Anchorage independent growth and plasminogen activator production by bovine endothelial cells.

Endothelial cells obtained from the aortae of 1- to 2-d-old calves were cloned at high efficiency using fibrin-coated dishes. Primary cultures as well as clones derived from them produced high fibrinolytic activity when grown on 125I-fibrin-coated dishes which was 90% dependent upon the presence of plasminogen. High plasminogen-dependent proteolytic activity was also demonstrated in endothelial cell lysates and in the culture medium of the cells. The production and secretion of the plasminogen activator(s) were found to increase during the log phase of cell growth and to reach a maximum level at confluence. These endothelial cells exhibited morphological phenotypes comparable to those of transformed cells when grown in the presence of acid-treated fetal calf, dog, or human serum. Furthermore, they demonstrated anchorage independent growth, and large colonies were formed in semisolid media. Spontaneous neoplastic transformation of these cells was excluded by karyotypic analysis, lack of tumorigenicity in athymic nude mice, and limited lifespan in culture. Cell clones isolated from colonies grown in agarose demonstrated the same growth characteristics and proteolytic activity as before plating in agarose. High fibrinolytic activity, morphological changes in the appropriate serum, and growth in semisolid media may therefore be indicative of the migratory and/or invasive capacity of both nontransformed endothelial cells as well as tumor cells.

A non-invasive, in vivo technique for monitoring vascular status of glioblastoma during angiogenesis.

The growth of solid tumors dependent on the process of angiogenesis in which growth factors secreted by tumor and stromal cells promote endothelial cell proliferation, migration, and maturation. This process generates a tumor-specific vascular supply and enables small or dormant tumors to grow rapidly with exponential increases in tumor volume. Determination of tumor oxygenation at the microvascular level will provide important insight into tumor growth, angiogenesis, necrosis, and therapeutic response, and will facilitate to develop protocols for studying tumor behavior. A non-invasive multi-modality approach based on near infrared spectroscopy (NIRS) technique, namely: Steady State Diffuse Optical Spectroscopy (SSDOS) along with Magnetic Resonance Imaging (MRI) is applied for monitoring the concentration of oxyhemoglobin, deoxyhemoglobin and water within tumor region and for studying the vascular status of tumor and the patho-physiological changes that occur during angiogenesis. Since, the growth of solid tumors depends on the formation of new blood vessels, an association between intramural microvessel density (MVD) and tumor oxygenation is also investigated. The relative decrease in oxygenation value with tumor growth indicates that though blood vessels infiltrate and proliferate the tumor region, a hypoxic trend is clearly present.

Inhibition of human tumor cell-associated fibrinolysis by vascular bovine smooth muscle cells.

Fibrinolysis induced by human tumor cells producing either urokinase (u-PA) or tissue-type plasminogen activators (t-PA) was inhibited by growth in vitro of the tumor cells together with aortic bovine smooth muscle cells (SMC). Both types of plasminogen activators secreted by the tumor cells were partially or completely neutralized in such cocultivation experiments. The fibrinolysis inhibitory activities were found in living SMC, homogenates of the cells, and medium conditioned by the SMC. Incubation of medium conditioned by the SMC with media conditioned by the tumor cells followed by polyacrylamide gel electrophoresis and regular fibrin zymography resulted in sodium dodecyl sulfate-resistant inhibition of fibrinolysis. Gel electrophoretic analysis of medium conditioned by SMC followed by reverse fibrin autography revealed the presence of a single zone of lysis inhibition that corresponded to a relative molecular weight of 51,500 +/- 1,700. These results demonstrate that SMC produce and secrete a powerful inhibitor of u-PA- and t-PA-induced fibrinolysis, which may alter the invasive and degradative potential of tumor cells.

Inhibition by bovine endothelial cells of degradation by HT-1080 fibrosarcoma cells of extracellular matrix proteins.

