growth rates of treated tumors.Conflict of Interest: VisualSonics has licensed 3D reconstruction, visualization, and segmentation software from our laboratory.