Blockade of vascular endothelial growth factor receptor 2 inhibits intraplaque haemorrhage by normalization of plaque neovessels.

BACKGROUND: Plaque angiogenesis is associated with atherosclerotic lesion growth, plaque instability and negative clinical outcome. Plaque angiogenesis is a natural occurring process to fulfil the increasing demand of oxygen and nourishment of the vessel wall. However, inadequate formed, immature plaque neovessels are leaky and cause intraplaque haemorrhage. OBJECTIVE: Blockade of VEGFR2 normalizes the unbridled process of plaque neovessel formation and induces maturation of nascent vessels resulting in prevention of intraplaque haemorrhage and influx of inflammatory cells into the plaque and subsequently increases plaque stability. METHODS AND RESULTS: In human carotid and vein graft atherosclerotic lesions, leaky plaque neovessels and intraplaque haemorrhage co-localize with VEGF/VEGFR2 and angiopoietins. Using hypercholesterolaemic ApoE3*Leiden mice that received a donor caval vein interposition in the carotid artery, we demonstrate that atherosclerotic vein graft lesions at t28 are associated with hypoxia, Hif1α and Sdf1 up-regulation. Local VEGF administration results in increased plaque angiogenesis. VEGFR2 blockade in this model results in a significant 44% decrease in intraplaque haemorrhage and 80% less extravasated erythrocytes compared to controls. VEGFR2 blockade in vivo results in a 32% of reduction in vein graft size and more stable lesions with significantly reduced macrophage content (30%), and increased collagen (54%) and smooth muscle cell content (123%). Significant decreased VEGF, angiopoietin-2 and increased Connexin 40 expression levels demonstrate increased plaque neovessel maturation in the vein grafts. VEGFR2 blockade in an aortic ring assay showed increased pericyte coverage of the capillary sprouts. CONCLUSION: Inhibition of intraplaque haemorrhage by controlling neovessels maturation holds promise to improve plaque stability.

The protective role of Toll-like receptor 3 and type-I interferons in the pathophysiology of vein graft disease.

BACKGROUND: Venous grafts are commonly used as conduits to bypass occluded arteries. Unfortunately, patency rates are limited by vein graft disease (VGD). Toll like receptors (TLRs) can be activated in vein grafts by endogenous ligands. This study aims to investigate the role of TLR3 in VGD. METHODS: Vein graft surgery was performed by donor caval vein interpositioning in the carotid artery of recipient Tlr2-/-, Tlr3-/-, Tlr4-/- and control mice. Vein grafts were harvested 7, 14 and 28d after surgery to perform immunohistochemical analysis. Expression of TLR-responsive genes in vein grafts was analysed using a RT2-profiler PCR Array. mRNA expression of type-I IFN inducible genes was measured with qPCR in bone marrow-derived macrophages (BMM). RESULTS: TLR2, TLR3 and TLR4 were observed on vein graft endothelial cells, smooth muscle cells and macrophages. Tlr3-/- vein grafts demonstrated no differences in vessel wall thickening after 7d, but after 14d a 2.0-fold increase (p = 0.02) and 28d a 1.8-fold increase (p = 0.009) compared to control vein grafts was observed, with an increased number of macrophages (p = 0.002) in the vein graft. Vessel wall thickening in Tlr4-/- decreased 0.6-fold (p = 0.04) and showed no differences in Tlr2-/- compared to control vein grafts. RT2-profiler array revealed a down-regulation of type-I IFN inducible genes in Tlr3-/- vein grafts. PolyI:C stimulated BMM of Tlr3-/- mice showed a reduction of Ifit1 (p = 0.003) and Mx1 (p < 0.0001) mRNA compared to control. CONCLUSIONS: We here demonstrate that TLR3 can play a protective role in VGD development, possibly regulated via type-I IFNs and a reduced inflammatory response.

The role of CD27-CD70-mediated T cell co-stimulation in vasculogenesis, arteriogenesis and angiogenesis.

