Modulation of in vivo migratory function of alpha 2 beta 1 integrin in mouse liver.

We report herein that expression of alpha 2 beta 1 integrin increased human erythroleukemia K562 transfectant (KX2C2) cell movement after extravasation into liver parenchyma. In contrast, a previous study demonstrated that alpha 2 beta 1 expression conferred a stationary phenotype to human rhabdomyosarcoma RD transfectant (RDX2C2) cells after extravasation into the liver. We therefore assessed the adhesive and migratory function of alpha 2 beta 1 on KX2C2 and RDX2C2 cells using a alpha 2 beta 1-specific stimulatory monoclonal antibody (mAb), JBS2, and a blocking mAb, BHA2.1. In comparison with RDX2C2 cells, KX2C2 were only weakly adherent to collagen and laminin. JBS2 stimulated alpha 2 beta 1-mediated interaction of KX2C2 cells with both collagen and laminin resulting in increases in cell movement on both matrix proteins. In the presence of Mn2+, JBS2-stimulated adhesion on collagen beyond an optimal level for cell movement. In comparison, an increase in RDX2C2 cell movement on collagen required a reduction in its adhesive strength provided by the blocking mAb BHA2.1. Consistent with these in vitro findings, in vivo videomicroscopy revealed that alpha 2 beta 1-mediated postextravasation cell movement of KX2C2 cells in the liver tissue could also be stimulated by JBS2. Thus, results demonstrate that alpha 2 beta 1 expression can modulate postextravasation cell movement by conferring either a stationary or motile phenotype to different cell types. These findings may be related to the differing metastatic activities of different tumor cell types.

Comparison of biologic behavior and histology of transplanted mammary tumors in GR mice.

Mammary tumors that arose spontaneously in inbred GR mice were transplanted into syngeneic castrated males. The hormone responsiveness of the transplants was studied in mice treated with estrone and progesterone and was compared with the hormone responsiveness in mice that received no hormone treatment. Microscopic examination of hormone-responsive and hormone-independent tumors revealed similar histologic patterns in both groups. It was evident that pale cells, which are classically associated with hormone-responsive tumors, may also be present in transplanted hormone-independent tumors in this strain. No correlation was found between the histologic pattern of these transplanted tumors and the biologic behavior, hormonal status, or presence of a specific murine mammary tumor virus (MuMTV) proviral fragment. Mammary tumors also appeared capable of undergoing differentiation into more than one morphologic type. Two cotransplanted tumors (derived from the same parental tumor) had markedly different histologic patterns; however, analysis of MuMTV proviral fragments indicated that the MuMTV-infected cells were of the same parentage.

Early steps in hematogenous metastasis of B16F1 melanoma cells in chick embryos studied by high-resolution intravital videomicroscopy.

BACKGROUND: There are few techniques that permit direct observation of tumor metastasis. The ability to observe steps in this process as they occur in experimental animals would complement studies on molecular mechanisms. PURPOSE: We have developed a novel procedure using high-resolution intravital videomicroscopy to permit direct observation of cells as they arrest in the microcirculation, extravasate, and form micrometastases. We used this procedure to study early steps in experimental metastasis in immune-deficient chick embryos, permitting us to develop this technique in a relatively accessible respiratory organ and in the absence of host immune responses. Our goals were to develop techniques applicable to this host and to other hosts and to clarify the process of hematogenous tumor spread in this host. METHODS: We injected fluorescently labeled B16F1 melanoma cells into the circulation of 11- to 13-day chick embryos, and using intravital videomicroscopy, we observed the cells in the chorioallantoic membrane over time. RESULTS: The majority of injected cells were trapped initially in orifices to the chorioallantoic membrane capillary plexus or in tapering ends of arterioles leading to the plexus. During the first 2 hours, cells were found only in vessel lumina. After 8 hours, 83% of cells had extravasated, and the rest were in the process of extravasation. Cell shape changes and pseudopodial extensions were seen during extravasation and tumor development. Tumor cell division was seen only after extravasation. Tumors tended to develop near microvessels and were often wrapped around them. CONCLUSIONS: Intravital videomicroscopy can provide new information about steps in metastasis. This procedure is applicable to other hosts and can be used in future studies to test hypotheses about molecular mechanisms of tumor spread.

