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Ole Miss Football Coach Ed Orgeron scheduled some third-down situation work for his squad today and was pleased with what he saw out of Rebel QB Brent Schaeffer.
"I thought Brent worked well in the pocket and made some big plays. We tried to rush him with four men and it's difficult to do against him with his scrambling ability," Coach O noted. "We tried to blitz him some too and he picked those up as well. He really had a good day today.
"The offensive line also did a good job of protecting him. I was pleased with those aspects."
The Rebels will begin two-a-days tomorrow and will hold their first scrimmage Saturday, according to Coach O.
"I'm looking forward to the two-a-days. I think we are ready for it. There have been some lulls in a couple of practices so far, but for the most part our practices have been pretty steady. I think we are prepared for two-a-days," Coach O said.
Coach O on freshman LB John Cornell: "He looks really good - fast. It's just a matter of him picking up the scheme," Coach O said. "We are throwing a lot at the freshmen right now and some of them are starting to swim a little bit, but as soon as he picks things up, I think he will be great. He's an outside linebacker right now."
On the attention span of the freshmen: "We are not having a difficult time keeping their attention because 20 of the freshmen were here all summer working out with the team," he continued. "Almost all of them all summer. They are in fantastic shape. That is the most freshmen I have ever had work out with a team I was with in the summer. Their conditioning is a lot further along than most freshmen classes."
On the competitive nature of the frosh: "We have been preaching to them since we have been recruiting them for them to come in here and compete immeidately and I love that they have," Coach O commented. "Competition's part of our program and they understand that and welcome it. Again, it helps that they were with the team over the summer. It's not like they just reported so they are familiar with their teammates."
On frosh OL John Jerry: "He's coming along. He's athletic, he's physical. I believe his run blocking is a little ahead of his pass protection now, fundamentally. He seems to be picking up the offense at a pretty good rate," O assessed. "He's on the way to being a starter. I don't know if he can start the first game or not, but he's talented enough to. I know Michael Oher did it last year and I know John is talented enough to do it this year. It all depends on his August progress."
On SS B. Brown: "He's coming off a little camp injury, but he's doing fine now. He will play for us this year," Coach O added. "The difference in the secondary this year and last is confidence. The second year in a defense, they start understanding lining up right and offensive formations and the checks needed. They are further ahead than the rest of our units because we have three returning starters there."
On his frosh defensive ends: "Marcus Tillman and Kentrell Lockett both got first team snaps today and did very well with their chances," said O.
On MLB Patrick Willis' progress: "Patrick is fine," O noted.
On DE Viciente DeLoach: "He's doing OK," said Orgeron.
On injuries: "We're doing pretty well. Normal camp injuries, nothing unusual," said O. "I credit some of that to (S & C Coach) Aaron Ausmus. There's no doubt he is fantastic."
On depth chart movement: "There hasn't been much, nothing significant to talk about," he said. "We are taking our time with that because of all the installation and the new guys we've got. We don't want a lot of moving around and jockeying around from practice to practice right now. If a guy is not worthy of a first-team jersey, he's not getting one. We have some guys practicing with the first team right now who don't have a first team jersey."
On his philosophy of players playing both ways: "I wish I had about six guys who could do it, but in reality I think it sounds good, but is very difficult to achieve," he continued. "I think some skill players could do it, but that's about it."
On the use of the fullback in Dan Werner's package: "The fullback is very important in our offensive package. Those guys are hard to come by because you are looking for an athletic guy who is not going to get the ball much and is also big enough to block. He's like a mix between a tailback and a tight end. We are happy with Jason Cook and freshman Reggie Hicks there," Orgeron explained. "They are athletic enough to catch the ball in the flat and smart enough to pick up all the protections. We do give the ball to the fullback every once in a while here. Our offense is geared to two backs and the FB is important to us. They are hard to come by so some teams don't use the fullback."
The Rebs' first scrimmage will be Saturday.
During two-a-days, our practice reports will consist of one article after the second practice of each day. There will be no practice report after the morning practice during two-a-days.
Practice Report: Wednesday
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Arts, Literature, Performing Arts, Visual ArtsFollow
Separate stories: Join Michael Cathcart and Sarah Kanowski for ABC Radio National's Books and Arts: Australia's only national broadcast devoted to literature and the arts. Published Mondays to Saturdays.
April 10th, 2015
Episode 320 of 1440 episodes
Over the past four years John Wolseleyhas explored the wetlands of Australia in a series of new works commissioned by Sir Roderick Carnegie for the National Gallery of Victoria.
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Welcome to the daily audio workout for your head.

subset: commoncrawl


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Laptop Battery
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- Materials Metal, Porcelain, Sisal, Plastic, Rope
- Minimum Order Qty 4 EA min
- Measurements of Product 2.28 in H x 2.68 in W x 6.18 in L
- UPC Code 045544607230
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- 1 EA Sleeve Box
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Rated K is show broadcast on ABS-CBN. It is aired every Sunday after Wansapanataym and before Pinoy Big {Sibling|Buddy|Close friend}: Lucky 7 in the Philippines. {It really is|It truly is|It can be} hosted by Korina Sanchez. The program is similar in style and presentation with the defunct magazine program Balitang K which was also hosted by Sanchez.Watch Pinoy TV Online, Pinoy Tambayan, Pinoy TV Channel, Pinoy Teleserye, Pinoy Telebyuwers, Pinoy Lambingan at Teleserye, Telebyuwers ,Magtvna Pinoy Channel,Phnoy TV, MagTVna, Pinoy TV Replay
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If you have private health insurance the hospital part is pretty cheep anyway, it is the OB fees and other specalists that make up most of the cost. Our hospital cost was $0, but OB $4k, and anesthesiologist 1k :-0
Wowee!!! So expensive :-(
I'll have to have a chat with my partner .. Although I would love to see a special OB / I doubt we can afford it. I'm still in shock of how much lol
It crazy, some are cheaper, some more expensive. If there is someone you are interested in give them a call and ask their fees. Do it early though, the good ones book out quickly.
Wonderful thanks for your advise :-)
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Xiamen Zhenlihua Industry & Trade Co., Ltd
Jacquard, Computerized Jacquard, Computerized Jacquard Machine,
Type: Computerized Jacquard Knitting Machine.
Component: Cylinder, 3 way actuator.
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Speed: 12 - 18 RPM.
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3. Cam system which is specially developed has less needle loss and high rotating speed.
If you have any questions, please visit our website: http://www.zhenlihuamachine.com/
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Leading China Circular Knitting Machine Manufacturer, Supplier & Exporter, Xiamen Zhenlihua Industry & Trade Co., Ltd. was founded in 2000. After more than ten years' development, it has successively set up the sales and market service sites in Fujian, Zhejiang, Jiangsu, Guangdong and Shandong province in China. The company was committed to the researching, developing, producing and selling of Circular Knitting machines.
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Opposing roles of CXCR4 and CXCR7 in breast cancer metastasis
DOI: 10.1186/bcr3074
© Hernandez et al.; licensee BioMed Central Ltd. 2011
Received: 12 December 2010
Accepted: 9 December 2011
Published: 9 December 2011
CXCL12-CXCR4 signaling has been shown to play a role in breast cancer progression by enhancing tumor growth, angiogenesis, triggering cancer cell invasion in vitro, and guiding cancer cells to their sites of metastasis. However, CXCR7 also binds to CXCL12 and has been recently found to enhance lung and breast primary tumor growth, as well as metastasis formation. Our goal was to dissect the contributions of CXCR4 and CXCR7 to the different steps of metastasis - in vivo invasion, intravasation and metastasis formation.
We overexpressed CXCR4, CXCR7 or both in the rat mammary adenocarcinoma cell line MTLn3. Stable expressors were used to form tumors in severe combined immunodeficiency (SCID) mice, and in vivo invasiveness, intravital motility, intravasation, and metastasis were measured.
We found that CXCR4 overexpression increased the chemotactic and invasive behavior of MTLn3 cells to CXCL12, both in vitro and in vivo, as well as in vivo motility and intravasation. CXCR7 overexpression enhanced primary tumor growth and angiogenesis (as indicated by microvessel density and VEGFA expression), but decreased in vivo invasion, intravasation, and metastasis formation. In vitro, expression of CXCR7 alone had no effect in chemotaxis or invasion to CXCL12. However, in the context of increased CXCR4 expression, CXCR7 enhanced chemotaxis to CXCL12 but decreased invasion in response to CXCL12 in vitro and in vivo and impaired CXCL12 stimulated matrix degradation. The changes in matrix degradation correlated with expression of matrix metalloproteinase 12 (MMP12).
We find that CXCR4 and CXCR7 play different roles in metastasis, with CXCR4 mediating breast cancer invasion and CXCR7 impairing invasion but enhancing primary tumor growth through angiogenesis.
There are currently two known receptors for CXCL12: CXCR4 and CXCR7 [1, 2], which belong to the family of G-protein coupled receptors (GPCRs). CXCR4 is expressed in several human cancers including glioma [3], neuroblastoma [4], pancreatic [5] and breast [6], with overexpression of CXCR4 in breast cancer correlating with poor patient prognosis [7–9]. CXCL12/CXCR4 signaling has been reported to stimulate growth of several tumors including breast [10–13], with carcinoma-associated fibroblasts (CAFs) being an important source of CXCL12 in the tumor microenvironment [14]. CAFs can enhance tumor growth in a paracrine manner, with secreted CXCL12 directly stimulating growth of CXCR4 expressing breast cancer cells, and in an endocrine manner, recruiting endothelial progenitor cells (EPCs) to the primary tumors, thus enhancing angiogenesis [15]. CXCL12, also known as SDF-1, belongs to the CXC family of chemokines. CXCL12 functions as a growth factor for B cell progenitors [16], a chemotactic factor for both T cells and monocytes, a regulator of hematopoiesis and as a chemoattractant for tissue-committed stem cells [17, 18]. Importantly, CXCL12 has been found to be expressed in many human solid tumors including breast, pancreas and prostate cancers, and glioblastoma [17], with high levels of CXCL12 expression correlating with poor prognosis of breast cancer patients [19].
