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New hurricane menaces Caribbean
Filed under: Entertainment News, Latest, Politics News — Tags: airport, alert, arrival of Hurricane Ike, Atlantic, Atlantic coast, Bahamas, British, British territory to the north of Haiti, Caicos, capital, Caribbean, Caribbean nations, Carolina, catastrophic, Category Four, Ceballos, city of Gonaives, Civil defense director Colonel Juan Manuel Mendez, Cuba, Dominican Republic, Dominican troops, eastern provinces, Fay, floodwater, floodwaters, Georgia, Gloria, Gloria Ceballos, Gonaives, Grand Turk Island, Gustav, Haiti, Haitian officials, Hanna, Hardest hit by Hanna, HAVANA, heavy rainfall, hurricane, Hurricane Gustav, Hurricane Ike, Ike, island, island of Hispaniola, islands, Juan, Juan Manuel Mendez, Manuel, menaces, Mendez, meteorological official Gloria Ceballos, National Hurricane Center, New hurricane menaces Caribbean, New Jersey, NHC, north, north of Haiti, Oxfam, Parnell, police, Providenciales, reports, residents, South Carolina, storm, Tropical Storm Fay, tropical storm Hanna, Turks, UN World Food Program, UN's World Food Programme, US, US south-east coast, visitors, water, WFP — expressyoureself @ 3:41 am
Caribbean nations are bracing for another major storm, Hurricane Ike, coming just days after Tropical Storm Hanna passed through the region.
Ike has regained strength after weakening, with winds of up to 135mph (215km/h) as it nears the Turks and Caicos islands and the Bahamas.
Cuba has issued a hurricane watch for its eastern provinces.
Haitian officials have said that at least 500 people have been found dead as floodwater's caused by Hanna recede.
That storm has hit the US south-east coast and is dropping torrential rain on North and South Carolina.
Storm warnings are in force along the Atlantic coast from Georgia to New Jersey.
'Major hurricane'
Hurricane Ike gained strength to Category Four on the Saffir Simpson scale – an "extremely dangerous hurricane" – after weakening slightly earlier on Saturday, said the Florida-based National Hurricane Center (NHC).
As of 2100 GMT, Ike was tracking west south-west, moving at 15mph about 90 miles (145km) east of Grand Turk Island.
SAFFIR-SIMPSON SCALE
Cat 1: Winds 74-95mph (119-153km/h). No real damage to buildings
Cat 2: Winds 96-110mph (154-177km/h). Storm surge 6-8 feet (1.8-2.8 metres) above normal
Cat 3: Winds 111-130mph (178-209km/h). Major hurricane. Coastal flooding destroys smaller structures
Cat 4: Winds 131-155mph (210-249km/h). Large storm surge and widespread damage to smaller buildings
Cat 5: Winds greater than 155mph (249km/h). Small buildings blown away, roofs on large buildings destroyed. All trees and signs knocked down. Widespread coastal flooding.
Source: US National Hurricane Center
The NHC said the storm was expected to pass near or over the Turks and Caicos islands and the south-eastern Bahamas late on Saturday or early Sunday.
After Hanna pummeled the low-lying Turks and Caicos, a British territory to the north of Haiti, earlier in the week, many residents and visitors decided to leave.
Authorities decided to close the airport in Providenciales at mid-day on Saturday.
Ike should hit the northern coast of eastern Cuba by late Sunday or early Monday, according to the NHC forecast.
If it stays on its projected course, Ike will cut across the island from east to west, putting the crumbling colonial buildings of the capital, Havana, at risk.
A storm surge of up to 12ft (3.6m) is expected along with "large and dangerous battering waves" and heavy rainfall, the NHC said.
The center of the hurricane is forecast to pass to the north of Haiti, which shares the island of Hispaniola with the Dominican Republic.
But Haiti, already reeling from three major storms in as many weeks, will not be spared, with up to 12in (30cm) of rain due to fall.
As floodwaters caused by Tropical Storm Hanna receded, Haitian officials said more than 500 people had been killed.
Hurricane Gustav last week and Tropical Storm Fay two weeks ago killed about 120 people.
Hardest hit by Hanna was the city of Gonaives, which was flooded with up to 16ft of water that has only now begun to recede.
The devastation there has been described as catastrophic.
Police said 500 people were confirmed dead but that others are still missing and the number could rise higher.
The UN's World Food Program (WFP) said hundreds of thousands of people had been displaced by the flooding.
The WFP has begun distributing food aid but a spokesperson said the scale of the disaster was putting their resources to the test.
Other aid workers say people's spirits are running low after the successive storms.
"Food supplies and water are scarce and the price of the food that's left is rising," said Parnell Denis from Oxfam in Gonaives.
"The morale of people staying in the shelters is so very low; I am afraid to tell them that another storm is on its way."
More bad weather will hamper the aid effort even further.
In the Dominican Republic, which shares the island of Hispaniola with Haiti, there have been no reports of major damage.
However, preparations are under way for the arrival of Hurricane Ike.
"The ground is saturated and some of the dams in the south-east region are fairly close to their maximum capacity," said meteorological official Gloria Ceballos.
Civil defense director Colonel Juan Manuel Mendez said Dominican troops had been put on alert.
Are you in the Caribbean? Have you been affected by the storms? What preparations have you made to deal with the adverse weather? Send us your comments and experiences
'Hundreds' killed by Haiti storm
Filed under: Latest, Politics News — Tags: 'Hundreds' killed by Haiti storm, alert, appeal, Atlantic coast, Bahamas, Caicos, Carolina, Civil defense, coasts of North and South Carolina, Dominican, Dominican Republic, Dominican troops, Dorfeuille, Ernst Dorfeuille, floodwaters, Georgia, Gloria, Gloria Ceballos, Gonaives, Haiti, Haiti storm, Hanna, helicopter, Hispaniola, hurricane, Hurricane Ike, International Federation of Red Cross, island of Hispaniola, Joel Boutrioue, Juan, Juan Manuel Mendez, Manuel, Mendez, Michele Pierre-Louis, New Jersey, Polie, Prime Minister Michele Pierre-Louis, Puerto Rico, Rainfall, Red Crescent Societies, Red Cross, reports, South Carolina, south-eastern Bahama, storm, torrential rainfall, tropical storm Hanna, tropical storm warnings, Turks, Turks Caicos Islands, U.N, UN's humanitarian co-ordinator in Haiti, US, US National Hurricane Center, Weather — expressyoureself @ 7:31 am
Floods in Gonaives left people stranded and in desperate need of help
Almost 500 bodies have been found in the port city of Gonaives, Haiti, after floodwaters caused by recent storms receded, according to reports.
Polie commissioner Ernst Dorfeuille said 495 bodies had been found and the toll could get higher.
A ship carrying 33 tons of UN aid arrived in Haiti on Friday to help an estimated 600,000 people struggling in the wake of tropical storm Hanna.
The storm is heading for the US, while Hurricane Ike threatens the Bahamas.
See Ike's predicted path
The US National Hurricane Center says Ike is a Category Three hurricane, with winds of up to 185 km/h (115mph).
The smell of the dead is very unpleasant in Gonaives
Commissioner Ernst Dorfeuille
It is expected to pass near or over the Turks and Caicos Islands and south-eastern Bahamas late on Saturday or early Sunday.
By then it could be a major hurricane, forecasters say.
Tropical storm Hanna is causing heavy squalls off the coasts of North and South Carolina and tropical storm warnings are in force along the Atlantic coast from Georgia to New Jersey.
In Haiti, the devastation from the storm in Gonaives has been described as catastrophic.
Prime Minister Michele Pierre-Louis said her newly-installed government would take the necessary measures to help victims.
Stench of death
Commissioner Dorfeuille told Reuters new agency: "The weather is calm now and we are discovering more bodies. We have found 495 bodies so far and there are 13 people missing.
"The smell of the dead is very unpleasant in Gonaives. The death toll could be even higher."
Hanna dumped massive amounts of rain on the country over four days, blowing down fruit trees and swamping tin-roofed houses.
The port city of Gonaives bore the brunt of the storm, forcing thousands of people to seek shelter on rooftops and balconies as flood waters rose.
The UN's humanitarian co-ordinator in Haiti, Joel Boutrioue, told the it was still difficult to get aid to thousands of people. Roads are cut off and access to some areas is only possible by air – which is limited by the available number of helicopters.
The International Federation of Red Cross and Red Crescent Societies has also launched an appeal, asking for $3.4m in aid.
Haiti was first drenched by Tropical Storm Fay, before Hurricane Gustav wreaked havoc last week, with torrential rainfall over heavily deforested and hilly terrain causing floods and mudslides.
Earlier, Hanna was also blamed for two deaths in Puerto Rico.
Are you in Haiti? Have you been affected by the storms? Are you based elsewhere in the region? What preparations have you made to deal with the adverse weather? Send us your comments and experiences
McCain vows to fight to change US
Filed under: Latest, Politics News — Tags: Add new tag, Alaska, Alaska Governor, Alaska Governor Sarah Palin, America, American, American people, Americans, Arizona, arizona senator, attacking the Democrats, barack obama, bipartisan, Bush, Bush years, Carolina, Cindy, Convention, corruption, Democrat Geraldine Ferraro, Democrat Geraldine Ferraro in 1984, Democratic, Democratic rival Barack Obama, diplomatic, economic, family values, female, George W Bush, Geraldine Ferraro, Hariprasad, Hariprasad Bhusal, independents, India, john mccain, leadership, man, maverick, McCain, McCain administration, McCain vows to fight to change US, military, Minnesota, Minnesota Governor Tim Pawlenty, Mr McCain, Mr McCain's wife, Mrs Palin, national convention, Palin, Pawlenty, Politics, President, President George W Bush, prisoner of war in Vietna, Reagan, Republican, Republican Party, Republican politics, Roosevelt, Sarah Palin, senator, Senator Lindsey Graham, South Carolina, South Carolina Senator Lindsey Graham, speech, St Paul, The party of Lincoln, the Republican Party's first female vice-presidential c, Tim, Tim Pawlenty, US, US vice-presidency, video, Vietnam, war in Vietnam, Washington, Washington politics, White House, woman — expressyoureself @ 2:49 pm
