Epithelial requirement for in vitro proliferation and xenograft growth and metastasis of MDA-MB-468 human breast cancer cells: oncogenic rather than tumor-suppressive role of E-cadherin.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is associated with downregulated E-cadherin and frequently with decreased proliferation. Proliferation may be restored in secondary metastases by mesenchymal-to-epithelial transition (MET). We tested whether E-cadherin maintains epithelial proliferation in MDA-MB-468 breast cancer cells, facilitating metastatic colonization in severe combined immunodeficiency (SCID) mice. METHODS: EMT/MET markers were assessed in xenograft tumors by immunohistochemistry. Stable E-cadherin manipulation was effected by transfection and verified by Western blotting, immunocytochemistry, and quantitative polymerase chain reaction (qPCR). Effects of E-cadherin manipulation on proliferation and chemomigration were assessed in vitro by performing sulforhodamine B assays and Transwell assays, respectively. Invasion was assessed by Matrigel outgrowth; growth in vivo was assessed in SCID mice; and EMT status was assessed by qPCR. Hypoxic response of E-cadherin knockdown cell lines was assessed by qPCR after hypoxic culture. Repeated measures analysis of variance (ANOVA), one- and two-way ANOVA with posttests, and paired Student's t tests were performed to determine significance (p < 0.05). RESULTS: EMT occurred at the necrotic interface of MDA-MB-468 xenografts in regions of hypoxia. Extratumoral deposits (vascular and lymphatic inclusions, local and axillary nodes, and lung metastases) strongly expressed E-cadherin. MDA-MB-468 cells overexpressing E-cadherin were more proliferative and less migratory in vitro, whereas E-cadherin knockdown (short hairpin CDH1 [shCDH1]) cells were more migratory and invasive, less proliferative, and took longer to form tumors. shCDH1-MDA-MB-468 xenografts did not contain the hypoxia-induced necrotic areas observed in wild-type (WT) and shSCR-MDA-MB-468 tumors, but they did not exhibit an impaired hypoxic response in vitro. Although vimentin expression was not stimulated by E-cadherin knockdown in 2D or 3D cultures, xenografts of these cells were globally vimentin-positive rather than exhibiting regional EMT, and they expressed higher SNA1 than their in vitro counterparts. E-cadherin suppression caused a trend toward reduced lung metastasis, whereas E-cadherin overexpression resulted in the reverse trend, consistent with the increased proliferation rate and predominantly epithelial phenotype of MDA-MB-468 cells outside the primary xenograft. This was also originally observed in WT xenografts. Furthermore, we found that patients with breast cancer that expressed E-cadherin were more likely to have metastases. CONCLUSIONS: E-cadherin expression promotes growth of primary breast tumors and conceivably the formation of metastases, supporting a role for MET in metastasis. E-cadherin needs to be reevaluated as a tumor suppressor.

18F-FDG PET-CT uptake is a feature of both normal diameter and aneurysmal aortic wall and is not related to aneurysm size.

PURPOSE: Aortic metabolic activity is suggested to correlate with presence and progression of aneurysmal disease, but has been inadequately studied. This study investigates the 2-[(18)F] fluoro-2-deoxy-D-glucose ((18)F-FDG) uptake in a population of infra-renal abdominal aortic aneurysms (AAA), compared to a matched non-aneurysmal control group. METHODS: The Positron Emission Tomography - Computed Tomography (PET/CT) database was searched for infra-renal AAA. Exclusion criteria were prior repair, vasculitis, and saccular/mycotic thoracic or thoraco-abdominal aneurysms. Matching of 159 non-aneurysmal (<3 cm diameter) controls from the same population was assessed. Infra-renal aortic wall FDG uptake was assessed using visual analysis; maximum standardized uptake value (SUVmax) and target to background mediastinal blood pool ratio (TBR) were documented. Predictors of FDG uptake (age, sex, aortic diameter, hypertension, statin use, and diabetes) were assessed using univariate analysis. Follow-up questionnaires were sent to referring clinicians. RESULTS: Aneurysms (n = 151) and controls (n = 159) were matched (p > 0.05) for age, sex, diabetes, hypertension, smoking status, statin use, and indication for PET/CT. Median aneurysm diameter was 5.0 cm (range 3.2-10.4). On visual analysis there was no significant difference in the overall numbers with increased visual uptake 24% (36/151) in the aneurysm group vs. 19% (30/159) in the controls, p = ns. SUVmax was slightly lower in the aneurysm group vs. controls (mean (2 SD) 1.75(0.79) vs. 1.84(0.58), p = 0.02). However there was no difference in TBR between the AAA group and controls (mean (2 SD) 1.03 (0.46) vs. 1.05(0.31), p = 0.36). During a median 18 (interquartile range 8-35) months' follow-up 20 were repaired and four were confirmed ruptured. CONCLUSIONS: The level of metabolic activity as assessed by (18)F-FDG PET/CT in infra-renal AAA does not correlate with aortic size and does not differ between aneurysms and matched controls.