CXCL12/CXCR4 signaling has been shown to stimulate the chemotactic and invasive behavior of breast cancer cells in vitro and in vivo [6, 10, 19–21], and has been proposed to serve as a homing mechanism for cancer cells to sites of metastasis. CXCL12 is expressed at high levels in the bone marrow, lung, liver, and lymph nodes, common sites of breast cancer metastasis, with protein extracts from these organs stimulating chemotaxis of breast cancer cells in a CXCR4-dependent manner [6]. Furthermore, downregulation of CXCR4 signaling using a neutralizing antibody or miRNA, decreases spontaneous and experimental lung metastasis formation of MDA-MB-231 cells [6, 20].
Like CXCR4, CXCR7 is also expressed in different human cancers, including breast, being highly expressed in the tumor vasculature [22, 23]. CXCR7 is considered an atypical GPCR because ligand binding does not result in intracellular Ca2+ release [2, 24], and there are conflicting reports on the ability of CXCR7 to activate phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase (MAPK) signaling, and to promote cell motility. Binding of CXCL12 or interferon-inducible T-cell alpha chemoattractant (I-TAC/CXCL11), the other known CXCR7 ligand, to CXCR7 activates PI3K and MAPK signaling in astrocytes, Schwann cells, gliomas, rhabdomyosarcoma, and pancreatic cancer cells [23–26]. Moreover, CXCR7 has been reported to mediate CXCL12 chemotaxis in T cells [1] and rhabdomyosarcoma cells [26], and to promote hepatocellular carcinoma invasion in vitro [27]. However, other studies have shown that CXCR7 does not play a role in bare filter migration but in transendothelial migration [28], and that CXCR7 plays no role in T cell chemotaxis or MAPK/PI3K signaling [29]. Although the interaction of CXCR7 with G proteins is controversial, new studies have found that CXCR7 binds to β-arrestin 2, with this interaction resulting in receptor internalization [28, 30, 31], and mediating chemotaxis to I-TAC in vascular smooth muscle cells [32]. Furthermore, CXCR4 and CXCR7 can form both homodimers and heterodimers with heterodimer formation suggested to modulate CXCR4 signaling both positively, and negatively [33–35]. Most recently, CXCR4+CXCR7+ MDA MB 231 cells have been shown to chemotax in response to CXCL12 stimulation better than 231 cells expressing only CXCR4, with this chemotactic response being dependent on β-arrestin 2 [36].
CXCR7 has been implicated in enhancing cancer cell adhesion to fibronectin and endothelial cells [2, 23, 27]; increasing cell survival by decreasing apoptosis [2, 23] and promoting primary tumor growth of lymphoma, lung, breast, prostate and hepatocellular cancer cells [2, 22, 23, 27]. CXCR7 expression has been reported to contribute to tumor angiogenesis through the secretion of angiogenic factors such as vascular endothelial growth factor (VEGF) [23, 27], as well as to promote experimental metastasis formation of breast cancer cells [22].
Although CXCL12 signaling has been implicated in breast cancer metastasis as a homing mechanism for cancer cells to common sites of metastasis, not much is currently known about the role of CXCL12 signaling in the early steps of metastasis within the primary tumor. Also, the role of CXCR7 in breast cancer cell motility, tumor growth and metastasis is still unclear, with the effect of coexpression of CXCR4 and CXCR7 in these processes mostly unknown. With research suggesting that both CXCR4 and CXCR7 alone can enhance metastasis, we set out to dissect the roles of CXCR4 and CXCR7 in the different steps of metastasis (invasion, intravasation, and metastasis formation) by overexpressing CXCR4, CXCR7, or both receptors in the rat mammary adenocarcinoma cell line MTLn3. Here we report that CXCR4 overexpression increases the chemotactic and invasive behavior of MTLn3 cells, in vitro and in vivo, to CXCL12, as well as their motile behavior within the primary tumor. Furthermore, although CXCR4 overexpression had no effect on primary tumor growth, it enhanced intravasation without affecting spontaneous lung metastasis formation. CXCR7 overexpression alone did not result in CXCL12-induced chemotaxis or invasion in vitro; however, in the context of high CXCR4 expression it further increased the in vitro chemotactic response of MTLn3 CXCR4 cells to CXCL12, while reducing invasion and matrix degradation. In vivo, CXCR7 increased primary tumor growth while it impaired invasion to CXCL12, intravasation and spontaneous lung metastasis formation. CXCR7 overexpression downregulated the effects of CXCR4 in motility within the primary tumor, intravasation, and spontaneous lung metastasis formation.
Materials and methods
Cell lines
All MTLn3 cell lines were grown in alpha MEM supplemented with 5% FBS (100-106; Gemini Bio-Products, West Sacramento, CA, USA) and 0.5% penicillin/streptomycin (15140-122; Invitrogen, Grand Island, NY, USA). To create the human CXCR4 expressors, hCXCR4 was transferred from the pDNR-Dual hCXCR4 vector (Harvard Institute of Proteomics, Boston, MA, USA), to the JP1520 retroviral vector following the Creator Cloning protocol, using Cre recombinase (Clontech, Mountain View, CA, USA) and Max Efficiency DH5alpha bacteria (Life Technologies, Grand Island, NY, USA) grown in 7% sucrose, 30 μg/ml chloramphenicol plates. Colonies were picked and correct insertion of human CXCR4 verified by sequencing. The human CXCR7 sequence was digested out from the pcDNA 3.1+ plasmid (kindly provided by ChemoCentryx, Mountain View, CA, USA) using NotI, the ends blunted using DNA Polymerase I, large Klenow fragment (NEB, Ipswich, MA, USA), to insert into JP1520, which was digested with BamHI and BbsI removing the loxP site, ends blunted as above and treated with Antarctic phosphatase (NEB, Ipswich, MA, USA). Both insert and vector were gel purified using the Qiagen Gel extraction kit, then ligated using a Rapid DNA Ligation kit (Roche, Branchburg, NJ, USA). Subcloning efficiency DH5alpha bacteria (Life Technologies, Grand Island, NY, USA) were transformed with the ligated vector and colonies screened for correct insertion of hCXCR7 using differential enzyme digestions, followed by verification using sequencing analysis. MTLn3-GFP (MTLn3 cells expressing green fluorescent protein) cells were transduced with either the empty JP1520 vector, JP1520-CXCR4, JP1520-CXCR7, or both CXCR4 and CXCR7, by first transfecting Phoenix packaging cells with 2 μg of each vector using lipofectamine (Invitrogen, Grand Island, NY, USA), collecting virus and transducing MTLn3-GFP cells seeded at 60% confluency. Transduced cells were selected with 1 μg/ml puromycin. MTLn3 CXCR7 and MTLn3 CXCR4-CXCR7 cells were subsequently fluorescence-activated cell sorting (FACS) sorted in a DakoCytomation MoFlo to obtain a homogenous population of CXCR7 expressing cells. MDA MB 435 cell lines were grown in DMEM (10-013 CV, Cellgro, Manassas, VA, USA) supplemented with 10% FBS (S11550, Atlanta Biologicals, Lawrenceville, GA, USA) and 0.5% penicillin/streptomycin. 435 cells seeded at 60% confluency, were transduced with either the empty JP1520 vector, JP1520-CXCR4 or JP1520-CXCR7 using premade virus, with transductants selected using 1 μg/ml puromycin. MDA MB 435 CXCR7 cells were FACS sorted in a DakoCytomation (Carpinteria, CA, USA) MoFlo to obtain a homogenous population of CXCR7 expressing cells. The 435 double overexpressors, CXCR4-CXCR7, were made by transducing sorted 435-CXCR7 cells with JP1520-CXCR4 virus and then FACS sorted for high CXCR4 expression. MDA MB 435 CXCR4 cells were sorted at the same time to obtain cell lines with homogenous CXCR4 expression.
Reverse transcription and PCR
MTLn3 cells grown to 70 to 85% confluency were used for RNA isolation using the Qiagen RNeasy Mini kit with DNase I treatment (Valencia, CA, USA). A 1 μg sample of total RNA was used for reverse transcription using Superscript III and random hexamers in a 20 μl reaction volume. A 2 μl aliquot of the reaction was used for PCR using Taq polymerase for 30 cycles. Primers used were: rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (PPR06557A Superarray), rat CXCR4 (5' AGGAACTGAACGCTCCAGAA 3' and 5' AACCACACAGCACAACCAAA 3'), human CXCR4 (5' CTCCAAGCTGTCACACTCCA 3' and 5' TCGATGCTGATCCCAATGTA 3'), human and rat CXCR7 (5' GCACTACATCCCGTTCACCT 3' and 5'AAGGCCTTCATCAGCTCGTA 3'). PCR products were run in a 1.5% agarose gel containing ethidinium bromide. For quantitation of endogenous expression of rat CXCR4, the collected cDNA was used for quantitative real-time PCR with SYBR Green (PA-012, SuperArray Biosciences, Frederick, MD, USA) and an Applied Biosystems 7900HT (Carlsbad, CA, USA). To evaluate the expression of matrix metalloproteinases (MMPs) in the different MTLn3 transductants, the cell lines were grown to 80 to 90% confluency in six-well plates, starved overnight in alpha-MEM/0.35% BSA in a 37°C incubator, and then stimulated for four hours with 10 nM CXCL12 (460-SD; R&D systems, Minneapolis, MN, USA) in alpha-MEM/0.35% BSA or just alpha-MEM/0.35% BSA at 37°C. RNA was extracted using the Qiagen RNeasy Mini kit with DNase I treatment. A 2 μg sample of total RNA was used for reverse transcription using a Superscript First Strand kit (11904-018, Invitrogen, Grand Island, NY, USA) and cDNA used for real time PCR with SYBR Green (PA-012) on an Applied Biosystems 7900HT. Rat specific primers for MMPs were obtained from real-time primers (Elkins Park, PA, USA). RNA expression of MMPs was normalized to GAPDH.
