Ian C. MacDonald

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.

Metastasis: 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.

Multistep Nature of Metastatic Inefficiency : Dormancy of Solitary Cells after Successful Extravasation and Limited Survival of Early Micrometastases

In cancer metastasis, only a small percentage of cells released from a primary tumor successfully form distant lesions, but it is uncertain at which steps in the process cells are lost. Our goal was to determine what proportions of B16F1 melanoma cells injected intraportally to target mouse liver 1) survive and extravasate, 2) form micrometastases (4 to 16 cells) by day 3, 3) develop into macroscopic tumors by day 13, and 4) remain as solitary dormant cells. Using in vivo videomicroscopy, a novel cell accounting assay, and immunohistochemical markers for proliferation (Ki-67) and apoptosis (TUNEL), we found that 1) 80% of injected cells survived in the liver microcirculation and extravasated by day 3, 2) only a small subset of extravasated cells began to grow, with 1 in 40 forming micrometastases by day 3, 3) only a small subset of micrometastases continued to grow, with 1 in 100 progressing to form macroscopic tumors by day 13 (in fact, most micrometastases disappeared), and 4) 36% of injected cells remained by day 13 as solitary cancer cells, most of which were dormant (proliferation, 2%; apoptosis, 3%; in contrast to cells within macroscopic tumors: proliferation, 91%; apoptosis/necrosis, 6%). Thus, in this model, metastatic inefficiency is principally determined by two distinct aspects of cell growth after extravasation: failure of solitary cells to initiate growth and failure of early micrometastases to continue growth into macroscopic tumors.

Steps in tumor metastasis: new concepts from intravital videomicroscopy

SummaryMetastases 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.

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.

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.

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.

Early interactions of cancer cells with the microvasculature in mouse liver and muscle during hematogenous metastasis: videomicroscopic analysis

Biomechanical interactions of cancer cells with the microvasculature were studied using high resolution intravital videomicroscopy. We compared initial arrest of murine B16F10 melanoma and D2A1 mammary carcinoma cells fluorescently labelled with calcein-AM, in low pressure (liver) vs high pressure (cremaster muscle) microvascular beds. Cells were arrested due to size restriction at the inflow side of the microcirculation, penetrating further and becoming more deformed in muscle than liver [median length to width ratios of 3.3 vs 1.3 for D2A1 cells, and 2.5 vs 1.2 for B16F10, at 1 min post-injection (p.i.)]. During the next 2 h many cells became stretched, giving maximum length to width ratios of 68 vs 22.1 (D2A1) and 28 vs 5.6 (B16F10) in muscle vs liver. Ethidium bromide exclusion demonstrated that over 97% of the cells maintained membrane integrity for > 2 h p.i. (In contrast, when an acridine orange labelling procedure was used, membrane disruption of B16F10 cells occurred within 15 min p.i.) Our experiments do not indicate the ultimate fate of the cancer cells, but if cell lysis occurs it must be on a time scale of hours rather than minutes. We report a process of ‘clasmatosis’ in cancer cells arrested in the microcirculation: large membrane-enclosed fragments (>3 µm in diameter) became ‘pinched off’ from arrested cells, in both liver and muscle, often within minutes or even seconds of arrest. The significance of this process is not yet understood. In this study intravital videomicroscopy has thus provided a valuable clarification of the interactions of cancer cells with vessel walls during metastasis.

Mammary carcinoma cell lines of high and low metastatic potential differ not in extravasation but in subsequent migration and growth.

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, invasivenessin vitroand 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 usedin vivovideomicroscopy 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.