BACKGROUND: T cells have a distinctive role in neovascularization, which consists of arteriogenesis and angiogenesis under pathological conditions and vasculogenesis under physiological conditions. However, the role of co-stimulation in T cell activation in neovascularization has yet to be established. The aim of this study was to investigate the role T cell co-stimulation and inhibition in angiogenesis, arteriogenesis and vasculogenesis. METHODS AND RESULTS: Hind limb ischemia was induced by double ligation of the left femoral artery in mice and blood flow recovery was measured with Laser Doppler Perfusion Imaging in control, CD70-/-, CD80/86-/-, CD70/80/86-/- and CTLA4+/- mice. Blood flow recovery was significantly impaired in mice lacking CD70 compared to control mice, but was similar in CD80/86-/-, CTLA4+/- and control mice. Mice lacking CD70 showed impaired vasculogenesis, since the number of pre-existing collaterals was reduced as observed in the pia mater compared to control mice. In vitro an impaired capability of vascular smooth muscle cells (VSMC) to activate T cells was observed in VSMC lacking CD70. Furthermore, CD70-/-, CD80/86-/- and CD70/80/86-/- mice showed reduced angiogenesis in the soleus muscle 10 days after ligation. Arteriogenesis was also decreased in CD70-/- compared to control mice 10 and 28 days after surgery. CONCLUSIONS: The present study is the first to describe an important role for T cell activation via co-stimulation in angiogenesis, arteriogenesis and vasculogenesis, where the CD27-CD70 T cell co-stimulation pathway appears to be the most important co-stimulation pathway in pre-existing collateral formation and post-ischemic blood flow recovery, by arteriogenesis and angiogenesis.

A protective role of IRF3 and IRF7 signalling downstream TLRs in the development of vein graft disease via type I interferons.

BACKGROUND: Toll like receptors (TLR) play an important role in vein graft disease (VGD). Interferon regulatory factors (IRF) 3 and 7 are the transcriptional regulators of type I interferons (IFN) and type I IFN responsive genes and are downstream factors of TLRs. Relatively little is known with regard to the interplay of IRFs and TLRs in VGD development. The aim of this study was to investigate the role of IRF3 and IRF7 signaling downstream TLRs and the effect of IRF3 and IRF7 in VGD. METHODS AND RESULTS: In vitro activation of TLR3 induced IRF3 and IRF7 dependent IFNß expression in bone marrow macrophages and vascular smooth muscle cells. Activation of TLR4 showed to regulate pro-inflammatory cytokines via IRF3. Vein graft surgery was performed in Irf3-/- , Irf7-/- and control mice. After 14 days Irf3-/- vein grafts had an increased vessel wall thickness compared to both control (P = 0.01) and Irf7-/- (P = 0.02) vein grafts. After 28 days, vessel wall thickness increased in Irf3-/- (P = 0.0003) and Irf7-/- (P = 0.04) compared to control vein grafts and also increased in Irf7-/- compared to Irf3-/- vein grafts (P = 0.02). Immunohistochemical analysis showed a significant higher influx of macrophages after 14 days in Irf3-/- vein grafts and after 28 days in Irf7-/- vein grafts compared to control vein grafts. CONCLUSIONS: The present study is the first to describe a protective role of both IRF3 and IRF7 in VGD. IRFs regulate VGD downstream TLRs since Irf3-/- and Irf7-/- vein grafts show increased vessel wall thickening after respectively 14 and 28 days after surgery.

T-cell co-stimulation by CD28-CD80/86 and its negative regulator CTLA-4 strongly influence accelerated atherosclerosis development.

OBJECTIVE: T-cells are central to the immune response responsible for native atherosclerosis. The objective of this study is to investigate T-cell contribution to post-interventional accelerated atherosclerosis development, as well as the role of the CD28-CD80/86 co-stimulatory and Cytotoxic T-Lymphocyte Antigen (CTLA)-4 co-inhibitory pathways controlling T-cell activation status in this process. METHODS AND RESULTS: The role of T-cells and the CD28-CD80/86 co-stimulatory and CTLA-4 co-inhibitory pathways were investigated in a femoral artery cuff mouse model for post-interventional remodeling, with notable intravascular CTLA-4+ T-cell infiltration. Reduced intimal lesions developed in CD4(-/-) and CD80(-/-)CD86(-/-) mice compared to normal C57Bl/6J controls. Systemic abatacept-treatment, a soluble CTLA-4Ig fusion protein that prevents CD28-CD80/86 co-stimulatory T-cell activation, prevented intimal thickening by 58.5% (p=0.029). Next, hypercholesterolemic ApoE3*Leiden mice received abatacept-treatment which reduced accelerated atherosclerosis development by 78.1% (p=0.040) and prevented CD4 T-cell activation, indicated by reduced splenic fractions of activated KLRG1+, PD1+, CD69+ and CTLA-4+ T-cells. This correlated with reduced plasma interferon-γ and elevated interleukin-10 levels. The role of CTLA-4 was confirmed using CTLA-4 blocking antibodies, which strongly increased vascular lesion size by 66.7% (p=0.008), compared to isotype-treated controls. CONCLUSIONS: T-cell CD28-CD80/86 co-stimulation is vital for post-interventional accelerated atherosclerosis development and is regulated by CTLA-4 co-inhibition, indicating promising clinical potential for prevention of post-interventional remodeling by abatacept.