Fluorescent activated cell sorting (FACS)
To analyze the levels of CXCR4 and CXCR7 expression in the MTLn3 and MDA MB 435 cell lines, cells were grown to 80% confluency, detached at 37°C using PBS without Ca2+/Mg2+ +2 mM ethylenediaminetetraacetic acid (EDTA) and resuspended in 1 ml cold PBS without Ca2+/Mg2+ supplemented with 0.2% BSA. Cells were labeled with either control mouse IgG antibody (MAB002; R&D systems, Minneapolis, MN, USA), anti-human CXCR4 antibody (MAB172; R&D systems, Minneapolis, MN, USA) or anti-human CXCR7 antibody (11G8; ChemoCentryx, Mountain View, CA, USA) for 45 minutes at 4°C. Unbound primary antibody was removed by washing and bound antibody was detected with APC antimouse secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). Expression of rat CXCR4 protein was evaluated in the MTLn3 transductants using a rat specific CXCR4 antibody (ab7199, Abcam, Cambridge, MA, USA) with a rabbit IgG as a control (011-000-003, Jackson ImmunoResearch, West Grove, PA, USA) and anti-rabbit DyLight 649 as the secondary antibody (111-496-144, Jackson ImmunoResearch, West Grove, PA, USA). Fluorescently labeled cells were evaluated using a Becton Dickinson LSRII (Franklin Lakes, NJ, USA). FCS files were analyzed using FlowJo software (Ashland, OR, USA).
In vitro chemotaxis
Chemotaxis was evaluated using a 48-well microchemotaxis chamber (Neuroprobe, Gaithersburg, MD, USA) and PVP-free 8 μm pore polycarbonate filters (Neuroprobe, Gaithersburg, MD, USA) coated with 27 μg/ml rat tail collagen type I (BD Bioscience, Franklin Lakes, NJ, USA). Cell lines were starved in L15 medium supplemented with 0.35% BSA for three hours at 37°C, detached using PBS without Ca2+/Mg2+ + 2 mM EDTA and resuspended in L15-0.35% BSA to plate 2 × 104 cells per well for MTLn3 transductants, or 1.5 × 104 cells per well for the MDA MB 435 transductants. CXCL12 solutions were prepared in L15-0.35% BSA and placed in the bottom wells with cells plated in the top wells of the assembled chamber. To inhibit CXCL12 binding to CXCR7, we added 10 nM I-TAC (572-MC; R&D systems, Minneapolis, MN, USA), or used the CXCR7 inhibitors CCX733 (ChemoCentryx, Mountain View, CA, USA) and CCX771 (ChemoCentryx, Mountain View, CA, USA) added to both top and bottom wells, including vehicle (DMSO) as a control. To inhibit CXCR4, we added AMD3100 (Sigma, St. Louis, MO, USA) to both top and bottom wells. After a four-hour incubation at 37°C, filters were placed in 10% formalin solution to fix the cells for 30 minutes, cells on the top of the filter, non-migrating cells, were removed using a cotton swab, and migrating cells subsequently stained overnight in hematoxylin. The number of cells crossing the filter in one representative 10× field was counted per well for the MTLn3 transductants, using a Nikon Labophot light microscope (Melville, NY, USA), and corresponding wells averaged per experiment. To determine MDA MB 435 chemotaxis, the number of cells that crossed each well were counted and corresponding wells averaged per experiment.
In vitro invasion
MTLn3 transductants grown to 70 to 85% confluency were starved for three hours in alpha-MEM supplemented with 0.35% BSA in a 37°C incubator. Cells were detached using PBS without Ca2+/Mg2+ containing 2 mM EDTA, resuspended in alpha-MEM supplemented with 0.35% BSA to plate 1 × 105 cells in a 500 μl volume on top of Matrigel-precoated 8 μm pore-filters (354480; BD Biosciences transwells, Franklin Lakes, NJ, USA) that had been equilibrated for one hour with alpha-MEM/0.35% BSA in a 37°C incubator. Cells were allowed to invade overnight in a 37°C incubator in response to either alpha-MEM/0.35% BSA alone or containing 10 nM CXCL12. The filters were fixed in 10% formalin for 30 minutes and stained with crystal violet for 15 minutes. Cells that had not invaded were removed with a cotton tip applicator from the top of the filter, filters removed, placed in a coverslip and the total number of invading cells present in a filter counted using a Nikon Labophot light microscope with a 10× objective.
Matrix degradation assay
MTLn3 CXCR4 or MTLn3 CXCR4-CXCR7 GFP labeled cells were plated overnight on MatTek dishes, at 1 × 105 cells/dish, over a thin Alexa 405-gelatin matrix in the presence of the protease inhibitor GM6001 (10 μm). Cells were subsequently starved for three hours, washed three times in starvation media [37] and stimulated with 5 nM CXCL12 for six hours. At the end of the incubation time, cells were fixed in 3.7% paraformaldehyde and imaged. The images were processed using the ImageJ Spot enhancing filter 2D (3.0 pixels Gaussian filter) and the threshold levels set to select only degradation areas. The degradation area was normalized to the cell coverage area in the GFP channel. Alexa405 (A30000, Invitrogen, Grand Island, NY, USA) was conjugated to gelatin (G2500, Sigma, St. Louis, MO, USA) and thin matrix Alexa405-gelatin matrix was prepared as previously described [38]. Results are reported as the degraded area/cell area per field normalized to the MTLn3 CXCR4 unstimulated levels.
In vivo invasion
All animal procedures were conducted observing the National Institutes of Health regulations on the use and care of experimental animals. Our animal protocol was approved by the Albert Einstein College of Medicine animal use committee. Female severe combined immunodeficiency (SCID) mice aged four to seven weeks from NCI were used for all experiments. To form primary tumors, MTLn3 transductants grown to 70 to 85% confluency were detached using PBS without Ca2+/Mg2+ + 2 mM EDTA, resuspended in cold PBS supplemented with 0.2% BSA to inject 5 × 105 cells per animal in a 100 μl volume. Cells were injected into the fourth mammary fat pad and tumors allowed to grow until they reached an average volume of 1,300 mm3 for the in vivo invasion assay. Mice were anesthetized using isoflurane and blocking needles placed into the primary tumors using micromanipulators (MN-151; Narishige, East Meadow, NY, USA). Hamilton 33 gauge needles were loaded with a mixture of EDTA, 10% matrigel and CXCL12 dissolved in L15-0.35% BSA, and these experimental needles used in place of the blocking needles after the animal was appropriately setup. Detailed information about this assay can be found in [39]. To block the colony stimulating factor 1 (CSF-1) receptor on the mouse tumor associated macrophages we used the anti-mouse CSF-1R antibody (AFS98) [40] at 15 μg/ml; to block epidermal growth factor (EGF)-derived from the mouse tumor associated macrophages from binding to EGF receptor (EGFR) we used a neutralizing EGF antibody at a concentration of 20 μg/ml (AF2028; R&D systems, Minneapolis, MN, USA). An isotype IgG antibody (012-000-007 or 111-005-144, Jackson ImmunoResearch, West Grove, PA, USA) was used at the same concentration as the blocking/neutralizing antibodies as a control. To inhibit CXCR4 we used 100 nM AMD3100. Cells were allowed to invade into the needles for four hours and the contents of the needles were subsequently extruded into coverslips, invasive cells stained with 4',6-diamidino-2-phenylindole (DAPI) and counted using an Inverted Olympus IX70 microscope (Center Valley, PA, USA).
Intravital imaging
Primary tumors of an average volume of 1,300 mm3 were used for intravital imaging. Mice were anesthetized with isoflurane and a skin-flap surgery carefully performed to expose the primary tumor while minimizing damage to tissue and blood vessels. Animals were placed on the stage of an inverted microscope and the GFP-labeled carcinoma cells imaged using an Olympus Fluoview FV1000-MPE microscope (Center Valley, PA, USA) at an excitation of 880 nm with a 25 × 1.05 NA water objective. Collagen fibers were visualized by second harmonic generation. Time-lapse Z-series were taken at 5 μm steps for a total of 100 μm into the tumor over 30 minutes at two-minute intervals. Movies were analyzed using Image J http://imagej.nih.gov/ij/. A cancer cell was considered to be motile when it had protruded/translocated at least half a cell length, and the total number of motile cancer cells in a 50 μm Z-stack time-lapse movie was determined. A more detailed description of this protocol can be found in [41].
Spontaneous metastasis and intravasation
Primary tumors were allowed to grow until they reached an average volume of 1,500 mm3 to perform end-point metastasis assays. At this time, mice were anesthetized using isoflurane and blood collected via cardiac puncture from the right side of the heart to obtain cancer cells that had intravasated. The blood drawn was then plated into a 10 cm dish with alpha MEM supplemented with 5% FBS/0.5% P/S, and cancer cell colonies allowed to grow for a week in a 37°C incubator followed by counting using a light microscope. Tumor blood burden is reported as the total number of cancer cell colonies present in a dish normalized to the volume of blood plated. Lungs and primary tumors were harvested and fixed in 10% formalin solution. The lungs were paraffin-embedded, sectioned and stained with H&E to count the number of lung metastasis present in all lobes of a single section using a light microscope with a 10× objective. To evaluate lymph node metastasis, both axillary and inguinal lymph nodes were removed from tumor-bearing mice, fixed in 10% formalin solution, paraffin-embedded, sectioned and stained with H&E. The presence of metastases was assessed using a Nikon Labophot light microscope (Melville, NY, USA). To estimate bone marrow metastasis, the femur ipsilateral to the site of primary tumor growth was dissected and bone marrow was flushed using 1 ml syringes with 25-gauge needles into a 10 cm plate containing alpha MEM supplemented with 5% FBS/0.5% P/S. Plates were incubated at 37°C for a week and tumor colonies then counted.