Medium conditioned by bovine arterial endothelial cells inhibited the degradation by human fibrosarcoma cells of living cultures of rat smooth muscle cells or their cell-free extracellular matrices. Endothelial cell-conditioned medium had no effect on the growth kinetics of fibrosarcoma cells, and the inhibitory influence of conditioned medium on matrix degradation was greatest with low numbers of tumor cells. Conditioned medium inhibited the production of tumor cell plasminogen activators, enzymes previously found to play a role in matrix glycoprotein degradation. The endothelial factor was heat- and acid-stable and non-dialyzable, and mixing experiments showed that it did not directly inactivate the tumor cell plasminogen activator. Endothelial cells may therefore modulate the production of proteolytic enzymes important in the implantation stage of tumor metastasis.

Relationship between fibrinolysis of cultured cells and malignancy.

Cells cultured from various human and nonhuman malignant and normal tissues as well as mammalian cells transformed in vitro were examined for their ability to induce fibrinolysis. Generally, except for normal cells derived from lung or kidney, malignant cells had a greater ability to induce fibrinolysis than did their normal counterparts. A correlation existed between the abilities of the cells to induce fibrinolysis, grow in soft agar, and form tumors in immunosuppressed hosts.

Inhibition of tumor cell collagenolytic activity by bovine endothelial cells.

The ability of cellular vascular components including endothelial cells, smooth muscle cells, and fibroblasts to interact with the collagenolytic activity of invasive human tumor cell lines has been investigated. The human HT1080 fibrosarcoma and Bowe melanoma cells, which rapidly digest collagenous proteins in vitro, failed to dissolve them when cocultivated with bovine endothelial cells. This inhibition was not dependent on the ability of endothelial cells to form a monolayer separating the tumor cells from the collagenous substrate. In contrast, little collagenolysis inhibitory activity was detected in bovine vascular smooth muscle cells and human fibroblasts when compared to endothelial cells. Serum-free medium conditioned by endothelial cells inhibited tumor cell-mediated collagenolysis. Our data further suggest that this inhibition was mediated by secreted collagenase inhibitors, since endothelial cell-conditioned medium did not suppress the production of metalloproteinases by the tumor cells but inhibited the activities of collagenases derived from tadpole, rabbit, and human fibroblasts. Treatment of the endothelial cells with cycloheximide suppressed the collagenase inhibitory activity, demonstrating active production of collagenase inhibitors by the cells. Gel filtration chromatography of endothelial cell-conditioned medium allowed the separation of two distinct peaks with inhibitory activities for vertebrate collagenase in the molecular weight range of 70,000 to 75,000 and 30,000 to 35,000, respectively. While the inhibitor with an approximate molecular weight of 30,000 to 35,000 shared many properties with the tissue inhibitor of metalloproteinases, the high-molecular-weight inhibitor demonstrated characteristics not yet described for any collagenase inhibitor. The production and secretion of inhibitors of vertebrate collagenase by bovine endothelial cells may be of importance in the local control of collagen turnover under physiological as well as pathological conditions.

Differential regulation of plasminogen activators by epidermal growth factor in normal and neoplastic human urothelium.

The regulation of urokinase (u-PA) and tissue plasminogen activator (t-PA) in cultured normal and neoplastic urothelium was examined because plasminogen activators (PAs) are thought to be important in malignancy. Both activators were synthesized by normal urothelial cells grown in vitro under chemically defined conditions. The level of t-PA activity decreased when normal urothelial cells reached saturation density, but was stimulated more than 10-fold by the addition of epidermal growth factor (EGF) to the culture medium. Northern blot analysis showed that the regulation occurred at the transcriptional level. On the other hand u-PA activity was regulated to a lower degree by EGF and was not affected by cell density. Immunohistochemical examination of urothelial cells in histology specimens showed that t-PA was present only in the apical cells facing the lumen, suggesting that the expression of the activity might be a marker for end-stage differentiation in vivo. In contrast to normal cells, tumor cell lines made only u-PA under all conditions tested, and the levels of its expression were either unaffected or slightly decreased by EGF. Tumor cells and normal cells therefore showed substantial differences in protease regulation by EGF.

Effects of X-irradiation on artificial blood vessel wall degradation by invasive tumor cells.

