Search

Metastasis is responsible for most cancer deaths. A better under standing 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.

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.

During our recent studies of the capillaries in exocrine pancreas of mouse, numerous local constrictions which reduced the luminal diameter were observed both by scanning electron microscopy of corrosion casts and by in vivo microscopy. In the present study we have identified the features responsible for the constrictions and compared the diameters of vessels and constrictions measured using the two methods. A simple theoretical model was used to predict the effects of such constrictions on blood flow in the acinar capillaries of the pancreas. Intravital observations revealed that bulging endothelial cells were primarily responsible for the constrictions. For samples of 100 measurements, good agreement was found between the mean capillary diameters from casts (6.3 microns +/- 0.50 SD) and in vivo (6.2 microns +/- 0.53 SD), but the mean diameter measurement at constrictions was greater (P < 0.01) in casts (3.9 microns +/- 0.84 SD) than in vivo (3.5 microns +/- 1.05 SD). Topical application of norepinephrine caused endothelial nuclear regions to bulge into the capillary lumen, decreasing the mean diameter at these locations to 3.3 microns +/- 0.9 (SD, n = 21). Based on the 100 in vivo measurements, the theoretical model predicted that, on average, the constrictions would reduce flows to 51% of those in fully open vessels. It is unlikely, however, that the constrictions observed in acinar capillaries of the pancreas of mouse would result in significant blockage of the vessels by red blood cells.

The chick embryo is a useful model for studying hematogenous metastasis. Cancer cells injected into veins of the chorioallantoic membrane (CAM) circulate briefly through all tissues but form metastases predominantly in the CAM. This respiratory organ is particularly suitable for intravital microscope because of its accessibility without the need for surgery and the density and planar configuration of its vessels (which we confirmed by microcorrosion casting). Using an inverted microscope with oblique transillumination for high-resolution images and epifluorescence to identify labeled B16F1 melanoma cells, we studied successive stages of metastasis formation in the CAM in vivo. By 2 min postinjection (pi) all cancer cells had become arrested within the microvasculature. This initial arrest appeared to be due to size restriction, based on measurements of cell and vessel diameters. At 15-60 min pi, trapped cells were seen in tapering arterioles (27%), orifices from arterioles to the capillary plexus (61%), or in the plexus itself (12%). Some cells had extravasated into the underlying mesenchyme by 3 hr (pi), and at 24 hr all cancer cells had completed this process. The mean rate of migration out of capillary lumens was approximately 1 micron/hr. Micrometastases grew in a planar configuration just beneath the capillary plexus, with a cell doubling time of approximately 24 hr. Our technique is also applicable to other tumor types and host animals and provides a powerful tool to complement studies on the molecular basis of metastasis.

The spleen concentrates blood to twice arterial hematocrit, but how it does so is poorly understood. We hypothesize that hemoconcentration results from percolation of blood through the reticular meshwork (RM) between capillary endings and venous channels. The RM has a large flow cross section, producing low shear rates, and an enormous surface area for red blood cell (RBC) adhesion. If these conditions cause slowing of RBCs with respect to plasma, increased hematocrits will occur (inverse Fahraeus effect). Movements of individual RBCs through the RM were studied from intravital microscopic videorecordings of Ringer-perfused mouse spleens. Analysis of three 70-microns paths showed characteristic "stop and go" motion, RBCs spending 0.015 to 9.71 see in any 7-microns segment, despite steady (+/- 15%) perfusate velocity. At some locations RBCs adhered to reticular cells/fibers by point attachment and at others they became caught over fibers. In general, RBCs were detained in the RM by surface interactions rather than by narrow channel. Perfusate ("plasma") velocity was estimated to be at least that of the fastest RBCs in each segment. Ratios of overall mean RBC and plasma velocities for three paths were 0.19, 0.20, and 0.37. Applying these velocity ratios to a feed hematocrit of 40% leads to predicted splenic hematocrits up to 78%. We conclude that this slowing of RBCs within the reticular meshwork provides the primary mechanism for generating the high intrasplenic hematocrit.

It is generally assumed that when a muscle is shortened or extended the total length of capillaries does not change, implying that capillaries are nondistensible, longitudinally. On the basis of stereological estimates of capillary anisotropy versus sarcomere length, we propose that as long as capillaries are in a tortuous configuration muscle extension will merely decrease the tortuosity, leaving vessel length unaltered. Once capillaries have been pulled into a straight configuration, further extension of the muscle will cause the vessels to stretch. By means of intravital videomicroscopy we have demonstrated that stretching of individual capillaries does indeed occur over a sarcomere length range of 2.1 to 2.9 μm in rat extensor digitorum longus muscle. In vivo measurements of the lengths of six capillaries together with the sarcomere lengths of adjacent fibers were made in muscles positioned at various degrees of extension. Normalized data indicated that four capillaries stretched to the same degree as the muscle, one stretched more and another less. This may reflect differences in distensibility or tortuosity of capillaries in series with one another. The elastic stretching of capillaries during muscle activity may have important consequences in terms of shifts in permeability and increases in capillary surface area.

We have videorecorded spontaneous cyclic contractions of capillary walls which often stopped the flow of blood cells, in spleens of rat and mouse. An inverted microscope and oblique lighting were key elements in obtaining images in which the boundaries of cells composing vessel walls were clearly distinguishable. Using slow motion replay, we measured the widths of endothelial cells ( C ), pericytes ( P , when present) and capillary lumens ( L ; at the site of constriction; U ; 15–20 μm upstream), throughout 11–12-min sequences containing many contraction/relaxation cycles. In roughly 50% of contractions L decreased to 0–1 μm, such luminal “closures” occurring within 2–12 sec and lasting for 1 min. Intervals between contractions ranged from 12 sec to 3 min (average 1 min). Documentation of one such cycle by sequential photographs from the video monitor is presented, showing dramatic bulging of an endothelial cell into the lumen. Comparison of records of L and C versus time showed that almost invariably when L decreased C increased, and vice versa; highly significant correlations existed between C and L in every case ( P C were responsible for 18–77% of the total variance in L , whereas P contributed only 0–4%; changes in U , covariance between C , P , and U , and unexplained variance were responsible for 0–20, 11–30, and 11–53%, respectively, of the total variance in L . We conclude that these spontaneous capillary contractions were primarily due to endothelial contractility.