The spleen represents the only lymphatic tissue specialized to filter the blood, and its microcirculation is probably the most complex of any organ in the body. Arteries and veins enter and leave the spleen along the hilus of its concave surface and pass within trabeculae into the interior of the organ. Unlike other organs studied from a microcirculatory standpoint, the spleen is not concerned with transcapillary exchange in relation to metabolism. Its two primary functions concern immunologic reactions and the filtration of cells and particulate matter from the blood. It has long been recognized that the spleen concentrates normal blood cells to twice arterial hematocrit, and harbors more than 30% of all circulating platelets in the body. The spleen is rarely the primary site of disease, but is affected by a wide range of disease processes originating elsewhere in the body. Microvascular corrosion casting may be applied to spleens of humans as well as laboratory animals.

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.

Proc Amer Assoc Cancer Res, Volume 47, 2006 3974 Introduction: Metastasis is the main cause of death in breast cancer. A better understanding of the metastatic process is needed if these deaths are to be prevented. Breast Cancer Metastasis Suppressor 1 gene (BRMS1) inhibits the ability of multiple human and murine cancer cell lines to metastasize to lymph node (LN) and lung. Comparison of mRNA expression in metastatic MDA-MB-435 human breast carcinoma cells (435) and metastasis-suppressed BRMS1 transfectants (435/BRMS1) showed a marked (>90%) reduction of osteopontin (OPN) mRNA and protein expression. OPN expression is associated with disease progression in patients, with higher levels of OPN produced by cancer cells associated with poorer patient survival. This work tests the hypothesis that restoration of OPN levels in 435/BRMS1 would be accompanied by regaining of metastatic capability. Methods: Neomycin-resistant 435/BRMS1 cells were transfected with either full-length OPN cDNA or a vector only plasmid (pcDNA3.1+) and single colonies were selected by hygromycin resistance. Stable colonies were selected to create pooled populations of OPN over-expressing (435/BRMS1/OPN) and vector control (435/BRMS1/VC) cell lines, which were assessed in vitro (colony formation in soft agar). In vivo , parental 435, 435/BRMS1/VC, and 435/BRMS1/OPN cells were implanted into the left thoracic mammary fat pad of female athymic mice. Primary tumors were allowed to grow for 8 wk prior to resection. The mice were then left for a further 8 weeks or until moribund. Metastatic tumor burden (tumor area/organ total area) was calculated from analysis of histological slides using Image software from the National Institutes of Health. Results: Re-expression of OPN in BRMS1 expressing breast cancer cells was achieved and resulted in increased number and size of colonies in soft agar. Quantification of total metastatic burden within LN and lung revealed that re-expression of OPN significantly increased (p < 0.05) metastatic tumor burden within the LN but not in lung. In LN, metastatic burden was 76% for 435, 2% for 435/BRMS1, and 35% for 435/BRMS1/OPN cells. In lung, metastatic burden was 38% for 435, 1% for 435/BRMS1, and 3% for 435/BRMS1/OPN cells. Conclusion: Re-expression of OPN in 435/BRMS1 cells increased number and size of colonies in soft agar, thus OPN down-regulation by BRMS1 may be responsible for decreased anchorage independent growth in vitro . Mice injected with 435/BRMS1/OPN cells developed significantly more LN, but not lung, metastases than 435/BRMS1 cells. Thus, OPN down-regulation by BRMS1 may be responsible, in part, for decreased LN metastatic burden but did not appear to be involved in decreased lung metastatic burden. These novel studies clarify one mechanism by which BRMS1 can suppress metastasis, which may lead to new therapeutic approaches for suppressing breast cancer metastasis based on this understanding.

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.

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.

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. BioEssays 24:885–893, 2002. © 2002 Wiley Periodicals, Inc.

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.

Metastasis is responsible for most cancer deaths. A better understanding of the process provides opportunities to develop new treatments to prevent metastasis. This article summarizes findings from experimental in vivo videomicroscopy and quantitative studies on metastatic inefficiency, which indicate that early steps in hematogenous metastasis may be quite efficient, but that regulation of cancer cell growth in secondary sites determines metastatic outcome. The authors have identified three key stages of this growth regulation: survival of a subset of single cells, proliferation of a subset of these cells to form preangiogenic micrometastases, and persistence of growth of a subset of these to form vascularized metastases. Formation of clinically relevant metastases is determined by the proportion of cells that proceeds successfully through each stage, and surviving single cells and preangiogenic micrometastases both represent possible sources of tumor dormancy.