John McCain has accepted the Republican Party's candidacy for the White House in a speech to cheering supporters at the party's national convention.
He vowed to bring change to government, restore the people's trust in the party and to fight for a better nation.
Praising his running mate Sarah Palin, he said she was the right person to help him bring change to Washington.
The Arizona senator said he respected Democratic rival Barack Obama and would seek a bipartisan approach to politics.
Presenting himself once again as a maverick, he pledged to fight corruption, whether Democratic or Republican, and make sure that he worked for the good of the American people.
"Let me offer an advance warning to the old, big spending, do nothing, me first, country second Washington crowd: change is coming," Mr McCain told the crowds in St Paul, Minnesota.
Again and again, I've worked with members of both parties to fix problems that need to be fixed
In a criticism of his own party, he said he would "fight to restore the pride and principles" of the party, damaged after some Republicans gave in to "the temptations of corruption".
"We're going to recover the people's trust by standing up again for the values Americans admire," he said. "The party of Lincoln, Roosevelt and Reagan is going to get back to basics."
Mr McCain then turned to attacking the Democrats over taxes and spending, saying they would seek to raise taxes whereas he would keep them low and cut them where possible.
Going into some policy specifics, he pledged create new jobs, improve education and to reduce a "dangerous dependence on foreign oil" by producing more energy at home, including by drilling new offshore oil wells.
Mr McCain promised to take a bipartisan approach to resolving the nation's problems, saying: "Again and again, I've worked with members of both parties to fix problems that need to be fixed.
McCAIN'S MOST-USED WORDS
"That's how I will govern as president. I will reach out my hand to anyone to help me get this country moving again.
"I have that record and the scars to prove it. Barack Obama does not."
After speaking of the five years he spent as a prisoner of war in Vietnam and how that experience had inspired his love of his country, he called on his fellow Americans to fight with him to make it a better one.
"Stand up, stand up, stand up and fight. Nothing is inevitable here. We're Americans and we never give up. We never quit. We never hide from history. We make history."
The almost hour-long speech, which ended in the traditional shower of confetti and red, white and blue balloons, brought to a close the party's four-day event.
'Tested and true'
Mr McCain's speech was measured and entirely lacking in the sarcasm and vitriol which have been levelled at Mr Obama over the past couple of nights.
He said he hated war and would use all America's tools – diplomatic, military and economic – to build what he called a stable and enduring peace, as well as shaking up Washington and including Democrats and independents in a McCain administration.
Mrs McCain praised her husband as a great father and devoted American
It was all a rather different tone to the Republican politics of the past eight years, and to many of the other speakers at this Republican convention, our correspondent says.
There was very little of President George W Bush in this speech, our correspondent adds, as Mr McCain tries to show that he is his own man and can signify a break with the Bush years.
Mr McCain's wife, Cindy, in her speech praised her husband's family values, strength of character, war service and leadership.
"If Americans want straight talk and the plain truth, they should take a good close look at John McCain… a man tested and true, who's never wavered in his devotion to our country," she said, after arriving on stage flanked by their seven children.
Her speech followed the convention's formal nomination of Mrs Palin – the Republican Party's first female vice-presidential candidate.
Mrs Palin becomes only the second woman, the first being Democrat Geraldine Ferraro in 1984, to run for the US vice-presidency.
'Integrity and courage'
Speaking ahead of Mr McCain's address, senior Republicans praised his courage and leadership.
I have to say, from my vantage point next to the DC delegation, my overall impression was that the audience in the hall were disappointed
BBC North America editor Justin Webb
Minnesota Governor Tim Pawlenty, at one point hotly tipped to be Mr McCain's running mate choice, described the Arizona senator's life as "a testimony to service, duty, courage and common sense".
"In this time, we don't need a president who can just read a poll or momentarily thrill a crowd. We don't need rhetoric or empty promises," he said.
"We need a president who has the integrity and courage to make the tough choices so America will be stronger and safer."
South Carolina Senator Lindsey Graham hailed Mr McCain's determination to back the Bush administration's "surge" strategy in Iraq despite the political risks.
McCain is an experienced person and his speech impressed me
Hariprasad Bhusal, India
He introduced a video presenting Alaska Governor Sarah Palin as a "maverick" moose-hunter from Alaska who was joining "the original maverick" Mr McCain to bring change to Washington politics.
In a well-received speech on Wednesday, Mrs Palin praised Mr McCain and attacked Mr Obama as having talked of change, but done nothing of substance.
President George W Bush has also strongly endorsed John McCain as the best man to succeed him in the White House.
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Establishment and Characterization of a CTC Cell Line from Peripheral Blood of Breast Cancer Patient
Pan Zhao1*, Wenbin Zhou2*, Chang Liu1,3*, Huirong Zhang1, Zhiqiang Cheng4,5, Weiqing Wu6, Kaisheng Liu1, Hong Hu2, Caineng Zhong2, Yayuan Zhang2, Dongxian Zhou2, Feiyuan Liu1, Yong Dai1 , Jianhong Wang1,5 , Chang Zou1,5
1. Clinical Medical Research Center, the Second Clinical Medical College of Jinan University, the First Affiliated Hospital of Southern University, Shenzhen People's Hospital, Shenzhen 518020, China
2. Department of breast and thyroid surgery, the Second Clinical Medical College of Jinan University, the First Affiliated Hospital of Southern University, Shenzhen People's Hospital, Shenzhen 518020, China
3. Central Laboratory, Dalian Municipal Central Hospital, Dalian 116033, China
4. Department of Pathology, the Second Clinical Medical College of Jinan University, the First Affiliated Hospital of Southern University, Shenzhen People's Hospital, Shenzhen 518020, China
5. Shenzhen Public Service Platform on Tumor Precision Medicine and Molecular Diagnosis, the Second Clinical Medical College of Jinan University, Shenzhen People's Hospital, Shenzhen 518020, China
6. Department of Health Management, the Second Clinical Medical College of Jinan University, the First Affiliated Hospital of Southern University, Shenzhen People's Hospital, Shenzhen 518020, China
* These authors contribute equally to this work
✉ Corresponding author: Chang Zou, Email: zouchang.cuhkcom, Fax: 86 (755) 22942850, Clinical Medical Research Center, The Second Clinical Medical College of Jinan University, The first Affiliated Hospital of Southern University, Shenzhen People's hospital, Shenzhen 518020, China More
Zhao P, Zhou W, Liu C, Zhang H, Cheng Z, Wu W, Liu K, Hu H, Zhong C, Zhang Y, Zhou D, Liu F, Dai Y, Wang J, Zou C. Establishment and Characterization of a CTC Cell Line from Peripheral Blood of Breast Cancer Patient. J Cancer 2019; 10(24):6095-6104. doi:10.7150/jca.33157. Available from https://www.jcancer.org/v10p6095.htm
Background: Circulating tumor cell (CTC)-based patient-derived cells are ideal models for investigating the molecular basis of cancer. However, the rarity and heterogeneity of CTCs as well as the difficulties of primary culture limit their practical application. Establishing efficient in vitro culture methods and functionally characterizing CTCs is essential for cancer studies. To this end, we developed an experimental protocol for the isolation, expansion, and identification of breast cancer CTCs.
Methods: The CTC-3 cell line was established from peripheral blood cells of a breast cancer patient. A karyotype analysis was performed. The molecular profile was assessed by flow cytometry, quantitative real-time PCR, and western blot. The characteristics of tumors formed by CTC-3 cells were evaluated by cell growth and tumor sphere formation assays and in a mouse xenograft model. The tumors were analyzed by immunohistochemistry, immunofluorescence analysis, and hematoxylin and eosin staining.
Results: The CTC-3 cell line showed more aggressive growth both in vitro and in vivo than the widely used MCF-7 breast cancer cell line. CTC-3 cells were also more resistant to chemotherapeutic agents, and gene profiling indicated higher expression levels of the epithelial-to-mesenchymal transition and stemness markers as compared to MCF-7 cells.
Conclusions: CTC-3 cells are a better model for investigating the malignant behavior of breast cancer than existing cell lines.
Keywords: Circulating tumor cell, Breast cancer, Cancer stem cell, Primary culture
Breast cancer is the most prevalent malignancy among women worldwide. Metastasis to bone, brain, liver, and lung accounts for the majority of breast cancer-related deaths [1]. Tumor cell dissemination involves several steps starting with detachment from the primary tissue followed by vascular entry and travel to distant tissue where a secondary tumor can be initiated [2].
Established breast cancer cell lines are invaluable tools for identifying and characterizing the molecules and pathways that control tumor growth and metastasis [3]. Since the establishment of the first human breast carcinoma cell line in 1958 [4], the most widely used breast cancer cell lines were originally from Caucasians or African Americans including T47D, MDA-MB-231, and MCF-7 [3]. However, since tumor cells are typically isolated from a single site in the tumor by biopsy, the resultant cell line does not exhibit the heterogeneity of the primary tumor. Moreover, tumor cells do not reflect the molecular changes that occur during cancer progression. The translational relevance of cell lines is also questionable because prolonged culture and multiple passaging can induce epigenetic and molecular phenotypes that are not representative of the original tumor [5].