Occupational radiation exposure during endovascular aortic procedures.

OBJECTIVES: To measure the radiation exposure of the operating team during endovascular aortic procedures, and to determine factors that predict high exposures. MATERIALS AND METHODS: Electronic dosimeters placed over and under protective lead garments, were used to prospectively record radiation exposure during endovascular aortic repairs performed in a designated interventional radiology suite. Univariate and multivariate linear regression analyses of predictors of radiation exposure were performed. RESULTS: A total of 26 infra-renal and 10 thoracic endovascular cases were studied. Median (IQR) patient age and body mass index were 76.0 (70.0-81.8) years and 26.2 (23.9-28.9) kg/m(2) respectively. Over-lead exposure to the operator was higher for thoracic than for infra-renal procedures (421.0 [233.8-597.8] µSv vs. 52.5 [27.8-179.8] µSv, p = .0003), reflecting a significant exposure to unprotected parts of the body. Under-lead exposures for operator and assistant were 5.5 (2.0-14.2) µSv and 1.0 (0.0-2.3) µSv respectively, which for an average caseload would comply with total body effective dose limits. Type of case and percentage of digital subtraction angiography (DSA) time in left anterior oblique angulations predicted dose to the operator (p < .0001). CONCLUSIONS: Thoracic procedures, DSA runs and obliquity of the C-arm are strong predictors of radiation exposure during endovascular aortic repairs. Understanding scatter radiation dynamics and instigating measures to minimise radiation exposure should be mandatory.

Predictors of outcome after endovascular repair for chronic type B dissection.

OBJECTIVES: To assess the durability of endovascular repair (TEVAR) in chronic type B dissection (CD) and identify factors predictive of outcome. DESIGN: Retrospective analysis of a prospective database. MATERIALS: Patients undergoing TEVAR for CD at a tertiary referral centre 2000-2010. METHODS: Analysis of pre-operative characteristics, operative outcome, false lumen thrombosis, aortic diameter and survival. RESULTS: 58 consecutive patients were included (49 elective, 9 urgent, mean age 66 years). Mean aortic diameter was 6.4 cm (Standard deviation SD 1.3 cm). Three patients died perioperatively (5%, 1 urgent, 2 elective). Complications included retrograde type A dissection (n = 3), paraplegia (1), and transient ischaemic attack (1). Estimated survival (Kaplan-Meier) was 89% (1-year) and 64% (3-years). Forty-seven patients had mid-term imaging follow-up at mean 38 months. Reintervention rate was 15% at 1-year and 29% at 3-years. Aortic diameter decreased in 24, was stable in 15 and increased in 8. Mid-term survival was higher in patients with aortic remodelling (reduction of aortic diameter >0.5 cm; 3-year 89%) than without (54%; Log Rank p = 0.005). Remodelling occurred with extensive false lumen thrombosis. CONCLUSION: Satisfactory mid-term outcome after TEVAR for CD remains a challenge. Survival is associated with aortic remodelling, which is related to persistence of flow in the false lumen.