Metastasis is responsible for most cancer deaths. Therapeutic strategies to prevent development of metastases thus have potential to impact on cancer mortality. Development of these therapies requires a better understanding of the biology and molecular events of the metastatic process. Metastasis is usually defined, clinically and experimentally, by evidence of the endpoint of the process, that is, the presence of metastatic tumors. Endpoint assays are suitable for determining if a therapeutic approach is effective, but can provide little information on how a treatment works in vivo and what steps in metastasis are affected. We describe here two methodological advances in the study of metastasis as a process: in vivo videomicroscopy, which permits direct observation of steps in metastasis, and a "cell accounting" technique that permits quantification of the fate of cells over time. These procedures have provided new and unexpected insights into the biology of the metastatic process. Based on these insights, we consider which steps in the metastatic process are biologically and clinically most appropriate as therapeutic targets for development of anti-metastasis therapies. We conclude that the most promising stage of the metastasis process for therapeutic targeting is the growth phase, after cancer cells have arrested in the microcirculation in secondary sites and have completed extravasation. Earlier phases in the process are either biologically inappropriate or clinically inaccessible, except in specific cases (e.g., chemoprevention strategies). The role of "seed" and "soil" in determining organ-specific metastasis is also discussed. The metastatic growth phase fortunately is a clinically broad target, and any treatment that limits growth of metastases prior to their causing irreversible harm to the patient has the potential to be clinically useful. A variety of therapeutic approaches to target this phase are under active development, including inhibition of angiogenesis or signal transduction pathways needed to support the growth of metastatic cells.

Matrix metalloproteinases (MMPs) have been shown to contribute functionally to tumor metastasis. MMP inhibitors are thus being assessed for clinical utility as anti-metastatic therapeutics. Batimastat (BB-94) is a synthetic MMP inhibitor that has been shown to inhibit tumor growth and metastasis in mice. Here we assessed the ability of batimastat to inhibit liver metastases of murine B16F1 cells, after injection of cells in mice via mesenteric vein to target the liver. We then determined which of the sequential steps in metastasis were affected by batimastat, in order to identify its mechanism of action in vivo. Intravital videomicroscopy was used to assess the effect on extravasation, and a 'cell accounting' procedure was used to determine the effect on initial survival of cells. Stereological quantification of functional blood vessels was used to determine the effect on tumor vascularity, thereby avoiding problems associated with immunohistochemical detection of liver sinusoidal endothelial cells. We found that batimastat (50 mg/kg i.p. 5 h prior to and after cell injection, daily thereafter) resulted in a 23% reduction in mean diameter of liver metastases (equivalent to a 54% reduction in tumor volume), while not reducing the number of metastases. Extravasation of cells from the liver circulation was not affected: at 8, 24 and 48 h after injection of cells, the same proportion of cells had extravasated from treated vs. control mice. Batimastat also did not inhibit early survival of cells. However, batimastat-treated mice had a significantly reduced percentage vascular volume within liver metastases, indicating inhibition of angiogenesis. This study demonstrates in vivo that the mechanism by which batimastat limits growth of B16F1 metastases in liver is not by affecting extravasation, but by inhibiting angiogenesis within metastases. This finding suggests that MMP inhibitors may be appropriate for use in patients with metastatic cells that have already extravasated in secondary sites.

Carboxyamidotriazole (CAI), an inhibitor of calcium-mediated signal transduction, is a promising new cytostatic anti-cancer drug which has entered Phase II clinical trials, and for which multiple modes of action have been proposed. We tested the hypothesis that CAI can inhibit tumor angiogenesis in vivo. The ability of orally administered CAI to inhibit experimental metastases of B16F1 melanoma cells in mouse liver was assessed. A computer-assisted stereological technique was then used to analyze images from histological sections of CAI-treated vs. control livers; the vascular volume percentage (percentage of tumor volume consisting of functional microvessels) was determined to assess the effect of CAI on tumor angiogenesis. CAI treatment significantly reduced the size (8 × reduction in volume; P = 0.02) but not the number of metastases. In association with this reduction in tumor size, CAI significantly decreased the vascular volume percentage within metastases by at least a factor of two (P = 0.001). A reduction in both number of microvessels/mm2 and microvessel size (cross-sectional area) was found to contribute to this decrease. CAI treatment did not affect the vascular volume percentage of normal liver tissue surrounding metastases (P = 0.8). This study documents for the first time that CAI can inhibit tumor angiogenesis within metastases in vivo.