Artificial vessel wall cultures, constructed by growing arterial endothelial cells on preformed layers of rat smooth muscle cells, were used to evaluate the effects of X-irradiation on tumor cell-induced tissue degradation. Bovine endothelial cells had radiation sensitivities similar to those of rat smooth muscle cells. Preirradiation of smooth muscle cells, before the addition of human fibrosarcoma (HT 1080) cells, did not increase the rate of degradation and destruction by the invasive cells. However, the degradation rate was decreased if the cultures were irradiated after the addition of HT 1080 cells. The presence of bovine endothelial cells markedly inhibited the destructive abilities of fibrosarcoma cells, but preirradiation of artificial vessel walls substantially decreased their capabilities to resist HT 1080-induced lysis. These findings suggest that the abilities of blood vessels to limit extravasation may be compromised by ionizing radiation.

Inhibition of invasion of HT1080 sarcoma cells expressing recombinant plasminogen activator inhibitor 2.

Plasminogen activators (PA) elaborated by tumor cells play an important role in the complex process of tissue invasion and metastasis. In the present study the effect of the PA inhibitor type 2 (PAI-2) on tissue invasion in vitro and in vivo was investigated. Clones either expressing (B-) or not expressing the endogenous PAI-2 gene (C+) were isolated from the human HT1080 fibrosarcoma cell line and transfected with full-length PAI-2 cDNA. Recombinant PAI-2 (rPAI-2) expressed by these cells completely inhibited receptor-bound urokinase activity and partially neutralized secreted PA activity. Degradation of extracellular matrix proteins by these transfected cells was markedly decreased when compared to mock or untransfected control cells. The rPAI-2-expressing cells did not penetrate a multilayer of rat smooth muscle cells in vitro, which was readily invaded and destroyed by control cells. The PAI-2 transfectants remained tumorigenic in athymic/nude mice, but tumors originating from these cells showed the presence of a thick, collagenous capsule absent in tumors formed by control cells. Thus, expression of rPAI-2 in HT1080 cells resulted in neutralization of receptor-bound urokinase with subsequent inhibition of matrix protein degradation and invasion in vitro and induction of a thick, peritumoral capsule in vivo.

The plasminogen-plasminogen activator (PA) system in neuroblastoma: role of PA inhibitor-1 in metastasis.

Proteases of the plasminogen-plasminogen activator (PA) system play an important role in cancer metastasis. We have examined the expression of these proteases and their cell surface receptors and inhibitors in neuroblastoma, a tumor that originates in cells of the neural crest and is the second most common solid tumor in children. This analysis was performed in seven established human cell lines and 20 primary tumor specimens. Urokinase PA and, in particular, tissue-type PA were expressed in cell lines and in tumor tissues; however, their levels of expression did not correlate with clinical stage. There was little evidence suggesting that neuroblastoma cells concentrate PA activity at their cell surface because urokinase-type PA receptor mRNA was detected in two cell lines and in 5 of 20 tumor samples by reverse transcription-PCR only. PA inhibitor (PAI)-2 was absent in all cell lines and tumor tissue samples examined. However, PAI-1, which was not expressed by the cell lines, was expressed by stromal cells and, specifically, endothelial cells in tumor tissue. By extending the analysis of PAI-1 expression in 64 primary tumor specimens, we found that high PAI-1 expression paradoxically correlated with metastatic stage and tumor recurrence. In vitro experiments indicated that the expression of PAI-1 by human microvascular endothelial cells was stimulated in the presence of SK-N-BE(2) human neuroblastoma cells and neuroblastoma culture medium. Recombinant PAI-1 also promoted SK-N-BE(2) cell detachment from vitronectin and migration from vitronectin toward fibronectin. From these data, we conclude that the up-regulation of PAI-1 expression in endothelial cells may promote rather than inhibit metastasis in neuroblastoma.

Matrix metalloproteinases-2 and -9 are expressed in human neuroblastoma: contribution of stromal cells to their production and correlation with metastasis.

