fibrin and Melanoma

Topics: Animals; Aorta; Cells, Cultured; Culture Techniques; Endothelium; Fibrin; Fibrinolysis; Humans; Kidney; Melanoma; Plasminogen Activators; Rats; Stress, Physiological; Swine; Thrombosis; Urokinase-Type Plasminogen Activator

Most metastases in patients occur as a result of hematogenous dissemination of tumor cells. This process of metastasis is complex and consists of several steps, foremost of which is the arrest of circulating emboli in capillary beds and the formation of a thrombus at that site. Thrombus formation in the metastasis of human cancer was described first by Billroth in 1878. It was reported that the organization of tumor cell emboli, and the subsequent penetration of tumor cells into the capillary wall, was the first stage of metastasis. Since then, many investigations and observations have been made clinically as well as experimentally to clarify the process (or mechanisms) of tumor cell arrest and how to inhibit it. Coagulative and fibrinolytic pathways were believed to have a main role in thrombus formation. However, other factors responsible for the relationship between tumor cells and the host must be also considered. Elegant and extensive studies by Fidler and Kripke demonstrated that development of metastasis is not a random process, but a selection process of specialized subpopulations of highly metastatic cells within the primary tumors. Biochemical constituents and ionic properties on cell surfaces, deformability or locomotive activities of tumor cells, as well as thrombo-plastic-fibrinolytic activities, are also important factors determining the arrest patterns of circulating tumor cells. On the other hand, host defense factors against tumor cells in the bloodstream have been attracting much attention recently in tumor immunology. Host defense factors relating the arrest of tumor cells to the establishment of metastatic foci seemed difficult to define, since many studies showed contradictory data concerning the influence of immune response on tumor cell arrest. Hemodynamic abnormality may also influence the arrest of tumor cells in the circulation. Hypercoagulability induced from host tissues is greatly associated with the arrest patterns. Platelet activities might affect thrombus formation. Nevertheless, exact explanations of the process or mechanisms inhibiting or enhancing the arrest of tumor cells after hematogenous dissemination have not been obtained. In any event, for cancer treatment, it is important to determine which substances inhibit the arrest of circulating tumor cells and how to prevent hematogenous metastasis. In this review, we will focus upon coagulative and fibrinolytic processes and then upon substances that inhibit the arrest of

Topics: Animals; Anticoagulants; Blood Coagulation; Fibrin; Fibrinolysis; Fibrinolytic Agents; Humans; Immunity, Cellular; Killer Cells, Natural; Melanoma; Mice; Neoplasm Metastasis; Neoplasms; Neoplastic Cells, Circulating; Platelet Aggregation; Rats; T-Lymphocytes, Cytotoxic

An increasing number of studies suggest inflammation stimulates tumour invasion. In melanoma, despite recent advances in targeted therapy and immunomodulatory therapies, this cancer remains difficult to treat. Our previous studies show melanoma cells interact with skin cells in their invasion into tissue engineered skin and suggest inflammation stimulates invasion. The aim of this study was to investigate the use of an anti-inflammatory on melanoma invasion. To do this we developed a wounded and inflamed in vitro 3D melanoma model in which to investigate the use of an anti-inflammatory on melanoma invasion. The tissue engineered skin model was based on human de-epidermised acellular dermis to which keratinocytes, fibroblasts and three different melanoma cell lines were added in various combinations. A simple incisional wound was made in the model and TNF-α and fibrin were added to simulate conditions of inflammation. Topical ibuprofen in a hydrogel was added and the extent of melanoma invasion into the dermis was assessed under the various conditions. The results showed that penetration of two of the cell lines (HBL and A375SM) into the tissue engineered skin was exacerbated by wounding and ibuprofen significantly decreased invasion of A375SM cells and slightly reduced invasion of HBL cells. A third cell line, C8161, was aggressively invasive under all conditions to an extent that was not influenced by wounding, TNF-α or the addition of ibuprofen. In summary, the results for one these cell lines (and a trend for a second cell line) support the hypothesis that a wound environment is conducive to melanoma invasion but the local addition of an anti-inflammatory drug such as ibuprofen may attenuate invasion.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Fibrin; Fibroblasts; Humans; Ibuprofen; Keratinocytes; Melanoma; Models, Biological; Neoplasm Invasiveness; Skin Neoplasms; Tissue Engineering; Tumor Necrosis Factor-alpha

Fibrin gels are attractive biomaterials for local delivery of a variety of agents, from drugs to proteins. Similarly, polymer-anticancer-drug conjugates and nanoparticles are emerging as potential candidates for cancer treatment. Combining these different approaches, we have studied the efficacy of fibrin gels loaded with cisplatin (DDP) and a complex of DDP with hyaluronate (DDP-HA) for tumor growth inhibition in a melanoma model. Loaded gels prepared at relatively high fibrinogen concentration (22 mg/ml) showed good in vitro antiproliferative activities, prolonged release of the anticancer drug, and a long persistence (10-15 days) in vivo when implanted subcutaneously (sc) in immunodeficient mice. Gels loaded with DDP or DDP-HA containing 1/3 or even 1/6 of their systemic dose (6 mg/kg) and positioned under the tumor mass in mice bearing a sc human SK-Mel-28 tumor showed an antitumor activity better than that of the original parent compound given intraperitoneally (ip). Moreover, in an additional experiment in vivo, fibrin gels loaded with N-trimethyl chitosan-based nanoparticles containing a DDP-HA complex were assayed, resulting in a further 8 % improvement of anticancer activity, with lesser adverse systemic toxic effects. Taken together, these results suggest that the combination of fibrin gels and drugs complexed with suitable macromolecules holds great promise for loco-regional anticancer therapy of melanoma and other surgically removable cancer types.

Topics: Animals; Cisplatin; Female; Fibrin; Gels; Humans; Hyaluronic Acid; Melanoma; Mice; Mice, Nude; Xenograft Model Antitumor Assays

Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of cancer cells critical in cancer progression. However, the underlying mechanisms of how TRCs maintain their self-renewing capability remain elusive. Here we show that relatively undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening, histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability. In contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is unresponsive to matrix stiffness or applied forces. Silencing H3K9 methyltransferase G9a or SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner. Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both in vitro and in vivo. Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC self-renewal.

