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Cell and Tumor Biology |
Departments of 1 Medical Biophysics, 2 Biomedical Engineering Graduate Program, and 3 Electrical and Computer Engineering, The University of Western Ontario; 4 London Regional Cancer Program; and 5 Robarts Research Institute, London, Ontario, Canada
Requests for reprints: Ann F. Chambers, London Regional Cancer Program, 790 Commissioners Road East, London, Ontario, Canada, N6A 4L6. Phone: 519-685-8652; Fax: 519-685-8646; E-mail: Ann.Chambers{at}Lrcc.on.ca.
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Introduction |
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A multitude of preclinical imaging modalities are under development, including magnetic resonance imaging (MRI), X-ray computed tomography (CT), positron emission tomography (PET), and fluorescent and bioluminescent imaging, yet no single modality should be considered a comprehensive solution for cancer microimaging applications. Each modality possesses a unique combination of strengths and weaknesses that affect their selection for use in a particular study. In general, desirable characteristics in a noninvasive imaging modality would be high resolution to allow detection of minimal disease, cost-effectiveness and rapid image acquisition to facilitate throughput, inherent contrast between the liver parenchyma and tumor to avoid genetically encoded or endogenously given contrast agents, and applicability to a range of liver metastasis models. MRI offers high-resolution imaging yet may be time consuming and relatively expensive to purchase and operate (1). X-ray CT also offers high resolution, but poor soft tissue contrast necessitates the use of radiopaque contrast agents and radiation dosage may limit longitudinal imaging (1). The resolution of PET does not match that of MRI or CT, and the requirement for production and containment of radionuclides can make costs prohibitive (1). Fluorescent and bioluminescent imaging offer a relatively cost-effective way to study liver metastases but suffer from poor resolution and the requirement to transfect endogenous reporter genes into the cell line of interest (1, 2). The expression of foreign reporter proteins may lead to increased immunogenicity and thus must be carefully examined for their effect on the metastatic model being studied (3–5).
Ultrasound is an attractive option for preclinical imaging due to the cost and time efficiencies of this modality. Previous studies using high-frequency ultrasound imaging of murine cancer models showed the feasibility of this technique to track s.c. tumor progression (6). That study concluded that further application of ultrasound imaging would require a fast method for generating three-dimensional images. A new high-frequency scanner that employs three-dimensional image acquisition methods and reconstruction software developed in our laboratory has addressed this limitation (7). These developments raised the possibility of using high-frequency ultrasound in the evaluation of clinically relevant metastatic models, which are difficult to study in a noninvasive fashion.
We report here the application of a high-frequency (40 MHz) ultrasound system with three-dimensional imaging capabilities to the study of murine liver metastasis. We showed that the resolution of high-frequency ultrasound allowed detection of liver metastases at a minimum size that compared favorably with that of MRI, CT, or optical methods (8–11). The applicability of this technique was shown by identifying and tracking liver metastases from four tumor cell lines of different tumor origins. The importance of three-dimensional volumetric imaging to reduce uncertainty in volume determination was established by comparison of three-dimensional segmented volumes with the commonly assumed ellipsoidal volume calculated from diameter measurements. The utility of three-dimensional high-frequency ultrasound in the evaluation of chemotherapeutics was shown in a preclinical trial with doxorubicin. These results show that the cost and time efficiencies of traditional ultrasound coupled with the three-dimensional capabilities and high resolution of this high-frequency ultrasound system make this modality particularly well suited to the study of liver metastasis in a wide range of preclinical models.