Neuroblastoma, the second most common solid childhood tumor, can be a highly invasive and metastatic form of cancer. To assess the role of matrix-degrading proteases in this cancer, we have examined the expression of matrix metalloproteinases (MMPs) and their corresponding tissue inhibitors of metalloproteinases (TIMPs) in 7 human neuroblastoma cell lines and 24 primary untreated tumors. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) were the only two MMPs expressed. MMP-2 was detected predominantly in an inactive proform in all tumor cell lines and tumor tissue extracts. The lack of MMP-2 activation in cell lines was attributed to the absence of expression of a membrane-type MMP (MT1-MMP), which activates proMMP-2, and to the abundant expression of TIMPs, particularly TIMP-2. Immunohistochemical analysis of tumor tissue samples indicated that MMP-2 was present in both tumor cells and stromal cells. In contrast, MMP-9 was not expressed by neuroblastoma cell lines but was present in inactive and active forms in extracts from tumor tissues. Immunohistochemical analysis of positive specimens indicated that MMP-9 was predominantly present in stromal, vascular, and perivascular cells surrounding nests of tumor cells. There was no correlation between the levels of these MMPs and the MYCN copy number or the histopathological phenotype. However, there were higher levels of MMP-2 and MMP-9 in stage IV (metastatic) disease when compared with stages I and II (noninvasive and nonmetastatic) or IV-S (P < 0.05).

In vitro correlates of transformation in C3H/10T1/2 clone 8 mouse cells.

Various potential in vitro correlates of malignancy were studied in four chemically transformed C3H/10T1/2 Clone 8 mouse cell lines and were compared with controls cells. The degree of tumorigenicity was best predicted by the relative plating efficiencies of the morphologically transformed cells in soft agar. All transformed cells also showed an increase in extracellular fibrinolytic activity which may be an additional marker for transformation. Intracellular fibrinolytic activity and loss of 125I-labeled cell surface protein (M.W. 250,000) were not correlated with morphological transformation or tumorigenicity in these cells.

Invasive properties of primary pediatric neoplasms in vitro.

Primary solid tumors were mechanically and/or enzymatically disassociated, and the resulting suspensions of single cells and small clumps of cells were seeded onto three different substrates, i.e., tissue culture plastic, rat smooth muscle cells (SMCs), and SMC-derived extracellular matrix. Tests of the relative effectiveness of these substrates in supporting the survival and/or growth of ten different neoplasms demonstrated that only two explants remained viable for longer than 2 weeks when seeded onto tissue culture plastic while nine of the ten survived on biological substrates for 1 month or longer. Thus, tissue culture plastic was a poor substrate for primary pediatric neoplasms. In general, more than 80% of the most common solid neoplasms in childhood (brain tumor, neuroblastoma, renal tumor, rhabdomyosarcoma, osteogenic sarcoma, and Ewing's sarcoma) routinely survived or grew in long-term cultures when cultured onto SMCs or their matrix. Both substrates were effective in promoting survival and/or growth; however, cells of neuroblastomas and certain brain tumors showed a preference for a living smooth muscle substrate. Tumor cells maintained their characteristic cellular and subcellular morphology when compared with the histology of the in vivo neoplastic lesions. Light and electron microscopy of selected neoplasms cultured on SMCs for various time periods demonstrated areas of distinct cellular invasion and/or partial destruction of the SMC multilayers which correlated with the invasive potentials of the neoplasms in patients. Invasion and destruction of the SMCs were also noticed with quiescent tumor cell cultures, indicating that growth was not a necessary property of invasion. Several neoplasms were also capable of the degradation of connective tissue proteins as indicated by the hydrolysis of radiolabeled SMC matrices, but simple correlations between the extent of matrix degradation and invasive ability could not be drawn. The culture system described consistently provided for the survival and/or growth of the most common pediatric tumors for long time periods. Thus, basic biological properties of primary tumors, e.g., growth, invasive potentials, and differentiation capabilities, could be investigated routinely.

Morphology, growth, chromosomal pattern and fibrinolytic activity of two new human neuroblastoma cell lines.