Topics: Animals; Biosensing Techniques; Cell Line, Tumor; Cell Proliferation; Disease Progression; DNA Methylation; Female; Fibrin; Fluorescence Resonance Energy Transfer; Gene Silencing; Histone-Lysine N-Methyltransferase; Histones; Integrin beta1; Lysine; Melanoma; Melanoma, Experimental; Methylation; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Stem Cells; Promoter Regions, Genetic; RNA, Small Interfering; Skin Neoplasms; SOXB1 Transcription Factors; Time Factors

Elevated soluble fibrin (sFn) levels are characteristic of melanoma hematogeneous dissemination, where tumor cells interact intimately with host cells. Melanoma adhesion to the blood vessel wall is promoted by immune cell arrests and tumor-derived thrombin, a serine protease that converts soluble fibrinogen (sFg) into sFn. However, the molecular requirement for sFn-mediated melanoma-polymorphonuclear neutrophils (PMNs) and melanoma-endothelial interactions under physiological flow conditions remain elusive. To understand this process, we studied the relative binding capacities of sFg and sFn receptors e.g., α(v)β(3) integrin and intercellular adhesion molecule-1 (ICAM-1) expressed on melanoma cells, ICAM-1 on endothelial cells (EC), and CD11b/CD18 (Mac-1) on PMNs. Using a parallel-plate flow chamber, highly metastatic melanoma cells (1205Lu and A375M) and human PMNs were perfused over an EC monolayer expressing ICAM-1 in the presence of sFg or sFn. It was found that both the frequency and lifetime of direct melanoma adhesion or PMN-facilitated melanoma adhesion to the EC in a shear flow were increased by the presence of sFn in a concentration-dependent manner. In addition, sFn fragment D and plasmin-treated sFn failed to increase melanoma adhesion, implying that sFn-bridged cell adhesion requires dimer-mediated receptor-receptor cross-linking. Finally, analysis of the respective kinetics of sFn binding to Mac-1, ICAM-1, and α(v)β(3) by single bond cell tethering assays suggested that ICAM-1 and α(v)β(3) are responsible for initial capture and firm adhesion of melanoma cells. These results provide evidence that sFn enhances melanoma adhesion directly to ICAM-1 on the EC, while prolonged shear-resistant melanoma adhesion requires interactions with PMNs.

Topics: CD11b Antigen; CD18 Antigens; Cell Adhesion; Cell Communication; Cell Line, Tumor; Endothelial Cells; Endothelium; Fibrin; Humans; Integrin alphaVbeta3; Intercellular Adhesion Molecule-1; Kinetics; Ligands; Melanoma; Neutrophils; Stress, Mechanical

Fibrin (Fn) deposition defines several type 1 immune responses, including delayed-type hypersensitivity and autoimmunity in which polymorphonuclear leukocytes (PMNs) are involved. Fn monomer and fibrinogen are multivalent ligands for a variety of cell receptors during cell adhesion. These cell receptors provide critical linkage among thrombosis, inflammation, and cancer metastasis under venous flow conditions. However, the mechanisms of Fn-mediated interactions among immune cells and circulating tumor cells remain elusive. By using a cone-plate viscometer shear assay and dual-color flow cytometry, we demonstrated that soluble fibrinogen and Fn had different abilities to enhance heterotypic aggregation between PMNs and Lu1205 melanoma cells in a shear flow, regulated by thrombin levels. In addition, the involvement of integrin α(v)β(3), ICAM-1, and CD11b/CD18 (Mac-1) in fibrin(ogen)-mediated melanoma-PMN aggregations was explored. Kinetic studies provided evidence that ICAM-1 mediated initial capture of melanoma cells by PMNs, whereas α(v)β(3) played a role in sustained adhesion of the two cell types at a shear rate of 62.5 s(-1). Quantitative analysis of the melanoma-PMN interactions conducted by a parallel-plate flow chamber assay further revealed that at a shear rate of 20 s(-1), α(v)β(3) had enough contact time to form bonds with Mac-1 via Fn, which could not otherwise occur at a shear rate higher than 62.5 s(-1). Our studies have captured a novel finding that leukocytes could be recruited to tumor cells via thrombin-mediated Fn formation within a tumor microenvironment, and α(v)β(3) and ICAM-1 may participate in multistep fibrin(ogen)-mediated melanoma cell adhesion within the circulation.

Topics: Animals; Blood Flow Velocity; CD11b Antigen; CD18 Antigens; Cell Adhesion; Cell Communication; Cell Line, Tumor; Fibrin; Fibrinogen; Humans; Inflammation Mediators; Integrin alphaVbeta3; Intercellular Adhesion Molecule-1; Melanoma; Mice; Neutrophil Infiltration; Thrombosis

In primary human melanoma, the membrane-type matrix metalloproteinase, MT3-MMP, is overexpressed in the most aggressive nodular-type tumors. Unlike MT1-MMP and MT2-MMP, which promote cell invasion through basement membranes and collagen type I-rich tissues, the function of MT3-MMP in tumor progression remains unclear. Here, we demonstrate that MT3-MMP inhibits MT1-MMP-driven melanoma cell invasion in three-dimensional collagen, while yielding an altered, yet MT1-MMP-dependent, form of expansive growth behavior that phenocopies the formation of nodular cell colonies. In melanoma cell lines originating from advanced primary or metastatic lesions, endogenous MT3-MMP expression was associated with limited collagen-invasive potential. In the cell lines with highest MT3-MMP expression relative to MT1-MMP, collagen-invasive activity was increased following stable MT3-MMP gene silencing. Consistently, MT3-MMP overexpression in cells derived from less advanced superficially spreading melanoma lesions, or in the MT3-MMP knockdown cells, reduced MT1-MMP-dependent collagen invasion. Rather than altering MT1-MMP transcription, MT3-MMP interacted with MT1-MMP in membrane complexes and reduced its cell surface expression. By contrast, as a potent fibrinolytic enzyme, MT3-MMP induced efficient invasion of the cells in fibrin, a provisional matrix component frequently found at tumor-host tissue interfaces and perivascular spaces of melanoma. Since MT3-MMP was significantly upregulated in biopsies of human melanoma metastases, these results identify MT3-MMP as a matrix-dependent modifier of the invasive tumor cell functions during melanoma progression.