Assessment of the accuracy of AortaScan for detection of abdominal aortic aneurysm (AAA).

BACKGROUND: AortaScan AMI 9700 is a portable 3D ultrasound device that automatically measures the maximum diameter of the abdominal aorta without the need for a trained sonographer. It is designed to rapidly diagnose or exclude an AAA and may have particular use in screening programs. Our objective was to determine its accuracy to detect AAA. METHODS: Subjects from our AAA screening and surveillance programs were examined. The aorta was scanned using the AortaScan and computed tomography (CT). RESULTS: Ninety-one subjects underwent imaging (44 AAA on conventional ultrasound surveillance and 47 controls). The largest measurement obtained by AortaScan was compared against the CT-aortic measurement. The mean aortic diameter was 2.8 cm. The CT scan confirmed the diagnosis of AAA in 43 subjects. There was one false positive measurement on conventional ultrasound. AortaScan missed the diagnosis of AAA in eight subjects. There were thirteen false positive measurements. The sensitivity, specificity, positive and negative predictive values were 81%, 72%, 72% and 81% respectively. CONCLUSION: A device to detect AAA without the need for a trained operator would have potential in a community-based screening programme. The AortaScan, however, lacks adequate sensitivity and significant technical improvement is necessary before it could be considered a replacement for trained screening personnel.

Techniques of assessing hypoxia at the bench and bedside.

Tissues require an adequate supply of oxygen in order to maintain normal cell function. Low oxygen tension (hypoxia) is characteristic of a number of conditions, including cancer, atherosclerosis, rheumatoid arthritis, critical limb ischaemia, peripheral vascular disease, and ischaemic heart disease. Tissue hypoxia is found in tumours, atherosclerotic plaque, and ischaemic myocardium. There is a growing interest in methods to detect and assess hypoxia, given that hypoxia is important in the progression of these diseases. Hypoxia can be assessed at the level of the whole organ, tissue, or cell, using both invasive and non-invasive methods, and by a range of immunohistochemical, biochemical, or imaging techniques. This review describes and critiques current methods of assessing hypoxia that are used at the bench and in clinical practice.

Predictors of stroke and paraplegia in thoracic aortic endovascular intervention.

BACKGROUND: Endoluminal repair of thoracic aortic pathology has become established in clinical practice, but is associated with significant neurological complications. The aim of this study was to identify factors that were predictive of stroke and paraplegia. METHODS: Prospective data was collected for a cohort of 293 consecutive patients having thoracic aortic endovascular repair between August 1997 and September 2009. Patient and procedural characteristics were related to the incidence of stroke and paraplegia using multivariate logistic regression analysis. RESULTS: The median age was 68 years (18-87), there were 191 men and 102 women. Mortality was 5.1% for 195 elective and 13.4% for 98 urgent patients. Stroke affected 16 (5.5%) patients: 11 affected the anterior and 5 the posterior circulation. Coverage of the left subclavian artery with no revascularisation was the only significant factor predictive of stroke (OR 5.34 (1.42-20.40) P = 0.01). Paraplegia affected 16 patients (5.5%) but no independent risk factor was identified: 12 were identified perioperatively and 4 were delayed by up to 6 months. CONCLUSION: Covering the left subclavian artery without revascularisation increases the risk of stroke following endoluminal repair of thoracic pathology. Paraplegia appears to be more complex and no independent precipitating factor was identified.

The role of endothelial cells and their progenitors in intimal hyperplasia.

Intimal hyperplasia leading to restenosis is the major process that limits the success of cardiovascular intervention. The emergence of vascular progenitor cells and, in particular, endothelial progenitor cells has led to great interest in their potential therapeutic value in preventing intimal hyperplasia. We review the mechanism of intimal hyperplasia and highlight the important attenuating role played by a functional endothelium. The role of endothelial progenitor cells in maintaining endothelial function is reviewed and we describe how reduced progenitor cell number and function and reduced endothelial function lead to an increased risk of intimal hyperplasia. We review other potential sources of endothelial cells, including monocytes, mesenchymal stem cells and tissue resident stem cells. Endothelial progenitor cells have been used in clinical trials to reduce the risk of restenosis with varied success. Progenitor cells have huge therapeutic potential to prevent intimal hyperplasia but a more detailed understanding of vascular progenitor cell biology is necessary before further clinical trials are commenced.