For microvessel density evaluation, formalin-fixed, paraffin-embedded sections from MTLn3 JP, MTLn3 CXCR4, MTLn3 CXCR7, and MTLn3 CXCR4-CXCR7 primary tumors were deparaffinized, rehydrated, blocked in donkey serum and stained with rat antimouse CD34 antibody (CL8927AP; Cedarlane labs, Burlington, NC, USA) at a 1:400 dilution for one hour. Slides were washed and subsequently stained with a biotinylated antirat secondary antibody for 50 minutes. The slides were rinsed and exposed to ABC-HRP (PK-6100, Vector, Burlingame, CA, USA) for 20 minutes, washed and exposed to diaminobenzidine (DAB) for one to four minutes (SK-4100, Vector, Burlingame, CA, USA), and subsequently counterstained with Harris hematoxylin (s212, Poly-scientific, Bay Shore, NY, USA), rinsed and mounted. Mean vessel density was determined by counting the number of blood vessels present per field seen in a light microscope using a 10× objective. A total of three different primary tumors were used per cell line, counting five fields per tumor. For VEGFA evaluation, samples for immunohistochemistry (IHC) were sectioned at 5 μm, deparaffinized in xylene followed by graded alcohols. Antigen retrieval was performed in 10 mM sodium citrate buffer at pH 6.0, heated to 96C, for 20 minutes. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide in PBS for 10 minutes. Blocking was performed by incubating sections in 5% normal donkey serum with 2% BSA for one hour. The primary antibody to VEGFA, (PAB12284, ABNOVA, Walnut, CA, USA) was used at 1:250 for 1.5 hours at room temperature. The primary species (rabbit IgG) was substituted for the primary antibody to serve as a negative control. The sections were stained by routine IHC methods, using HRP rabbit polymer conjugate (Invitrogen, Grand Island, NY, USA), for 20 minutes to localize the antibody bound to antigen, with diaminobenzidine as the final chromogen. All immunostained sections were lightly counterstained with hematoxylin.
Statistical analysis
Analysis of variance (ANOVA) was used to demonstrate that there were significant differences between conditions when there were more than two conditions, and paired analyses were performed using either student t-test, or Mann-Whitney test in order to identify the conditions that were significantly different. Correlation of MMP12 expression with CXCR4 and CXCR7 was performed using Oncomine with the following databases: Bittner Breast, Bonnefoi Breast, Desmedt Breast, Ginestier Breast, Gluck Breast, Hess Breast, Ivshina Breast, Loi Breast, and van't Veer Breast. SPSS was used to determine correlation coefficients and their significance from the downloaded expression data. The Oncomine database was also used to identify clinical parameters with which MMP12 mRNA levels were significantly correlated. The following parameters were examined: estrogen receptor (ER) positive, triple negative, high grade, metastasis, recurrence and survival. The probabilities of overexpression or underexpression of MMP12 for all breast cancer datasets containing more than 40 samples for which the parameters were provided by Oncomine were downloaded. For each parameter, the number of datasets in which the probability of MMP12 overexpression or underexpression was less than 0.05 was identified and the binomial cumulative probability distribution was used to determine the likelihood of that number occurring by chance using SPSS (IBM, Armonk, NY).
CXCR7 enhances in vitrochemotaxis to CXCL12 in the presence of high CXCR4
Having engineered MTLn3 cell lines overexpressing either CXCR4, CXCR7, or both receptors, we compared their chemotaxis to CXCL12 using a microchemotaxis chamber (Figure 1c). The MTLn3 CXCR4 cell line showed significantly increased CXCL12-induced chemotaxis (P < 0.005) compared with the control cell line, MTLn3 JP. The MTLn3 CXCR7 cell line showed no chemotactic response to CXCL12 (P > 0.1). Furthermore, overexpression of both CXCR4 and CXCR7 resulted in a significantly increased chemotactic response to CXCL12 compared with that of the MTLn3 CXCR4 cell line (P < 0.005). This migration phenotype suggests that although CXCR7 alone does not mediate CXCL12-induced motility, in the presence of CXCR4, CXCR7 augments CXCL12-stimulated motility. This was confirmed in the cancer cell line MDA-MB-435 previously shown to express low endogenous levels of CXCR4 [42] [see Additional data file 2], which also showed the CXCR4-CXCR7 double overexpressors to have the most chemotaxis to CXCL12.
To test whether the increased motility observed in the MTLn3 CXCR4-CXCR7 cell line was dependent on CXCR4, we added the CXCR4 inhibitor, AMD3100, at a concentration that specifically inhibits CXCR4 without acting as an agonist of CXCR7 [43]. Addition of AMD3100 significantly decreased CXCL12-induced chemotaxis of both MTLn3 CXCR4 and MTLn3 CXCR4-CXCR7 cells (P < 0.005) (Figure 1d) suggesting that CXCR4 is important for CXCL12-induced chemotaxis in the double overexpressors. Addition of I-TAC (CXCL11), a chemokine that binds to CXCR7 [2, 22], failed to act as a chemoattractant or impair CXCL12-induced chemotaxis for either MTLn3 CXCR4, or MTLn3 CXCR4-CXCR7 cells (P > 0.5) (Figure 1e). Similarly, addition of the CXCR7 inhibitors CCX771 and CCX733 did not inhibit chemotaxis to CXCL12 in the MTLn3 CXCR4-CXCR7 cells (P > 0.4) (Figure 1f). These results show that in vitro, CXCR7 alone does not mediate CXCL12-induced chemotaxis. However, when expressed in cells with high levels of CXCR4, CXCR7 augments CXCR4-mediated chemotaxis to CXCL12, in agreement with recent studies using MDA-MB 231 cells [36]. Importantly, the chemotaxis response of the double overexpressors, CXCR4-CXCR7, was impaired in the presence of CXCR4 inhibitor, AMD3100, but not upon addition of ITAC or the CXCR7 inhibitors CCX771 or CCX733 suggesting that binding of CXCL12 to CXCR4 but not CXCR7 is needed for chemotaxis to occur.
CXCR7 inhibits invasion to CXCL12
To measure the effects of CXCR4 and CXCR7 overexpression on invasion in response to CXCL12 in vivo, we injected MTLn3 JP, MTLn3 CXCR4, MTLn3 CXCR7, and MTLn3 CXCR4-CXCR7 cell lines labeled with GFP into the fourth mammary fat pads of SCID mice and allowed the tumors to grow until they reached an average volume of 1,300 mm3 for in vivo invasion analysis. FACS analysis of the primary tumors confirmed that overexpression of CXCR4 and CXCR7 in the respective cell lines was conserved in vivo [see Additional data file 3]. Needles containing Matrigel and CXCL12 as a chemoattractant were inserted in the primary tumors and invasive cells collected for a four-hour period. Overexpression of CXCR4 dramatically increased the in vivo invasive behavior of MTLn3 cells to CXCL12, with peak invasion shifted to lower concentrations (Figure 2b). CXCR7 overexpression alone, however, did not enhance the ability of MTLn3 cells to invade in response to CXCL12, as they failed to invade in vivo at the highest concentration of CXCL12 (62.5 nM) tested compared with buffer levels, a concentration which induced a three-fold increase in invasion above background in the MTLn3 JP tumors. Similarly, expression of CXCR7 in the presence of CXCR4 also impaired the invasive response to CXCL12 at all concentrations, with the peak response at 15.6 nM being significantly reduced in the MTLn3 CXCR4-CXCR7 tumors (P < 0.005). Invasion in response to 6.25 nM CXCL12 was not tested for the MTLn3 CXCR7 strain since responses to the higher concentrations were similar to the buffer response and the in vitro chemotaxis data did not demonstrate any response at lower concentrations. These results are consistent with the in vitro invasive behavior of these cell lines, confirming that CXCR7 plays a negative role in CXCL12-induced invasion. The relatively weak in vivo invasive response seen at 15.6 nM CXCL12 in the MTLn3 CXCR4-CXCR7 cell line is significantly impaired upon addition of the CXCR4 inhibitor AMD3100 (P < 0.05; Figure 2c), indicating the remaining response is still mediated by CXCR4. As there is no increase in CXCR4 expression seen in the MTLn3 JP tumor cells [see Additional data file 3], the response seen in MTLn3 JP tumors to high levels of CXCL12 may reflect initiation of the paracrine loop by tumor-associated macrophages, which express CXCR4 [44–46].
