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.

Time-course characterization of the computed tomography contrast enhancement of an iodinated blood-pool contrast agent in mice using a volumetric flat-panel equipped computed tomography scanner.

OBJECTIVE: The objective of this study was to determine the time-course of computed tomography (CT) contrast enhancement of an iodinated blood-pool contrast agent. METHODS: Five C57BL/6 mice were anesthetized, imaged at baseline, and given an iodinated blood-pool contrast agent. Micro-CT scans were acquired at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after injection. The mean CT number was determined in a region of interest in 7 organs. RESULTS: The CT contrast enhancement was plotted as a function of time for each organ. We identified an imaging window immediately after injection suitable for visualizing the vascular system and a second imaging window at 24 hours for visualizing liver and spleen. CONCLUSIONS: A single injection of the blood-pool contrast agent can be used for dual-phase investigations of the vasculature (t = 0 hours) and liver (t = 24 hours), which can be applied to studies of liver tumors or disease.

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.

Mapping of the functional microcirculation in vital organs using contrast-enhanced in vivo video microscopy.

A functional microcirculation is vital to the survival of mammalian tissues. In vivo video microscopy is often used in animal models to assess microvascular function, providing real-time observation of blood flow in normal and diseased tissues. To extend the capabilities of in vivo video microscopy, we have developed a contrast-enhanced system with postprocessing video analysis tools that permit quantitative assessment of microvascular geometry and function in vital organs and tissues. FITC-labeled dextran (250 kDa) was injected intravenously into anesthetized mice to provide intravascular fluorescence contrast with darker red blood cell (RBC) motion. Digitized video images of microcirculation in a variety of internal organs (e.g., lung, liver, ovary, and kidney) were processed using computer-based motion correction to remove background respiratory and cardiac movement. Stabilized videos were analyzed to generate a series of functional images revealing microhemodynamic parameters, such as plasma perfusion, RBC perfusion, and RBC supply rate. Fluorescence contrast revealed characteristic microvascular arrangements within different organs, and images generated from video sequences of liver metastases showed a marked reduction in the proportion of tumor vessels that were functional. Analysis of processed video sequences showed large reductions in vessel volume, length, and branch-point density, with a near doubling in vessel segment length. This study demonstrates that postprocessing of fluorescence contrast video sequences of the microcirculation can provide quantitative images useful for studies in a wide range of model systems.

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 approximately 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.

Ineffectiveness of doxorubicin treatment on solitary dormant mammary carcinoma cells or late-developing metastases.

Breast cancer is noted for long periods of tumor dormancy and metastases can occur many years after treatment. Adjuvant chemotherapy is used to prevent metastatic recurrence but is not always successful. As a model for studying mechanisms of dormancy, we have used two murine mammary carcinoma cell lines: D2.0R/R cells, which are poorly metastatic but form metastases in some mice after long latency times, and D2A1/R cells, which form more numerous metastases much earlier. Previously we identified a surprisingly large population of dormant but viable solitary cells, which persisted in an undivided state for up to 11 weeks after injection of D2.0R/R cells. Dormant cells were also detected for D2A1/R cells, in a background of growing metastases. Here we used this model to test the hypothesis that dormant tumor cells would not be killed by cytotoxic chemotherapy that targets actively dividing cells, and that the late development of metastases from D2.0R/R cells would not be inhibited by chemotherapy that effectively inhibited D2A1/R metastases. We injected mice with D2A1/R or D2.0R/R cells via a mesenteric vein to target liver. We developed a doxorubicin (DXR) treatment protocol that effectively reduced the metastatic tumor burden from D2A1/R cells at 3 weeks. However, this treatment did not reduce the numbers of solitary dormant cells in mice injected with either D2A1/R or D2.0R/R cells. Furthermore, DXR did not reduce the metastatic tumor burden after an 11-week latency period in mice injected with D2.0R/R cells. Thus, apparently effective chemotherapy may spare non-dividing cancer cells, and these cells may give rise to metastases at a later date. This study has important clinical implications for patients being treated with cytotoxic chemotherapy.

Cancer spread and micrometastasis development: quantitative approaches for in vivo models.