Clinical relevance of biologic factors in male breast cancer.

There is ample information on the clinical role of biologic factors in female breast cancer: urokinase-type plasminogen activator (uPA), its receptor uPAR, its inhibitors PAI-1 and PAI-2, cathepsin D and pS2-protein. However such reports are missing or very rare for male breast cancer. We determined the cytosolic levels of oestrogen receptor (ER), progesterone receptor (PgR), cathepsin D, pS2-protein, uPA, uPAR, PAI-1 and PAI-2 of the primary tumour tissues from 40 male breast cancer patients. The tumour levels were compared with those of 180 matched females and 4114 historic females with breast cancer. In male breast tumours the level of PgR was higher, those of uPA, PAI-1, PAI-2 and cathepsin D lower. The tumour level of ER in men was similar to those in the matched and postmenopausal women, but much higher than those in the historic women. Male breast cancer seems to be biologically different from female breast cancer. Correlation of the eight cell biologic factors with disease outcome showed that PAI-1 (p = 0.03) was the only independent predictive factor for poor prognosis in male breast cancer.

High tumor levels of vascular endothelial growth factor predict poor response to systemic therapy in advanced breast cancer.

Vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been reported to be associated with a poor prognosis in primary breast cancer and in several other cancer types. In the present study, we have measured with ELISA the levels of VEGF in cytosolic extracts of 845 primary breast tumors of patients who developed a recurrence during follow-up. All of the patients received tamoxifen (n = 618) or cyclophosphamide, methotrexate, 5-fluorouracil (CMF) or 5-fluorouracil, Adriamycin, cyclophosphamide (FAC) chemotherapy (n = 227) as first-line systemic therapy after diagnosis of advanced disease. VEGF levels were not related to age or menopausal status but were negatively related to the cytosolic levels of estrogen receptor and progesterone receptor (P < 0.0001). In patients who relapsed within 1 year after primary surgery, tumor VEGF levels were higher than in patients who showed a longer disease-free interval (P = 0.0005). In patients with a first relapse in the viscera, VEGF levels were higher compared with those that relapsed to the bone or soft tissue (P = 0.0004). In univariate analysis for response to first-line tamoxifen therapy, patients with high or intermediate levels showed a poor rate of response, compared with patients with low tumor-VEGF levels (P = 0.0001). Similarly, in multivariate analysis for response to tamoxifen treatment, corrected for age, site of relapse, disease-free interval, and estrogen receptor and progesterone receptor status, VEGF status was an independent predictive factor (P = 0.009). In concordance, higher levels of VEGF were associated with a short progression-free survival and postrelapse overall survival (both, P < 0.0001). On first-line chemotherapy, the rate of response decreased with higher tumor levels of VEGF, both in univariate (P = 0.003) and in multivariate analysis (P = 0.004). Furthermore, higher VEGF levels were associated with a short progression-free survival (P = 0.003) and postrelapse overall survival (P = 0.001). In conclusion, the tumor VEGF level is an important independent marker that predicts a poor efficacy of both tamoxifen and chemotherapy in advanced breast cancer. Knowledge of the tumor level of VEGF might be helpful in selecting individual patients who may benefit from treatments with antiangiogenic agents combined with conventionally used drugs.

The urokinase-type plasminogen activator system in resected non-small-cell lung cancer. Rotterdam Oncology Thoracic Study Group.

BACKGROUND: Urokinase-type plasminogen activator (uPA), its receptor (uPAR) and plasminogen activator inhibitors (PAI-1 and PAI-2), all play important roles in tumour invasion and metastasis. The tumour levels of the components of the urokinase-type plasminogen activator system (uPA-system) may help to identify individuals with a poor prognosis in postoperative non-small-cell lung cancer (NSCLC) patients. PATIENTS AND METHODS: The levels of uPA, uPAR PAI-1 and PAI-2 were measured by enzyme-linked immunosorbent assay (ELISA) in triton-extracts, prepared from 88 NSCLC tissues (stage I-IIIa) and 74 normal lung tissues from the same patients. RESULTS: The expression levels of uPA, uPAR, PAI-1 and PAI-2 were significantly higher in tumour tissues as compared to their normal equivalents (all, P < 0.0001). Significant relations were found between gender and uPA (P = 0.04) or uPAR (P < 0.001), and between PAI-2 and pathological stage (P = 0.03). For none of the studied factors of the uPA-system a significant relation with survival was found, neither in all patients, nor in the subgroups of patients with squamous-cell lung carcinoma or adenocarcinoma. CONCLUSIONS: The expression levels of the components of the uPA-system were higher in NSCLC tissue as compared to normal lung tissue, but there were no significant relationships between their levels and survival.