These data show that CXCR7 expression alone plays no role in CXCL12-induced motility (chemotaxis or invasion). However, CXCR7 expression in the context of high levels of CXCR4, while enhancing chemotaxis to CXCL12, impairs invasion in vitro and in vivo to CXCL12. These results raised the possibility that CXCR7 inhibits the ability to degrade extracellular matrix in response to CXCL12 stimulation and hence invasion. To address this, we measured the ability of MTLn3 CXCR4 and MTLn3 CXCR4-CXCR7 cells to degrade fluorescently labeled matrix (Figure 2d). Although there was no significant difference in the ability of these cell lines to degrade matrix in the absence of stimulation, when exposed to CXCL12 only the CXCR4 overexpressors showed significantly increased degradation (P < 0.05), while the double overexpressors showed no increase, indicating that CXCR7 expression impairs CXCL12-induced invasion by suppressing CXCL12-induced matrix degradation. Evaluation of the expression of different MMPs (MMP1, MMP2, MMP3, MMP7, MMP9-14) at the mRNA level revealed that MMP12 was significantly higher in the CXCR4 line compared with the other lines after stimulation with CXCL12 (P < 0.01 by ANOVA) [see Additional data file 4]. Western blotting after CXCL12 stimulation also indicated that MMP12 expression was highest in the CXCR4 line [see Additional data file 4]. These results suggest that there is differential regulation of MMPs in the MTLn3 CXCR4 and CXCR4-CXCR7 cells upon CXCL12 stimulation with the CXCR4-expressing cells showing increased expression of MMPs such as MMP3, MMP10, and MMP12. Evaluation of breast cancer data present in the Oncomine database indicates that only MMP12 expression is significantly correlated with CXCR4 expression: from the nine breast cancer databases evaluated, the average correlation was 0.31 with an average P value for the correlation of less than 0.04. In the same databases, the average correlation of MMP12 with CXCR7 was -0.03, which was not significant (P < 0.46).
We had previously reported that CXCL12 could induce in vivo invasion in the MMTV-PyMT transgenic breast cancer model, and that this invasion was dependent on the EGF/CSF-1 paracrine loop between cancer cells and macrophages where CSF-1 is secreted by cancer cells and stimulates macrophages to produce EGF [21]. To test whether CXCL12 induced in vivo invasion in the MTLn3 CXCR4 model was also dependent on EGF/CSF-1 signaling, we tested the ability of these cells to invade in vivo in response to CXCL12 in the presence of either a neutralizing EGF antibody or a blocking CSF-1R antibody [40]. The MTLn3 CXCR4 invasive response to CXCL12 was significantly impaired in the presence of either antibody [see Additional data file 5], indicating EGF/CSF-1 signaling is required for CXCL12 induced in vivo invasion in this model. Thus CXCR4 overexpression did not override the dependency of these breast cancer cells on the EGF/CSF-1 paracrine loop for in vivo invasion.
CXCR4 overexpression stimulates cancer cell motility within the primary tumor
CXCR7 overexpression enhances primary tumor growth while CXCR4 enhances intravasation
We next evaluated the effects of CXCR4 and CXCR7 overexpression on the process of intravasation, by doing intracardiac punctures prior to euthanization of mice carrying tumors of an average volume of 1,500 mm3. As shown in Figure 4d, MTLn3-CXCR4 cells were significantly more efficient in entering the bloodstream compared with MTLn3-JP and CXCR4-CXCR7. Both MTLn3 CXCR7 and MTLn3 CXCR4-CXCR7 tumor models showed low numbers of intravasated cancer cells. These results agree with the increased invasive and motile behavior of the CXCR4 overexpressing MTLn3 cells seen within the primary tumor, and the impaired ability of the CXCR7 overexpressors to invade in vivo.
To compare the ability of the MTLn3 transductants to spontaneously metastasize from the primary tumor to the lung, we evaluated lung metastasis formation in animals that had been injected in the mammary fat pad with the transduced MTLn3 cells. Lungs were harvested when the primary tumors reached an average volume of 1,500 mm3, fixed, paraffin-embedded and sectioned to count the number of metastases present per H&E section. The number of spontaneous lung metastases present in the MTLn3 CXCR4 model was not significantly different to the number of lung metastases formed in the empty vector control model, MTLn3 JP (P = 0.3 student's t-test and P = 0.8 Mann-Whitney; Figure 4e), nor was there any difference in the size of the metastases (data not shown). This was an unexpected result as MTLn3 CXCR4 cells were more efficient in leaving the primary tumor and entering the bloodstream compared with MTLn3 JP cells, thus we looked for metastasis formation in the bone and lymph nodes but no difference was observed at these sites (P = 0.7 and P = 0.8, respectively) [see Additional data file 10]. However, the MTLn3 CXCR7 tumors gave rise to significantly fewer lung metastases compared with the empty vector model, MTLn3 JP, (a, P < 0.005) and likewise, the double overexpressors, MTLn3 CXCR4-CXCR7, showed decreased lung metastasis compared with both MTLn3 JP (b, P < 0.005) and MTLn3 CXCR4 (c, P < 0.005; Figure 4e). No significant difference in size of lung metastasis, or in bone or lymph node metastasis was observed in the different CXCR7 transductants compared with control MTLn3 JP or MTLn3 CXCR4 (data not shown) [see Additional data file 10].
Previous studies have reported that both CXCR4 and CXCR7 play roles in breast cancer growth and metastasis, with both receptors being implicated in primary tumor growth, invasion and metastasis formation [2, 6, 10, 12, 22]. However, distinguishing the roles of these receptors in the early steps of metastasis has not been performed. Using the rat mammary adenocarcinoma cell line MTLn3, we studied the effects of overexpression of CXCR4 and CXCR7 on CXCL12-induced chemotaxis and invasion, as well as in vivo motility, intravasation and metastasis formation. We show that CXCR4 overexpression increases chemotactic and invasive behavior, both in vitro and in vivo, in response to a CXCL12 gradient, as well as enhances the motile behavior of tumor cells within the primary tumor and their ability to intravasate. Expression of CXCR7 alone had no effect on chemotaxis or invasion in vitro, but suppressed CXCL12-induced invasion in vivo, as well as intravasation and metastasis. Expression of both CXCR4- and CXCR7-enhanced chemotaxis to CXCL12 in vitro, but CXCL12-induced invasion in vivo and in vitro was reduced compared with that of cells expressing CXCR4 alone, and metastasis was also reduced.
The increased chemotactic response seen in vitro upon expression of CXCR4 is consistent with many previous studies demonstrating that CXCR4 can mediate chemotaxis to CXCL12 [47]. The literature on the ability of CXCR7 to mediate chemotactic responses is mixed, with some reports suggesting that CXCR7 can mediate chemotactic responses [1] and others indicating that it cannot [2, 29]. Our data are consistent with the latter studies; using two cell lines (MTLn3 and MDA-MB-435) that show little chemotactic response to CXCL12 on their own, we find that expression of CXCR7 alone does not enhance chemotactic responses to CXCL12. However, coexpression of CXCR7 and CXCR4 resulted in increased chemotaxis towards CXCL12 compared with cells expressing CXCR4 alone. These data agree with a recent study showing that increased expression of CXCR7 in MDA-MB-231 cells results in enhanced chemotaxis to CXCL12 [36]. AMD3100, a CXCR4 selective inhibitor, inhibited CXCL12-induced chemotaxis and invasion of both MTLn3 CXCR4 and MTLn3 CXCR4-CXCR7 cells, while inhibition of CXCL12 binding to CXCR7 using I-TAC, CCX733 or CCX771 had no effect on CXCL12-induced chemotaxis. This indicates that CXCL12 binding to CXCR4 is needed for the chemotactic response but that binding of CXCL12 to CXCR7 is not necessary. The potentiation of the chemotactic response by CXCR7 is potentially through regulation of downstream signaling by CXCR4. CXCR7 has been shown to heterodimerize with CXCR4 [33–35] and to regulate recruitment of β-arrestin 2, as well as enhance ERK and p38 signaling in response to CXCL12 stimulation [36, 48]. Our results suggest that although CXCR7 has been shown to alter Gαi coupling to CXCR4 [34], the enhancement of β-arrestin signaling by CXCR7 [36] is more significant, resulting in enhanced chemotactic responses. It has been shown that CXCR7 can act as a scavenger receptor that internalizes CXCL12 and in that way decreases binding of CXCL12 to CXCR4 [49, 50], therefore downregulating CXCR4 signaling. This has been proposed as a mechanism for suppression of chemotaxis to CXCL12 at low concentrations of CXCL12 [34]. Under our chemotaxis conditions, the double overexpressors showed increased chemotactic behavior in vitro to CXCL12 compared with the CXCR4 overexpressors even at low CXCL12 concentrations, suggesting that the scavenging function was not reducing chemotaxis in vitro. It is possible that under our in vitro conditions (in which there is a large volume in the attractant well, which is unlikely to be depleted during the time scale of the experiment) the scavenging function could actually increase chemotactic responses by reducing the amount of CXCL12 that leaks past the cells into the buffer side, and thereby maintaining a steeper gradient [51].
However, we found that coexpression of CXCR7 with CXCR4 did impair CXCL12-induced invasion in vitro of MTLn3 CXCR4 cells. CXCR7 potentially could modulate CXCR4 regulated gene expression by signaling through β-arrestin 2 [30, 32, 34]. This might result in decreased ability to degrade extracellular matrix, which could translate into a defect in invasion but not chemotaxis. Indeed this seems to be the case as the double overexpressors showed significantly reduced matrix degradation in response to CXCL12 treatment compared with the CXCR4 overexpressors. Evaluation of MMP expression in the MTLn3 transductants showed increased MMP12 mRNA expression upon CXCL12 stimulation in the MTLn3 CXCR4 cell line compared with the other transductants including the CXCR4-CXCR7 double expressor. Although MMP3 has been reported to be induced by CXCR7 [52], we did not observe that in the MTLn3 lines. Examination of breast cancer studies in the Oncomine database supports the possibility that CXCR4 can regulate MMP12 expression: MMP12 expression correlated significantly with CXCR4 expression, and not with CXCR7 expression. In summary, we propose that stimulation of CXCR4 can induce expression of MMP12 to increase invasiveness, and simultaneous expression of CXCR7 may suppress this induction.