Death from cancer is usually due to metastasis. Fortunately, most cells that escape from a primary tumor fail to form metastases. Identifying reasons for this failure will help development of anti-metastatic therapies. Intravital videomicroscopy (IVVM) can be used to observe cancer cells injected into live animals. Co-injected microspheres can be used to assess cell survival. These techniques have been used to show that circulating tumor cells generally arrest in the microcirculation and may extravasate with high efficiency. While many tumor cells may survive in a secondary site, only a small subset form micrometastases and only a subset of these micrometastases persist to form vascularized macrometastases. Furthermore, solitary tumor cells may remain dormant for long periods of time in secondary sites. These findings suggest that metastatic growth and angiogenesis are prime targets for anti-metastatic therapy.

Dissemination and growth of cancer cells in metastatic sites.

Metastases, rather than primary tumours, are responsible for most cancer deaths. To prevent these deaths, improved ways to treat metastatic disease are needed. Blood flow and other mechanical factors influence the delivery of cancer cells to specific organs, whereas molecular interactions between the cancer cells and the new organ influence the probability that the cells will grow there. Inhibition of the growth of metastases in secondary sites offers a promising approach for cancer therapy.

Persistence of solitary mammary carcinoma cells in a secondary site: a possible contributor to dormancy.

Tumors can recur years after treatment, and breast cancer is especially noted for long periods of dormancy. The status of the cancer during this period is poorly understood. As a model to study mechanisms of dormancy, we used murine D2.0R mammary carcinoma cells, which are poorly metastatic but form occasional metastases in liver and other organs after long latency. Highly metastatic D2A1 cells provided a positive, metastatic control. Our goals were to learn how the cell lines differ in survival kinetics in a secondary site and to seek evidence for the source of D2.0R dormancy. In spontaneous metastasis assays from mammary fat pad injections, we found evidence for dormancy because of a persistence of large numbers of solitary cells in the liver. To quantify the fate of cells after arrival in liver, experimental metastasis assays were used. To permit identification of cells that had not divided, cells were labeled before injection with fluorescent nanospheres, which were diluted to undetectable levels by cell division. Cancer cells were injected i.v. to target them to the liver and coinjected with reference microspheres to monitor cell survival. Dormancy was defined as retention of nanosphere fluorescence in vivo, as well as negative staining for the proliferation marker Ki67. A large proportion of D2.0R cells persisted as solitary dormant cells. No metastases formed, but viable cells could be recovered from the liver 11 weeks after injection. Large numbers of solitary, dormant, Ki67-negative D2A1 cells were also detected against a background of progressively growing metastases. Thus, this study identified a possible contributor to tumor dormancy: solitary, dormant cells that persist in tissue. If such cells are present in patients, they could contribute to tumor recurrence and would not be susceptible to current therapeutic strategies targeting proliferating cells.

Activated ras regulates the proliferation/apoptosis balance and early survival of developing micrometastases.

Mutant, activated ras oncogenes are found in many human cancers. Experimental studies have shown that Ras enhances metastatic ability in several cell types. However, the biological mechanisms by which Ras contributes to metastasis remain poorly understood. Our goal was to determine which steps in the formation of macroscopic metastases were affected by Ras. Green fluorescent protein-transfected NIH 3T3 and T24 H-ras-transformed (PAP2) fibroblasts were injected via mesenteric vein to target mouse liver. The proportion of cells that survived at each step of the metastatic process (at 60 min to 14 days after injection) were quantified. We found that Ras did not enhance the ability of cells to extravasate from liver sinusoids or to survive as solitary undivided cells in liver tissue. Furthermore, we found that a subset of cells from both cell lines initiated growth to form micrometastases by day 3. Only micrometastases formed by ras-transformed cells, however, persisted to form macroscopic metastases by day 14, whereas most NIH 3T3 micrometastases disappeared. We investigated this difference in maintenance of developing metastases by quantifying apoptosis and proliferation within the micrometastases. PAP2 metastases had a significantly higher proportion of proliferating cells as compared with apoptosing cells, whereas NIH 3T3 metastases had low proliferation and high apoptosis levels. Whereas the ability of Ras to induce vascular endothelial growth factor has suggested one way that Ras might affect metastatic ability (through induction of angiogenesis), our study provides in vivo evidence for a direct role for Ras in maintenance of metastatic growth via a shift in proliferation/apoptosis balance to favor metastatic growth.