Topics: Catalysis; Cell Line, Tumor; Cell Membrane; Collagen; Disease Progression; Fibrin; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lymphatic Metastasis; Matrix Metalloproteinase 16; Melanoma; Neoplasm Invasiveness; Polymerase Chain Reaction; Prognosis; Skin Neoplasms

Two hundred and four accessible cases of malignant melanoma from the Grampian region of Scotland, collected over a period of 4 years, with minimum follow-up of 2 years, were studied for coagulation factors and vascular endothelial growth factor (VEGF) expression as potential prognostic markers. The aim was to allow comparison with previous work using microvessel density on the same cases.. Immunohistochemistry for VEGF, tissue factor (TF), fibrin and protease-activated thrombin receptor (PAR)-1 in 204 cases of melanoma was performed, and intensity of expression scored. Chalkley microvessel counts (MVD) were obtained for the tumour edge. TF expression and presence of fibrin correlated well with Breslow thickness and ulceration, reaching statistical significance, but surprisingly not for metastatic recurrence. Fibrin was variably present in over half the cases, located at the invasive edge, ulcerated surface and between tumour cell surfaces. In a few cases fibrin was within tumour cells, typically co-located with melanin and confirmed by electron microscopy. In contrast, immunohistochemistry for PAR-1 produced statistically significant results, correlating expression with Breslow thickness (P < or = 0.001), ulceration (P = 0.001) and recurrence (P < or = 0.005). Intensity of reactivity of VEGF correlated significantly with Breslow thickness, Clark level, ulceration and MVD, but not for metastatic recurrence.. It appears paradoxical that VEGF expression is not more predictive of recurrence, but even low expression may be sufficient for tumour angiogenesis and other factors must govern tumour aggression. Antagonism of VEGF may still prove a successful adjunct in future therapeutic trials. Both MVD and PAR-1 can be used as adjuncts to Breslow thickness and ulceration as prognostic indicators for melanoma, as they appear to give independent information for all thicknesses. PAR-1 expression is the best antibody marker of recurrence risk from those studied. It remains to be seen whether this methodology can predict response to novel antiangiogenic therapies currently entering trial.

Topics: Biomarkers, Tumor; Fibrin; Humans; Immunohistochemistry; Melanocytes; Melanoma; Melanosomes; Microscopy, Electron, Transmission; Neoplasm Recurrence, Local; Neovascularization, Pathologic; Prognosis; Receptor, PAR-1; Skin Neoplasms; Thromboplastin; Vascular Endothelial Growth Factor A

Although attempts to develop any viable chemotherapeutic approaches to combat metastatic cancers have largely failed, potential genetic targets to halt metastatic progression continue to be identified. As drugs are developed to address these targets, there is a need for high-throughput systems that accurately reproduce in vivo microenvironments to gauge their efficacy. Accordingly, we have developed a three-dimensional in vitro culture system representative of the environment present upon secondary metastasis to quantitatively measure tumor cell invasion in this setting three-dimensionally. Culturing melanomas of different metastatic capacities within the system showed that each cell type invades the matrix in a manner commensurate to its known metastatic potential in vivo. Moreover, the developed quantitative schemes were put to use to characterize the effect of microenvironmental influences (i.e., matrix components, interstitial cell presence) on planar and vertical melanoma invasion. We propose this novel, quantitative system as a useful tool to assess the effects of pharmacologic and/or microenvironmental influences on tumor cell invasion at a metastatic site.

Topics: Collagen; Epidermal Cells; Epidermis; Extracellular Matrix; Fibrin; Green Fluorescent Proteins; Humans; Image Processing, Computer-Assisted; Melanoma; Models, Biological; Neoplasm Invasiveness; Skin Neoplasms

Angiogenesis is a process required not only for embryonal development but is encountered in wound healing and in pathological situations such as tumour growth. In vitro, formation of capillary-like structures can be induced by seeding human microvascular endothelial cells (HDMECs) on top of a fibrin matrix in the presence of phorbol 12-myristate 13-acetate (PMA) as a stimulating agent. In this study, we show that supernatants collected from high-invasive melanoma cells (BLM) induce the formation of tubular structures similar to PMA treatment whereas supernatants from low-invasive cells (WM164) did not. Analysis of proteins secreted into the supernatant of both melanoma cell lines identified differential expression of several pro-angiogenic proteins in high- and low-invasive melanoma cells. Vascular endothelial growth factor (VEGF) was strongly expressed by high- but not by low-invasive melanoma cells. Neutralisation of VEGF as well as inhibition of matrix metalloproteases (MMPs) using the broad spectrum MMP inhibitor 1,10-phenanthroline, both strongly reduced the melanoma-induced tube formation. PMA treatment of HDMECs on a fibrin matrix stimulated MT1-MMP synthesis, indicating that this protease is involved in PMA-induced angiogenesis. In addition, stimulation of HDMECs by supernatants of BLM melanoma cells resulted in a strong induction of ADAM-15, which is known to act as a metalloproteinase. In conclusion, these results show that VEGF released by melanoma cells is an important mediator of neo-vascularisation and that this process depends on the presence of metalloproteinases.

Topics: Endothelium, Vascular; Fibrin; Gels; Humans; Melanoma; Metalloendopeptidases; Metalloproteases; Neovascularization, Pathologic; Phenanthrolines; Protease Inhibitors; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

An international collaborative study was organised to replace the 2nd International Standard (IS) for tissue plasminogen activator (tPA). The 2nd IS for tPA (86/670) was used to calibrate the replacement Standard, which was selected from two candidate materials included in the collaborative study. Participants were provided with five sets of four samples (A, B, C, D) and asked to use sample A (2nd IS, 86/670, 850 IU/ml) to determine the activity of B (86/624, approximately 850 IU/ml), C and D (coded duplicates of the same material, 98/714 approximately 11,000 IU/ml). A total of 14 laboratories returned results from Europe, USA, Japan and Australia, providing data from 60 independent assays. Four laboratories used a reference method based on a published monograph from the European Pharmacopoeia for Alteplase for Injection, 1998, and the remaining 10 used their own method. Fibrin was used as promoter of tPA activity by 12 out of the 14 laboratories, the remaining two used kits where fibrinogen fragments were the promoter. Data from this collaborative study and the previous study to establish the 2nd IS for tPA show that tPA from melanoma cells and recombinant tPA from CHO cells are both suitable materials as International Standards. It was agreed that sample C, D, recombinant tPA, 98/714, be established as the 3rd International Standard for tPA with a potency of 10,000 IU per ampoule, calculated as the mean value from laboratories using fibrin as a promoter of tPA activity. The standard was established by WHO in November 2000.