An epitope on VLA-6 (alpha6beta1) integrin involved in migration but not adhesion is required for extravasation of murine melanoma B16F1 cells in liver.

VLA-6 (alpha6beta1) integrin represents the major receptor for interaction with laminin substrate. It has been proposed that VLA-6 mediates tumor cell adhesion to the endothelium during extravasation. We have further explored this possibility using mouse melanoma B16F1 cells, which express VLA-6 as the principal laminin receptor, and two VLA-6 monoclonal antibodies (mAbs), MA6 and GoH3. Adhesion is a prerequisite of cell movement on matrix proteins. Thus, GoH3, which inhibited VLA-6-mediated adhesion, blocked cell movement on laminin. The recently prepared alpha6 integrin-specific mAb MA6 bound to an epitope in close proximity to GoH3, but it had no effect on VLA-6-mediated cell adhesion. We report here that although MA6 did not affect adhesion, it blocked mouse melanoma B16F1 cell movement on laminin to the same extent as GoH3. Results therefore demonstrate an active role of VLA-6 in providing cell movement as well as the initial adhesive event on laminin. In addition, mAb MA6 had no effect on the induction of tyrosine phosphorylation of focal adhesion kinase upon adhesion of B16F1 cells to laminin. Therefore, inhibition of cell movement by MA6 involved mechanism(s) other than an interference of VLA-6 signaling events leading to phosphorylation of focal adhesion kinase. The epitopes of GoH3 and MA6 may represent spatially and temporally related sites on VLA-6 that are involved during cell movement, or, alternatively, MA6 may inhibit the interaction of VLA-6 with associated cell surface molecules required for cell movement. In vivo videomicroscopy experiments also revealed that an inhibition of VLA-6 migratory function by MA6 resulted in a reduction in the ability of B16F1 to extravasate during hematogenous metastasis in the liver.

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.

Overexpression of metalloproteinase inhibitor in B16F10 cells does not affect extravasation but reduces tumor growth.

It is widely accepted that a major role of matrix metalloproteinases in the metastatic process is degradation of basement membrane during cancer cell invasion. We tested the hypothesis that the reduction in metastatic potential which has been demonstrated for B16F10 melanoma cells genetically engineered to overexpress tissue inhibitor of metalloproteinase-1 (TIMP-1) is caused by a decrease in their ability to extravasate. Using intravital videomicroscopy of chick embryo chorioallantoic membrane, we studied extravasation of B16F10 cells and B16F10 cells transfected to overexpress TIMP-1. More than 800 cells in 36 chick embryos were analyzed for each cell line during 72 h postinjection. TIMP-1 upregulation had no effect on the time course of extravasation, virtually all cells from both cell lines having extravasated by 36 h. We also studied the morphology of micrometastases at days 3 and 7. Lack of contact between cancer cells within micrometastases at day 3 and reduction in size and number of tumors at day 7 were observed for TIMP-1 overexpressor cells compared to B16F10. Our findings illustrate that the imbalance between TIMP and metalloproteinases created by overexpression of TIMP-1 in B16F10 cells reduces their metastatic ability in vivo by affecting tumor growth postextravasation.

Temporal progression of metastasis in lung: cell survival, dormancy, and location dependence of metastatic inefficiency.