Circulating tumor cells (CTCs) are rare tumor cells that leave the primary tumor and circulate into the blood of cancer patients [5, 6]. Detection of CTCs may be useful for early breast cancer diagnosis since it bypasses the need for ionizing radiation (mammography) or invasive biopsy [7, 8]. CTCs could also embody the molecular heterogeneity of a primary cancer and their metastasis is more tolerable to patients [9, 10]. The potential of CTCs as minimally invasive biomarkers for predicting patient prognosis and monitoring recurrence and as targets for precision therapeutics has been demonstrated in several studies [11-14].
Several breast cancer CTC cell lines have been generated from cancer patients and used to monitor response to anti-cancer drugs [15]. Drug sensitivity measurements were concordant with clinical history including sensitivity to paclitaxel and capecitabine and resistance to fulvestrant, doxorubicin, and olaparib [16]. CTCs were used to develop a drug sensitivity test and screening system that could guide drug discovery for personalized treatment [17]. However, the practical application of CTCs is hindered by their rarity and heterogeneity and the difficulties of primary culture. Developing more efficient in vitro culture methods is essential for establishing CTC cell lines that recapitulate the characteristics and behavior of the original tumor.
In this study we describe the establishment of a CTC cell line derived from naturally transformed breast cancer cells obtained from a 42-year-old Chinese woman diagnosed with breast carcinoma. Our cell enrichment technique is based on the removal of red blood cells by chemical lysis and the magnetic depletion of normal hematopoietic cells labeled with an anti-CD45 antibody/magnetic nanoparticle complex. The novel CTC-3 cell line was characterized in terms of biological and molecular features and karyotype, and tumorigenic potential was evaluated in vitro and in mice. We also analyzed the response of CTC-3 cells to different first-line drugs for the treatment of breast cancer.
Patient samples and blood collection
After obtaining informed consent, peripheral blood was collected from patients with advanced metastatic breast cancer. Blood was collected in EDTA tubes (10 ml) and was used for in vitro CTC culture (Supplementary Materials and Methods).
MCF-7, T47D, and MDA-MB-231 breast cancer cell lines were cultured as described in the Supplementary Materials and Methods.
Immunofluorescence analysis
Immunofluorescence labeling was performed using fluorescein isothiocyanate (FITC)-conjugated anti-pan cytokeratin (CK) (ab215838) and phycoerythrin (PE)-conjugated anti-cluster of differentiation (CD)45 (ab10558) antibodies and Fluoroshield Mounting medium with 4',6-diamidino-2-phenylindole (DAPI; ab104139) (all from Abcam, Cambridge, MA, USA). Cells were fixed and permeabilized by incubation for 20 min in 4% paraformaldehyde and 0.2% Triton X-100, respectively. A mixture of 10 μg/ml anti-CD45 and 10 μg/ml anti-CK antibodies and 500 nM DAPI were added to a microfluidic device followed by incubation for 20 min. After washing, the device was examined and only cells that were positive for DAPI and CK and negative for CD45 (DAPI+/CK+/CD45-) with appropriate size and morphology were counted as CTCs [17].
Karyotyping
The karyotyping protocol is described in the Supplementary Materials and Methods.
Subcutaneous tumorigenicity assay
To compare the tumorigenicity of CTC cells to that of MCF-7 cells, female immunodeficient mice (8 weeks old, n = 12; Medical Laboratory Animal Center, Guangdong Province) were divided into two groups that were subcutaneously injected in the left and right shoulders with 106 CTC-3 and MCF-7 cells, respectively, resuspended in 100 µl medium. Tumor growth was monitored and tumor volume (mm3) was measured weekly using electronic calipers and calculated with the formula (length × width × height)/2. Tumor growth (mean ± SD of three independent animals) was plotted as a function of time. Animal experiments were performed in accordance with the guidelines for laboratory animal use and were approved by the Animal Experimentations Ethnics Committee.
Cell growth analysis
CTC-3 and MCF-7 cells were cultured in complete growth medium. When they reached 70%-80% confluence, the cells were trypsinized and resuspended at a density of 5.5 × 103 cells/ml; a 1-ml cell suspension was added to each well of a 24-well plate. The cells were trypsinized and counted on days 3, 5, and 7 of culture (n = 3).
Tumor sphere formation assay
CTC-3 and MCF-7 cells were cultured in complete growth medium. When they reached 70%-80% confluence, the cells were trypsinized and resuspended in cancer stem cell (CSC) medium (Gibco, Grand Island, NY, USA) consisting of Dulbecco's Modified Eagle's Medium (DMEM)/F12 supplemented with epidermal growth factor, basic fibroblast growth factor, insulin, B27, and knock-out serum. The cells were seeded at a density of 1 × 104 cells/well in ultra-low attachment 6-well plates. After 14 days of culture under normoxic conditions with replenishment of the medium every 3 days, tumor spheres were formed.
Immunocytochemistry
Paraffin-embedded tumor tissue samples including primary tumor and lymph node biopsies from breast cancer patients and subcutaneous CTC-3 cell xenografts in immunodeficient mice were cut into 3 mm-thick sections and analyzed for estrogen receptor (ER), progesterone receptor (PR), erbB-2, E-cadherin and Ki-67 expression using appropriate antibodies (Supplementary Materials and Methods).
Western blot analysis
CTC-3, T47D, MBA-MD-231, and MCF-7 cells were lysed in lysis buffer containing protease and phosphatase inhibitors (Keygentec, Nanjing, China). Protein concentrations were quantified by BCA kit (Keygentec). Equal amount of protein was loaded in each lane. Constant voltage electrophoresis was carried out with 10% polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Merk, Massachusetts, USA). POVDF membranes were blocked with 3% BSA (Sigma) and hybridized with Estrogen Receptor (1:1000, Abcam, Cambridge, UK), Progesterone Receptor (1:1000, Abcam), Her-2 (1:1000, Abcam), or β-actin (1:2000, Abcam) primary antibodies overnight at 4 ˚C. After washed with TBST (Keygentec), the PVDF membranes were incubated with secondary antibodies (ZSGB-bio, Beijing, China) at room temperature for 60 minutes, followed by washing with TBST. PVDF membranes were covered with 3,3-diaminobenzidine (DAB) for the display of specific protein bands.
Drug resistance test
CTC-3 cells seeded at an initial density 1 × 104 cells/well were maintained in complete medium at 37°C in a humidified atmosphere of 5% CO2. After 12 h, the cells were treated with cyclophosphamide, paclitaxel, or pirarubicin for 24, 48, and 72 h at concentrations of 0.01, 0.1, 1, 10, and 100 μg/ml. The viability of surviving cells was evaluated with the Cell Counting Kit 8 assay [18].
Flow cytometry was performed with PE-conjugated mouse anti-human CD44 and FITC-conjugated mouse anti-human CD24 antibodies (both from BD Biosciences, San Diego, CA, USA) on a FACS Canto II instrument (BD Biosciences, San Diego, CA, USA), and data were analyzed with Cell Quest software (BD Biosciences).
RNA-sequencing (RNA-seq)
Total RNA was isolated from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using the NanoPhotometer spectrophotometer (Implen, Westlake Village, CA, USA); RNA concentration and integrity were estimated using the Qubit 2.0 flurometer (Life Technologies, Carlsbad, CA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, San Diego, CA, USA), respectively. Polyadenylated RNAs were isolated using Next Magnetic Oligo dT 25 beads (New England Biolabs, Ipswich, MA, USA), and first-strand synthesis was performed using the Next RNA First Strand Synthesis Module (New England Biolabs). Directional second strand synthesis was performed using Next Ultra Directional Second Strand Synthesis Module (New England Biolabs). The Next DNA Library Prep Master Mix Set for Illumina (New England Biolabs) was used to prepare expression libraries according to the manufacturer's protocol. Accurate quantification for sequencing applications was performed using the quantitative real-time qRT-PCR-based KAPA Biosystems Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA). Paired-end sequencing (75 bp) was performed on a NextSeq 500 sequencer (Illumina, San Diego, CA, USA). RNA-seq reads were aligned to the human genome (hg19) using TopHat2 [16], and the number of reads mapped to each gene was calculated with HTseq (http://htseq.readthedocs.io/). Differentially expressed genes between CC and CC-CR were detected with edgeR based on a negative binomial distribution. P values were adjusted with Benjamini and Hochberg's multiple test correction procedure. Differential expression was determined based on |log2 (fold-change)| > 1 and false discovery rate < 0.01. Data analysis is described in Supplementary Materials and Methods.
qRT-PCR analysis
Total RNA was isolated from cells using an RNA purification kit (Corning Inc., Corning, New York, USA) according to the manufacturer's instructions. Reverse transcription was performed using the PrimeScript RT Reagent Kit (TaKaRa, Shiga, Japan). qRT-PCR was carried out with SYBR Premix Ex Taq (Perfect Real Time) (Takara Bio) on a LightCycler 96 System (Roche, Basel, Switzerland). The primers used in this study are listed in Doc S1. GAPDH was used as an internal control. The results are presented as the expression ratio of target sample to control sample for each group, which was calculated with the cycle threshold method (2-∆∆CT).
Cell invasion assay
