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.
Death from cancer is usually the result of dissemination of cancer cells from a primary tumor to secondary vital organs, and the formation of metastases. This process involves a series of steps, each of which have become targets of anticancer therapies such as intravasation of cancer cells into the bloodstream or lymphatics, delivery to organs (e.g., liver, lung, bone, brain, and lymph nodes), extravasation of cells into the organ parenchyma, cell proliferation to form secondary tumors, and development of new blood vessels to sustain continued growth (1). Importantly, single metastatic cells (2,3) or prevascular micrometastases (4) may also remain dormant within an organ, persisting until conditions are suitable for proliferation. Therefore, while surgical treatment of the primary tumor may be successful, undetectable dormant single metastatic cells or prevascular micrometastases can remain clinically silent for long periods and may eventually result in tumor formation and patient relapse (3,4).Metastasis to the brain can occur with many tumor types, including breast cancer, lung cancer, and melanoma. For breast cancer patients, the prevalence of brain metastases was historically estimated at 10 -16% with a 1-year survival rate of 20% (5). More recent studies, however, have demonstrated the prevalence of brain metastases in breast cancer patients to be closer to 22-30% (6), suggesting that its incidence may be increasing as a sanctuary site as systemic control improves. Brain metastases are typically treated with stereotactic radiosurgery or surgery with whole-brain radiation, supplemented with corticosteroid therapy for symptomatic relief. Patchell et al. (7) reported that surgery and whole-brain radiation can cure up to 90% of solitary brain metastases, which suggests that undiagnosed micrometastases or dormant cells are responsible for treatment failure. Thus, identification of micrometastatic and dormant brain metastatic tumor cells may facilitate an understanding of their biology and development of therapeutic interventions.For brain metastases of breast cancer, only a handful of experimental model systems have been reported. Yoneda et al. (8) performed six rounds of selection of human MDA-MB-231 breast carcinoma cells for brain metastasis in mice, followed by excision of the lesion and establishment of a cell culture. The resulting MDA-MB-231BR "brain-seeking" clone metastasized to the brain following intracardiac injection in 100% of the mice. Metastasis was identified histologically, which provided only one time point per animal. Clearly, studies of the metastatic process would greatly benefit from techniques that could dynamically monitor metastases from their earliest stage to endstage growth throughout entire organs or animals. This
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.
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