The control cell line MTLn3 JP, although failing to chemotax or invade in vitro in response to CXCL12 stimulation, showed enhanced invasion in vivo at high concentrations of CXCL12. In addition, expression of CXCR7 alone reduced the invasion seen at high concentrations of CXCL12. As MTLn3 JP did not show chemotaxis to CXCL12 at any concentration in vitro and we did not observe upregulation of CXCR4 expression in vivo in this control cell line, we believe that the in vivo invasion at high concentration is a result of stimulating macrophages (which express CXCR4 [44–46]) within the tumor microenvironment, which can promote cancer cell invasion through the paracrine loop [21]. The reduced in vivo invasion of the CXCR7 expressing cells could be due to the scavenger function of CXCR7. For the in vivo invasion assay, CXCL12 diffusion would be constrained in the compact microenvironment to the spaces between cells. CXCR7 expressed on the tumor cells could then scavenge CXCL12, resulting in suppression of the activation of the invasion response.
The CXCR4 overexpressing line showed a strong in vivo invasion response to CXCL12, consistent with its strong chemotaxis and invasion responses in vitro. The invasion response was mediated by the paracrine loop with macrophages, as demonstrated by inhibition of invasion by either blocking EGF or CSF1R signaling. The CXCR4-CXCR7 line showed a reduced in vivo invasion response, which could reflect a CXCR7-induced reduction in matrix degradation (as we demonstrated for in vitro invasion), scavenging of CXCL12 by CXCR7, or both.
Consistent with previous reports, overexpression of CXCR7 resulted in a small but statistically significant increase in primary tumor growth possibly due to increased angiogenesis [22]. This was correlated with increased microvessel density and increased VEGFA expression. VEGFA has been shown to be upregulated by CXCR7 in a number of tumor cells [23, 27, 53], and thus it is likely that the increase in VEGF in the CXCR7 expressing lines leads to increased angiogenesis and tumor growth. We did not see an effect of CXCR4 expression on primary tumor growth as previously reported in other breast cancer models [11, 12], suggesting that this effect might be cell line specific.
The intravasation efficiencies of the various lines correlated with their in vivo invasiveness, with the CXCR4 overexpressing lines showing significantly more intravasation than the other lines. This suggests that CXCL12 signaling could contribute to the intravasation process. Indeed, perivascular macrophages have been shown to express CXCL12 [54], and thus a local gradient of CXCL12 leading towards blood vessels could stimulate directed invasion around vessels. Thus the CXCR7 lines, which show reduced invasion to CXCL12 gradients, would also be reduced in their intravasation capability.
A paradoxical result is our finding that CXCR4 overexpression did not result in increased spontaneous lung metastasis formation despite enhancing invasion and intravasation. This result disagrees with previous studies suggesting that wild-type or mutant CXCR4 mediates spontaneous metastasis of breast cancer cells to the lungs [6, 55, 56]. However, in those studies, CXCR4 signaling was shown to have a significant effect on primary tumor growth (or primary tumor size was not provided), and metastasis was compared at equal times rather than equal primary tumor sizes, leaving open the possibility that the increased metastasis seen in those studies reflects the increased intravasation from a larger primary tumor. Alternatively, it is possible that the effects of CXCR4 expression on metastasis varies with the particular model used, similar to the varying effects on growth. Our results cannot be explained by impaired cell survival of intravasated cancer cells since the intravasation assay used in this study evaluated viable cells. One possibility is that there might be gene expression changes occurring in MTLn3 CXCR4 cells within the tumor microenvironment that provide an advantage in entering the circulation but impair the ability to extravasate or seed lung metastases. Indeed, MMP12, which we find upregulated in CXCR4 cells stimulated with CXCL12, has been shown to be antiangiogenic [57–59]. Thus it is possible that although MMP12 is helpful in enabling tumor cells to invade, its antiangiogenic effects suppress the ability of tumor cells to extravasate and successfully seed metastases in the lung.
In summary, our studies provide insight into the complexity of the contributions of CXCR4 and CXCR7 to tumor cell invasion and metastasis. CXCR4 can enhance local invasion and intravasation due to CXCL12-induced chemotaxis and matrix degradation. The ligand scavenging function of CXCR7 may have contrasting effects on chemotaxis and invasion depending upon the diffusion constraints imposed upon CXCL12. CXCR7 can affect tumor growth through increased angiogenesis. We have identified MMP12 as a potential mediator of CXCR4-enhanced invasion, and further work will be needed to test its contributions to the different steps of metastasis.
We have found that CXCR4 and CXCR7 in breast cancer cells can make distinct contributions to tumor malignancy. CXCR4 expression increases tumor cell invasiveness and motility. CXCR7 expression inhibits invasion and metastasis, potentially through suppression of CXCR4 induced expression of MMP12. However, CXCR7 expression can stimulate VEGFA expression, microvessel density, and primary tumor growth.
analysis of variance
bovine serum albumin
cancer-associated fibroblast
colony stimulating factor 1
ethylenediaminetetraacetic acid
epidermal growth factor
endothelial progenitor cells
estrogen receptor
fluorescence-activated cell sorting
glyceraldehyde 3-phosphate dehydrogenase
G-protein coupled receptor
hematoxylin and eosin
interferon-inducible T-cell alpha chemoattractant
mitogen-activated protein kinase
matrix metalloproteinase
phosphate buffered saline
phosphatidylinositol 3-kinase
reverse transcription polymerase chain reaction
severe combined immunodeficiency
vascular endothelial growth factor.
We thank ChemoCentryx for reagents and members of the Condeelis and Cox labs for helpful comments. Special thanks to Tatiana Smirnova and Evanthia Roussos for valuable help with intracardiac punctures, Rani Sellers, Barbara Cannella and Hong Zhang for aid with analysis of microvessel density and VEGFA staining, and Jeffrey Wyckoff for training in the multiphoton microscope. JES is the Betty and Sheldon Feinberg Senior Faculty Scholar in Cancer Research. Funding was provided by CA110269 (LH) and CA100324 (JES and JC).
Authors' Affiliations
- Balabanian K, Lagane B, Infantino S, Chow KY, Harriague J, Moepps B, Arenzana-Seisdedos F, Thelen M, Bachelerie F: The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. J Biol Chem. 2005, 280: 35760-35766. 10.1074/jbc.M508234200.View ArticlePubMedGoogle Scholar
- Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, Penfold ME, Sunshine MJ, Littman DR, Kuo CJ, Wei K, McMaster BE, Wright K, Howard MC, Schall TJ: A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med. 2006, 203: 2201-2213. 10.1084/jem.20052144.View ArticlePubMedPubMed CentralGoogle Scholar
- Sehgal A, Keener C, Boynton AL, Warrick J, Murphy GP: CXCR-4, a chemokine receptor, is overexpressed in and required for proliferation of glioblastoma tumor cells. J Surg Oncol. 1998, 69: 99-104. 10.1002/(SICI)1096-9098(199810)69:2<99::AID-JSO10>3.0.CO;2-M.View ArticlePubMedGoogle Scholar
- Geminder H, Sagi-Assif O, Goldberg L, Meshel T, Rechavi G, Witz IP, Ben-Baruch A: A possible role for CXCR4 and its ligand, the CXC chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma. J Immunol. 2001, 167: 4747-4757.View ArticlePubMedGoogle Scholar
- Koshiba T, Hosotani R, Miyamoto Y, Ida J, Tsuji S, Nakajima S, Kawaguchi M, Kobayashi H, Doi R, Hori T, Fujii N, Imamura M: Expression of stromal cell-derived factor 1 and CXCR4 ligand receptor system in pancreatic cancer: a possible role for tumor progression. Clin Cancer Res. 2000, 6: 3530-3535.PubMedGoogle Scholar
- Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, Barrera JL, Mohar A, Verastegui E, Zlotnik A: Involvement of chemokine receptors in breast cancer metastasis. Nature. 2001, 410: 50-56. 10.1038/35065016.View ArticlePubMedGoogle Scholar
- Salvucci O, Bouchard A, Baccarelli A, Deschenes J, Sauter G, Simon R, Bianchi R, Basik M: The role of CXCR4 receptor expression in breast cancer: a large tissue microarray study. Breast Cancer Res Treat. 2006, 97: 275-283. 10.1007/s10549-005-9121-8.View ArticlePubMedGoogle Scholar