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Materials and Methods |
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MEM with L-glutamine, ribonucleosides, and deoxyribonucleosides (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma, Mississauga, Ontario, Canada). PAP2 cells (13, 14) were maintained in DMEM (Invitrogen) supplemented with 10% FCS (Invitrogen). MDA-MB-435/HAL cells were maintained in EMEM (Invitrogen) supplemented with 2 mmol/L L-glutamine, 100 µmol/L nonessential amino acids, 25 mmol/L HEPES buffer, 1 mmol/L sodium pyruvate (Invitrogen), and 1x MEM vitamin solution (Sigma). The MDA-MB-435/HAL line was derived from the MDA-MB-435 cell line by an in vivo selection procedure for increased metastatic potential (15). All animals were cared for in accordance to the Canadian Council on Animal Care, under a protocol approved by the University of Western Ontario Council on Animal Care. For experimental metastasis assays, mice were anesthetized with an i.p. injection of xylazine/ketamine (2.6 mg ketamine and 0.13 mg xylazine per 20 g body mass). As described previously, anesthetized mice received mesenteric injections of 0.1 mL of cells suspended in their respective growth media to target the liver (16). For B16F1 cells, a suspension of 3 x 105 cells was injected into C57BL/6 mice (Harlan, Indianapolis, IN). For HT-29 cells, a suspension of 2 x 106 cells was injected into NIH III mice (Charles River, Wilmington, MA). For PAP2 cells, a suspension of 2 x 105 cells was injected into SCID mice (Charles River). For MDA-MB-435/HAL cells, a suspension of 3 x 105 cells was injected into SCID mice (Charles River). All mice were female and 7 to 11 weeks old at the time of cell injection. Ultrasound imaging. For ultrasound imaging, the Vevo 660 high-frequency ultrasound system (VisualSonics, Inc., Toronto, Ontario, Canada) was used. The Vevo 660 is the second generation of a system described previously (17). The Vevo 660 ultrasound probe has a 40-MHz center frequency with a 6-mm focal depth. The spatial resolution at the focus is 40 x 80 x 80 µm3. Before the first imaging session, the mouse's abdomen was depilated with commercial hair removal cream. During imaging, the mouse was kept under anesthesia with 1.5% isoflurane in oxygen and restrained on a heated stage. During imaging with the immunodeficient NIH III and SCID mice, the animals were handled and imaged in a HEPA-filtered workstation (Microzone Corp., Ottawa, Ontario, Canada). Ultrasound is strongly reflected by the ribcage, which hinders imaging of any tissue located beneath the ribs, such as the lungs and a portion of the liver. Thus, the volume of liver tissue accessible for ultrasound imaging may vary between animals and between imaging sessions for the same animals. In general, we found that a significant volume of the left lateral, left medial, and right medial liver lobes were routinely accessible for imaging. During imaging, two-dimensional images were acquired in the sagittal plane after ultrasound contact gel was applied to the abdomen. For three-dimensional imaging, parallel two-dimensional images were acquired by stepping the transducer in 30-µm intervals in the out-of-plane dimension. Using software developed in our laboratory, two-dimensional images were interpolated and reconstructed online to create a three-dimensional volumetric image (7). The system can acquire and produce a typical three-dimensional image in <20 seconds. The three-dimensional reconstruction software is available through VisualSonics, or through the authors for research purposes.
Volume measurements. To determine tumor volume, the boundaries of a metastasis were outlined within parallel planes separated by 50 µm in the volumetric image. The total metastasis volume was calculated by summing the outlined areas and multiplying by the interslice distance (18). Segmented volumes were compared with ellipsoid volumes estimated using the formula V = 
abc / 6. The measurements for diameters a, b, and c were obtained from the three-dimensional volumetric images. The sagittal plane showing the greatest tumor diameter was selected, and the greatest diameter a measured. The diameter b, perpendicular to a, was then measured. The volume was then rotated and the transverse plane showing the largest tumor diameter was selected. The diameter c, perpendicular to both a and b, was then measured. To determine the % difference between the ellipsoid and three-dimensional segmented volumes the following formula was used: 100% x (ellipsoid volume – segmented volume)/{(ellipsoid volume + segmented volume) / 2}.
Longitudinal growth measurements. For longitudinal imaging, the initial imaging time point was based on previous indications of when micrometastases could first be detected by ultrasound. Individual liver metastases were identified and a three-dimensional image recorded. Individual liver metastases were identified on successive imaging dates by their particular liver lobe location, tumor shape, and proximity to landmark structures such as major blood vessels or the liver edges. Landmarks were all internal to the liver, because the liver lobes move in relation to any external landmarks such as the ribs. Animals were sacrificed due to escalating tumor burden, as assessed by ultrasound imaging, or when at least four imaging time points had been acquired to construct a growth curve. Approximately 5 minutes was required to locate, identify and image the liver metastases of each mouse. If the time spent on setup, animal handling, anesthesia, and recovery is included, the average duration of an imaging session was 15 minutes per mouse.
Treatment protocols. The B16F1 liver metastasis model was used to assess the ability of high-frequency ultrasound to evaluate cytotoxic chemotherapeutic agents. At day 7 after cell injection, the first treatment with doxorubicin was given. Doxorubicin (Pharmacia, Mississauga, Ontario, Canada) was given at a previously described treatment schedule (1 mg/kg, 0.1 mL, i.p.) every second day until day 17 after cell injection, for a total of six treatments (19). Control animals received saline control injections (0.1 mL, i.p.). Ultrasound imaging was done from day 8 after cell injection, the earliest time B16F1 liver metastases could be detected in the images, until the end of the experiment.
