Eric E. Schmidt

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.

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.

Intravital videomicroscopy of the chorioallantoic microcirculation: A model system for studying metastasis

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.

Kinetics of red blood cell passage through interendothelial slits into venous sinuses in rat spleen, analyzed by in vivo microscopy

Sequential photomicrographs of RBCs passing through interendothelial slits (IES) in walls of venous sinuses in rat spleen were obtained by video recording in vivo microscopic views. Kinetics of RBC passage were analyzed by slow-motion playback of recordings. An inverted microscope and oblique lighting from a water-cooled fiber optic light source were key elements in obtaining images of sufficient quality for analysis. The direction of RBC passage through IES was, invariably, from reticular spaces of the red pulp into venous sinuses. RBC flow through an individual IES occurred as brief discontinuous bursts, separated by periods of zero, or near zero, flow. Mean rates of RBC flow through six IES analyzed in normal relaxed spleen ranged from 1.4 to 9.1 cells/15 sec, the total RBCs studied being 1523 and the total combined period of observation 98 min. The maximum instantaneous rate was 10 RBCs/sec. RBC transit times ranged from 0.02 to 60.5 sec, even for a single IES; the distribution was highly skewed: median 0.23 sec, mean 1.7 sec. Analysis of RBC flow through two closely adjacent IES simultaneously, for 30 min, showed that most bursts were asynchronous. The results indicate that changes in caliber of IES are primarily responsible for the observed pattern of flow. It was estimated that only 19% of the total IES present anatomically actually allowed passage of RBCs during any 5-min period.

Preclinical Assessment of Anti-cancer Therapeutic Strategies using in vivo Videomicroscopy

Preclinical in vivo studies of agents targeted against metastasis have to date been based primarily on end-point assays. Such assays can determine whether a treatment affects the number or size of metastases in an organ at a given time, but are poorly suited to determining how and at what stage in the process the treatment affected the end point. High resolution in vivo videomicroscopy permits direct observation of the process of metastasis as it occurs in living animals over time. Studies based on this technique and a cell accounting procedure we have devised, have shown that early steps in the metastatic process (survival in the circulation, extravasation) contribute relatively little to cell loss and metastatic inefficiency. Steps that occur after extravasation appear to be primarily responsible for the significant losses that result in metastatic inefficiency, and these steps may represent good targets for the design of new antimetastatic therapies. Matrix metalloproteinases have been implicated functionally in metastasis, and are viewed as an appropriate target in the development of inhibitors of metastasis. Using both endogenous and synthetic exogenous metalloproteinase inhibitors, we have shown that the inhibition of metastasis which these agents produce is not due to inhibition of cell extravasation from the circulation into the tissue, but to reduction of angiogenesis within metastases. A similar conclusion was reached concerning the mechanism of action, on metastasis, of carboxyamidotriazole, an inhibitor of calcium-mediated signal transduction which is currently in Phase II single agent clinical trials. In vivo videomicroscopy of sequential steps in metastasis, coupled with methods that allow precise quantification of cell loss at specific steps in the metastatic process, as well as standard histological assessment at stages identified as crucial, allow characterization of the details of metastasis as an ongoing process. This provides a powerful complement to end-point assays, for it allows mechanistic information to be obtained from in vivo experiments, an approach which provides better understanding of how and when a drug may function in vivo to inhibit metastasis.

Microcirculation in mouse spleen (nonsinusal) studied by means of corrosion casts

Corrosion casts of mouse spleen, examined by scanning electron microscopy, enabled vascular pathways of the arterial, intermediate, and venous circulations to be traced over considerable distances. The arterial tree is surrounded by white pulp immediately upon entering at the hilus, and relatively few arterioles extend into red pulp. A profusion of capillaries is present in both periarterial lymphatic sheaths and lymphatic nodules, arranged as bifurcating systems (rather than anastomosing networks) terminating in the marginal sinus (MS) and marginal zone (MZ). The MS, which is situated between white pulp and MZ, consists of a discontinuous layer of flattened anastomosing spaces which are up to six times as large as those in rat spleen. Extensive filling of the entire MZ took place before appreciable filling of surrounding red pulp occurred. Capillary terminations in red pulp are always continuous with reticular meshwork, i.e., no evidence for a “closed” circulation was found. Casts of the venous origins support the classification “pulp venules” rather than “venous sinuses” and show major morphological differences from the richly anastomosing system of sinuses in rat. In the subcapsular region of mouse spleen large anastomosing veins ramify over the surface, with reticular meshwork occupying extensive areas between adjacent veins. For in vivo microscopy this arrangement offers advantages over that found in rat spleen (accompanying paper), where almost the entire surface is densely covered with venous sinuses.

Interactions of leukocytes with vessel walls and with other blood cells, studied by high-resolution intravital videomicroscopy of spleen.

Visualization of circulating leukocytes in vivo is difficult because their optical density differs so little from that of plasma. We have obtained intravital microscopic images of leukocytes at high resolution in spleens of rats and mice by means of an inverted microscope with a 100 x oil-immersion lens; oblique lighting gave improved contrast. Photographic evidence and quantitative data describing the behaviour of lymphocytes, polymorphonuclear leukocytes (PMNs), and macrophages within the microvasculature are presented. Mean numbers of marginated lymphocytes in venous vessels ranged from 0.1 to 4.5 per 1000 microns 2 of wall surface, and speeds of rolling from 11 to 20 microns/sec. Adherence times of leukocytes to vessel walls were log normally distributed, median values being 30, 130, and 560 sec, for lymphocytes, PMNs, and macrophages, respectively. Mean speeds of migration along luminal surfaces were similar (7-19 microns/min) for all three types of cells. Lymphocyte migration outward through the venular wall, observed on two occasions, took 1-2 min. Median values for duration of adherence of RBCs and lymphocytes to macrophages were 1 and 42 sec, respectively. Phagocytosis of a lymphocyte was observed and took 3 min. Macrophages often underwent dramatic changes in shape, including formation of a pseudopod up to 155 microns in length. High-resolution intravital videomicroscopy of spleen has great potential for studying leukocyte behaviour, e.g., homing and migration of lymphocytes, and immunologically related macrophage-lymphocyte interactions in vivo.

Microcirculation in rat spleen (Sinusal), studied by means of corrosion casts, with particular reference to the intermediate pathways

Blood vessels and their connections in rat spleen were traced over considerable distances by scanning electron microscopy of microcorrosion casts prepared by injection of minimal amounts of casting material. The periarterial lymphatic sheath and lymphatic nodules are highly developed, containing an abundance of capillaries which terminate in the marginal sinus (MS) and marginal zone (MZ). The MS, which consists of a series of discontinuous flattened vascular spaces interconnected by short capillaries, shows circumferential filling followed by flow radially outward into the MZ. Contrary to the generally accepted view, many venous sinuses begin as open‐ended tubes at the MS or MZ, allowing free entry of blood into the venous system, thereby bypassing the reticular meshwork of the red pulp. The majority of arterial capillaries terminate in the reticular meshwork (“open” circulation), but evidence for direct connections between capillaries and venous sinuses (“closed” circulation) was also obtained. Casts of the subcapsular region reveal an elaborate system of venous sinuses in fan‐shaped arrays, superimposed on an extensive network of capillaries draining into flattened reticular spaces; such casts provide a three‐dimensional map useful in interpreting light microscopic observations of red cell flow in vivo. Sphincter‐like constrictions in venous sinuses, at points of connection with larger sinuses, indicate that these are possible sites for control of intrasplenic flow distribution.