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.

Inhibition of angiogenesis in liver metastases by carboxyamidotriazole (CAI).

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 x 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.

Fate of melanoma cells entering the microcirculation: over 80% survive and extravasate.

Metastasis is an inefficient process; only a few cancer cells are able to form tumors after being released into the circulation. We studied the fate of cancer cells after injection into the circulation, quantifying their survival and ability to extravasate by 1 day later. B16F10 cells, parental or transfectants overexpressing tissue inhibitor of metalloproteinases 1, were injected i.v. into chorioallantoic membrane of chick embryos and analyzed by intravital videomicroscopy. Cell survival was quantified in two ways: (a) 15-microns microspheres were injected with cancer cells, and proportions of viable cells to microspheres were compared before and after injection; and (b) individual cancer cells were monitored continuously for 0.5-8-h intervals covering the first 24 h. Both methods showed virtually no destruction of cells. Greater than 80% of injected cells survived and extravasated by 24 h, indicating that growth after extravasation is a key stage of metastatic control.