The urokinase system of plasminogen activation and prognosis in 2780 breast cancer patients.

The antigen levels of components of the urokinase-type plasminogen activator (uPA) system of plasminogen activation are correlated with prognosis in several types of cancers, including breast cancer. In the present study involving 2780 patients with primary invasive breast cancer, we have evaluated the prognostic importance of the four major components of the uPA system [uPA, the receptor uPAR (CD87), and the inhibitors PAI-1 and PAI-2]. The antigen levels were determined by ELISA in cytosols prepared from primary breast tumors. The levels of the four factors significantly correlated with each other; the Spearman rank correlation coefficients (r(s)) ranged from 0.32 (between PAI-2 and PAI-1 or uPAR) to 0.59 (between uPA and PAI-1). The median duration of follow-up of patients still alive was 88 months. In the multivariate analyses for relapse-free survival (RFS) and overall survival (OS), we defined a basic model including age, menopausal status, tumor size and grade, lymph node status, adjuvant therapy, and steroid hormone receptor status. uPA, uPAR, PAI-1, and PAI-2 were considered as categorical variables, each with two cut points that were established by isotonic regression analysis. Compared with tumors with low levels, those with intermediate and high levels showed a relative hazard rate (RHR) and 95% confidence interval (95% CI) of 1.22 (1.02-1.45) and 1.69 (1.39-2.05) for uPA, and 1.32 (1.14-1.54) and 2.17 (1.74-2.70) for PAI-1, respectively, in multivariate analysis for RFS in all patients. Compared with tumors with high PAI-2 levels, those with intermediate and low levels showed a poor RFS with a RHR (95% CI) of 1.30 (1.14-1.48) and 1.76 (1.38-2.24), respectively. Similar results were obtained in the multivariate analysis for OS in all patients. Furthermore, uPA and PAI-1 were independent predictive factors of a poor RFS and OS in node-negative and node-positive patients. PAI-2 also added to the multivariate models for RFS in node-negative and node-positive patients, and in the analysis for OS in node-negative patients. uPAR did not further contribute to any of the multivariate models. A prognostic score was calculated based on the estimates from the final multivariate model for RFS. Using this score, the difference between the highest and lowest 10% risk groups was 66% in the analysis for RFS at 10 years and 61% in the analysis for OS. Moreover, separate prognostic scores were calculated for node-negative and node-positive patients. In the 10% highest risk groups, the proportion of disease-free patients was only 27 +/- 6% and 9 +/- 3% at 10 years for node-negative and node-positive patients, respectively. These proportions were 86 +/- 4% and 61 +/- 6% for the corresponding 10% lowest risk groups of relapse. We conclude that several components of the uPA system are potential predictors of RFS and OS in patients with primary invasive breast cancer. Knowledge of these factors could be helpful to assess the individual risk of patients, to select various types of adjuvant treatment and to identify patients who may benefit from targeted therapies that are currently being developed.

Prognostic value of tissue-type plasminogen activator (tPA) and its complex with the type-1 inhibitor (PAI-1) in breast cancer.

The prognostic value of tissue-type plasminogen activator (tPA) measured in samples derived from 865 patients with primary breast cancer using a recently developed enzyme-linked immunosorbent assay (ELISA) was evaluated. Since the assay could easily be adapted to the assessment of the complex of tPA with its type-1 inhibitor (PAI-1), it was investigated whether the tPA:PAI-1 complex also provides prognostic information. To this end, cytosolic extracts and corresponding detergent extracts of 100,000 g pellets obtained after ultracentrifugation when preparing the cytosolic fractions for routine steroid hormone receptor determination were assayed. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient r(s) = 0.75, P < 0.001 for tPA and r = 0.50, P < 0.001 for tPA:PAI-1 complex). In both Cox univariate and multivariate analysis elevated levels of (total) tPA determined in the pellet extracts, but not in cytosols, were associated with prolonged relapse-free (RFS) and overall survival (OS). In contrast, high levels of the tPA:PAI-1 complex measured in cytosols, but not in the pellet extracts, were associated with a poor RFS and OS. The prognostic information provided by the cytosolic tPA:PAI-1 complex was comparable to that provided by cytosolic (total) PAI-1. Furthermore, the estimated levels of free, uncomplexed tPA and PAI-1, in cytosols and in pellet extracts, were related to patient prognosis in a similar way as the (total) levels of tPA and PAI-1 respectively. Determination of specific forms of components of the plasminogen activation system, i.e. tPA:PAI-1 complex and free, uncomplexed tPA and/or PAI-1, may be considered a useful adjunct to the analyses of the separate components (tPA and/or PAI-1) and provide valuable additional prognostic information with respect to survival of breast cancer patients.