Topics: Animals; CHO Cells; Cooperative Behavior; Cricetinae; Fibrin; Humans; International Cooperation; Melanoma; Recombinant Proteins; Reference Standards; Tissue Plasminogen Activator

In vivo tumor cells interact with a variety of host cells such as endothelial cells and platelets, and these interactions are mediated by integrins GPIIb/IIIa and alphavbeta3. We used chimeric (c) 7E3 Fab (ReoPro) and murine (m) 7E3 F(ab')(2) to elucidate the role of these integrins in angiogenesis, tumor growth, and metastasis. These antibodies are potent inhibitors of GPIIb/IIIa and alphavbeta3. c7E3 Fab inhibited alphavbeta3-mediated human umbilical vein endothelial (HUVEC) and melanoma cell adhesion, migration, invasion, and basic fibroblast growth factor stimulated proliferation of HUVECs (IC(50) values range from 0.15 to 5 microg/ml for different assays). In an in vitro angiogenesis assay, c7E3 Fab inhibited basic fibroblast growth factor and platelet-stimulated capillary formation of HUVECs (IC(50) = 10 microg/ml and 15 microg/ml, respectively), demonstrating that endothelial alphavbeta3 is important for sprouting, and platelet-stimulated sprouting is mediated by GPIIb/IIIa. In an experimental metastasis assay, a single pretreatment of human melanoma cells with c7E3 Fab (2.5 microg/ml) inhibited lung colonization of the tumor cells in severe combined immunodeficient mice. In vivo, m7E3 F(ab')(2) partially inhibited growth of human melanoma tumors in nude mice compared with control-treated animals. These data suggest that tumor cell-expressed integrins are important but not the only component involved in tumor growth. Because c7E3 Fab and m7E3 F(ab')(2) do not cross-react with murine integrins, this inhibition of metastasis and tumor growth is attributable to direct blockade of human tumor alphavbeta3 integrins. m7E3 F(ab')(2) completely blocked tumor formation and growth of human melanoma tumors growing in nude rats. In this xenograft model, m7E3 F(ab')(2) simultaneously binds to both human tumor and host platelet GPIIb/IIIa and endothelial alphavbeta3 integrins, thus participating as an antiangiogenic and an antitumor agent. Collectively, these results indicate that combined blockade of GPIIb/IIIa and alphavbeta3 affords significant antiangiogenic and antitumor benefit.

Topics: Abciximab; Animals; Antibodies, Monoclonal; Apoptosis; Blood Platelets; Cattle; Cell Adhesion; Cell Division; Cell Movement; Endothelium, Vascular; Extracellular Matrix Proteins; Female; Fibrin; Fibrinolytic Agents; Fibroblast Growth Factor 2; HT29 Cells; Humans; Immunoglobulin Fab Fragments; Melanoma; Mice; Mice, Nude; Neovascularization, Pathologic; Platelet Glycoprotein GPIIb-IIIa Complex; Rats; Receptors, Vitronectin; Xenograft Model Antitumor Assays

Directional migration of capillaries towards tumour implants is generally assumed to be regulated by chemotaxis. Preliminary evidence has also been presented for the existence of a reverse chemotactic signalling pathway, with capillaries attracting tumour cells via paracrine factors. By using a variety of endothelial cell types and tumour cell lines, this study has systematically investigated chemotaxis between endothelial cells and tumour cells in two- and three-dimensional systems. Checkerboard analysis revealed faint attraction of human umbilical vein endothelial cells (HUVECs), but not porcine aortic endothelial cells (PAECs), by tumour cells. In reverse, both PAECs and HUVECs potently induced chemotactic migration of tumour cells. Using a microcarrier-based fibrin gel assay, directional migration of endothelial cells towards tumour cells was not observed. In reverse, tumour cells were strongly attracted by endothelial cells. Identification of endothelium-derived chemotactic molecules may provide a valuable approach for the treatment of tumour metastasis.

Topics: Animals; Capillaries; Cell Communication; Cell Culture Techniques; Chemotaxis; Culture Media, Conditioned; Endothelium, Vascular; Fibrin; Glioblastoma; Humans; Melanoma; Microscopy, Phase-Contrast; Neoplasms; Neovascularization, Pathologic; Swine; Tumor Cells, Cultured

Metastasizing tumor cells invade host tissues by degrading extracellular matrix constituents. We report here that the highly sulfated glycosaminoglycans, heparin and heparan sulfate, as well as the sulfated polysaccharide, fucoidan, significantly enhanced tumor cell invasion in vitro into fibrin, the basement membrane extract, Matrigel, or through a basement membrane-like extracellular matrix. The enhancement of tumor cell invasion was due to a stimulation of the proteolytic cascade of plasminogen activation since the effect required plasminogen activation and was abolished by inhibitors of urokinase-type plasminogen activator (uPA) or plasmin. Sulfated polysaccharides enhanced five reactions of tumor-cell initiated plasminogen activation in a dose-dependent manner. They amplified plasminogen activation in culture supernatants up to 70-fold by stimulating (i) pro-uPA activation by plasmin and (ii) plasminogen activation by uPA. (iii) In addition, sulfated polysaccharides partially protected plasmin from inactivation by alpha 2-antiplasmin. Sulfated polysaccharides also stimulated tumor-cell associated plasminogen activation, e.g., (iv) cell surface pro-uPA activation by plasmin and (v) plasminogen activation by cell surface uPA. These results suggest that sulfated glycosaminoglycans liberated by tumor-cell mediated extracellular matrix degradation in vivo might amplify pericellular plasminogen activation and locally enhance tumor cell invasion in a positive feedback manner.