Cancer metastasis is an inefficient process. The steps in metastasis responsible for this inefficiency and how metastatic inefficiency can vary in different locations within an organ remain poorly understood. B16F10 cells were injected to target mouse lung, and at sequential times thereafter we quantified in lung the time course of: (a) overall cell survival and metastatic development; and (b) local cell survival and growth with respect to the lung surface and specific interior structures. We found high rates of initial survival of cells trapped in the lung circulation, extravasation into lung tissue, and subsequent survival of extravasated solitary cells (74% at day 3) before metastasis formation. However, at the time of initial replication of metastatic cells a major loss of cells occurred. Although only a small proportion of injected cells started to form metastases, most of these developed into macroscopic tumors. Solitary cells found at later times were dormant. Thus, overall metastatic inefficiency was largely due to postextravasation events affecting solitary cells. Regionally within the lung, cells and metastases were randomly distributed to day 4, but by day 10 preferential tumor growth was found along the lung surface and around arterial and venous vessels. Thus, trapping and early growth of injected cells was unaffected by location within the lung, whereas subsequent metastatic growth was enhanced in specific microenvironments. This study: (a) quantifies early temporal and spatial progression of metastasis in lung; (b) documents persistence of solitary dormant cells; and (c) shows that metastatic inefficiency depends on the initiation of growth in a subset of extravasated cells, whereas continued growth of metastases occurs preferentially in specific tissue environments.

Integrin VLA-2 (alpha2beta1) function in postextravasation movement of human rhabdomyosarcoma RD cells in the liver.

It is now known that members of the selectin and integrin families are critical in the initial interaction of cells in circulation with endothelial surfaces. Also, platelet/endothelial cell adhesion molecule-1 has been shown to be involved in transendothelial migration of extravasating cells. Little is known about adhesion molecules involved in subsequent postextravasation events. In this study, the significance of VLA-2 (alpha2beta1) integrin in the movement of human rhabdomyosarcoma RD cells in the liver was characterized by in vivo videomicroscopy. Results show that after extravasation, the mock-transfected RDpF cells were able to migrate to the subcapsular region of the liver. Although the RDX2C2 transfectant expressing VLA-2 integrin extravasated equally well, a majority of RDX2C2 cells remained in close proximity to blood vessels and failed to reach the subcapsular region. The functional involvement of VLA-2 in affecting the ability of RD cells to reach the subcapsular region was verified by the preparation of an RD transfectant [RDX2C2(I-)] expressing a nonfunctional variant of VLA-2 lacking the inserted (I)-domain of alpha2 subunit. In vivo microscopy showed that RDX2C2(I-) cells migrated in a manner similar to control RDpF cells. To demonstrate that RDX2C2 cells that remained in dose proximity to blood vessels were due to VLA-2 function, a blocking monoclonal antibody against VLA-2 (BHA2.1) was prepared. Mice were injected with BHA2.1 or control monoclonal antibody P3 at the time when RDX2C2 cells completed their extravasation. Treatment with BHA2.1 increased the number of RDX2C2 cells that reached the subcapsular region and subsequently formed tumor foci. Therefore, VLA-2 integrin expression has major roles in postextravasation movement and affects tumor foci formation at the liver surface.

Clinical targets for anti-metastasis therapy.

Metastasis is responsible for most cancer deaths. Therapeutic strategies to prevent development of metastases thus have potential to impact on cancer mortality. Development of these therapies requires a better understanding of the biology and molecular events of the metastatic process. Metastasis is usually defined, clinically and experimentally, by evidence of the endpoint of the process, that is, the presence of metastatic tumors. Endpoint assays are suitable for determining if a therapeutic approach is effective, but can provide little information on how a treatment works in vivo and what steps in metastasis are affected. We describe here two methodological advances in the study of metastasis as a process: in vivo videomicroscopy, which permits direct observation of steps in metastasis, and a "cell accounting" technique that permits quantification of the fate of cells over time. These procedures have provided new and unexpected insights into the biology of the metastatic process. Based on these insights, we consider which steps in the metastatic process are biologically and clinically most appropriate as therapeutic targets for development of anti-metastasis therapies. We conclude that the most promising stage of the metastasis process for therapeutic targeting is the growth phase, after cancer cells have arrested in the microcirculation in secondary sites and have completed extravasation. Earlier phases in the process are either biologically inappropriate or clinically inaccessible, except in specific cases (e.g., chemoprevention strategies). The role of "seed" and "soil" in determining organ-specific metastasis is also discussed. The metastatic growth phase fortunately is a clinically broad target, and any treatment that limits growth of metastases prior to their causing irreversible harm to the patient has the potential to be clinically useful. A variety of therapeutic approaches to target this phase are under active development, including inhibition of angiogenesis or signal transduction pathways needed to support the growth of metastatic cells.