Breast-conserving therapy: proteases as risk factors in relation to survival after local relapse.

PURPOSE: To evaluate whether cathepsin D, urokinase-type plasminogen activator (uPA), its inhibitor, plasminogen activator inhibitor-1 (PAI-1), or clinical factors can predict which patients are at risk for developing distant metastases after local recurrence (LR). PATIENTS AND METHODS: Of 1,630 patients treated with breast-conserving surgery and radiotherapy of the breast between 1980 and 1992, LR developed in 171 as a first event. From the available primary tumor tissues, we determined the cytosolic levels of cathepsin D, uPA and PAI-1. RESULTS: In patients with LR, a short (< or = 2 years) disease-free interval (DFI) and skin involvement of LR were associated with poor postrelapse distant metastasis-free survival (PR-DMFS, P = .001, both) and postrelapse overall survival (PR-OS; P < .0001 and P < .0002, respectively). The primary tumor levels of uPA and PAI-1 were elevated for patients with a short DFI (P < .01), but such a relation was not observed for patients with skin involvement. In univariate analyses, high levels of uPA and PAI-1 in the primary tumor were associated with poor PR-OS (P = .038 and P = .040, respectively) but not PR-DMFS. In Cox multivariate analyses for PR-DMFS and PR-OS, only a short DFI and skin involvement of the LR were independently associated with a poor clinical outcome. CONCLUSION: In patients treated with breast-conserving therapy who had LR as a first event, a short DFI and skin involvement were strong indicators for poor PR-DMFS and PR-OS. The proteases studied did not contribute significantly to the final multivariate model.

Prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) in cytosols and pellet extracts derived from 892 breast cancer patients.

To evaluate the clinical relevance of urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-1) measured by a recently developed enzyme-linked immunosorbent assay (ELISA), we analysed both components in samples derived from 892 patients with primary breast cancer (median follow-up 99 months). The assays were performed in cytosolic extracts as well as in corresponding detergent extracts of pellets obtained after ultracentrifugation, which was carried out when preparing the cytosolic fractions for routine steroid hormone receptor determination. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient r = 0.60, P < 0.0001 for uPA and r = 0.65, P < 0.0001 for PAI-1). Furthermore, strong correlations were found between the levels of both uPA (r = 0.85, P < 0.0001) and PAI-1 (r = 0.90, P< 0.0001) in the cytosols and their levels previously measured with ELISAs based on commercial reagents. In both Cox univariate and multivariate analysis, high cytosolic levels of uPA or PAI-1 were significantly associated with increased rates of relapse and death. The levels of uPA and PAI-1 in the pellet extracts also provided prognostic information, although to a lesser extent compared with the cytosolic extracts. The prediction of prognosis on the basis of uPA and PAI-1 assessed by an alternative ELISA once again emphasizes the established prognostic role and usefulness of these parameters in selection of breast cancer patients at high or low risk of recurrence.

Urokinase-type-plasminogen-activator (uPA) production by human breast (myo) fibroblasts in vitro: influence of transforming growth factor-beta(1) (TGF beta(1)) compared with factor(s) released by human epithelial-carcinoma cells.

The urokinase-type plasminogen activator (uPA) may be considered as a key enzyme in the processes of cancer cell invasion and metastasis. Evidence has been presented that, in breast stroma, uPA is expressed predominantly by myofibroblasts located at the invasive areas of the tumor. To examine whether transforming growth factor type-1 (TGF beta(1)) produced by breast-carcinoma cells is a candidate responsible for the induction of uPA-producing myofibroblasts, we studied in vitro the capacity of normal and tumor-derived human breast fibroblasts to express uPA and the myofibroblast marker alpha-smooth-muscle actin in response to TGF beta(1). Next, we compared these influences with those elicited by factor(s) released by epithelial-cancer cells. In all 8 fibroblast strains tested, TGF beta(1) induced a similar concentration-dependent increase in the fraction of alpha-smooth-muscle-actin-positive fibroblasts. While uPA expression was decreased by TGF beta(1) in most of the fibroblast strains, 2 strains were relatively insensitive to TGF beta(1) in this respect. Although factors present in media conditioned by non-uPA-producing epithelial-tumor cells could trigger fibroblasts to become potent producers of uPA, the TGF beta(1) content of the conditioned media were linked to the differential effects of externally added TGF beta(1) with respect to uPA expression. The data demonstrate that, although fibroblasts may utilize TGF beta(1) secreted by tumor cells to differentiate into myofibroblasts, tumor cells secrete factor(s) other than TGF beta(1) ultimately responsible for the generation of powerful uPA-producing fibroblasts.

