Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization.

Acquisition of invasive/metastatic potential through protease expression is an essential event in tumor progression. High levels of components of the plasminogen activation system, including urokinase, but paradoxically also its inhibitor, plasminogen activator inhibitor 1 (PAI1), have been correlated with a poor prognosis for some cancers. We report here that deficient PAI1 expression in host mice prevented local invasion and tumor vascularization of transplanted malignant keratinocytes. When this PAI1 deficiency was circumvented by intravenous injection of a replication-defective adenoviral vector expressing human PAI1, invasion and associated angiogenesis were restored. This experimental evidence demonstrates that host-produced PAI is essential for cancer cell invasion and angiogenesis.

Direct evidence of the importance of stromal urokinase plasminogen activator (uPA) in the growth of an experimental human breast cancer using a combined uPA gene-disrupted and immunodeficient xenograft model.

Several studies have indicated an interaction between tumor cells and infiltrating stromal cells regarding the urokinase plasminogen activation (uPA) system. By developing combined uPA gene-disrupted and immunodeficient mice, we have studied the role of stromal uPA for the growth of the MDA-MB-435 BAG human tumor xenograft. Subcutaneous tumor growth and lung metastasis were compared between wild-type immunodeficient mice and mice with the combined deficiencies. Tumor growth was evaluated by volume measurements and plasma beta-galactosidase activity and metastasis was evaluated by counting lung surface metastases. Although no differences appeared in primary tumor take between the two groups of mice, a significant difference was observed in primary tumor growth, with tumors in uPA-/- mice growing significantly more slowly. In addition, a nonsignificant trend toward fewer lung metastases in uPA-/- mice was observed. The present data points to a critical role of stromal-derived uPA in the primary tumor growth of MDA-MB-435 BAG xenografts, whereas only a trend toward fewer lung metastases in uPA gene-disrupted mice was found.

MCF7/LCC2: a 4-hydroxytamoxifen resistant human breast cancer variant that retains sensitivity to the steroidal antiestrogen ICI 182,780.

The development of resistance to the antiestrogen tamoxifen occurs in a high percentage of initially responsive patients. We have developed a new model in which to investigate acquired resistance to triphenylethylenes. A stepwise in vitro selection of the hormone-independent human breast cancer variant MCF-7/LCC1 against 4-hydroxytamoxifen produced a stable resistant population designated MCF7/LCC2. MCF7/LCC2 cells retain levels of estrogen receptor expression comparable to the parental MCF7/LCC1 and MCF-7 cells. Progesterone receptor expression remains estrogen inducible in MCF7/LCC2 cells, although to levels significantly lower than observed in MCF-7 and MCF7/LCC1 cells. MCF7/LCC2 cells form tumors in ovariectomized nude mice without estrogen supplementation, and these tumors are tamoxifen resistant but can be estrogen stimulated. Significantly, MCF7/LCC2 cells have retained sensitivity to the steroidal antiestrogen ICI 182,780. These data suggest that some breast cancer patients who acquire resistance to tamoxifen may not develop cross-resistance to treatment with steroidal antiestrogens.

MCF7/LCC9: an antiestrogen-resistant MCF-7 variant in which acquired resistance to the steroidal antiestrogen ICI 182,780 confers an early cross-resistance to the nonsteroidal antiestrogen tamoxifen.