Topics: Cell Adhesion; Cell Movement; Collagen; Culture Media, Conditioned; Drug Combinations; Enzyme Activation; Extracellular Matrix; Fibrin; Fibrinolysin; Heparin; Heparitin Sulfate; Humans; Laminin; Melanoma; Models, Biological; Neoplasm Invasiveness; Plasminogen; Polysaccharides; Proteoglycans; Recombinant Fusion Proteins; Stimulation, Chemical; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Platelet integrin alpha IIb beta 3 (GPIIb-IIIa) plays important roles in platelet-mediated clot retraction. However, little is known about the mechanisms of clot retraction mediated by nucleated cells. In this report, we demonstrate that another member of the beta 3 integrin family, alpha v beta 3, is involved in clot retraction mediated by nucleated cells. Retraction of fibrin clots was observed using a human melanoma cell line, C32TG, which contains no alpha IIb beta 3 complex. This retraction was inhibited by RGD-containing peptide, monoclonal anti-beta 3, and anti-alpha v beta 3 antibodies. Immunoelectron microscopic studies revealed a direct interaction between beta 3 integrin and fibrin fibers at an early stage of clot retraction. We found that another human embryonal cell line, 293, which is known to express alpha v beta 1, but no alpha v beta 3, lacks fibrin gel retractile activity. Upon transfection of beta 3 DNA into 293 cells, the beta 3 subunit formed a complex with an endogenous alpha v subunit. The beta 3-bearing transfectants were found to retract fibrin gels, which was specifically inhibited by anti-beta 3 antibody. In addition, a point mutation at Asp119 in the beta 3 ligand binding domain abolished the clot retractile activity of 293 transfectants, indicating the requirement of alpha v beta 3 ligand-binding activity. Our findings suggest that alpha v beta 3 is involved in mediating the interaction between the three-dimensional fibrin network and nucleated cells and in promoting "post-receptor occupancy" events.

Topics: Antibodies, Monoclonal; Blood Platelets; Cell Line; Clot Retraction; Embryo, Mammalian; Fibrin; Humans; Integrins; Melanoma; Microscopy, Electron; Oligopeptides; Platelet Glycoprotein GPIIb-IIIa Complex; Platelet Membrane Glycoproteins; Receptors, Cytoadhesin; Receptors, Vitronectin; Recombinant Proteins; Transfection; Tumor Cells, Cultured

Fibrin clot retraction may be important in resolution of thrombi and, in platelets, is mediated by integrin alpha IIb beta 3 (GPIIb-IIIa). Nucleated cells that lack alpha IIb beta 3 can retract fibrin clots, and we now report that integrin alpha v beta 3 can support this process. In addition, we compared the capacities of recombinant beta 3 integrins to mediate clot retraction in Chinese hamster ovary and M21 melanoma cells. We found that alpha v beta 3, but not alpha IIb beta 3, could spontaneously support retraction. Transferring the cytoplasmic domain of alpha v to alpha IIb enabled the resulting chimeric alpha IIb beta 3 to support clot retraction. The capacity of the alpha v cytoplasmic domain to support clot retraction was not caused by activation of the ligand binding function of alpha IIb beta 3 or by enhancement of alpha IIb beta 3's capacity to stimulate the formation of focal adhesions or the tyrosine phosphorylation of pp125FAK. These experiments define requirements for alpha IIb beta 3-mediating clot retraction, establish the capacity of alpha v beta 3 to mediate this process, and suggest differing functional roles of the alpha v and alpha IIb cytoplasmic domains.

Topics: Animals; Antibodies; Antigens, CD; Cell Line; CHO Cells; Clot Retraction; Cricetinae; Endothelium, Vascular; Fibrin; Fibrinogen; Gene Expression; Humans; Immunosorbent Techniques; Integrin beta3; Integrins; Melanoma; Platelet Glycoprotein GPIIb-IIIa Complex; Receptors, Cytoadhesin; Receptors, Vitronectin; Recombinant Proteins; Transfection; Tumor Cells, Cultured

The three-dimensional growth of cultured tumor cell lines (HT29, a colon adenocarcinoma cell line; M21, a melanoma cell line; KB, a nasopharyngeal carcinoma cell line) has been investigated in an agar culture system with a fibrin matrix in vitro. The tumor cells developed to tumors 3 x 3 mm in diameter after 10 days in culture in vitro. This size was large enough to allow histologic examination. The tumor cells located in the surface area of the three-dimensional tumor seemed to grow well. However, the tumor cells in the center degenerated or did not proliferate, indicating a lack of nutrition and/or anoxia in the center. The histologic comparison between the xenografted tumors on nude mice and the three-dimensional tumors in vitro suggests that the structures of the three-dimensional tumors were comparable, especially in the surface area, to the xenografted tumors. Furthermore, the antitumor effect of mitomycin C on the three-dimensional tumors was found to be dose-dependent.

Topics: Adenocarcinoma; Agar; Animals; Cell Division; Cell Hypoxia; Colonic Neoplasms; Culture Techniques; Extracellular Matrix; Fibrin; Humans; KB Cells; Melanoma; Mice; Mice, Nude; Mitomycin; Transplantation, Heterologous; Tumor Cells, Cultured

Topics: Bone and Bones; Fibrin; Humans; Melanocytes; Melanoma; Osteosarcoma; Skin Neoplasms

Tissue-type plasminogen activator (t-PA) binds to heparin with an association constant of 2.4 x 10(4) l/mol at pH 7.4. The binding increases at lower pH-values and reaches maximal values below pH 6.0. Sodium chloride above 0.5 mol/l abolishes t-PA binding to heparin at neutral pH. t-PA binds to lysine maximally at pH 6 to pH 9. At neutral pH the association constant is 1.8 x 10(5) l/mol. Sodium chloride concentrations of 1 mol/l reduce interaction of the enzyme to lysine by about 60% at pH 7.4. Binding of t-PA to fibrin and to partially plasmin-degraded fibrin takes place maximally at pH 6 to 9. The interaction of t-PA with plasmin-degraded fibrin is less sensitive to addition of lysine than the interaction of t-PA and fibrin. Sodium chloride concentrations of 1 mol/l reduce the interaction of t-PA to fibrin by about 60% at pH 7.4.