Neuroblastoma cell lines LA-N-1 and LA-N-2 were extablished from neuroblastoma cells in the bone marrow and in the primary tumor, respectively, of two children with metastatic neuroblastoma. Morphology, growth in vitro and in athymic nude mice, chromosomal patter, and fibrinolytic activity of these cell lines and of previously extablished human neuroblastoma cell lines IMR-32, SK-N-MC, and SK-N-SH were compared. Most LA-N-1 cells were tear-drop shaped, small cells with processes; they tended to grow in clusters. LA-N-2 was comprised of elongated cells and small round cells, the latter growing in dense clumps on the former. Electron microscopy revealed numerous cytoplasmic dense cores in many LA-N-1 cells but none in LA-N-2 CELLS. During logarithmic growth in vitro, doubling times for LA-N-1, LA-N-2, SK-N-MC, SK-N-SH, and IMR-32 cells were 32,56, 23, 36, and 26 hr, respectively. Cells of all lines formed colonies in soft agar, and, after variable latency periods, LA-N-1, LA-N-2, SK-N-MC, and IMR-32 cells formed tumors in athymic nude mice. The marker chromosome(s) characteristic of each cell line was present in more than 90% of cells of given line. Significant plasminogen-dependent fibrinolytic activity was present in cells of all lines. These studies indicate that LA-N-1 and LA-N-2 cells arose from single but different aberrant progenitor cells and that they have properties of neuroblastoma cells. They also demonstrate that cell lines derived from human neuroblastomas are heterogenous as are the tumors in children.

Plasminogen activator inhibitor-1 promotes angiogenesis by stimulating endothelial cell migration toward fibronectin.

Increased expression of plasminogen activator inhibitor-1 (PAI-1) in cancer patients is associated with unfavorable outcome, and the reason for this paradox has been poorly understood. We have previously reported elevated levels of PAI-1 in primary tumors of advanced neuroblastomas (Y. Sugiura et al., Cancer Res., 59: 1327-1336, 1999). Here we demonstrate that PAI-1 is coexpressed with the angiogenesis marker alpha(v)beta3 integrin in blood vessels of primary neuroblastoma tumors, suggesting that PAI-1 plays a role in angiogenesis. Using human brain microvascular endothelial cells (HBMECs), we found that PAI-1 inhibits alpha(v)beta3 integrin-mediated cell adhesion to vitronectin but promotes alpha5beta1-mediated migration from vitronectin toward fibronectin. Inhibition of vitronectin adhesion by PAI-1 did not induce HBMEC apoptosis. PAI-1 also inhibited endothelial tube formation on Matrigel in the presence of vitronectin but had a stimulatory effect in the presence of fibronectin. This effect of PAI-1 on microvascular endothelial cells is primarily related to the ability of PAI-1 to bind to vitronectin via its NH2-terminal domain and to interfere with cell adhesion to vitronectin. We propose that PAI-1 acts as a positive switch for angiogenesis by promoting endothelial cell migration away from their vitronectin-containing perivascular space toward fibronectin-rich tumor tissue. These observations provide a novel explanation for the enhancing effect of PAI-1 in cancer progression.

Elevated levels of plasminogen activator inhibitor-1 may account for the altered fibrinolysis by keloid fibroblasts.

Using a 3-dimensional fibrin gel model system simulating fibroplasia of wound repair, we investigated the interaction between keloid fibroblasts and fibrin matrix and compared it with that of normal fibroblasts. Normal skin fibroblasts caused fibrin gel degradation under serum-free conditions, whereas keloid fibroblasts did not cause microscopically detectable gel degradation. Fibrin gel degradation occurred through plasmin-mediated fibrinolysis, which was initiated by fibroblasts exhibited high uPA but low plasminogen activator inhibitor-1 (PAI-1) activities, and transforming growth factor-beta 1 prevented fibrinolysis of normal fibroblasts by upregulating PAI-1 while downregulating uPA activities. In contrast, keloid fibroblasts exhibited an intrinsically high level of PAI-1 and a low level of uPA. This change in the ratio of activator and inhibitor activities was attributed to altered fibrin degradation by keloid fibroblasts. The PAI-1 increase was also demonstrated at the RNA level by Northern analysis. In terms of the pivotal role of the plasmin/plasminogen activator system in matrix remodeling, the elevated PAI-1 level exhibited by keloid fibroblasts may have significant consequences not only in altered fibrin degradation, but also in subsequent repair steps that lead to keloids and fibrosis.