Acquired resistance to antiestrogens is a major problem in the clinical management of initially endocrine responsive metastatic breast cancer. We have shown previously that estrogen-independent and -responsive MCF7/LCC1 human breast cancer cells selected for resistance to the triphenylethylene tamoxifen produce a variant (MCF7/LCC2) that retains sensitivity to the steroidal antiestrogen ICI 182,780 (N. Brunner et al., Cancer Res., 53: 3229-3232, 1993). We have now applied stepwise selections in vitro from 10 pM to 1 microM ICI 182,780 against MCF7/LCC1 and obtained a stable ICI 182,780-resistant variant designated MCF7/LCC9. In contrast to 4-hydroxytamoxifen-selected MCF7/LCC2 cells, MCF7/LCC9 cells exhibit full cross-resistance to tamoxifen, despite never having been exposed to this drug. Significantly, tamoxifen cross-resistance arose early in the selection, appearing following selection against only 0.1 nM ICI 182,780. Although limited resistance to ICI 182,780 also was observed, full ICI 182,780 resistance was not detected until the selective pressure increased to 100 nM ICI 182,780. Cross-resistance to tamoxifen persisted throughout these additional selections. Despite their antiestrogen cross-resistance, MCF7/LCC9 cells retain a level of estrogen receptor expression comparable to that of their parental MCF7/LCC1 cells. Whereas MCF7/LCC1 cells retain an estrogen-inducible expression of progesterone receptors, MCF7/LCC9 cells exhibit an up-regulated expression of both progesterone receptor mRNA and protein that is no longer estrogen responsive. Estrogen-independent and -responsive components of the MCF7/LCC9 phenotype are apparent in vivo. These cells form slowly growing tumors in ovariectomized athymic nude mice but respond mitogenically upon estrogenic supplementation. The in vivo growth of MCF7/LCC9 tumors is not affected by treatment with ICI 182,780. Although there is some evidence of tamoxifen stimulation of tumor growth, this did not reach statistical significance. If this pattern of cross-resistance occurs in some breast cancer patients, administering triphenylethylene antiestrogens as a first-line therapy with a cross-over to steroidal compounds upon recurrence may be advantageous.

Urokinase-type plasminogen activation in three human breast cancer cell lines correlates with their in vitro invasiveness.

In order to invade and spread cancer cells must degrade extracellular matrix proteins. This degradation is catalysed by the concerted action of several enzymes, including the serine protease plasmin. Several experimental studies have shown that inhibition of plasmin formation reduces cancer cell invasion and metastasis, indicating a critical role of this proteolytic pathway in these processes. In order to further study the role of plasmin in cancer progression, we have characterized urokinase-type plasminogen activator (uPA) mediated plasmin formation in three human breast cancer cell lines. Using monoclonal antibodies against uPA and its receptor uPAR, we have investigated the contribution of uPA and uPAR to invasive capacity in an in vitro invasion assay. MDA-MB-231 BAG cells were found to express high protein levels of uPA, uPAR and PAI-1. MDA-MB 435 BAG cells produced low amounts of uPA, PAI-1 and moderate amounts of uPAR, whereas MCF-7 BAG cells showed low levels of uPA, uPAR and PAI-1 protein. In a plasmin generation assay MDA-MB-231 BAG cells were highly active in mediating plasmin formation, which could be abolished by adding either an anticatalytic monoclonal antibody to uPA (clone 5) or an anti-uPAR monoclonal antibody (clone R3), which blocks binding of uPA to uPAR. The two other cell lines lacked the capacity to mediate plasmin formation. In the Matrigel invasion assay the cells showed activity in this order: MCF-7 BAG < MDA-MB-435 BAG < MDA-MB-231 BAG. Testing MDA-MB-231 BAG cells in the Matrigel invasion assay revealed that invasion could be inhibited in a dose-dependent manner either by the clone 5 uPA antibody or by the clone R3 uPAR antibody, suggesting that the cell surface uPA system is actively involved in this invasive process. It is concluded that these three cell lines constitute a valuable model system for in vitro studies of the role of cell surface uPA in cancer cell invasion and has application in the search for novel compounds which inhibit mechanisms involved in uPA-mediated plasmin generation on cancer cells.

Measurement of tumor load and distribution in a model of cancer-induced osteolysis: a necessary precaution when testing novel anti-resorptive therapies.