Topics: Chromatography, Agarose; Electrophoresis, Polyacrylamide Gel; Fibrin; Fibrinolysin; Heparin; Humans; Hydrogen-Ion Concentration; Kinetics; Lysine; Melanoma; Sodium Chloride; Tissue Plasminogen Activator; Tumor Cells, Cultured

This study evaluates the contribution of two types of plasminogen activators (PAs; tissue-type PA (tPA) versus urokinase-type PA (uPA) toward the invasiveness of human melanoma cells in a novel in vitro assay. We identified two human melanoma cell lines, MelJuso and MeWo, expressing uPA or tPA as shown at mRNA, protein, and enzyme activity level. MelJuso cells produced uPA as well as plasminogen activator inhibitor-1 (PAI-1). The latter was, however, not sufficient to neutralize the cell-associated or secreted uPA activity. MeWo cells secreted tPA, but the enzyme was not found to be cell-associated. PAI-1 production by these cells was not detectable. Plasminogen activation and fibrinolytic capacity of both cell lines were reduced by anticatalytic monoclonal antibodies specific for the respective type of PA or by aprotinin. In a novel in vitro invasion assay, antibodies to PA as well as aprotinin decreased the invasiveness of both cell lines into a fibrin gel, Matrigel, or intact extracellular matrix. Our results confirm the importance of uPA-catalyzed plasminogen activation in tumor cell invasiveness. Furthermore, we provide evidence that tPA, beyond its key role in thrombolysis, can also be involved in in vitro invasion of human melanoma cells.

Topics: Collagen; Drug Combinations; Extracellular Matrix; Fibrin; Humans; Laminin; Melanoma; Neoplasm Invasiveness; Plasminogen Activators; Plasminogen Inactivators; Proteoglycans; Tissue Plasminogen Activator; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

A comparative kinetic analysis of the enzymatic activities of one-chain and two-chain tissue-type plasminogen activator (t-PA) demonstrates that two-chain t-PA catalyzes the hydrolysis of the peptide substrate D-Val-Leu-Arg-pNA about 4-fold more effectively than one-chain t-PA. The difference is accounted for almost entirely by a corresponding difference is the kcat values of the enzymes, whereas the Km values are similar. The amidolytic activity of two-chain t-PA is not enhanced by intact or partially plasmin-degraded fibrin. In contrast, the activity of one-chain t-PA is stimulated up to 3.7-fold by intact fibrin and up to 4.7-fold by plasmin-degraded fibrin (fibrin X-fragment). The stimulatory effects are realized via increases in the kcat values. It appears thus that in the presence of fibrin the intrinsically inferior catalytic properties of one-chain t-PA become similar to the properties of two-chain t-PA. The dependency of the activity of one-chain t-PA on the concentration of fibrin monomer is consistent with a single association site of both proteins and an association constant of Kass = 6.25 x 10(6) l/mol. Stimulation of one-chain t-PA by plasmin-degraded fibrin is more complex and appears to involve two different binding sites with association constants of Kass = 0.67 x 10(9) l/mol and Kass = 3.85 x 10(6) l/mol, respectively. The stimulatory effects of fibrin and partially plasmin-degraded fibrin on one-chain t-PA are suppressed by epsilon-aminocaproic acid and by a monoclonal antibody directed against the lysine binding site of t-PA. The latter findings support the notion that fibrin activation of one-chain t-PA is mediated by the lysine binding site on kringel domains of the enzyme.

Topics: Amino Acid Sequence; Fibrin; Fibrinogen; Fibrinolysin; Humans; Kinetics; Melanoma; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Substrate Specificity; Tissue Plasminogen Activator; Tumor Cells, Cultured

Tissue-type plasminogen activator (t-PA), when isolated from human colon fibroblast (hcf) cells, is N-glycosylated differently than when isolated from the Bowes melanoma (m) cell line (Parekh et al., 1988). Both hcf- and m-t-PA can be separated into type I t-PA (with three occupied N-glycosylation sequons, at Asn-117, -184, and -448) and type II t-PA (with two occupied sequons, at Asn-117 and -448). Oligosaccharide analysis of each of these types of t-PA indicates that hcf-t-PA and m-t-PA have no glycoforms in common, despite having the same primary amino acid sequence. We have therefore compared in vitro the enzymatic activities and fibrin binding of type I and type II hcf- and m-t-PA with those of aglycosyl t-PA isolated from tunicamycin-treated cells. Plasminogen activation kinetics were determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. In the absence of stimulator, there was little difference in activity between type I and type II t-PA, but the activity of aglycosyl t-PA was 2-4-fold higher than that of the corresponding glycosylated t-PA. In the presence of a fibrinogen fragment stimulator, the Kcat value of type II t-PA was approximately 5-fold that of type I t-PA from the same cell line, while the Km values for activation of Glu-plasminogen were similar (0.13-0.18 microM). The stimulated activity of glycosyl t-PA was similar to that of type II t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cells, Cultured; Colon; Enzyme Activation; Fibrin; Fibroblasts; Glycosylation; Humans; Melanoma; Plasminogen; Tissue Plasminogen Activator; Tumor Cells, Cultured; Tunicamycin

Rapid in vivo growth of cultured human cancer or leukemia cells was achieved by implantation into the subrenal capsule of mice. A solid structure, necessary for accurate implantation and measurement of tumor growth in this model, was provided by stepwise addition of fibrinogen and thrombin to the tumor cells, leading to rapid enzymatic formation of a solid tumor-fibrin matrix. Human leukemia and epithelial cancers increased in volume between 6- and 40-fold when measured 6-10 days after implantation into normal or immunosuppressed mice. Immunosuppression of host CD-1 mice was achieved by cyclosporine given daily after tumor implantation, cyclophosphamide given preimplantation combined with cyclosporine, or whole-body irradiation given preimplantation. Confirming the validity of tumor measurements, tumor histology in the immunosuppressed mice revealed cell proliferation, invasion, and neovascularization. Similarly, no artifactual measurement of tumor growth was observed by nonviable cancer cells, implanted after in vitro exposure to a known cytotoxic concentration of thiotepa. This model provides an economical, short-term technique for the in vivo study of human tumor growth, for the evaluation of new cancer therapies, and for in vitro - in vivo drug activity correlations in specific types of human cancer or leukemia cell lines.