- Yasuoka H, Tsujimoto M, Yoshidome K, Nakahara M, Kodama R, Sanke T, Nakamura Y: Cytoplasmic CXCR4 expression in breast cancer: induction by nitric oxide and correlation with lymph node metastasis and poor prognosis. BMC Cancer. 2008, 8: 340-10.1186/1471-2407-8-340.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu Y, Ji R, Li J, Gu Q, Zhao X, Sun T, Wang J, Du Q, Sun B: Correlation effect of EGFR and CXCR4 and CCR7 chemokine receptors in predicting breast cancer metastasis and prognosis. J Exp Clin Cancer Res. 2010, 29: 16-10.1186/1756-9966-29-16.View ArticlePubMedPubMed CentralGoogle Scholar
- Lapteva N, Yang AG, Sanders DE, Strube RW, Chen SY: CXCR4 knockdown by small interfering RNA abrogates breast tumor growth in vivo. Cancer Gene Ther. 2005, 12: 84-89. 10.1038/sj.cgt.7700770.View ArticlePubMedGoogle Scholar
- Smith MC, Luker KE, Garbow JR, Prior JL, Jackson E, Piwnica-Worms D, Luker GD: CXCR4 regulates growth of both primary and metastatic breast cancer. Cancer Res. 2004, 64: 8604-8612. 10.1158/0008-5472.CAN-04-1844.View ArticlePubMedGoogle Scholar
- Darash-Yahana M, Pikarsky E, Abramovitch R, Zeira E, Pal B, Karplus R, Beider K, Avniel S, Kasem S, Galun E, Peled A: Role of high expression levels of CXCR4 in tumor growth, vascularization, and metastasis. FASEB J. 2004, 18: 1240-1242.PubMedGoogle Scholar
- Rubin JB, Kung AL, Klein RS, Chan JA, Sun Y, Schmidt K, Kieran MW, Luster AD, Segal RA: A small-molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors. Proc Natl Acad Sci USA. 2003, 100: 13513-13518. 10.1073/pnas.2235846100.View ArticlePubMedPubMed CentralGoogle Scholar
- Orimo A, Weinberg RA: Stromal fibroblasts in cancer: a novel tumor-promoting cell type. Cell Cycle. 2006, 5: 1597-1601. 10.4161/cc.5.15.3112.View ArticlePubMedGoogle Scholar
- Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, Carey VJ, Richardson AL, Weinberg RA: Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell. 2005, 121: 335-348. 10.1016/j.cell.2005.02.034.View ArticlePubMedGoogle Scholar
- Nagasawa T, Kikutani H, Kishimoto T: Molecular cloning and structure of a pre-B-cell growth-stimulating factor. Proc Natl Acad Sci USA. 1994, 91: 2305-2309. 10.1073/pnas.91.6.2305.View ArticlePubMedPubMed CentralGoogle Scholar
- Kryczek I, Wei S, Keller E, Liu R, Zou W: Stroma-derived factor (SDF-1/CXCL12) and human tumor pathogenesis. Am J Physiol Cell Physiol. 2007, 292: C987-995.View ArticlePubMedGoogle Scholar
- Maksym RB, Tarnowski M, Grymula K, Tarnowska J, Wysoczynski M, Liu R, Czerny B, Ratajczak J, Kucia M, Ratajczak MZ: The role of stromal-derived factor-1--CXCR7 axis in development and cancer. Eur J Pharmacol. 2009, 625: 31-40. 10.1016/j.ejphar.2009.04.071.View ArticlePubMedPubMed CentralGoogle Scholar
- Kang H, Watkins G, Parr C, Douglas-Jones A, Mansel RE, Jiang WG: Stromal cell derived factor-1: its influence on invasiveness and migration of breast cancer cells in vitro, and its association with prognosis and survival in human breast cancer. Breast Cancer Res. 2005, 7: R402-410. 10.1186/bcr1022.View ArticlePubMedPubMed CentralGoogle Scholar
- Liang Z, Wu H, Reddy S, Zhu A, Wang S, Blevins D, Yoon Y, Zhang Y, Shim H: Blockade of invasion and metastasis of breast cancer cells via targeting CXCR4 with an artificial microRNA. Biochem Biophys Res Commun. 2007, 363: 542-546. 10.1016/j.bbrc.2007.09.007.View ArticlePubMedGoogle Scholar
- Hernandez L, Smirnova T, Kedrin D, Wyckoff J, Zhu L, Stanley ER, Cox D, Muller WJ, Pollard JW, Van Rooijen N, Segall JE: The EGF/CSF-1 paracrine invasion loop can be triggered by heregulin beta1 and CXCL12. Cancer Res. 2009, 69: 3221-3227. 10.1158/0008-5472.CAN-08-2871.View ArticlePubMedPubMed CentralGoogle Scholar
- Miao Z, Luker KE, Summers BC, Berahovich R, Bhojani MS, Rehemtulla A, Kleer CG, Essner JJ, Nasevicius A, Luker GD, Howard MC, Schall TJ: CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature. Proc Natl Acad Sci USA. 2007, 104: 15735-15740. 10.1073/pnas.0610444104.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang J, Shiozawa Y, Wang Y, Jung Y, Pienta KJ, Mehra R, Loberg R, Taichman RS: The role of CXCR7/RDC1 as a chemokine receptor for CXCL12/SDF-1 in prostate cancer. J Biol Chem. 2008, 283: 4283-4294.View ArticlePubMedGoogle Scholar
- Odemis V, Boosmann K, Heinen A, Kury P, Engele J: CXCR7 is an active component of SDF-1 signalling in astrocytes and Schwann cells. J Cell Sci. 2010, 123: 1081-1088. 10.1242/jcs.062810.View ArticlePubMedGoogle Scholar
- Hattermann K, Held-Feindt J, Lucius R, Muerkoster SS, Penfold ME, Schall TJ, Mentlein R: The chemokine receptor CXCR7 is highly expressed in human glioma cells and mediates antiapoptotic effects. Cancer Res. 2010, 70: 3299-3308. 10.1158/0008-5472.CAN-09-3642.View ArticlePubMedGoogle Scholar
- Grymula K, Tarnowski M, Wysoczynski M, Drukala J, Barr FG, Ratajczak J, Kucia M, Ratajczak MZ: Overlapping and distinct role of CXCR7-SDF-1/ITAC and CXCR4-SDF-1 axes in regulating metastatic behavior of human rhabdomyosarcomas. Int J Cancer. 2010, 127: 2554-2568. 10.1002/ijc.25245.View ArticlePubMedPubMed CentralGoogle Scholar
- Zheng K, Li HY, Su XL, Wang XY, Tian T, Li F, Ren GS: Chemokine receptor CXCR7 regulates the invasion, angiogenesis and tumor growth of human hepatocellular carcinoma cells. J Exp Clin Cancer Res. 2010, 29: 31-10.1186/1756-9966-29-31.View ArticlePubMedPubMed CentralGoogle Scholar
- Zabel BA, Wang Y, Lewen S, Berahovich RD, Penfold ME, Zhang P, Powers J, Summers BC, Miao Z, Zhao B, Jalili A, Janowska-Wieczorek A, Jaen JC, Schall TJ: Elucidation of CXCR7-mediated signaling events and inhibition of CXCR4-mediated tumor cell transendothelial migration by CXCR7 ligands. J Immunol. 2009, 183: 3204-3211. 10.4049/jimmunol.0900269.View ArticlePubMedGoogle Scholar
- Hartmann TN, Grabovsky V, Pasvolsky R, Shulman Z, Buss EC, Spiegel A, Nagler A, Lapidot T, Thelen M, Alon R: A crosstalk between intracellular CXCR7 and CXCR4 involved in rapid CXCL12-triggered integrin activation but not in chemokine-triggered motility of human T lymphocytes and CD34+ cells. J Leukoc Biol. 2008, 84: 1130-1140. 10.1189/jlb.0208088.View ArticlePubMedGoogle Scholar
- Luker KE, Gupta M, Steele JM, Foerster BR, Luker GD: Imaging ligand-dependent activation of CXCR7. Neoplasia. 2009, 11: 1022-1035.View ArticlePubMedPubMed CentralGoogle Scholar
- Luker KE, Steele JM, Mihalko LA, Ray P, Luker GD: Constitutive and chemokine-dependent internalization and recycling of CXCR7 in breast cancer cells to degrade chemokine ligands. Oncogene. 2010, 29: 4599-4610. 10.1038/onc.2010.212.View ArticlePubMedPubMed CentralGoogle Scholar
- Rajagopal S, Kim J, Ahn S, Craig S, Lam CM, Gerard NP, Gerard C, Lefkowitz RJ: Beta-arrestin- but not G protein-mediated signaling by the "decoy" receptor CXCR7. Proc Natl Acad Sci USA. 2010, 107: 628-632. 10.1073/pnas.0912852107.View ArticlePubMedGoogle Scholar
- Sierro F, Biben C, Martinez-Munoz L, Mellado M, Ransohoff RM, Li M, Woehl B, Leung H, Groom J, Batten M, Harvey RP, Martinez AC, Mackay CR, Mackay F: Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7. Proc Natl Acad Sci USA. 2007, 104: 14759-14764. 10.1073/pnas.0702229104.View ArticlePubMedPubMed CentralGoogle Scholar
- Levoye A, Balabanian K, Baleux F, Bachelerie F, Lagane B: CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling. Blood. 2009, 113: 6085-6093. 10.1182/blood-2008-12-196618.View ArticlePubMedGoogle Scholar
- Luker KE, Gupta M, Luker GD: Imaging chemokine receptor dimerization with firefly luciferase complementation. FASEB J. 2009, 23: 823-834. 10.1096/fj.08-116749.View ArticlePubMedPubMed CentralGoogle Scholar
- Decaillot FM, Kazmi MA, Lin Y, Ray-Saha S, Sakmar TP, Sachdev P: CXCR7/CXCR4 heterodimer constitutively recruits {beta}-arrestin to enhance cell migration. J Biol Chem. 2011, 286: 32188-32197. 10.1074/jbc.M111.277038.View ArticlePubMedPubMed CentralGoogle Scholar
- Oser M, Yamaguchi H, Mader CC, Bravo-Cordero JJ, Arias M, Chen X, Desmarais V, van Rheenen J, Koleske AJ, Condeelis J: Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation. J Cell Biol. 2009, 186: 571-587. 10.1083/jcb.200812176.View ArticlePubMedPubMed CentralGoogle Scholar
- Artym VV, Zhang Y, Seillier-Moiseiwitsch F, Yamada KM, Mueller SC: Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function. Cancer Res. 2006, 66: 3034-3043. 10.1158/0008-5472.CAN-05-2177.View ArticlePubMedGoogle Scholar
- Hernandez L, Smirnova T, Wyckoff J, Condeelis J, Segall JE: In vivo assay for tumor cell invasion. Methods Mol Biol. 2009, 571: 227-238. 10.1007/978-1-60761-198-1_15.View ArticlePubMedPubMed CentralGoogle Scholar