The invasiveness of CTC-3 cells was evaluated with the Matri-gel invasion assay as previously described [19] using transwell inserts (8 μm pore size) (BD Biosciences) in a 24 well plate. CTC-3 and MCF-7 cells (105 cells/insert) were seeded on top of the Matri-gel in serum-free minimal essential medium (MEM) and DMEM/F12. The lower chamber was filled with MEM/10% fetal bovine serum (FBS) and DMEM/F12/10% FBS. After incubation at 37°C for 48 h, CTC-3 and MCF-7 cells that had migrated through the membrane were stained with crystal violet and counted under a microscope.
Data are expressed as the mean ± SD of three independent experiments. Statistical analysis was performed using Prism v.7.0 (GraphPad, La Jolla, CA, USA), Origin 8 (OriginLab, Northampton, MA, USA), and Excel (Microsoft, Redmond, WA, USA) software. Results are expressed as mean ± SD of at least three independent experiments. The significance of differences among groups was assessed with the two-tailed Student's t test for normally distributed data, and P < 0.05 and < 0.01 were considered significant and highly significant, respectively.
In vitro culture of CTCs isolated from metastatic breast cancer patients
CTCs began to proliferate and form clusters after 14 days of culture (Fig. 1A). Immunocytochemical analysis revealed DAPI+ cells that expressed the epithelial marker CK and were negative for the leukocyte marker CD45 (DAPI+/CK+/CD45-; > 95% of cells), indicating that CTCs were of epithelial origin according to the standard definition (Fig. 1B). This stable breast cancer CTC line was named CTC-3, and has been growing in culture for 2 years with a short doubling time. CTC-3 cells formed tumor spheres when encapsulated in alginate gel beads (Fig. 1C). Besides when cultured in an ultra-low attachment 6-well plate in CSC medium, the cells formed tumor spheroids with a mean diameter of 200 μm (Fig. 1D). Karyotyping revealed that the chromosome number of CTC-3 varied from 56 to 60 and that the cells were aneuploid (Fig. 1E). Moreover, short tandem repeat (STR) analysis indicated that CTC-3 cells and peripheral blood mononuclear cells (PBMCs) from the donor showed > 83% similarity (Fig. 1F). The CTC-3 cell line can be frozen, banked, and thawed and successfully regrown.
Comparison of CTC-3 cells with patient tumor and xenograft tissues
To assess their tumorigenic potential, CTC-3 and MCF-7 cells were subcutaneously injected into immunodeficient mice. Her-2, ER, and PR expression in the xenografts as well as in the primary tumor and lymph node of breast cancer patients was examined. In primary tumors, the rates of positivity were 90% for ER, 80% for PR, and 2+ for Her-2 (Figs. 2A), which was similar with that of the lymph node metastasis (Figure S1A). In contrast, xenografts from mice were negative for ER and PR and positive for Her-2 expression (Fig. 2A). All tissues presented the features of differentiated adenocarcinoma cells, including low nucleus-to-cytoplasm ratio, irregular nuclear shape, and vacuoles. E-cadherin and Ki-67 were detected in primary tumors and xenografts (Fig. 2A), while lymph nodes and xenografts expressed CK (Fig. S1B). Additionally, xenografts were positive for carcinoembryonic antigen (CEA) and negative for p63 (Fig. S1C). Immunofluorescence analysis revealed that CTC-3 cells were negative for ER, PR, and Her-2 (Fig. 2B), which is similar to the negative control MDA-MB-231 cells. On the other hand, the positive control T47D cells expressed both ER and PR.
Growth characteristics of CTC-3 cells
The growth rate of CTC-3 and MCF-7 cells was compared in vitro and in vivo. Both cell lines grew rapidly, with cell numbers that were 29 and 20.56 times higher, respectively, than the initial values after 7 days of culture. However, the number of CTC-3 cells was higher than that of MCF-7 cells on day 7 (Fig. 3A). In addition, compared with the MCF-7, the number of CTC-3 cells migrated through Matri-gel-coated membranes was increased by 6.53-fold (Fig. 3C, D). The results of the xenograft assay showed that the tumorigenic potential of CTC-3 and MCF-7 cells was comparable (Fig. 3E), although CTC-3 cells formed a greater number of tumor spheres that were more compacted when cultured in CSC medium (Fig. 3B).
Drug sensitivity of CTC-3 cells
We compared the drug sensitivity of CTC-3 and MCF-7 cells to the first-line anti-breast cancer drugs pirarubicin, paclitaxel, and cyclophosphamide. The rate of growth inhibition rate was higher for MCF-7 than for CTC-3 cells treated with at 0.01, 0.1, 1, 10 μg/ml pirarubicin for 48 h, with significant differences observed at concentrations of 1 and 10 μg/ml (70.22% ± 1.52% vs. 45.83% ± 3.12% and 95.63% ± 1.70 % vs. 86.59% ± 0.53%, respectively) (Fig. 4A). However, at 100 μg/ml pirarubicin, the inhibition rate was 100% for both cell lines. The calculated the half-maximal inhibitory concentration (IC50) of CTC-3 and MCF-7 was 2.178 and 0.654 μg/ml, respectively (Fig. 4A).
The inhibition rates of MCF-7 cells treated with 0.01 and 0.1 μg/ml paclitaxel (63.38% ± 2.57% and 71.45% ± 1.86%, respectively) were higher than those of CTC-3 (49.24% ±1.41% and 52.89% ± 3.76%, respectively) (Fig. 4A). However, at 1 μg/ml, the rate was higher for CTC-3 than for MCF-7 cells (91.43% ± 0.57% vs. 78.37% ± 2.06%) (Fig. 4A). There were no significant differences between the two cell lines at paclitaxel concentrations of 1 and 10 μg/ml. The IC50 values of CTC-3 and MCF-7 were 0.3304 and 1.537 μg/ml, respectively (Fig. 4A).
The inhibition rate of MCF-7 and CTC-3 cells treated with cyclophosphamide did not differ significantly at concentrations of 0.01, 0.1, 1, and 10 μg/ml (Fig. 4A). However, at 100 μg/ml cyclophosphamide, the inhibition rate was higher for CTC-3 than for MCF-7 (51.51% ± 1.43% vs. 36.60% ± 3.61%) (Fig. 4A). The calculated IC50 values of CTC-3 and MCF-7 cells were 0.646 and 0.620 μg/ml, respectively (Fig. 4A). These results indicate that CTC-3 cells are more resistant to pirarubicin and cyclophosphamide but more sensitive to paclitaxel than MCF-7 cells.
In vitro expansion of breast cancer CTCs. (A) Morphology of the first generation of CTC-3 cells which grown as a single clone. (B) Immunocytochemical detection of DAPI+/CK+/CD45- CTC spheres. (C) Morphology of CTC-3 cell spheres embedded in alginate hydrogel. (D) In vitro tumor sphere formation assay of CTC-3 cells. (E) Karyotyping of CTC-3 cell line. (F) STR analysis of CTC-3 cells and PBMCs from the same patient.
Comparison of the CTC-3 cells with patient tumor tissues and mouse xenografts. (A) Hematoxylin and eosin staining and immunocytochemical detection of ER, PR, Her-2, E-cadherin, and Ki-67 in primary tumor tissues and xenografts. (B) Immunofluorescence analysis of ER, PR, and Her-2 expression in the TD47, MDA-MB-231, MCF-7, and CTC-3 cells.
Gene expression profile of CTC-3 cells
We performed RNA-seq of CTC-3 and MCF-7 cells to evaluate global changes in the expression of genes related to CSCs, drug resistance, and metastasis. We found that 17 genes were upregulated and two were downregulated in CTC-3 as compared to MCF-7 cells (Fig. 4B). In addition, the qRT-PCR results showed that the levels of CSC markers Oct3/4, Nanog, and especially CD44 were higher in CTC-3 cells than in MCF-7 (Fig. 4C). ATP-binding cassette super-family G member (ABCG)2 is closely related to drug resistance of tumor cells; here we found that ABCG2 was more highly expressed in CTC-3 than in MCF-7 cells, although the difference was non-significant. Interestingly, the levels of the epithelial-to-mesenchymal transition (EMT)-related markers E-cadherin was significantly lower and Vimentin was significantly higher, respectively, in CTC-3 as compared to MCF-7 cells, indicating that the former undergo EMT and thus have higher metastatic potential.
CSC-related marker expression in CTC-3 cells
Cells with high CD44 and low CD24 expression (CD44+/CD24-) define a CSC-like population in breast tumors [20] that exhibits high tumorigenic potential and drug resistance. The ratio of CSC-like cells in CTC-3 and MCF-7 cell lines was compared by flow cytometry analysis of CD44+ and CD24- cell ratio. In the CTC-3 cell line, 42.9% ± 1.98% of cells were CD44+ and 78.85% ± 15.34% were CD24- (Fig. 4D). In contrast, only 21.15% ± 15.34% MCF-7 cells were CD44+ but nearly all were CD24+ (99.15% ± 0.92%) (Fig. 4D). These results indicate that the CSC population is larger in CTC-3 than in MCF-7 cells.
CTCs are seed cells disseminated from the original tumor via the circulation that are responsible for metastasis and relapse in cancer [21-23]. Detection of CTCs and/or CTC clusters by immunolabeling or karyotyping has provided insight into the clinical significance of CTCs [24-26]. However, their correlation with tumor progression and patient prognosis remains unclear [27, 28]. CTCs are rare in blood and have polytrophic cell morphology and multiple cellular origins [29, 30]. For these reasons, CTCs are the ideal cell model for investigating the heterogeneity, metastasis, and stemness of solid tumors [31,32]. To this end, establishing CTC-based cell cultures and stable cell lines is critical for elucidating the functions of CTCs. In the present study, peripheral blood samples from 50 patients (16 breast cancer patients, 18 stomach cancer, 16 bowel cancer) were cultured (Doc S1), and we established the novel CTC-3 breast cancer cell from cells isolated from the peripheral blood of a breast cancer patient and characterized its features including tumorigenic potential in vitro and in vivo.
Proliferation and tumor sphere formation capacity of CTC-3 and MCF-7 cells. (A) Growth curve of CTC-3 and MCF-7 cells. Cells were counted every 2 days from three randomly selected replicates. (B) Gross morphology of tumors formed by CTC-3 and MCF-7 cells in vivo and change in tumor volume change over 60 days. (C) Matri-gel invasion assay of CTC-3 cells; the total number of migrated CTC-3 cells per transwell insert is shown. *P < 0.001 vs. control MCF-7 cells. (D) CTC-3 cells that have migrated through the transwell insert. Black arrows indicate cells stained with crystal violet. (E) Tumor sphere formation assay for CTC-3 and MCF-7 cells.
Drug sensitivity and gene expression profile of CTC-3 cells. (A) Drug sensitivity and IC50 of CTC-3 and MCF-7 cells treated with indicated concentrations of cyclophosphamide, paclitaxel, and pirarubicin for 48 h. (B) Gene expression profiles of CTC-3 and MCF-7 cells by RNA-seq. (C) qRT-PCR analysis of stemness markers octamer-binding transcription factor Oct 3/4, Nanog, CD44; drug resistance marker ABCG2; and EMT-related markers E-cadherin and Vimentin in CTC-3 and MCF-7 cells. *P < 0.05 vs. MCF-7 cells. Error bars represent SD of three replicates of three separate samples (n = 9). (D) Flow cytometry analysis of CD44 and CD24 expression in CTC-3 and MCF-7 cells.