Topics: Animals; Colonic Neoplasms; Female; Fibrin; Humans; Immunosuppression Therapy; Karyotyping; Kidney; Leukemia, Myeloid, Acute; Melanoma; Methods; Mice; Mice, Nude; Neoplasm Transplantation; Transplantation, Heterologous; Urinary Bladder Neoplasms; Vulvar Neoplasms

Topics: alpha-2-Antiplasmin; Animals; Cells, Cultured; Female; Fibrin; Fibrinolysis; Glycoproteins; Humans; Melanoma; Metabolic Clearance Rate; Molecular Weight; Plasminogen Inactivators; Protein Binding; Rabbits; Tissue Plasminogen Activator; Uterus

A potential synergic effect of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scu-PA) or urokinase on clot lysis was investigated in a whole human plasma system in vitro. The system consisted of a human plasma clot labeled with 125I-fibrinogen, immersed in citrated whole human plasma, to which the thrombolytic agents were added. Clot lysis was quantitated by measurement of released 125I, and activation of the fibrinolytic system in the surrounding plasma by measurements of fibrinogen and alpha 2-antiplasmin. t-PA, scu-PA and urokinase induced a dose-dependent and time-dependent clot lysis; 50 percent lysis after 2 h was obtained with 5 nM t-PA, 20 nM scu-PA and 12 nM urokinase. At these concentrations no significant activation of the fibrinolytic system in the plasma was observed with t-PA and scu-PA, whereas urokinase caused significant alpha 2-antiplasmin consumption and concomitant fibrinogen degradation. The shape of the dose-response curves was different; t-PA and urokinase showed a log linear dose-response whereas that of scu-PA was sigmoidal. Combinations of t-PA and scu-PA, of t-PA and urokinase or of scu-PA and urokinase at thrombolytic doses of each showed no synergism for thrombolysis. Fifty percent clot lysis in 2 h was obtained at total concentrations of the combined agents of 5 to 15 nM with molar ratios ranging from 1:4 to 4:1.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenocarcinoma; Drug Synergism; Fibrin; Fibrinolysis; Humans; Kinetics; Lung Neoplasms; Melanoma; Plasminogen Activators; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator

The rate of 'Glu'-plasminogen activation by tissue plasminogen activator was repeatedly determined during a fibrinolytic process. The process was found to proceed via two distinct phases. The kinetics of each phase obeyed Michaelis-Menten equation: First phase; kcat about 0.17 s-1 and Km about 1 microM, second phase; kcat about 0.13 s-1 and Km about 0.06 microM. Practically identical results were obtained with one-chain as with two-chain tissue plasminogen activator. Transition from first to second phase occurred when the system had been exposed to a certain degree of plasmin digestion. Electrophoretic analysis demonstrated time correlation between the appearance of minimally degraded fibrin (X-fragments) and the transition. No such correlation was found between transition and conversion of 'Glu'-plasminogen to 'Lys'-plasminogen. The effect can result in an acceleration (up to 13-fold) of the fibrinolytic process once a slight degradation of the fibrin has taken place. In vivo, the effect described may constitute a mechanism that protects a fibrin clot from premature lysis.

Topics: Animals; Cells, Cultured; Chemical Phenomena; Chemistry; Electrophoresis, Polyacrylamide Gel; Fibrin; Fibrinolysin; Fibrinolysis; Kinetics; Melanoma; Plasminogen; Plasminogen Activators

Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells were separated by Lysine Sepharose chromatography. The carbohydrate compositions of the forms were determined by gas-liquid chromatography. Variant I contained 12.8 g and variant II 7.1 g of carbohydrate per 100 g protein. Both variants contained N-acetylgalactosamine, suggesting O-glycosylation in addition to N-glycosylation. The possible role of N-linked oligosaccharides for the biological activity of t-PA was studied using t-PA secreted by melanoma cells in the presence of tunicamycin, an inhibitor of N-glycosylation. The latter t-PA showed the same plasminogen activating and fibrin binding properties as normally glycosylated t-PA, indicating that N-linked carbohydrate is not involved in the fibrinolytic activity of t-PA.

Topics: Carbohydrates; Cells, Cultured; Chromatography, Agarose; Fibrin; Humans; Melanoma; Tissue Plasminogen Activator; Tunicamycin

In order to evaluate the importance of the carbohydrate moiety of human tissue plasminogen activator (TPA), human melanoma (Bowes) cells were treated with a glycosylation inhibitor, tunicamycin (TM), and cellular fractions were assayed for fibrinolytic activity. Where glycosylation was inhibited by 90% and protein synthesis by 30%, TPA specific activity measured by fibrinolytic assays decreased 6-10-fold in the tissue culture medium and cell cytosol with a concomitant 2-fold increase in the 100000g microsomal pellet. In addition, TPA purified to apparent homogeneity was treated with endo-beta-N-acetylglucosaminidase H (Endo-H), producing a fraction that in contrast to native TPA did not adsorb to concanavalin A-Sepharose (Con A-Sepharose). This fraction represented TPA from which 85-90% of N-linked carbohydrate residues had been removed. Native TPA effectively activated plasminogen in the presence of fibrin (Km = 1 microM, kcat = 0.09 s-1) whereas saturation of the enzyme was not achieved at 100 microM plasminogen in the absence of fibrin. Glycosidase-treated and native TPA activated plasminogen at identical high rates in the presence and at identical negligible rates in the absence of fibrin. These studies indicate that the inhibition of glycosylation of TPA results in the inhibition of secretion of the molecule as has been observed for some other glycoproteins. The enzymatic removal of N-linked carbohydrate from purified TPA does not change its unique fibrin-directed properties.