Histology. At sacrifice, the mouse liver was excised and fixed in 10% neutral buffered formalin. Visual inspection validated the tumor size and location depicted by ultrasound imaging. For histologic confirmation, formalin-fixed, paraffin-embedded livers were sectioned (4 µm) and stained with H&E.
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Results |
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segmented volume) of 0.22 mm 0.01 mm3, 0.47 mm 0.03 mm3, 0.66 mm 0.08 mm3, and 0.78 mm 0.17 mm3 for B16F1, HT-29, MDA-MB-435/HAL, and PAP2 tumors, respectively. As a point of reference, a volume of 0.01 mm3 would be produced by 
700 cells, based on the assumption of a spherical cell volume and a cell diameter of 15 µm.
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Tracking the growth of individual liver metastases by noninvasive ultrasound imaging. To show the use of ultrasound imaging in the longitudinal study of liver metastases, mice were noninvasively imaged at 2- to 3-day intervals. At sacrifice, gross pathology and histologic sections verified tumor sizes and locations depicted during ultrasound imaging. The B16F1 metastases developed rapidly, forming detectable metastases as early as 10 days after cell injection in this experiment (Fig. 2A). The B16F1 metastases showed exponential growth with an average volume doubling time of 1.2 ± 0.2 (mean ± SD) days. The mean correlation coefficient for fitting an exponential curve was 0.966 ± 0.047. The HT-29 and MDA-MB-435/HAL liver metastases were much slower to develop, forming detectable metastases at a minimum of 33 days after cell injection (Fig. 2B). The HT-29 and MDA-MB-435/HAL metastases also showed exponential growth with doubling times ranging from 3.7 to 4.8 days for the HT-29 metastases and 5.7 to 10.4 days for MDA-MB-435/HAL metastases. The correlation coefficients ranged between 0.972 and 0.993 for HT-29 and between 0.635 and 0.889 for MDA-MB-435/HAL. The metastasis HT-29-4 was not included in the range of doubling times because its volume did not increase over the 10-day interval that it was imaged. Representative two-dimensional images from the longitudinal imaging of an individual B16F1 metastasis, B16F1-E, are shown (Fig. 2C). Individual PAP2 liver metastases could not be evaluated for longitudinal growth because in this highly aggressive model numerous metastases form and quickly fuse. In such cases, ultrasound could be used to monitor increasing tumor burden, instead of the growth of individual metastases, as an indicator of tumor progression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in this report, all four cell lines tested in experimental liver metastasis models, B16F1, HT-29, MDA-MB-435/HAL, and PAP2, showed inherent ultrasound contrast relative to the surrounding liver parenchyma. These cell lines are derived from different primary tumor types, murine melanoma, human colon carcinoma, human breast carcinoma, and oncogene-transformed murine fibroblasts, which shows the wide applicability of this technique. In all cases, the liver metastases were hypoechoic, appearing darker than the surrounding tissue on ultrasound images. Ultrasound imaging was shown highly sensitive with metastases from all four tumor models detected with maximum diameters of <0.78 mm. The consistent background image texture of normal liver parenchyma likely contributed to the detection of small tumors; thus, ultrasound seems particularly well suited for imaging liver metastasis models. To determine the absolute detection limit for any particular cell line, more frequent imaging on a greater number of mice would need to be done.
The ability to track the growth of individual liver metastases over time was shown for B16F1, HT-29, and MDA-MB-435/HAL tumor cell lines. The use of the human cell lines HT-29 and MDA-MB-435/HAL is particularly noteworthy because immunodeficient animals, which must be protected from infection, are used for these metastasis models. The ultrasound system was easily adapted to this requirement by restricting the mice and ultrasound probe to a HEPA-filtered environment. This process would not be possible with larger, less portable imaging modalities.