Analysis of JHM central nervous system infections in rats.

Intracerebral inoculation of murine coronavirus JHM into 2- to 3-day-old Wistar Furth rats causes an acute encephalomyelitis, while inoculations at 10 days of age usually result in hind leg paralysis. To examine the distribution of viral antigens within this infected central nervous system (CNS) tissue, we used the avidin-biotin-peroxidase method to detect monoclonal and polyclonal antibodies bound to JHM structural proteins; in addition we used the Western blot technique to detect viral proteins. Our study demonstrated the following characteristics: Infected neuronal and glial cells produced viral nucleocapsid and E2 glycoprotein. The synthesis of these viral structural proteins was not restricted to cells in any particular part of the central nervous system. While JHM E2 proteins could be detected in individual cells of JHM-infected CNS tissue, the relative level of detectable E2 protein in the total CNS tissue of infected rats was reduced by more than 13-fold compared with JHM-infected tissue culture cells. Hippocampus neuronal cells provided a sensitive indication of JHM infection. These cells invariably contained antigens in both acutely and chronically infected animals. The distribution of cells containing viral antigens differed markedly for JHM-induced acute encephalitis and chronic demyelinating disease. Acutely infected brains had large lesions containing low levels of viral antigen scattered throughout the brain. One percent to ten percent of histologically normal cells in many parts of the brain contained viral antigens; in addition, more neuronal cells than glial cells were observed to be antigen-positive. The hippocampus appeared normal with hematoxylin-eosin staining; however, a scattered infection of neuronal cells was apparent.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor progression and metastasis in murine D2 hyperplastic alveolar nodule mammary tumor cell lines.

We have examined tumor progression and metastatic properties of three clonal murine mammary tumor cell lines of recent origin (D2A1, D2.OR and D2.1). These lines were derived from spontaneous mammary tumors which originated from a D2 hyperplastic alveolar nodule (HAN) line. D2A1 cells were more malignant than D2.OR or D2.1 cells, whether measured by experimental metastasis assays after intravenous injection in nude mice or chick embryos, in vivo growth rate of primary tumors following mammary fat pad injection in nude mice, or spontaneous metastasis assay from primary tumors growing in mammary fat pads. D2A1 cells also were more invasive in vitro in a Matrigel invasion assay than D2.1 cells, while the D2.OR cells were non-invasive in this assay. The increased invasiveness and malignancy of D2A1 cells were associated with increased levels of mRNA for the cysteine proteinase cathepsin L. Levels of osteopontin (OPN), nm23, int-1 and int-2 mRNAs were also examined. Nm23 levels were highest in the most malignant cell line. These cell lines provide a model for studying the tumorigenic and metastatic ability of mammary tumor cells and offer several advantages: they were cloned from mammary tumors that originate from a common source of preneoplastic cells (D2HAN); they are of relatively recent origin; and they have spontaneously arrived at different stages of tumor progression.

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, invasiveness in vitro and 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 used in vivo videomicroscopy 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.

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 microns 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.

The matrix metalloproteinase inhibitor batimastat inhibits angiogenesis in liver metastases of B16F1 melanoma cells.