Preclinical in vivo studies of agents targeted against metastasis have to date been based primarily on end-point assays. Such assays can determine whether a treatment affects the number or size of metastases in an organ at a given time, but are poorly suited to determining how and at what stage in the process the treatment affected the end point. High resolution in vivo videomicroscopy permits direct observation of the process of metastasis as it occurs in living animals over time. Studies based on this technique and a cell accounting procedure we have devised, have shown that early steps in the metastatic process (survival in the circulation, extravasation) contribute relatively little to cell loss and metastatic inefficiency. Steps that occur after extravasation appear to be primarily responsible for the significant losses that result in metastatic inefficiency, and these steps may represent good targets for the design of new antimetastatic therapies. Matrix metalloproteinases have been implicated functionally in metastasis, and are viewed as an appropriate target in the development of inhibitors of metastasis. Using both endogenous and synthetic exogenous metalloproteinase inhibitors, we have shown that the inhibition of metastasis which these agents produce is not due to inhibition of cell extravasation from the circulation into the tissue, but to reduction of angiogenesis within metastases. A similar conclusion was reached concerning the mechanism of action, on metastasis, of carboxyamidotriazole, an inhibitor of calcium-mediated signal transduction which is currently in Phase II single agent clinical trials. In vivo videomicroscopy of sequential steps in metastasis, coupled with methods that allow precise quantification of cell loss at specific steps in the metastatic process, as well as standard histological assessment at stages identified as crucial, allow characterization of the details of metastasis as an ongoing process. This provides a powerful complement to end-point assays, for it allows mechanistic information to be obtained from in vivo experiments, an approach which provides better understanding of how and when a drug may function in vivo to inhibit metastasis.

Metastases are responsible for the majority of failures in cancer treatment. Clarifying steps in metastasis and their molecular mechanisms will be important for the development of anti-metastasis therapeutic strategies. Considerable progress has been made in identifying molecules involved in metastasis. However, because of the nature of assays that have been available, conclusions about steps in metastasis and their molecular bases have been drawn primarily from inference. In order to complete the picture of how metastases form, a technique is needed to directly watch the processin vivo as it occurs over time. We have developed an intravital videomicroscopy (IVVM) procedure to make such observations possible. Results from IVVM are providing us with new conceptual understanding of the metastatic process, as well as the nature and timing of the contributions of molecules implicated in metastasis (e.g. adhesion molecules and proteinases). Our findings suggest that early steps in metastasis, including hemodynamic destruction and extravasation, may contribute less to metastatic inefficiency than previously believed. Instead, our results suggest that the control of post-extravasation growth of individual cancer cells is a significant contributor to metastatic inefficiency. Thus, this stage may be an appropriate target for design of novel strategies to prevent metastases.

We examined the extravasation and subsequent migration and growth of murine mammary tumor cell lines (D2A1 and D2.OR) which differ in their metastatic ability in lung and liver, invasiveness in vitro and expression of the cysteine proteinase cathepsin L. In light of the differences in invasiveness and cathepsin L expression, we hypothesized that during hematogenous metastasis the two cell lines would differ primarily in their ability to extravasate. We used in vivo videomicroscopy of mouse liver and chick embryo chorioallantoic membrane to examine the process and timing of extravasation and subsequent steps in metastasis for these cell lines. In contrast to our expectations, no differences were found between the cell lines in either the timing or mechanism of extravasation, at least 95% of cells having extravasated by 3 days after injection. However, after extravasation, the more metastatic and invasive D2A1 cells showed a greater ability to migrate to sites which favor tumor growth and to replicate to form micrometastases. These studies point to post-extravasation events (migration and growth) as being critical in metastasis formation.