White blood cells and thrombocytes may promote the survival of CTCs [33]. To avoid loss of rare CTCs during separation, we seeded all PBMCs in a Matri-gel-coated 6-well plate after removing red blood cells from 6 ml of whole peripheral blood. Although debris from non-cancer cells could inhibit CTC growth, our results demonstrate that mimicking the in vivo tumor microenvironment as closely as possible may be important for successfully establishing CTC cultures.
Efforts to identify CTCs and their cellular origin have been hampered by the heterogeneity of solid tumors; to this end, descriptive studies of cell morphology, protein expression, karyotype, and STR identification are necessary. Functional analyses are equally important for characterizing CTCs; in this study, we carried out in vitro cell quantification and evaluation of anchorage-independent growth, spheroid formation, in vivo tumorigenicity, and drug resistance. Even after more than 13 passages, CTC-3 continued to be CK+/CD45- with a karyotype and STR profile that was similar to those of the patient's white blood cells. Moreover, hematoxylin and eosin staining revealed that CTC-3 cell xenografts showed pathological features that were similar to those of the patient tumor tissue. These results confirm that CTC-3 is a PDC line originating from breast cancer CTCs. The cell growth assay showed that CTC-3 has a more aggressive and CSC-like phenotype than MCF-7 cells, which are also derived from metastasis breast cancer tissue. Gene profiling by RNA-seq and qRT-PCR indicated that genes related to cell invasion and stemness were upregulated in the CTC-3 cell line relative to MCF-7 cells. Thus, CTC-3 cells may be a better cell model for studying breast cancer metastasis and stemness. In addition, the in vitro cell viability assay showed that CTC-3 cells are more resistant to first-line chemotherapies than MCF-7 cells, which was consistent with the observed upregulation of the multidrug-resistance gene ABCG2. Notably, the pathological diagnosis of the donor patient was ER+/PR+ and human epidermal growth factor receptor 2 (HER2) negative, and peripheral blood for CTC isolation was collected before any chemotherapy. On the other hand, western blot and flow cytometry analyses showed that the connective passages of CTC-3 cells were all triple-negative breast cancer cells, which was confirmed in CTC-3-derived xenografts by immunohistochemistry. These results underscore the heterogeneity of solid tumors and can potentially explain the failure of selective estrogen receptor modulator (SERM) treatment in ER-positive breast cancer patients. Following SERM treatment, the donor patient has achieved stable disease. However, based on our findings, in the case of relapse, first-line drugs may not be the best choice for this individual. We are currently attempting to isolate additional CTCs from this patient; once cultures are established, more in-depth analyses may be possible that could elucidate the molecular basis for relapse and metastasis.
Supplementary figures and tables.
This work was supported by the Science and Technology Foundation of Shenzhen (JCYJ20170412155231633, JCYJ20180305164128430), the Shenzhen Economic and Information Committee "Innovation Chain and Industry Chain" integration special support plan project(20180225112449943), the Guangdong Provincial Natural Science Foundation (2018A030310674), Shenzhen Public Service Platform on Tumor Precision Medicine and Molecular Diagnosis; and Shenzhen Cell Therapy Public Service Platform.
1. Nguyen DX, Bos PD. J. Massague, Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer. 2009;9(4):274-84
2. Kolbl AC, Jeschke U, Andergassen U. The significance of epithelial-to-mesenchymal transition for circulating tumor cells. Int J Mol Sci. 2016;17(8):27529216
3. Ali R, Samman N, Al Zahrani H, Nehdi A, Rahman S, Khan AL, Al Balwi M, Alriyees LA, Alzaid M, Al Askar A, Boudjela M. Isolation and characterization of a new naturally immortalized human breast carcinoma cell line, KAIMRC1. BMC Cancer. 2017;17(1):803
4. Umer M, Vaidyanathan R, Nguyen NT, Shiddiky MJA. Circulating tumor microemboli: Progress in molecular understanding and enrichment technologies. Biotechnol Adv. 2018;36(4):1367-89
5. Yu M, Bardia A, Wittner BS, Stott SL, Smas ME, Ting DT, Isakoff SJ, Ciciliano JC, Wells MN, Shah AM, Concannon KF, Donaldson MC, Sequist LV, Brachtel E, Sgroi D, Baselga J, Ramaswamy S, Toner M, Haber DA, Maheswaran S. Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science. 2013;339(6119):580-84
6. Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA, Yu M, Pely, Engstrom A, Zhu H, Brannigan BW, Kapur R, Stott SL, Shioda T, Ramaswamy S, Ting DT, Lin CP, Toner M, Haber DA, Maheswaran S. Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell. 2014;158(5):1110-22
7. Sato F, Saji S, Toi M. Genomic tumor evolution of breast cancer. Breast Cancer. 2016;23(1):4-11
8. Alix-Panabieres C, Pantel K. Clinical applications of circulating tumor cells and circulating tumor DNA as liquid biopsy. Cancer Discovery. 2016;6(5):479-91
9. Karagiannis GS, Goswami S, Jones JG, Oktay MH, Condeelis JS. Signatures of breast cancer metastasis at a glance. J Cell Sci. 2016;129(9):1751-8
10. Joosse SA, Gorges TM, Pantel K. Biology, detection, and clinical implications of circulating tumor cells. EMBO Mol Med. 2015;7(1):1-11
11. Alegre E, Fusco JP, Restituto P, Salas-Benito D, Rodriguez-Ruiz ME, Andueza MP, Pajares MJ, Patino-Garcia A, Pio R, Lozano MD, Gurpide A, Lopez-Picazo JM, Gil-Bazo I, Perez-Gracia JL, Gonzalez A. Total and mutated EGFR quantification in cell-free DNA from non-small cell lung cancer patients detects tumor heterogeneity and presents prognostic value. Tumour Biol. 2016;37(10):13687-94
12. Alix-Panabieres C, Schwarzenbach H, Pantel K. Circulating tumor cells and circulating tumor DNA. Annu Rev Med. 2012;63:199-215
13. Thiele JA, Bethel K, Kralickova M, Kuhn P. Circulating tumor cells: fluid surrogates of solid tumors. Annu Rev Pathol. 2017;12:419-47
14. Yu M, Bardia A, Aceto N, Bersani F, Madden MW, Donaldson MC, Desai R, Zhu H, Comaills V, Zheng Z, Wittner BS, Stojanov P, Brachtel E, Sgroi D, Kapur R, Shioda T, Ting DT, Ramaswamy S, Getz G, Iafrate AJ, Benes C, Toner M, Maheswaran S, Haber DA. Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility. Science. 2014;345(6193):216-20
15. Khoo BL, Grenci G, Jing T, Lim YB, Lee SC, Thiery JP, Han J, Lim CT. Liquid biopsy and therapeutic response: Circulating tumor cell cultures for evaluation of anticancer treatment. Sci Adv. 2016;2(7):1600274
16. Liu C, Liu Y, Xu XX, Wu H, Xie HG, Chen L, Lu T, Yang L, Guo X, Sun GW, Wang W, Ma XJ, He X. Potential effect of matrix stiffness on the enrichment of tumor initiating cells under three-dimensional culture conditions. Exp Cell Res. 2015;330(1):123-34
17. Albini A, Iwamoto Y, Kleinman HK. et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 1987;47(12):3239-45
18. Liu R, Wang X, Chen GY, Dalerba P, Gurney A, Hoey T, Sherlock G, Lewicki J, Shedden K, Clarke MF. The prognostic role of a gene signature from tumorigenic breast-cancer cells. N Engl J Med. 2007;356(3):217-26
19. Plaks V, Koopman CD, Werb Z. Cancer. Circulating tumor cells. Science. 2013;341(6151):1186-88
20. Garzon M, Villatoro S, Teixido C, Mayo C, Martinez A, de Los Llanos Gil M, Viteri S, Morales-Espinosa D, Rosell R. KRAS mutations in the circulating free DNA (cfDNA) of non-small cell lung cancer (NSCLC) patients. Transl Lung Cancer Res. 2016;5(5):511-16
21. Hench IB, Hench J, Tolnay M. Liquid biopsy in clinical management of breast, lung, and colorectal cancer. Front Med. 2018;5:9
22. Sheng Y, Wang T, Li H, Zhang Z, Chen J, He C, Li Y, Lv Y, Zhang J, Xu C, Wang Z, Huang C, Wang L. Comparison of analytic performances of Cellsearch and iFISH approach in detecting circulating tumor cells. Oncotarget. 2017;8(5):8801-6
23. Li Y, Zhang X, Ge S, Gao J, Gong J, Lu M, Zhang Q, Cao Y, Wang DD, Lin PP, Shen L. Clinical significance of phenotyping and karyotyping of circulating tumor cells in patients with advanced gastric cancer. Oncotarget. 2014;5(16):6594-602
24. Gao Y, Zhu Y, Zhang Z, Zhang C, Huang X, Yuan Z. Clinical significance of pancreatic circulating tumor cells using combined negative enrichment and immunostaining-fluorescence in situ hybridization. J Exp Clin Cancer Res. 2016;35:66
25. Xu L, Jia S, Li H, Yu Y, Liu G, Wu Y, Liu X, Liu C, Zhou Y, Zhang Z, Sheng Y. Characterization of circulating tumor cells in newly diagnosed breast cancer. Oncol Lett. 2018;15(2):2522-28
26. Bulfoni M, Turetta M, Del Ben F, Di Loreto C, Beltrami AP, Cesselli D. Dissecting the heterogeneity of circulating tumor cells in metastatic breast cancer: going far beyond the needle in the haystack. Int J Mol Sci. 2016;17(10):27783057
27. Ellsworth RE, Blackburn HL, Shriver CD, Soon-Shiong P, Ellsworth DL. Molecular heterogeneity in breast cancer: State of the science and implications for patient care. Semin Cell Dev Biol. 2017;64:65-72
28. Lu LC, Hsu CH, Hsu C, Cheng AL. Tumor heterogeneity in hepatocellular carcinoma: facing the challenges. Liver Cancer. 2016;5(2):128-38
29. Jordan NV, Bardia A, Wittner BS, Benes C, Ligorio M, Zheng Y, Yu M, Sundaresan TK, Licausi JA, Desai R, O'Keefe RM, Ebright RY, Boukhali M, Sil S, Onozato ML, Iafrate AJ, Kapur R, Sgroi D, Ting DT, Toner M, Ramaswamy S, Haas W, Maheswaran S, Haber DA. HER2 expression identifies dynamic functional states within circulating breast cancer cells. Nature. 2016;537(7618):102-6
30. Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, Hayes DF. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004;351(8):781-91
31. Khoo BL, Grenci G, Lim YB, Lee SC, Han J, Lim CT. Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device. Nat Protoc. 2018;13(1):34-58
Corresponding author: Chang Zou, Email: zouchang.cuhkcom, Fax: 86 (755) 22942850, Clinical Medical Research Center, The Second Clinical Medical College of Jinan University, The first Affiliated Hospital of Southern University, Shenzhen People's hospital, Shenzhen 518020, China