Prognostic significance of cathepsins B and L in primary human breast cancer.

PURPOSE: Evaluation of the clinical significance of cytosolic tumor levels of the lysosomal cysteine proteases cathepsin B (catB) and cathepsin L (catL) in patients with primary breast cancer. PATIENTS AND METHODS: CatB (n = 1,500) and catL (n = 1,391) levels were determined by enzyme-linked immunosorbent assay (ELISA) in cytosols routinely prepared from frozen-tissue samples that were submitted to our laboratory for the assessment of steroid-hormone-receptor status. The median duration of follow-up of patients still alive at the time of analysis was 93 months. RESULTS: Relating catB and catL levels with classical prognostic factors, the proteases were positively correlated with the number of positive lymph nodes (P < .01), and negatively with the level of steroid-hormone receptors (P < .01). We did not find a significant relationship between catB or catL levels with age and menopausal status of the patients or with the size of the primary tumor. The levels of catB and catL were positively correlated with each other and with the rates of relapse and death (all, P < .0001). In multivariate regression analysis for relapse-free survival (RFS) and overall survival (OS), corrected for the contribution of age/menopausal status, tumor size, the number of positive lymph nodes, and steroid-hormone-receptor status, catB and catL were significant predictors of the rates of relapse and death (all, P < .01). No statistically significant interactions of catB or catL with any of the classical prognostic factors or with each other were observed in their associations with the rates of relapse and death. CONCLUSION: CatB and catL levels measured in routinely prepared cytosols are strong parameters to predict the rate of relapse and the length of survival after treatment of the primary breast tumor.

Uptake and distribution of doxorubicin in hormone-manipulated human breast cancer cells in vitro.

BACKGROUND: Kinetic resistance to cytotoxic drugs may account for the moderate responsiveness of breast cancer to chemotherapy. In the present study the in vitro effects of estradiol-mediated DNA stimulation on the cellular uptake of the DNA intercalating drug doxorubicin (DOX) were examined in MCF-7 human breast cancer cells. METHODS: Using the fluorescent properties of the drug, the cellular uptake was investigated by high performance liquid chromatography (HPLC), and by flow cytometry. RESULTS: The uptake of DOX (0.01-2 microg/ml) by MCF-7 cells in suspension, incubated for 1 and 6 h, showed a strong correlation between the incubation concentration of DOX and the cellular uptake of the drug as measured by HPLC and flow cytometry. Simultaneous exposure of MCF-7 cells, in monolayer culture, to DOX (0.04-0.2 microg/ml) and estradiol (1 nM) for 1-24 h showed no significant difference in uptake of the drug compared to control cultures exposed to DOX in the absence of estradiol. Neither was there a significant difference in uptake of DOX when MCF-7 cells were pretreated with estradiol (1 nM) for 16-24 h followed by a 0.5, 1, 6, and 21/23 h incubation with DOX (0.01-2 microg/ml). Pretreatment with estradiol did not affect the retention of DOX as measured 24 h after a 0.5 h incubation with DOX (2 microg/ml). Furthermore, fluorescence microscopy revealed no difference in the cellular DOX distribution pattern of estradiol-stimulated MCF-7 cultures compared to unstimulated cultures. CONCLUSION: From this study we can conclude that, for the human MCF-7 breast cancer cells in vitro, the uptake, retention, and cellular distribution of DOX are not influenced by estrogenic manipulation.

The MTT tetrazolium salt assay scrutinized: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay is widely used for in vitro measurement of the metabolic viability of cell cultures subjected to different culture conditions. This convenient assay, which is based on the ability of viable cells to produce formazan, can be affected significantly by a number of conditions. These conditions can be roughly divided into groups, firstly influences which affect the spectrum of the produced formazan and secondly influences which affect the amount of formazan produced per cell. We studied the various chemical and biochemical aspects involved in the MTT assay. Our data indicated that microscopical viewing of cell cultures before and after performing the assay, a medium renewal with a well-defined MTT-incubation medium at the end of the culture period and regular cell counting are essential steps to ensure a reliable performance of the MTT assay. In conclusion, providing the necessary precautions are taken, the MTT assay can be used reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival.

Prognostic significance of the receptor for urokinase plasminogen activator in breast cancer.