Long-term surveillance with computed tomography after endovascular aneurysm repair may not be justified.

BACKGROUND: There is a common perception that a large number of secondary interventions are needed following endovascular aortic aneurysm repair. METHODS: Prospective data were collected for a cohort of 417 consecutive elective patients undergoing infrarenal aortic endograft repair between April 2000 and May 2008. The rate of secondary interventions, associated morbidity and need for reintervention following surveillance imaging were analysed. RESULTS: The male : female ratio was 11 : 1, median age 76 (range 40-93) years and median aneurysm diameter 6.1 (5.3-11) cm. The overall 30-day mortality rate was 1.7 per cent (seven of 417). Secondary interventions were performed in 31 patients (7.4 per cent), of which six (1.4 per cent) were detected by surveillance. Endoleaks requiring reintervention occurred in 12 patients (2.9 per cent; ten type I and two type III endoleaks). Limb ischaemia secondary to graft occlusion occurred in 17 patients (4.1 per cent); extra-anatomical bypass was needed in 15 patients (3.6 per cent) and the remaining two had an amputation. Graft explantation following late infection was required in two patients (0.5 per cent). CONCLUSION: Endoluminal repair of infrarenal aortic aneurysms can be performed with a low reintervention rate. The value of prolonged surveillance seems limited and current surveillance protocols may require revision.

Is endovascular repair of mycotic aortic aneurysms a durable treatment option?

OBJECTIVE: Endovascular repair for degenerative aortic aneurysms is well established, but its role in those with infective pathology remains controversial. This study aims to assess the durability of endovascular repair with a review of our midterm results. METHOD: A retrospective analysis of a prospectively maintained endovascular database (1998-2008) was conducted, which identified 673 consecutive patients with aortic aneurysms. RESULTS: Nineteen patients (2.8%) were identified with infected aortic aneurysms, in which there were a total of 23 separate aneurysms (16 thoracic and seven abdominal). Six patients (32%) presented with rupture. Eleven patients (58%) had received antibiotics preoperatively for a median duration of 11 days (1-54 days). Fifteen of the 19 (79%) had positive blood cultures, with Staphylococcus aureus being the most common organism. All 19 patients underwent endovascular repair. There were three Type I endoleaks (one requiring conversion to open repair) and two Type II endoleaks. One patient developed transient paraplegia, resolved by cerebrovascular fluid (CSF) drainage, and one patient had a stroke. The 30-day mortality was 11%, and survival at median follow-up of 20 months (0-83 months) was 73%. All eight deaths in the series were related to aneurysm. CONCLUSION: Endovascular treatment of infective aortic pathology provides an early survival benefit; however, concerns over on-going graft infection remain.

Adenovirus-mediated VEGF gene therapy enhances venous thrombus recanalization and resolution.

OBJECTIVE: Rapid thrombus recanalization reduces the incidence of post-thrombotic complications. This study aimed to discover whether adenovirus-mediated transfection of the vascular endothelial growth factor gene (ad.VEGF) enhanced thrombus recanalization and resolution. METHODS AND RESULTS: In rats, thrombi were directly injected with either ad.VEGF (n=40) or ad.GFP (n=37). Thrombi in SCID mice (n=12) were injected with human macrophages transfected with ad.VEGF or ad.GFP. Thrombi were analyzed at 1 to 14 days. GFP was found mainly in the vein wall and adventitia by 3 days, but was predominantly found in cells within the body of thrombus by day 7. VEGF levels peaked at 4 days (376+/-299 pg/mg protein). Ad.VEGF treatment reduced thrombus size by >50% (47.7+/-5.1 mm(2) to 22.0+/-4.0 mm(2), P=0.0003) and increased recanalization by >3-fold (3.9+/-0.69% to 13.6+/-4.1%, P=0.024) compared with controls. Ad.VEGF treatment increased macrophage recruitment into the thrombus by more than 50% (P=0.002). Ad.VEGF-transfected macrophages reduced thrombus size by 30% compared with controls (12.3+/-0.89 mm(2) to 8.7+/-1.4 mm(2), P=0.04) and enhanced vein lumen recanalization (3.39+/-0.34% to 5.07+/-0.57%, P=0.02). CONCLUSIONS: Treatment with ad.VEGF enhanced thrombus recanalization and resolution, probably as a consequence of an increase in macrophage recruitment.