redpajama_set_name: RedPajamaCommonCrawl


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Search - Brian Eno :: Desert Island Selection
Desert Island Selection
Genres: Dance & Electronic, Alternative Rock, Special Interest, New Age, Pop, Rock, Classic Rock
Here He Comes
Everything Merges With the Night
I'll Come Running (To Tie Your Shoes)
On Some Faraway Beach
Spirits Drifting
Back in Judy's Jungle
No One Receiving
Julie With...
Taking Tiger Mountain
1/1 - Brian Eno, Davies, Rhett
A greatest hits collection from one of the most influential artisits of our time after being out of print for more than a year. A collection of his best solo pop and vocal material from his most sought after period. Featur... more »ing tracks personally chosen by Eno from his first four albums, 'Here Comes The warm Jets', 'Taking Tiger Mountain By Strategy', 'Another Green World' & 'Before & After Science', as well as a selection from his highly praised ambient album 'Music For Airports'. 1986 release. Standard jewel case. « less
All Artists: Brian Eno
Title: Desert Island Selection
Label: Astralwerks
Album Type: Original recording reissued
Styles: Ambient, Electronica, Hardcore & Punk, Indie & Lo-Fi, Experimental Music, Progressive, Progressive Rock, Glam
UPCs: 017046151924, 077778718826, 0077778718857, 077778718857
A greatest hits collection from one of the most influential artisits of our time after being out of print for more than a year. A collection of his best solo pop and vocal material from his most sought after period. Featuring tracks personally chosen by Eno from his first four albums, 'Here Comes The warm Jets', 'Taking Tiger Mountain By Strategy', 'Another Green World' & 'Before & After Science', as well as a selection from his highly praised ambient album 'Music For Airports'. 1986 release. Standard jewel case.
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Non Ambient Eno
Randy Nicolosi | Shelton, CT United States | 11/23/2003
"I feel bad about the reviewers who downrated this CD because they were expecting ambient ENO. I've never been a big fan of his ambient works, preferring his vocal / rock works. That said, I think this is a good collection, but is too short; like having a great appetizer, but no dinner to follow. So, it's a good collection, but if you enjoy this style of ENO as I do, you should buy all the CDs."
The best introduction
www.mp3.com/wader | 04/16/2000
"If you're starting to poke around this strange figure "Brian Eno" that you've heard about but haven't made a purchase yet, BUY THIS. This cd contains incredible songs which will stay with you forever, and will make you buy his 4 early vocal albums they're from: Here come the warm jets, Taking tiger mountain by strategy, Another green world, and Before and after science."
A bit heavy on the slow stuff
ADP | Washington, DC USA | 05/17/2000
"This compilation of 70's recordings by Brian Eno leans too heavily on his slowest music, ignoring his noisy, Velvet-Underground-inspired rockers that are so deliriously brilliant. Still, Eno's slow songs come with plenty of their own pleasures, especially the almost-country "Here He Comes" and the breezy "St. Elmo's Fire," which could have been a hit single. "Everything Merges with the Night" may not move quickly, but it has genuine power, and it shows that high-pitched guitar sustain need not be confined to the soundtracks of sleazy movies. The bottom line: This is some of the best pop music anyone has ever made, in the 70's or any other decade. Even a mediocre compilation of it is a great experience."
Definite problem here...
DAC Crowell | 04/04/2000
"...and that problem is that one person's Eno "Desert Island Selection" isn't going to be another's! Brian Eno is just too multi-faceted to pin down to a single CD, really, and even as an introduction this fails, as there's certain whole albums that do a better job than a compilation ever could. Originally intended as a companion CD for the "More Dark than Shark" lyrics/art book collaboration with painter Russell Mills."


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You are here: Home » Place Names » Newport » River Ebbw and Nant Pencarn
The John Wallace Linton
River Ebbw and Nant Pencarn
River Ebbw and Nant Pencarn, Newport
At this point on the Gwent Levels, the Wales Coast Path crosses the river Ebbw on a footbridge. In medieval times, a stream called Nant Pencarn flowed somewhere in this area – perhaps where the river Ebbw is today.
The name Ebbw was recorded as Eboth in 1101, Ebouith c.1537 and Ebwy (its current Welsh spelling) in 1631. It comes from the earlier name Ebwydd, which probably means the river was likened to a wild horse. The eb- element relates to a horse (as in the modern Welsh ebol = "colt"). The suffix may come from gŵydd, meaning "wild". Several other Welsh river names are based on comparisons with animals.
The river gave its name to various places, including the town of Ebbw Vale (Glynebwy), beside its upper reaches. Near Newport, there was a manor of Eboth alias Greenfield.
In 1188, the Archbishop of Canterbury passed through this area on his tour of Wales to recruit for the third crusade. He was accompanied by Gerald of Wales, whose journal records that near Newport the party crossed Nant Pencarn.
The terrain has changed greatly since then, and nobody knows where that stream flowed. It could have taken approximately the course of the river Ebbw in this vicinity. It probably flowed through the Duffryn (or Dyffryn) area, to the west, where there were three farms and a manor called Pencarn. Pen = head, carn = mound or cairn.
Gerald wrote that Nant Pencarn could be crossed only at certain fords, because it ran through such marshy land. He quoted an earlier writer, Merlin Silvester, who prophesied that if a strong, freckled man crossed at Rhyd Pencarn during an invasion attempt, the Welsh would lose.
Gerald noted that in his own time, King Henry II had crossed at that ford, rather than a newer one nearby. Welshmen who had come to see where Henry would cross were dismayed. Gerald's tale probably relates to an invasion which resulted in Lord Rhys, ruler of South Wales, losing much of his territory to Henry in the 1160s.
With thanks to Richard Morgan, of the Welsh Place-Name Society


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Home/Leaders of Ummah/Qasem Suleimani/Iraqis remember Lt. Gen. Soleimani, Abu Muhandis on eve of martyrdom anniv.
Qasem SuleimaniIraq
Iraqis remember Lt. Gen. Soleimani, Abu Muhandis on eve of martyrdom anniv.
Iraqis gather from all over the Arab country for a march in remembrance of Lieutenant General Qassem Soleimani and Abu Mahdi al-Muhandis, senior Iranian and Iraqi anti-terror commanders, who were assassinated by the US two years ago.
According to Lebanon's al-Ahed news website, the mourners reached Baghdad from every Iraqi province on Sunday, before heading out on the march towards the exact place, where the commanders were assassinated in a United States' drone strike targeting the Baghdad International Airport on January 3, 2020.
Upon reaching the site of the assassinations, the participants began attending a ceremony that was held to honor the memory of the heroic figures.
The drone strike that assassinated the commanders was ordered by former American president Donald Trump. The atrocity came at the height of the heroes' popularity for their helping out Iraq and Syria defeat the Takfiri terrorist group of Daesh.
Washington had been using Daesh's presence to perpetuate its military interference in the Arab countries.
Following the atrocity, the Iraqi parliament voted overwhelmingly in favor of a law mandating full withdrawal of all foreign forces from the country's soil.
Around one million Iraqis had attended a similar commemoration ceremony in the country's capital on Saturday, vowing unanimously to stand firm on demanding complete expulsion of all American-led forces.
Imam Khomeini shattered superpowers' aura of invincibility: Imam Sayyed Ali Khamenei
Arab world views Imam Khomeini as great hero: Lebanese journalist
TEN DAYS 2022: Worldwide Islamic Revolution role model for all independent nations


text:
Woman (25) left fighting for life after being lured through dating app
She was left in an induced coma
Horrific details are emerging after a young woman was brutally assaulted in Dublin last week.
It has been claimed that the 25-year-old was lured to the attack through a dating app.
Detectives have been looking to speak to a man who was spotted on CCTV walking with the victim in the Windsor Terrace area of Queen's Road shortly before the brutal attack.
Police reportedly believe the woman may have been lured to the scene of the attack by a dating app.
A source told the Irish Sun: "This was a horrific attack on this woman, which left her with very serious injuries.
"Obviously the investigation is at an early stage and a motive is yet to be established
"The attack happened in broad daylight with a number of people doing their Christmas shopping nearby.
"This shows the viciousness of it and any eye witnesses should come forward."
(Image: Queens Street Dublin)
Heartache as friends who drowned trying to drive across flooded river named locally
Cops arrested a teenage boy after the woman was left fighting for her life when assaulted in the seaside town of Dun Laoghaire on Saturday afternoon
The woman is believed to have been stabbed with a broken bottle during the vicious assault.
She was rushed to hospital with serious injuries to her neck and arm.
The woman, who is understood to be Asian, was found close to the baths at Queen's Road in Dun Laoghaire at around 3.20pm
It looks like snow could be on the way today as temperatures set to drop to -2C in parts
The woman is recovering in St Vincent's Hospital where she remains in a serious but stable condition after being brought out of an induced coma.
Gardai are appealing for witnesses to the terrifying attack
Anyone with information or who was in the Queen's Road area of Dun Laoghaire this afternoon between 3pm and 4pm should contact Dun Laoghaire Garda Station on 01 666 5000.