The Arguello model of cancer metastasis to bone has been used extensively to study breast cancer-induced osteolytic disease. The effects of therapy on skeletal disease and on tumour burden in soft organs are traditionally measured using radiography and/or time-consuming histomorphometry, respectively. The purpose of this study was to develop a sensitive and efficient method for evaluating tumour burden in vivo using MDA-231 cells transduced with the E. coli lacZ gene (MDA-231BAG). Osteolysis was measured by radiography and tumour burden was measured histomorphometrically or biochemically. In untreated mice, measurements of tumour burden in bone extracts using human cytokeratin-associated tissue polypeptide antigen (TPA) ELISA or E. coli beta-galactosidase (beta-gal) activity immunoassay reflected the extent of osteolytic disease as measured by radiography; however, tumour load could be detected before onset of osteolysis. When monitoring the effect of therapy (0.2 mg/kg ibandronate/day), radiography alone proved to be insufficient. Mice treated with the bisphosphonate ibandronate from time of inoculation with cancer cells had no radiologically visible signs of osteolysis but significant tumour load was measured in the bone extracts using these assays. Furthermore, beta-gal activity could be used as a measurement of tumour load in soft organs, and unlike other human breast cancer markers expressed by the MDA-231 cells in vitro, beta-gal activity was detected in the serum of mice with progressive disease. In conclusion, we describe an efficient model of breast cancer-induced osteolysis to quantify the effect of therapy on disease load and distribution, which could be beneficial in evaluating novel therapies for the treatment of the disease.

Effect of NCAM-transfection on growth and invasion of a human cancer cell line.

A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial basement membrane. However, when injected into nude mice, both control and NCAM-expressing cell lines produced equally invasive tumors. Tumors generated from NCAM-transfected cells were heterogeneous, containing NCAM-positive as well as NCAM-negative areas, indicating the existence of host factors capable of modulating NCAM expression in vivo. In nude mice, NCAM-transfected cells developed tumors with longer latency periods and slower growth rates than tumors induced by NCAM-negative control cells, implying that NCAM may be involved not only in adhesive and motile behavior of tumor cells but also in their growth regulation. There was no indication of differences in cell proliferative characteristics between the different NCAM-transfected and the control transfected cells as determined by flow cytometric DNA analysis, suggesting an increased cell loss as the reason for decreased in vivo growth rate of the NCAM-transfected cells. The fact that NCAM expression influences growth regulation attributes a pivotal role to this cell adhesion molecule during ontogenesis and tumor development.

Segmentation of heart sound recordings by a duration-dependent hidden Markov model.

Digital stethoscopes offer new opportunities for computerized analysis of heart sounds. Segmentation of heart sound recordings into periods related to the first and second heart sound (S1 and S2) is fundamental in the analysis process. However, segmentation of heart sounds recorded with handheld stethoscopes in clinical environments is often complicated by background noise. A duration-dependent hidden Markov model (DHMM) is proposed for robust segmentation of heart sounds. The DHMM identifies the most likely sequence of physiological heart sounds, based on duration of the events, the amplitude of the signal envelope and a predefined model structure. The DHMM model was developed and tested with heart sounds recorded bedside with a commercially available handheld stethoscope from a population of patients referred for coronary arterioangiography. The DHMM identified 890 S1 and S2 sounds out of 901 which corresponds to 98.8% (CI: 97.8-99.3%) sensitivity in 73 test patients and 13 misplaced sounds out of 903 identified sounds which corresponds to 98.6% (CI: 97.6-99.1%) positive predictivity. These results indicate that the DHMM is an appropriate model of the heart cycle and suitable for segmentation of clinically recorded heart sounds.

Can new pulmonary gas exchange parameters contribute to evaluation of pulmonary congestion in left-sided heart failure?