We have recently described the urokinase-type plasminogen activator (uPA) and its type 1 inhibitor (PAI-1) as strong prognostic variables in breast cancer (J. A. Foekens et al., Cancer Res., 52: 6101-6105, 1992; J. Grondahl-Hansen et al., Cancer Res., 53: 2513-2521, 1993; J. A. Foekens et al., J. Clin. Oncol., 11: 899-908, 1994). A specific cell surface receptor (uPAR) binds uPA and strongly enhances plasmin generation, and the amount of uPAR in the tumor tissue might therefore be a rate-limiting factor in the extracellular proteolysis involved in tumor invasion. Here, we report on the prognostic value of uPAR in cytosolic (uPARc) and Triton (uPARt) extracts prepared from 505 primary breast tumors. The median observation time was 54 (range: 12-125) months. uPAR levels were determined by a sandwich ELISA. Univariate analysis showed that high uPAR levels (above the median value) were significantly associated with a shorter overall survival, showing a stronger discriminatory effect for uPARc [relative hazard rate (RHR): 1.47; P = 0.012)] as compared with uPARt (RHR, 1.33; P = 0.059), while no statistically significant differences were found for relapse-free survival. Multivariate analysis including all patients showed that when including other biochemical variables (estrogen receptor, progesterone receptor, PS2, cathepsin D, uPA, and PAI-1), the only retained independent variable via backward elimination was PAI-1 for both relapse-free survival and overall survival. When analyzed separately in clinically relevant subgroups, the prognostic value of uPAR was particularly strong in a subgroup of 201 node-positive postmenopausal women, showing considerably shorter overall (RHR: 2.39; P < 0.0001) and relapse free (RHR: 1.91; P = 0.0006) survival for patients with high uPARc content. High uPARt levels were also significantly associated with shorter overall survival in this subgroup of patients (RHR: 1.5; P = 0.047), but not with relapse-free survival (P = 0.64). Multivariate analysis, including the basic model, estrogen and progesterone receptors, PS2, cathepsin D, uPA, PAI-1, uPARc, and uPARt in the subgroup of postmenopausal node-positive patients, showed that only uPARc and PAI-1 were significant independent prognostic parameters, with respect to overall survival, RHRs being 2.72 (P < 0.0001) and 1.81 (P = 0.005), respectively. In multivariate analysis of relapse-free survival, uPARc, PAI-1, and uPA were independent parameters with respective relative relapse rates of 1.91 (P = 0.002) for uPARc, 1.68 (P = 0.02) for PAI-1, and 1.6 (P = 0.03) for uPA. These data lend support to the hypothesis that uPAR is an important molecule in plasmin-mediated extracellular matrix degradation leading to cancer cell dissemination and death of the patient.

Urokinase-type plasminogen activator and its inhibitor PAI-1: predictors of poor response to tamoxifen therapy in recurrent breast cancer.

BACKGROUND: Urokinase-type plasminogen activator (uPA) is a proteolytic enzyme thought to be involved in processes leading to tumor cell invasion of surrounding tissues. Its activity during metastasis may be regulated by an inhibitor, PAI-1. Previous work has shown that high levels of uPA and PAI-1 are associated with poor prognosis in primary breast cancers. PURPOSE: In this pilot study, we explored possible associations between the expression levels of uPA or PAI-1 and the efficacy of tamoxifen treatment in breast cancer patients with relapsed disease. METHODS: Levels of uPA, PAI-1, estrogen receptor (ER), and progesterone receptor (PgR) were assayed in cytosolic extracts derived from the primary breast tumors of 235 tamoxifennaive patients who had recurrent disease. The extracts were classified as positive or negative for each assayed factor. In some analyses, ER and PgR levels were evaluated together. In these analyses, three ER/PgR subsets were defined: low, intermediate, and high. All patients in the study received tamoxifen therapy upon relapse (median follow-up, 57 months). Association of the tested factors with the response to tamoxifen treatment was studied by logistic regression analysis. Association of the factors with progression-free and overall survival was further evaluated by Cox univariate and multivariate regression analyses. RESULTS: Patients with uPA-negative tumors exhibited a better response (tumor regression or stable disease, maintained for more than 6 months) to tamoxifen treatment than those with uPA-positive tumors (51% versus 26% response; odds ratio [OR] = 0.34; 95% confidence interval [CI] = 0.18-0.65). The response rate was also better for patients with PAI-1-negative tumors than for those with PAI-1-positive tumors (49% versus 35% response; OR = 0.57; 95% CI = 0.32-1.01). In addition, patients with uPA-positive or PAI-1-positive tumors showed shorter progression-free survival (P = .001 and P < .05, respectively) and total survival after relapse (P = .005 and P < .005, respectively). When patients were stratified by ER/PgR status, the only statistically significant association between uPA levels and reduced tamoxifen response was seen in the subset whose tumors possessed intermediate levels of ER/PgR (16% response in uPA-positive versus 60% response in uPA-negative tumors; OR = 0.13; 95% CI = 0.04-0.41). Overall, uPA status appeared independent of association with ER/PgR status in its ability to predict response to tamoxifen treatment. The association of PAI-1 expression and the response to tamoxifen was less pronounced when patients were stratified by ER/PgR status. CONCLUSION: Measurement of primary breast tumor uPA levels may be useful in predicting the overall response of metastatic disease to tamoxifen therapy.