My only question is how the hell did Branka and Felsi fall in love with him? The game goes out of its way to tell you how repulsive the man is. Beard matted with food? Check. Stinky feet? Check. Bad breath? Check. Constantly farting and burping? check.
As for my opinion of Oghren, I kinda appreciate the fact that he is one of the few characters who don't need the Warden to play psychologist. He's got his inner turmoil feeling guilty for Branka's fate and having lost his place in Orzammar, but he doesn't need you.
He has a better psychologist. True, that cause bad breath, burping, and vomit sometimes. But can help to forget (not Cole).
I felt a bit bad in Awakening to be constantly feeding his alcoholism till he keeled over. I suppose people might not see it like that in Thedas, however.


They explain that one of the reasons why "India's environmental laws fail to deliver the air quality improvements they promised" is because "environmental regulators are forced to overcome persistent deficits of information and authority in order to enforce these laws"….Government agencies that enforce India's Air and Water acts have only 200-300 technical staff to monitor at least 50,000 plants in some of the largest and most industrialized states." Because enforcement is so difficult, they suggest that electronic monitoring equipment be placed on factory smokestacks and power plants. "Convincing plants to turn on their pollution control equipment may be one of the quickest and cheapest ways to reduce pollution across India." Fair Observer. Read it on delhiair.org.


1(of a small bird or an insect) make a short, sharp, high-pitched sound.
1.1with direct speech (of a person) say something in a lively and cheerful way.
2South African with object Speak to (someone) in a taunting way.
A short, sharp, high-pitched sound.


text:
<?php

namespace JohnKrovitch\ORMBundle\Behavior;

trait Behaviors
{
    protected $behaviors = [];

    /**
     * @return array
     */
    public function getBehaviors()
    {
        return $this->behaviors;
    }

    /**
     * @param array $behaviours
     */
    public function setBehaviors($behaviours)
    {
        $this->behaviors = $behaviours;
    }

    public function addBehavior($behavior)
    {
        $this->behaviors[] = $behavior;
    }
}

redpajama_set_name: RedPajamaGithub


Finally you can have clear visibility across your organization and see measurable improvements for clients.
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We help you track progress and better involve clients in their treatment, which research has shown to increase engagement and improve outcomes.
Our Matching Program connects your team with pre-qualified and motivated new clients. In addition, access to real time data lets you identify and focus on clients that need the most support.
Running an evidence-based practice and implementing a consistent measurement process can be difficult and time consuming. Greenspace makes this process easy for mental health organizations and more engaging for your clients. Your team can supplement the objective measures with their own clinical progress notes.
Greenspace engages clients outside of sessions and creates a feedback loop that research identifies as crucial to achieving better outcomes.
The Greenspace platform automates much of the workflow and generates valuable outcome data for your organization.
Built on research that identifies consistent progress measurement as having a dramatic impact on client treatment outcomes, regardless of modality. Regularly tracking progress leads to better client engagement, improved communication and reduced bias in therapy.
For private mental health organizations, Greenspace can provide access to pre-qualified and motivated new clients that have been referred by their Primary Care Provider. Your team will receive a detailed intake report for these clients and can maintain full control over whether to accept or decline each new match. Once you accept a match, the Greenspace platform facilitates collaboration with a client's Primary Care Provider, leading to higher engagement and improved outcomes.
Organizations often struggle with visibility into their practice, without information on the outcomes being achieved. Greenspace collects this much needed information, which can then be used to support decision making, training and funding (or marketing).


text:
#pragma once
#include <aws/pinpoint/Pinpoint_EXPORTS.h>
#include <aws/core/utils/memory/stl/AWSString.h>

namespace Aws
{
namespace Pinpoint
{
namespace Model
{
  enum class Action
  {
    NOT_SET,
    OPEN_APP,
    DEEP_LINK,
    URL
  };

namespace ActionMapper
{
AWS_PINPOINT_API Action GetActionForName(const Aws::String& name);

AWS_PINPOINT_API Aws::String GetNameForAction(Action value);
} // namespace ActionMapper
} // namespace Model
} // namespace Pinpoint
} // namespace Aws


EurekaLog includes a "CustomWebFieldsRequest" event for interaction with the user program when a raised exception is handled by EurekaLog and the user want send a message to a Web server sending custom HTTP fields (via post) together the upload files.
Use the WebFields list to add all the needed Web fields using the form "WebFields.Add('FieldName=FieldValue')".
Note: to simplify management of your EurekaLog events, you can use the TEurekaLog.OnCustomWebFieldsRequest event.


Open Source Networking Days (OSN Days) is a series of regional, one-day events hosted and organized by local open source networking ecosystem members — including industry, service providers, academia, and start-ups — with support from LF Networking and its projects: FD.io, ONAP, OpenDaylight, OPNFV, PNDA, SNAS, and Tungsten Fabric. Attendees will hear from a roster of expert speakers on the state of the industry; the projects that make up the open source networking stack, the integration points between them, the use cases and business opportunities enabled by network transformation, how to get involved, and much more. There is no cost to attend. Register today!
Click on the locations above for event-specific programming, logistical information, and to register. Please send any questions to osndays@linuxfoundation.org.


text:
Eliot's Secret Plan to Crush Albany
By Azi Paybarah • 02/19/07 12:00am
Governor Eliot Spitzer has a secret plan to shrink Assembly Speaker Sheldon Silver into obscurity. It starts with a Democratic takeover of the State Senate.
The first step, according to a senior official in the Spitzer administration, will be an attempt to compel vulnerable Republican Senators to defect to the Democratic minority by threatening them with well-funded opponents.
"At this point, if some of the members in the majority decide to stay put, there's going to be no turning back," said the official. "We're at a fork in the road. The time for deals will be over very soon."
When, exactly?
"The ball can drop at any moment."
Accordingly, said the official, Mr. Spitzer's aides have begun to identify potential Democratic candidates now—nearly two years from the next legislative election—to run for State Senate in 2008, and to host fund-raisers featuring the Governor. "We'll put the full weight of the Governor behind them," the official said.
A shift of three more seats in the 62-member body away from the Republicans would give the Democrats control. The thinking from Mr. Spitzer's perspective is that having a friendly Democratic majority in the Senate would not only allow the Governor to circumvent the Democrats in the Assembly, but would create a clear contrast that would put pressure on Mr. Silver and his members to accede to a reform agenda.
Mr. Spitzer helped elect a Democrat in a just-concluded special election in Nassau, and is currently attempting to recruit his former opponent in the gubernatorial primary, Nassau County Executive Tom Suozzi to run for another seat there.
The Governor's threats will ostensibly leave Senate Republicans with the following options: retire; pledge to support Democratic Minority Leader Malcolm Smith over Republican Majority Leader Joe Bruno; or, for at least one lucky defector, take a job—like former Senator Michael Balboni of Nassau did—with the administration.
"There is one seat in the lifeboat left," the official said. "Who takes the last seat in the lifeboat?"
(Mr. Spitzer's predecessor famously opened up countless jobs in state departments and public authorities to party activists, a luxury that Mr. Spitzer doesn't have in trying to woo the three Senate Republicans he needs to give Democrats the majority in that house.)
Sound crazy? Maybe. But the way the Spitzer people figure it, their attempt to engineer a power shift in the Senate is the best shot they have of breaking the grip not only of the Republicans in the Senate, but of Mr. Silver.
"We're not sitting around talking to Assembly members about a coup. It wouldn't work; our tactics wouldn't make sense," the official said.
If a Senate majority takes power, "it'll make clear that there is a reform wing of the Democratic Party and a status quo wing of the party," the official continued. "Creating a reform-minded majority in the Senate is better than a coup against Shelly."
Filed Under: Home, Politics, Eliot Spitzer, Sheldon Silver, Thomas Suozzi, Nassau
SEE ALSO: How Pricey Co-ops Get Better Deals Than Rentals at Tax-Time