BACKGROUND: Assessment of pulmonary congestion in left-sided heart failure is necessary for guiding anticongestive therapy. Clinical examination and chest x-ray are semiquantitative methods with poor diagnostic accuracy and reproducibility. OBJECTIVES: To establish reference values, describe reproducibility, and investigate the diagnostic and monitoring properties in relation to pulmonary congestion of new pulmonary gas exchange parameters describing ventilation/perfusion mismatch (variable fraction of ventilation [fA2] or the drop in oxygen pressure from the mixed alveolar air of the two ventilated compartments to the nonshunted end-capillary blood [DeltaPO(2)]) and pulmonary shunt. METHODS: Sixty healthy volunteers and 69 patients requiring an acute chest x-ray in a cardiac care unit were included. The gas exchange parameters were estimated by analyzing standard bedside respiratory and circulatory measurements obtained during short-term exposure to different levels of inspired oxygen. Nine patients were classified as having pulmonary congestion using a reference diagnosis and were followed during 30 days of anticongestive therapy. Diagnostic and monitoring properties were compared with chest x-ray, N-terminal probrain natriuretic peptide (NT-proBNP), spirometry values, arterial oxygen tension, alveolar-arterial oxygen difference and venous admixture. RESULTS: The 95% reference intervals for healthy subjects were narrow (ie, fA2 [0.75 to 0.90], DeltaPO(2) [0.0 kPa to 0.5 kPa] and pulmonary shunt [0.0% to 8.2%]). Reproducibility was relatively good with small within subject coefficients of variation (ie, fA2 [0.05], DeltaPO(2) [0.4 kPa] and pulmonary shunt [2.0%]). fA2, DeltaPO(2) and NT-proBNP had significantly better diagnostic properties, with high sensitivities (100%) but low specificities (30% to 40%). During successful anticongestive therapy, fA2, DeltaPO(2), NT-proBNP and spirometry values showed significant improvements. CONCLUSIONS: The gas exchange parameter for ventilation/perfusion mismatch but not pulmonary shunt can have a possible role in rejecting the diagnosis of pulmonary congestion and in monitoring anticongestive therapy.

[Breast cancer and polypeptide growth factors]. / Cancer mammae og polypeptid-voekstfaktorer.

Growth factors are crucially involved in the growth of breast cancer cells and in disease progression, both as mediators of endocrine therapy, as co-factors in the development of hormonal resistance, and as regulators of the extracellular proteolysis that precedes invasion and metastasis. Better characterisation and understanding of the mechanisms of action of the various growth factors might open up new possibilities in therapeutic intervention. Thus it would be possible to include in future research the development of synthetic agonists to the inhibitory growth factors, inhibitors of the stimulatory growth factors, and the development of receptor antagonists to block the binding of stimulatory growth factors to their receptors.

Plasma Escherichia coli beta-galactosidase as a marker of tumor burden and response to experimental anti-neoplastic therapy in nude mice xenografted with lacZ transduced human tumor cells.

Genetic labeling of tumor cells with the Escherichia coli lacZ reporter gene, encoding the enzyme beta-galactosidase, is widely used for histochemical detection of micrometastases in mice. Recently, we have developed a novel, highly sensitive and specific immunocapture chemiluminescence assay for the quantitation of E. coli beta-galactosidase. This assay achieved a detection limit of 0.01 mU of E. coli beta-galactosidase per milliliter, and 97% signal recovery of purified enzyme added to mouse plasma. LacZ transduced MDA-MB-231 BAG human breast cancer cells grown in vitro released soluble beta-galactosidase into the culture medium, and the concentration found correlated with cell density. Growth of the same cells in nude mice produced readily measurable levels of E. coli beta-galactosidase enzyme activity in host plasma and a highly significant correlation could be demonstrated between the size of primary tumor xenografts and the host plasma level of E. coli beta-galactosidase activity. When mice bearing MDA-MB-231 BAG tumor xenografts were treated intravenously with a single injection of doxorubicin (5 mg/kg), the mean tumor volume after 16 days was reduced 4-fold in the group of doxorubicin-treated mice compared with saline-treated control mice, and the mean level of plasma E. coli beta-galactosidase was correspondingly reduced 3.8-fold in the doxorubicin-treated mice compared with control mice. Sensitive and specific measurement of soluble E. coli beta-galactosidase in blood, using an immunocapture chemiluminescence assay, thus provides objective assessment of tumor burden in mice xenografted with lacZ transduced human tumors. This assay may have important applications as a tool for determining the efficacy of new experimental anti-tumor agents.

Soluble urokinase receptor released from human carcinoma cells: a plasma parameter for xenograft tumour studies.