Plasminogen activator inhibitor-2: prognostic relevance in 1012 patients with primary breast cancer.

The antigen levels of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor (PAI) 1, as detected in tumor extracts by ELISA, have been reported to be correlated with a poor prognosis in primary breast cancer. In the present study we have characterized a novel PAI-2-specific ELISA, designed to measure PAI-2 antigen levels in tumor cytosols. We determined PAI-2 antigen levels along with those of uPA and PAI-1 in 1012 routinely prepared tumor cytosols of patients with primary breast cancer (median follow-up, 71 months). In the overall population there was no significant association between the level of PAI-2 and prognosis, while in tumors with high uPA values, PAI-2 (test for trend) was associated with a prolonged relapse-free survival, metastasis-free survival, and overall survival (for all analyses, P < 0.02). In Cox's multivariate analysis for relapse-free survival, metastasis-free survival, and overall survival in tumors with high uPA values (including patient's age, menopausal status, lymph node status, tumor size, estrogen and progesterone receptor status, uPA, and PAI-1), PAI-2 either dichotomized or, as a continuous variable, was independently associated with a favorable relapse-free survival, metastasis-free survival, and overall survival. We conclude that the PAI-2-specific ELISA described herein is well suited for the measurement of PAI-2 levels in cytosols routinely prepared for analysis of steroid hormone receptors. We speculate that PAI-2 may serve as an inhibitor for uPA in human primary breast cancers.

Plasminogen activator inhibitor-1 and prognosis in primary breast cancer.

PURPOSE: Evaluation of the relationship between plasminogen activator inhibitor-1 (PAI-1) and the metastatic potential of primary breast cancer, and to compare the prognostic impact of PAI-1 in multivariate analysis with those of conventional prognostic factors, including steroid-hormone receptors, and those of urokinase plasminogen activator (uPA), pS2-protein (PS2), and cathepsin D. PATIENTS AND METHODS: Cell biologic prognostic factors were analyzed in 657 cytosols routinely prepared from frozen-tissue biopsies that were submitted to our laboratory for the assessment of steroid-hormone receptor status. The median duration of follow-up in patients still alive at the time of analysis was 48 months. Estrogen receptor (ER) and progesterone receptor (PgR) status were assessed by radioligand binding assay, PS2, and cathepsin D by radiometric immunoassay, and uPA and PAI-1 by enzyme-linked immunosorbent assay (ELISA). RESULTS: PAI-1 levels were found to be strongly positively correlated with the rates of relapse (P < .0001) and death (P < .001). Relating the levels of PAI-1 with those of other cytosolic prognostic factors, we found a positive association with the metastasis-related proteases uPA (P < .0001) and cathepsin D (P < .0001). On the other hand, PAI-1 levels were found to be negatively correlated with ER (P < .005) and PgR (P < .001), and the estrogen-regulated pS2-protein (P < .001), which are proteins associated with a favorable prognosis. In multivariate regression analysis for 5-year relapse-free survival, and using an optimized cutoff point for discrimination between PAI-1-positive and -negative, independent predictors of the rate of relapse were found to be PAI-1 (P < .0001) and uPA (P = .01) of the cytosolic parameters, and tumor size, lymph node status, and premenopausal age of the clinical parameters. In multivariate analysis in patients with node-negative disease, only PAI-1 (P < .001) and tumor size (P = .03) were positively and premenopausal age negatively (P < .001) associated with the rate of relapse. In patients with node-positive disease, PAI-1 (P < .001), uPA (P = .02), tumor size (P < .001), and the number of positive lymph nodes (P < .001) were all positively associated with the rate of relapse. CONCLUSION: We conclude that the PAI-1 level measured in routinely prepared cytosols is an important parameter to predict the metastatic potential in both node-negative and node-positive human primary breast cancer.