Epithelial mesenchymal transition traits in human breast cancer cell lines.

Epithelial mesenchymal transition (EMT) has long been associated with breast cancer cell invasiveness and evidence of EMT processes in clinical samples is growing rapidly. Genome-wide transcriptional profiling of increasingly larger numbers of human breast cancer (HBC) cell lines have confirmed the existence of a subgroup of cell lines (termed Basal B/Mesenchymal) with enhanced invasive properties and a predominantly mesenchymal gene expression signature, distinct from subgroups with predominantly luminal (termed Luminal) or mixed basal/luminal (termed Basal A) features (Neve et al Cancer Cell 2006). Studies providing molecular and cellular analyses of EMT features in these cell lines are summarised, and the expression levels of EMT-associated factors in these cell lines are analysed. Recent clinical studies supporting the presence of EMT-like changes in vivo are summarised. Human breast cancer cell lines with mesenchymal properties continue to hold out the promise of directing us towards key mechanisms at play in the metastatic dissemination of breast cancer.

Imaging of deep vein thrombosis.

BACKGROUND: Deep vein thrombosis of the leg affects 1-2 per cent of the population with an annual incidence of 0.5-1 per 1000. It presents with non-specific symptoms and signs making clinical diagnosis difficult. Techniques to image and diagnose this condition are advancing rapidly. METHODS AND RESULTS: A literature review from 1980 to 2007 was undertaken using PubMed, The Cochrane Library, Medline and Embase. The most frequently used diagnostic test is duplex ultrasonography which is accurate above the knee and has a low cost, but is limited by inaccuracy when assessing the pelvic and distal veins and in diagnosing a new thrombosis in the post-thrombotic limb. Magnetic resonance imaging (MRI) and sonographic elasticity imaging are more recent techniques that have shown promise in overcoming these limitations. However, their availability is currently restricted because they are expensive. Computed tomography (CT) is sensitive, specific and provides good imaging of the pelvis. It has the advantage that it can be performed at the same time as CT pulmonary angiography. CONCLUSION: MRI has some specific advantages over duplex ultrasonography, but requires refinement before it can be used clinically. Venography or CT venography should be considered when duplex scanning is inadequate.

Effect of statins on proteolytic activity in the wall of abdominal aortic aneurysms.

BACKGROUND: The aim of this study was to examine the effect of statin treatment on the activity of proteases in the wall of abdominal aortic aneurysms (AAAs). METHODS: The activities of matrix metalloproteinases (MMPs) 9 and 3, cathepsins B, H, K, L and S, and the cystatin C level were measured in extracts of AAA wall taken from 82 patients undergoing AAA repair; 21 patients were receiving statin treatment before surgery. All values were standardized against soluble protein (SP) concentration in the extract, and reported as median (interquartile range) or mean(s.e.m.). RESULTS: The two groups had similar demographics. Reduced activity of MMP-9 (43 (34-56) versus 80 (62-110) pg per mg SP; P < 0.001), cathepsin H (183 (117-366) versus 321 (172-644) nmol 4-methylcoumarin-7-amide released per mg SP; P = 0.016) and cathepsin L (102 (51-372) versus 287 (112-816) micromol 7-amino-4-trifluoromethylcoumarin released per mg SP; P = 0.020) was found in the statin-treated aortas compared with AAAs from patients not taking a statin. The statin-treated group had lower MMP-3 activity, but this did not reach statistical significance (P = 0.053). Cystatin C levels were higher in statin-treated aortas than in controls (41.3(3.1) versus 28.9(2.1) ng per mg SP; P = 0.003). CONCLUSION: Statins decreased the activity of proteases that have been implicated in aneurysm disease.