Topics: Acetylglucosaminidase; Carbohydrates; Cell Line; Chromatography, Affinity; Enzyme Activation; Fibrin; Glucosamine; Humans; Kinetics; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Melanoma; Plasminogen; Plasminogen Activators; Tunicamycin

Hybridoma producing a monoclonal antibody IgG1 to one-chain tissue plasminogen activator (t-PA) derived from human melanoma cells was obtained by fusion of mouse myeloma cells (SP-1) and spleen cells of mice previously immunized with purified t-PA. The monoclonal antibody reacted only with t-PA derived from the human melanoma cells (Bowes), and not with plasminogen activator purified from porcine heart or from human urine. The monoclonal antibody obtained from mouse ascites demonstrated 50 times stronger antibody activity than that of polyclonal antibody obtained from mouse serum. The monoclonal antibody bound t-PA firmly, inhibited the fibrinolytic activity of t-PA, but did not inhibit the amidolytic activity of t-PA completely. The fibrin-binding ability of t-PA was not inhibited. The binding of monoclonal antibody to the non-reduced form of t-PA did not differ from that to the reduced form of t-PA.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Cell Line; Fibrin; Humans; Immunoglobulin G; Melanoma; Mice; Oxidation-Reduction; Plasminogen Activators; Plasminogen Inactivators

This paper reports studies on the interaction between human platelets, the plasma coagulation system, and two human tumor cell lines grown in tissue culture: Melanoma and breast adenocarcinoma. The interaction was monitored through the use of 125I-labelled fibrinogen, which measures both thrombin activity generated by cell-plasma interaction and fibrin/fibrinogen binding to platelets and tumor cells. Each tumor cell line activates both the platelets and the coagulation system simultaneously resulting in the generation of thrombin or thrombin-like activity. The melanoma cells activate the coagulation system through "the extrinsic pathway" with a tissue factor-like effect on factor VII, but the breast tumor seems to activate factor X directly. Both tumor cell lines activate platelets to "make available" a platelet-derived procoagulant material necessary for the conversion of prothrombin to thrombin. The tumor-derived procoagulant activity and the platelet aggregating potential of cells do not seem to be inter-related, and they are not specific to malignant cells.

Topics: Adenocarcinoma; Blood Platelets; Breast Neoplasms; Cell Line; Cells, Cultured; Factor VII; Factor X; Fibrin; Fibrinogen; Hemostasis; Humans; In Vitro Techniques; Iodine Radioisotopes; Melanoma

Comparison of the primary structures of high-Mr urokinase and tissue-type plasminogen activator reveals a high degree of structural homology between the two proteins, except that tissue activator contains a 43 residue long amino-terminal region, which has no counterpart in urokinase. We show that this segment is homologous with the finger-domains responsible for the fibrin-affinity of fibronectin. Limited proteolysis of the amino-terminal region of plasminogen activator was found to lead to a loss of the fibrin-affinity of the enzyme. It is suggested that the finger-domains of fibronectin and tissue-types plasminogen activator have similar functions and that the finger-domains of the two proteins evolved from a common ancestral fibrin-binding domain.

Topics: Amino Acid Sequence; Biological Evolution; Cell Line; Disulfides; Fibrin; Fibronectins; Humans; Melanoma; Molecular Weight; Peptide Fragments; Plasminogen Activators; Protein Binding; Protein Conformation; Urokinase-Type Plasminogen Activator

Topics: Chromogenic Compounds; Female; Fibrin; Fibrinolysin; Fibrinolysis; Humans; Melanoma; Plasminogen Activators; Thrombosis; Uterus

Topics: alpha-2-Antiplasmin; Animals; Dogs; Fibrin; Fibrinolysin; Fibrinolysis; Humans; Melanoma; Plasminogen; Plasminogen Activators; Rabbits

Discrimination between the physiological cleavage fragments of fibrinogen and fibrin offers an approach to differentiation between fibrinogenolytic processes and fibrinolysis after coagulation. By use of the cleavage-associated neoantigen of fibrinogen (fg-D(neo)) as a molecular marker, characteristic differences between the D regions of fibrinogen derivatives and fibrin derivatives can be demonstrated. The expression of fg-D(neo) by X, Y, D:E complex, and D-fragments of fibrinogen or fibrin is shown to be quantitative and unitary. Characteristic differences between fg-D(neo) sites present on fibrinogen cleavage fragments, as contrasted to fibrin cleavage fragments, are indicated by different competitive inhibition slopes, and appear to reflect differential binding affinity of selected anti-fg-D(neo) antibodies for the specific molecular site. There is a linear relationship between the slope of quantitative competitive inhibition and the relative molar ratio of fibrinogen and fibrin derivatives. Identical immunochemical expressions are observed in vitro and in vivo, and support the thesis that cleavage in vivo is produced by plasmin. The differential immunochemical features of fg-D(neo) expression may be the result of stable conformational and/or subtle structural differences between the D region of fibrinogen and fibrin cleavage fragments and suggest that precise changes in the D region are associated with the fibrin transition. These molecular features not only provide additional insight into the molecular immunology and structure of fibrinogen, but also appear to offer a new molecular approach to discrimination between fibrinogenolytic mechanisms as contrasted to fibrinolysis secondary to coagulation.

Topics: Abruptio Placentae; Acute Kidney Injury; Adenocarcinoma; Binding Sites, Antibody; Binding, Competitive; Blood Coagulation Disorders; Epitopes; Female; Fibrin; Fibrinogen; Fibrinolysin; Fibrinolysis; Humans; Immune Sera; Iodine Isotopes; Male; Melanoma; Meningococcal Infections; Peritonitis; Pregnancy; Prostatic Neoplasms; Radioimmunoassay; Structure-Activity Relationship

Topics: Anterior Chamber; Basement Membrane; Blood Platelets; Capillaries; Cell Nucleolus; Cell Nucleus; Choroid Neoplasms; Ciliary Body; Collagen; Cytoplasm; Cytoplasmic Granules; Endoplasmic Reticulum; Epithelium; Fibrin; Fibroblasts; Glaucoma; Humans; Hyalin; Macrophages; Melanoma; Microscopy, Electron; Microsurgery; Mitochondria; Mitosis; Osmium; Staining and Labeling

Topics: Aminocaproates; Animals; Coumarins; Fibrin; Heparin; Melanoma; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental; Neoplasms, Vascular Tissue; Tail

Topics: Adenocarcinoma; Adenoma; Adrenal Gland Neoplasms; Breast Diseases; Breast Neoplasms; Female; Fibrin; Fluorescent Antibody Technique; Genital Neoplasms, Female; Hodgkin Disease; Humans; Lymph Nodes; Lymphatic Diseases; Melanoma; Methods; Neoplasms; Neoplasms, Nerve Tissue; Ovarian Neoplasms; Sarcoma; Stomach Neoplasms; Thyroid Neoplasms; Tuberculosis