Matrix metalloproteinases (MMPs) have been shown to contribute functionally to tumor metastasis. MMP inhibitors are thus being assessed for clinical utility as anti-metastatic therapeutics. Batimastat (BB-94) is a synthetic MMP inhibitor that has been shown to inhibit tumor growth and metastasis in mice. Here we assessed the ability of batimastat to inhibit liver metastases of murine B16F1 cells, after injection of cells in mice via mesenteric vein to target the liver. We then determined which of the sequential steps in metastasis were affected by batimastat, in order to identify its mechanism of action in vivo. Intravital videomicroscopy was used to assess the effect on extravasation, and a 'cell accounting' procedure was used to determine the effect on initial survival of cells. Stereological quantification of functional blood vessels was used to determine the effect on tumor vascularity, thereby avoiding problems associated with immunohistochemical detection of liver sinusoidal endothelial cells. We found that batimastat (50 mg/kg i.p. 5 h prior to and after cell injection, daily thereafter) resulted in a 23% reduction in mean diameter of liver metastases (equivalent to a 54% reduction in tumor volume), while not reducing the number of metastases. Extravasation of cells from the liver circulation was not affected: at 8, 24 and 48 h after injection of cells, the same proportion of cells had extravasated from treated vs. control mice. Batimastat also did not inhibit early survival of cells. However, batimastat-treated mice had a significantly reduced percentage vascular volume within liver metastases, indicating inhibition of angiogenesis. This study demonstrates in vivo that the mechanism by which batimastat limits growth of B16F1 metastases in liver is not by affecting extravasation, but by inhibiting angiogenesis within metastases. This finding suggests that MMP inhibitors may be appropriate for use in patients with metastatic cells that have already extravasated in secondary sites.

Tumour metastasis to the liver, and the roles of proteinases and adhesion molecules: new concepts from in vivo videomicroscopy.

Most preclinical studies of tumour metastasis and effects of molecular interventions have been based on end point assays, and little is known about the fate of cells at sequential steps in the metastatic process. In vivo videomicroscopy permits direct observations of sequential steps in hematogenous metastasis as they occur in living animals over time. These steps include initial arrest of cells in the microcirculation, extravasation, postextravasation migration and growth in the target organ. In the mouse liver model, cells are arrested in periportal sinusoids based on size restriction, survive in the circulation and extravasate into the tissue by 48 to 72 h regardless of metastatic potential. Thereafter, cells may migrate to preferred sites for growth. Critical steps responsible for cell losses and metastatic inefficiency occur at the level of postextravasation cell growth. Many extravasated cells may remain dormant, and growth to form micrometastases is initiated in only a small subset of cells. Most early micrometastases may disappear after a few days, and only a small subset continue growth into macroscopic tumours. Angiogenesis is a prerequisite for continued growth of metastases, as shown previously by others. Integrin based interventions can modulate postextravasation cell migration and cell growth. Matrix metalloproteinase inhibitors can inhibit tumour angiogenesis and thus reduce growth. Key targets against which future therapeutic strategies should be directed include the initiation and maintenance of growth of micrometastases, and the activation of dormant solitary cells.

Threshold levels of ERK activation for chemotactic migration differ for NGF and EGF in rat pheochromocytoma PC12 cells.

In a previous study, we show that stimulation of chemotaxis in rat pheochromocytoma PC12 cells by nerve growth factor (NGF) and epidermal growth factor (EGF) requires activation of the RAS-ERK signaling pathway. In this study, we compared the threshold levels of ERK activation required for EGF and NGF-stimulated chemotaxis in PC12 cells. The threshold ERK activity required for NGF to stimulate chemotaxis was approximately 30% lower than that for EGF. PD98059 treatment inhibited EGF stimulation of growth and chemotaxis; however, stimulation of chemotaxis required an EGF concentration approximately 10 times higher than for stimulation of PC12 cell growth. Thus, ERK-dependent cellular functions can be differentially elicited by the concentration of EGF. Also, treatment of PC12 cells with the PI3-K inhibitor LY294002 reduced ERK activation by NGF; thus, higher NGF concentrations were required to initiate chemotaxis and to achieve the same maximal chemotactic response seen in untreated PC12 cells. Therefore, the threshold NGF concentration to stimulate chemotaxis could be adjusted by the crosstalk between the ERK and PI3-K pathways, and the contributions of PI3-K and ERK to signal chemotaxis varied with the concentrations of NGF used. In comparison, LY294002 treatment had no effect on ERK activation by EGF, but the chemotactic response was reduced at all the concentrations of EGF tested indicating that NGF and EGF differed in the utilization of ERK and PI3-K to signal chemotaxis in PC12 cells.