Vascular endothelial growth factor and basic fibroblast growth factor are found in resolving venous thrombi.

OBJECTIVE: Resolution of venous thrombi is accompanied by an ingrowth of capillaries, which appears to be analogous to angiogenesis in other tissues. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are major regulators of angiogenesis. The aim of this study was to determine the temporal changes and the location of VEGF and bFGF expression in a rat model of venous thrombus resolution. DESIGN AND METHODS: Thrombi were induced in the inferior venae cavae of rats by creating a stenosis to reduce blood flow by 80% to 90%. Thrombi, adjacent inferior vena cava wall, and systemic blood were collected at 1, 3, 7, 14, 21, and 28 days after thrombus generation (n = 9). Sham operations were performed (n = 5), and blood was collected at 1, 3, and 7 days. VEGF and bFGF were measured with specific immunoassays, and levels in tissues were expressed as picogram per milligram soluble protein. Tissues from two animals that were humanely killed 7 days after thrombus formation were prepared for histological examination. Immunohistochemistry was performed by the use of antibodies against VEGF, bFGF, and ED-1 (a monocyte/macrophage marker). RESULTS: Laminated thrombi were reliably produced with a median weight at 1 day of 39 mg (range, 23-63 mg). There was a significant increase in thrombus VEGF concentration between 1 day (median, 247; range, 0-514) and 7 days (median, 556; range, 254-1741) (P =.02). There was no difference between the seventh day and subsequent days. There was a positive linear correlation between thrombus bFGF concentration and time (R = 0.74, P <.0001), with a more than 300-fold increase in bFGF concentration over the 28 days of the study. VEGF and bFGF concentrations in the adjacent vena caval wall did not change significantly with time. The serum VEGF was significantly raised at 1 day (median, 5520 pg/mL; range, 4040-7912 pg/mL) and 3 days (median, 3880 pg/mL; range, 2564-7232 pg/mL) compared with 7 days (median, 1790 pg/mL; range, 232-3228 pg/mL) (P <.0001). Similar changes in the serum VEGF also developed in the sham-operated animals. The serum bFGF (day 1 median, 15.5 pg/mL; range, 1-42 pg/mL) did not change with time. Immunohistochemistry showed that the VEGF antigen was localized to monocytes, endothelial cells, and spindle-shaped cells within the thrombus. The bFGF antigen was localized to mononuclear cells and spindle-shaped cells and was also present in the extracellular matrix. CONCLUSION: VEGF and bFGF are found in organizing thrombi and have characteristic temporal expression patterns, which suggest that they have a role in thrombus resolution. Augmenting angiogenic growth factor expression may enhance thrombus recanalization, thus reducing long-term complications.

Mining and visualizing large anticancer drug discovery databases.

In order to find more effective anticancer drugs, the U.S. National Cancer Institute (NCI) screens a large number of compounds in vitro against 60 human cancer cell lines from different organs of origin. About 70,000 compounds have been tested in the program since 1990, and each tested compound can be characterized by a vector (i.e., "fingerprint") of 60 anticancer activity, or -[log(GI50)], values. GI50 is the concentration required to inhibit cell growth by 50% compared with untreated controls. Although cell growth inhibitory activity for a single cell line is not very informative, activity patterns across the 60 cell lines can provide incisive information on the mechanisms of action of screened compounds and also on molecular targets and modulators of activity within the cancer cells. Various statistical and artificial intelligence methods, including principal component analysis, hierarchical cluster analysis, stepwise linear regression, multidimensional scaling, neural network modeling, and genetic function approximation, among others, can be used to analyze this large activity database. Mining the database can provide useful information: (a) for the development of anticancer drugs; (b) for a better understanding of the molecular pharmacology of cancer; and (c) for improvement of the drug discovery process.