Time course of bioluminescent signal in orthotopic and heterotopic brain tumors in nude mice.

In vivo bioluminescence imaging is becoming increasingly popular. Quantification of bioluminescence signals requires knowledge of the variability and reproducibility of this technique. The objective of this study was to analyze the time course of luminescent signal emitted from firefly luciferase-expressing tumors in two locations, following luciferin injection and at different times after tumor cell implantation. Knowledge of the kinetics of the bioluminescent signals is required for the reliable quantification and comparison of signal during longitudinal studies. The kinetics of bioluminescence was evaluated in orthotopic and heterotopic brain tumors in mice using a human brain tumor cell line constitutively expressing luciferase. Tumor cells were implanted in the brains and flanks of the animals, and whole-body images revealing tumor location were obtained. Tumor burden was monitored over time by the quantitation of photon emission. The magnitude of bioluminescence measured in vivo varied with time after the injection of luciferin, as well as with dose, which necessitated that the comparison of the quantitative results take into consideration the time after injection. Heterotopic and orthotopic tumors exhibited significantly different time courses; however, time after implantation as characterized by kinetic studies performed on days 4 and 14 after cell implantation revealed no significant differences in orthotopic tumors. Future quantitative longitudinal studies must take into account the differences in the kinetics of different models.

Candidal splenic abscesses complicating acute leukemia of childhood treated by splenectomy.

Two patients with acute leukemia were found to have candidal splenic abscesses. Both patients were in remission and had normal granulocyte counts at the time the abscesses became evident. Both patients were treated with splenectomy and antifungal therapy with a definite response. The incidence and treatment of fungal splenic abscesses in leukemia patients is discussed with emphasis on the role of splenectomy.

Initial urinary catecholamine metabolite concentrations and prognosis in neuroblastoma.

Initial urinary catecholamine metabolite and amino acid excretion patterns were examined in 54 children with neuroblastoma. The relationships between prognosis and age at diagnosis, stage of disease, primary site, and histologic grade of tumor were similar in this population to those found in previous studies, but only age and stage were found to be independent prognostic variables. Prognosis in disseminated disease was found to correlate directly with the urinary vanilmandelic acid (VMA)/homovanillic acid (HVA) ratio but not with the absolute levels of HVA. The presence of the dopa metabolite, vanillactic acid, as well as increased amounts of cystathionine and/or low levels of VMA indicated poor prognosis. These results are consistent with the hypothesis that biochemically primitive neuroblastomas deficient in dopamine beta-hydroxylase are move virulent than their mature analogues which produce epinephrine, norepinephrine, and their metabolites.

Glucocorticoids inhibit plasminogen activator production by endothelial cells.

Confluent cultures of bovine aortic endothelial cells (BEC) were found to secrete both tissue type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA). Exposure of the cultures to increasing concentrations of dexamethasone resulted in a time and concentration dependent inhibition of cellular and secreted u-PA. Complete inhibition of u-PA production and secretion was found at dexamethasone concentrations of 10(-8) molar or higher. Several distinct PA forms with molecular weights ranging from 10,000-20,000 to greater than 200,000 were found in the conditioned medium of untreated BEC cultures. After addition of dexamethasone (10(-7) molar) to the culture medium the PAs with molecular weights of 117,000, 58,000, 47,000, were absent suggesting that they were u-PAs, whereas the PAs with molecular weights of greater than 200,000 and 75,000 remained unchanged suggesting their t-PA origins. The PAs with lower molecular weights of 35,000, 28,000 and 10,000 to 20,000 were most likely generated from the higher molecular weight forms by limited proteolysis since they were absent when the medium was conditioned in the presence of the protease inhibitor Trasylol. The two PA types may therefore be independently regulated in BEC.