text:
Home » Art Is Long, Money Is Short
Art Is Long, Money Is Short
A new study tallies the impact of Blagojevich's diminished arts funding.
by Deanna Isaacs April 10, 2008 August 19, 2021
Two days after Governor Rod Blagojevich delivered his budget address back in February, a rare e-mail alert was issued by the Illinois Arts Council, the government agency that doles out grants to arts organizations statewide. Signed by IAC chair Shirley Madigan—the wife of house speaker and Blago foe Mike Madigan—it warned that the governor's plan for fiscal 2009 won't undo the previous year's $4.5 million cut in arts funding. That message was followed a few days later by one from IAC executive director Terry Scrogum, confirming the bad news: the council's budget would remain more or less flat, at $15.2 million—23 percent less than the 2007 figure of $19.8 million.
Scrogum's tone was resigned. The budget process, with its inevitable negotiations and adjustments, is just beginning, he noted, but "this year may be an especially difficult one due to the condition of the nation's (and collaterally, the state's) economy. We are truly sorry to bring you this news."
Nevertheless, the next week a battle flag was raised. Another mass e-mail went out, this one from Ra Joy, head of the Illinois Arts Alliance, the nonprofit advocacy group. Under the headline "Take Action Now!" Joy called for a fight against the nearly 30 percent cut in arts grants that had been the end result of the arts-funding slash of 2008. He noted that Illinois now ranks behind hardscrabble states like Alabama and Louisiana in per capita funding for the arts; that we're one of only three states that cut arts spending in 2008; and that 73.4 percent of the 435 respondents to an online alliance survey "plan to reduce or eliminate their education, outreach, or free public programs" because of reduced IAC funding. He urged recipients to click through and send a letter to their lawmakers demanding that the IAC budget be increased to $24 million, or about $2 for each state resident. (Alliance communications coordinator Scarlett Swerdlow says the campaign generated 3,772 such messages.)
It's a familiar scenario: the cuts, the urgent call to action, the deluge of identical e-mails to legislators. The alliance followed up by releasing a report on the online survey, which was conducted in January and February. Longer on anecdotal quotes from respondents (72 percent of them in Chicago and its suburbs) than on hard numbers, the report points to everything from kids in Back of the Yards losing after-school dance classes to patrons paying more for their tickets to Light Opera Works shows in Evanston. And the smaller the organization, the report said, the bigger the impact. Chicago's fledgling New Leaf Theatre—part of the nearly 25 percent of surveyed nonprofits reporting budgets of under $50,000—told the alliance that further cuts in state grants may shut them down.
In most cases, IAC awards are capped at between 10 and 15 percent of an organization's budget. But they offer a couple of outsize benefits. First, as noted by both Olga Stefan of the Chicago Artists' Coalition (which got only half the IAC infusion it expected) and Lyle Allen of the League of Chicago Theatres (down 15 percent), the grants provide the hardest kind of money to come by: general operating funds. Unlike private donors, who usually want to spend their money on specific, high-profile programs, the IAC gives unrestricted money that can be put to work where it's needed most, even if that's somewhere as unglamorous as the boiler room. More important, the grants validate their recipients in the eyes of other potential donors. As a survey response from Theatre Building Chicago puts it, "Our IAC grant... positions us to find other, private funders. A continued reduction would lower our leverage."
Scrogum says no qualified organizations were dropped in FY2008; grants were simply reduced across the board. Operating money went to 801 organizations, and 490 additional grants—including 38 $7,000 fellowships—were given to individual artists or groups. Operating costs at the Illinois Arts Council, which were 10 percent of budget when the budget was close to $20 million, have not been reduced, according to Scrogum. "We're looking at ways to become more efficient," he says, "but you still have to evaluate everything."
Legislative wrangling over the budget will continue at least until May, and citizens got a chance to comment on it during a recent series of statewide hearings. One late-March meeting, in Lakeview at the Advocate Illinois Masonic Medical Center, drew half a dozen arts leaders along with nearly a hundred other advocates for various causes. Members of the arts contingent blamed the IAC cuts on everything from bad blood in Springfield to the national defense budget; the Pegasus Players' Arlene Crewdson and Juan Dies of the Sones de Mexico Ensemble each used their two minutes at the mic to argue that the arts return more to the state than they take. Meanwhile, the panel of legislators also heard that full-time caregivers for the disabled earn poverty-level wages of $17,000 a year and that the state Department of Human Services is so understaffed that, for example, the Northern Cook County office has only 56 workers to handle 70,000 cases. As one speaker put it, there was no shortage of "urgent moral matters." Gives you an idea of what the choices are.
Worth Its Weight in Cadmium Red
With the exception of the Public Broadcasting Corporation, which the Bush administration is looking to dump in two years, the picture on the national front is cheerier. The National Endowment for the Arts got a $20 million boost—its largest in three decades—to $144 million. And a bill to do something about a discriminatory tax situation that's been affecting artists for 40 years is being studied in Congress.
Ergo, another e-mail campaign. A recent message from Americans for the Arts (the IAA's national counterpart) calls on artists to send a prewritten letter urging their reps to vote for the Artist-Museum Partnership Act. As things stand now, anyone who owns a piece of art can donate it to a charity and take a nice fat tax deduction equivalent to its current market value. But if the artist who created the work donates it, only the cost of materials can be deducted. We're talking canvas and paint. This is a patently screwy arrangement, and local artists, who are often hit up to donate their work for charity auctions, have frequently lamented it.
On the upside, all kinds of art qualify under the proposed act, including "literary, musical, artistic, or scholarly compositions or similar property," so long as the work's value is established. An NEA report (based on a meeting hosted by NEA chair Dana Gioia and dominated by museum officials) makes the case for passage, arguing that the change will encourage artists to donate their work and especially aid small and medium-sized institutions.
But the way this legislation's written, an artist can earn the deduction only for donating works acquired to be retained—say, in a museum collection. So that painting you ponied up for the benefit auction at the food pantry? The answer's still no.v
Filmmakers' Move
Can't Buy Love/ Late Bloomer?
Counting for culture
Tagged: The Business, Vol. 37 No. 29


text:
A Full Day's Work
Directed by Jean-Louis Trintignant
Language: French with English subtitles
In this black-as-night French-Italian comedy, Jacques Dufilho (Nosferatu the Vampyre) plays a father who has carefully planned the execution of the nine jurors who sent his son to the gallows. Dressed in black, on a black motorcycle with his elderly mother (Luce Marquand, Last Tango in Paris) in the sidecar, the vengeful man calmly and methodically does away with each of the nine in the span of a single day—before the police or anyone else can stop him. Maestro Bruno Nicolai (All the Colors of the Dark, The Antichrist) scored this anarchic and audacious film featuring wildly imaginative murder set-pieces concocted by legendary actor Jean-Louis Trintignant (The Conformist, And Hope to Die) in his directorial debut.
Jacques Dufilho
Luce Marquand
Andrée Bernard
Franco Pesce
Albin Guichard


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Slide out trays are perfect for saving valuable desk top space from large printers and fax machines. Smooth metal side drawer glides ensure easy movement for heavy equipment while wire provisions are built-in behind the drawer to keep all cords organized and out of the way.
Create a professional work environment with this classically, elegant credenza. Equipped with ample storage space you can keep all of your most valuable resources organized and available right at your finger tips. Expertly crafted from pine solids with cherry and birch veneers this piece is bathed in a warm brown cherry finish to give your office a cozy feel. Stacked crown mouldings, and framed doors give this office piece a traditional design. Two doors conceal slide out drawers for printer and fax machines and along with three drawers are adorned with dark finished knob hardware.
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text:
Home Politics Foreign Policy Iran Says Ready to Build Power Plants in Azerbaijan
Iran Says Ready to Build Power Plants in Azerbaijan
Iran's President Hassan Rouhani has held talks on a wide range of issues with his Azeri counterpart Ilham Aliyev in Baku.
During the Thursday evening meeting, President Rouhani said, "With the efforts of senior officials of Iran and the Republic of Azerbaijan relations have increased to a satisfactory level."
He expressed satisfaction with the effective role of the economic cooperation commission of the two countries, saying, "In the field of industrial cooperation and auto industry, a good ground for cooperation has been provided and Iran is also ready to construct power plants in the Republic of Azerbaijan."
On the importance of completing the North-South Transit Route, he said, "Iran and the Republic of Azerbaijan have very good cooperation in this field, which we hope the final phases also pursued more quickly."
Rouhani also referred to the importance of creating preferential tariffs in trade for facilitating investment and joint ventures as a valuable step in further development of economic relations between Iran and the Republic of Azerbaijan.
He further stressed cementing relations between the two countries in the Caspian Sea as the sea of friendship, peace and development, adding, "Iran is always ready to develop maritime relations and cooperation with the Republic of Azerbaijan in the Caspian Zone in the fields of exploration and also environment".
On scientific and cultural cooperation between Iran and the Republic of Azerbaijan, the President continued, "Cooperation in scientific and cultural fields, as well as modern technologies should develop within the framework of cooperation among universities and research centres of the two countries."
Rouhani referred to the two countries' common views in regional and international fields, saying, "The Iranian government and nation have always been alongside the Azerbaijani government and nation in international communities, and the two countries can contribute to more regional and global peace and security."
"Tehran, Baku and Moscow should develop their trilateral and bilateral cooperation," he said, adding that the relations should deepen.
Rouhani also invited his Azerbaijani counterpart to make an official visit to Tehran.
At the meeting, the President of Azerbaijan described Rouhani's visit to Baku as a good opportunity for development of relations.
"The deep cultural and historical bonds between Iran and the Republic of Azerbaijan is a good ground for further deepening of relations, which have considerably increased in your term in office," he said.
President Aliyev described the 70-percent growth in trade between the two countries in one year as a praiseworthy record and said, "The two countries' cooperation within the framework of economic cooperation commission in the fields of energy, banking, industry and transportation have a good pace and the development of the North-South Corridor project is completely clear."
He also called on Iran to cooperate in constructing power plants in Azerbaijan and said, "Azerbaijan is ready to cooperate with Iran in maritime fields in the Caspian Zone, including in exploration."
The Azerbaijani President stressed the tripartite summit of Iran, Azerbaijan and Russia and called cooperation beneficial to the three countries.
On the importance of the Non-Aligned Movement (NAM) Summit, he said, "We want to defend all member states' interests within the framework of this movement."
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Ukraine Plane Crash Case Should Come to Special Court, President Says
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