The longitudinal imaging trials showed the importance of noninvasive imaging in allowing analysis on a per metastasis instead of a per mouse basis. For example, in contrast with the exponential growth seen with other liver metastases in the same animal, the metastasis HT-29-4 did not show a significant increase in tumor volume during the 10 days it was imaged. The volume of this metastasis was constant at 0.03 mm3. The identification of metastases with variable growth patterns would allow further investigation, such as microdissection and microarray analysis, to elucidate the molecular basis of such variations. The monitoring of dormant metastases would also permit the study of host-tumor interactions, such as the role of angiogenesis in the switch from a dormant to progressive phenotype (20). The detection sensitivity of high-frequency ultrasound allows the investigation of processes, such as tumor dormancy and angiogenesis, which may occur very early in the metastatic process.
The longitudinal imaging of B16F1 liver metastases revealed striking changes in ultrasound image texture during tumor development. The most obvious of these changes was the development of distinct anechoic regions that were shown to be areas of liquefactive necrosis. The ability to detect the formation of necrosis may be useful in the assessment of vascular targeting agents and antiangiogenic compounds (21, 22). Incorporating Doppler blood flow imaging into studies using the high-frequency ultrasound system may further enhance the assessment of tumor vasculature and hemodynamics (23). The cellular or structural characteristics that cause the more subtle changes in ultrasound backscatter are currently under investigation in several laboratories (24–26).
These studies have focused on imaging the development of individual metastases because of the unique research opportunities that this approach presents. In time, the models used here will form large coalescing metastases, which can no longer be monitored for individual growth characteristics. At this stage, ultrasound imaging could be used to measure relative tumor burden between animals. This application will require further study to determine how tumor burden in acoustically accessible liver areas represent the state of the entire liver.
Because s.c. tumor growth is frequently monitored by caliper measurement and calculation of an ellipsoid volume, we sought to determine if the approximation of an ellipsoid volume was sufficient for monitoring the growth of liver metastases. It was shown that tumor volumes calculated from three-dimensional and two-dimensional methods yielded vastly different results for many metastases. For B16F1 metastases, the mean percent volume difference between the two methods was –8.8 ± 23.5%. The large SD indicates that the two-dimensional method often gives large overestimations or underestimations of tumor volume when compared with the three-dimensional method. Because the true volume of the metastases could not be determined, it cannot be definitely stated that one method is more accurate than the other. However, it is reasonable to suggest that the three-dimensional method is more accurate because there is no assumption of a defined shape, and a three-dimensional image allows the operator greater time and control when defining tumor borders. Definition of tumor borders can be done offline with a three-dimensional image, whereas the operator of a two-dimensional system must identify maximum tumor diameters during imaging. Furthermore, previous work with a clinical ultrasound system has shown that the three-dimensional method is more accurate than the two-dimensional method when measuring the volume of regular and irregular shaped phantoms (18, 27). The inaccuracy and variability brought about by assuming a defined shape could hinder the ability to track volume changes in slowly growing metastatic models, the ability to track subtle responses to therapeutic treatment, and the ability to determine if a metastasis is going through a period of dormancy. The elimination of this uncertainty presents a compelling case for using an imaging modality with three-dimensional imaging capabilities.
The use of longitudinal ultrasound imaging in preclinical trials was shown with the anthracycline chemotherapeutic doxorubicin. Longitudinal assessment of individual liver metastases showed that doxorubicin significantly decreased tumor growth rate and tumor volume in the B16F1 liver metastasis model. Significant differences in tumor volume were evident at day 12 after cell injection, after only three doxorubicin treatments, when the average tumor volume in the control group was <1.00 mm3 and in the treated group was <0.25 mm3. The ability of high-frequency ultrasound to track the progression of micrometastases noninvasively allows the evaluation of therapeutic efficacy on sequential stages of tumor development, from early formation to the development of large vascularized metastases, in a single experiment.
In summary, this report is the first to describe the use of three-dimensional high-frequency (40 MHz) ultrasound imaging in the noninvasive detection and longitudinal evaluation of murine liver metastases. This development is significant in that ultrasound offers rapid, cost-effective, high-resolution imaging that can be applied to a wide range of liver metastasis models without the requirement for contrast agents. Compared with traditional histologic methods, ultrasound imaging may provide a more accurate assessment of tumor progression and chemotherapeutic response, which will open new avenues of investigation into dynamic processes such as tumor vascularization and tumor dormancy.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. David Griggs (Pfizer Global Research and Development, Chesterfield, MO) for providing the MDA-MB-435/HAL cell line, Mike Bygrave for technical support, Dr. Alan Tuck for expertise in pathology, and VisualSonics for technical support.
Received 2/ 9/05. Revised 3/16/05. Accepted 4/ 5/05.
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