The urokinase plasminogen activator receptor (uPAR) plays a critical role in urokinase-mediated plasminogen activation and thereby in the process leading to invasion and metastasis. Soluble urokinase receptor (suPAR) is released from tumours, and in cancer patients the blood level of soluble receptor is increased. Using an enzyme-linked, immunosorbent assay (ELISA)-specific for the human urokinase receptor, release of soluble receptor was measured in cultures of human breast carcinoma cells, in tumour extracts and in plasma from mice with xenografted human tumours. Soluble human urokinase receptor (shuPAR) was released into culture supernatant during the growth of the human breast cancer cell line MDA-MB-231 BAG, and the level of shuPAR in conditioned medium determined by ELISA was a linear function of both viable cell number and time of incubation. Western blotting showed that the form of shuPAR measured by ELISA in conditioned medium consisted virtually exclusively of the three-domain full-length protein, while uPAR in cell lysates consisted of full-length uPAR as well as the domains (2+3) cleavage product. shuPAR was also released into the plasma of nude mice during growth of MDA-MB-231 BAG, MDA-MB-435 BAG and HCT 116 cells as subcutaneously xenografted tumours. Western blotting demonstrated that the shuPAR released from the xenografted human tumours into plasma consisted of the three-domain full-length protein, despite the finding of some cleaved uPAR in detergent extracts of tumour tissue. The levels of shuPAR determined by ELISA in the plasma of host mice during the growth of xenografted cell lines were highly correlated with tumour volume.

Peptide-derived antagonists of the urokinase receptor. affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation.

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays an important role in pericellular plasminogen activation. Since proteolytic degradation of the extracellular matrix has an established role in tumor invasion and metastasis, the uPA-uPAR interaction represents a potential target for therapeutic intervention. By affinity maturation using combinatorial chemistry we have now developed and characterized a 9-mer, linear peptide antagonist of the uPA-uPAR interaction demonstrating specific, high-affinity binding to human uPAR (K(d) approximately 0.4 nM). Studies by surface plasmon resonance reveal that the off-rate for this receptor-peptide complex is comparable to that measured for the natural protein ligand, uPA. The functional epitope on human uPAR for this antagonist has been delineated by site-directed mutagenesis, and its assignment to loop 3 of uPAR domain III (Met(246), His(249), His(251), and Phe(256)) corroborates data previously obtained by photoaffinity labeling and provides a molecular explanation for the extreme selectivity observed for the antagonist toward human compared to mouse, monkey, and hamster uPAR. When human HEp-3 cancer cells were inoculated in the presence of this peptide antagonist, a specific inhibition of cancer cell intravasation was observed in a chicken chorioallantoic membrane assay. These data imply that design of small organic molecules mimicking the binding determinants of this 9-mer peptide antagonist may have a potential application in combination therapy for certain types of cancer.

Increased stromal expression of murine urokinase plasminogen activator in a human breast cancer xenograft model following treatment with the matrix metalloprotease inhibitor, batimastat.

The matrix metalloprotease (MMP) family of enzymes and the urokinase plasminogen activator (uPA) pathway have both been implicated in tumor invasion and metastasis and in poor prognosis of cancer. We have previously shown that treatment with batimastat, a synthetic MMP inhibitor, leads to significant retardation but not regression of tumor growth in a human breast cancer xenograft model. In addition, batimastat treatment did not inhibit local tumor invasion, nor did it encourage stromal encapsulation of the tumor, suggesting the additional involvement of non-MMP proteolytic mechanisms. To investigate the presence of an alternative extracellular matrix protease whose activity is known to be important in breast cancer, but which is not inhibited by batimastat, expression of murine and human uPA were examined by in situ hybridization and ELISA. No differences were observed between untreated and batimastat-treated tumors regarding human uPA mRNA and protein. In contrast, murine uPA mRNA expression was increased at the tumor-stromal junction in batimastat-treated tumors in comparison with the control tumors. In agreement with these results, batimastat treatment was shown to significantly induce murine uPA protein content in the tumors. Inoculating MDA435/LCC-6 cells into immunodeficient, uPA-deficient mice resulted in tumor growth retardation as compared to tumor growth in littermate wild-type controls, while addition of batimastat treatment to uPA-/- mice did not result in further growth inhibition. The increased expression of stromal uPA may represent a cellular response to MMP inhibition and may demonstrate a new level of plasticity in the malignant progression of the disease. These results may have important implications for the clinical applications of MMP inhibitors, as well as for development of other anti-invasion drugs.