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.

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.

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.

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 process in 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.