- Sudo T, Nishikawa S, Ogawa M, Kataoka H, Ohno N, Izawa A, Hayashi S: Functional hierarchy of c-kit and c-fms in intramarrow production of CFU-M. Oncogene. 1995, 11: 2469-2476.PubMedGoogle Scholar
- Kedrin D, Wyckoff J, Sahai E, Condeelis J, Segall JE: Imaging tumor cell movement in vivo. Curr Protoc Cell Biol. 2007, Chapter 19 (Unit 19 17):
- Liang Z, Wu T, Lou H, Yu X, Taichman RS, Lau SK, Nie S, Umbreit J, Shim H: Inhibition of breast cancer metastasis by selective synthetic polypeptide against CXCR4. Cancer Res. 2004, 64: 4302-4308. 10.1158/0008-5472.CAN-03-3958.View ArticlePubMedGoogle Scholar
- Kalatskaya I, Berchiche YA, Gravel S, Limberg BJ, Rosenbaum JS, Heveker N: AMD3100 is a CXCR7 ligand with allosteric agonist properties. Mol Pharmacol. 2009, 75: 1240-1247. 10.1124/mol.108.053389.View ArticlePubMedGoogle Scholar
- Qian BZ, Pollard JW: Macrophage diversity enhances tumor progression and metastasis. Cell. 2010, 141: 39-51. 10.1016/j.cell.2010.03.014.View ArticlePubMedPubMed CentralGoogle Scholar
- Rigo A, Gottardi M, Zamo A, Mauri P, Bonifacio M, Krampera M, Damiani E, Pizzolo G, Vinante F: Macrophages may promote cancer growth via a GM-CSF/HB-EGF paracrine loop that is enhanced by CXCL12. Molecular Cancer. 2010, 9: 273-10.1186/1476-4598-9-273.View ArticlePubMedPubMed CentralGoogle Scholar
- Welford AF, Biziato D, Coffelt SB, Nucera S, Fisher M, Pucci F, Di Serio C, Naldini L, De Palma M, Tozer GM, Lewis CE: TIE2-expressing macrophages limit the therapeutic efficacy of the vascular-disrupting agent combretastatin A4 phosphate in mice. J Clin Invest. 2011, 121: 1969-1973. 10.1172/JCI44562.View ArticlePubMedPubMed CentralGoogle Scholar
- Teicher BA, Fricker SP: CXCL12 (SDF-1)/CXCR4 Pathway in Cancer. Clin Cancer Res. 2010, 16: 2927-2931. 10.1158/1078-0432.CCR-09-2329.View ArticlePubMedGoogle Scholar
- Wang Y, Li G, Stanco A, Long JE, Crawford D, Potter GB, Pleasure SJ, Behrens T, Rubenstein JL: CXCR4 and CXCR7 have distinct functions in regulating interneuron migration. Neuron. 2011, 69: 61-76. 10.1016/j.neuron.2010.12.005.View ArticlePubMedPubMed CentralGoogle Scholar
- Naumann U, Cameroni E, Pruenster M, Mahabaleshwar H, Raz E, Zerwes HG, Rot A, Thelen M: CXCR7 functions as a scavenger for CXCL12 and CXCL11. PLoS One. 2010, 5: e9175-10.1371/journal.pone.0009175.View ArticlePubMedPubMed CentralGoogle Scholar
- Raz E, Boldajipour B, Mahabaleshwar H, Kardash E, Reichman-Fried M, Blaser H, Minina S, Wilson D, Xu QL: Control of chemokine-guided cell migration by ligand sequestration. Cell. 2008, 132: 463-473. 10.1016/j.cell.2007.12.034.View ArticlePubMedGoogle Scholar
- Torisawa YS, Mosadegh B, Bersano-Begey T, Steele JM, Luker KE, Luker GD, Takayama S: Microfluidic platform for chemotaxis in gradients formed by CXCL12 source-sink cells. Integrative biology: quantitative biosciences from nano to macro. 2010, 2: 680-686. 10.1039/c0ib00041h.View ArticleGoogle Scholar
- Zabel BA, Miao ZH, Lai NL, Wang Y, Lewen S, Berahovich RD, Jaen JC, Schall TJ: CXCR7 protein expression correlates with elevated MMP-3 secretion in breast cancer cells. Oncology Letters. 2010, 1: 845-847.View ArticlePubMedPubMed CentralGoogle Scholar
- Kollmar O, Rupertus K, Scheuer C, Nickels RM, Haberl GC, Tilton B, Menger MD, Schilling MK: CXCR4 and CXCR7 regulate angiogenesis and CT26.WT tumor growth independent from SDF-1. Int J Cancer. 2010, 126: 1302-1315.PubMedGoogle Scholar
- Sanchez-Martin L, Estecha A, Samaniego R, Sanchez-Ramon S, Vega MA, Sanchez-Mateos P: The chemokine CXCL12 regulates monocyte-macrophage differentiation and RUNX3 expression. Blood. 2011, 117: 88-97. 10.1182/blood-2009-12-258186.View ArticlePubMedGoogle Scholar
- Ma WF, Du J, Fu LP, Fang R, Chen HY, Cai SH: Phenotypic knockout of CXCR4 by a novel recombinant protein TAT/54R/KDEL inhibits tumors metastasis. Mol Cancer Res. 2009, 7: 1613-1621. 10.1158/1541-7786.MCR-09-0078.View ArticlePubMedGoogle Scholar
- Rhodes LV, Short SP, Neel NF, Salvo VA, Zhu Y, Elliott S, Wei Y, Yu D, Sun M, Muir SE, Fonseca JP, Bratton MR, Segar C, Tilghman SL, Sobolik-Delmaire T, Horton LW, Zaja-Milatovic S, Collins-Burow BM, Wadsworth S, Beckman BS, Wood CE, Fuqua SA, Nephew KP, Dent P, Worthylake RA, Curiel TJ, Hung MC, Richmond A, Burow ME: Cytokine receptor CXCR4 mediates estrogen-independent tumorigenesis, metastasis, and resistance to endocrine therapy in human breast cancer. Cancer Res. 2011, 71: 603-613. 10.1158/0008-5472.CAN-10-3185.View ArticlePubMedGoogle Scholar
- Cheng P, Jiang FH, Zhao LM, Dai Q, Yang WY, Zhu LM, Wang BJ, Xu C, Bao YJ, Zhang YJ: Human macrophage metalloelastase correlates with angiogenesis and prognosis of gastric carcinoma. Digestive Diseases and Sciences. 2010, 55: 3138-3146. 10.1007/s10620-010-1127-3.View ArticlePubMedGoogle Scholar
- D'Alessio S, Fibbi G, Cinelli M, Guiducci S, Del Rosso A, Margheri F, Serrati S, Pucci M, Kahaleh B, Fan P, Annunziato F, Cosmi L, Liotta F, Matucci-Cerinic M, Del Rosso M: Matrix metalloproteinase 12-dependent cleavage of urokinase receptor in systemic sclerosis microvascular endothelial cells results in impaired angiogenesis. Arthrits Rheum. 2004, 50: 3275-3285. 10.1002/art.20562.View ArticleGoogle Scholar
- Margheri F, Serrati S, Lapucci A, Anastasia C, Giusti B, Pucci M, Torre E, Bianchini F, Calorini L, Albini A, Ventura A, Fibbi G, Del Rosso M: Systemic sclerosis-endothelial cell antiangiogenic pentraxin 3 and matrix metalloprotease 12 control human breast cancer tumor vascularization and development in mice. Neoplasia (New York, NY). 2009, 11: 1106-1115.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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2 BHK Apartment - 1,094 sq ft
Being in a posh locality Shree Radhe brings you the calmness of country life along with it. The beautiful apartments are equipped with all the modern amenities of life, without compromising on the environmental factors that are essential for you. High tech electronic gadgets work closely with those aptly placed greenery placed around the house. The nature friendly fabric and furnishing across the home and elegant interior designing complements well with the modernized version of eco-friendly homes.
Every nook and corner of the house screams of lush and vibrancy of life, as comfort and luxury has no other name than Radhe. No one can beat the intricate interior and outdoor decor of the house, with a keen eye for beauty and detailing. For every step you take to explore the place Radhe brings a new reason to appreciate and fall in love with it.
SHREE RADHE is a new project offered by Shree Radhe construction. Large terrace facing park area and Nagar road with modern amenities like Multi-Instrumental Gym, Security : 24x7 Security Guard, Underground Parking Facility, Swimming Pool, Club house, Landscaped gardens.
Key Details
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Catalina McGregor is the founder and chair of the Cabinet Office CIO/CTO Council Green ICT Delivery Group. She was the guest speaker at last month's CW500 Club meeting. In case you missed it – and for those who aren't among the 500 – you can watch her being interviewed here by our web editor, James Garner, on policy and greening IT.
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Closeouts. Merrell's Encore Burst clogs deliver convenient, cozy comfort fit for cold weather with their water-repellent suede and wool upper, supersoft plush fleece lining, and a grippy Encore 2 rubber outsole that takes on slick, winter surfaces with confidence.
Do you own this product? Write a review and help others with their buying decisions!Write a review
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12 Thrifty Trash to Treasure Crafts
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Latest Comments
"As an avid knitter, I am always looking for new projects to make and often like to make gifts for everyone in my family. This hat and scarf set would make a great gift for the men in my life! The instructions are clear and should be easy to follow."
"Any brown paper will degrade over time unless it's coated with plastic or wax. Grocery store brown paper bags should work fine however, if you want the paper to degrade a little more quickly, most paper towels or paper napkins will work fine. The only thing I would worry about is that watering the plants will make both psper tubes and paper fall apart."
"Crafts requiring saws, blades or chemicals should be left to adults. Perhaps children could help with other aspects of the craft, such as painting or gluing."
"I agree with others who have said crafts involving chemicals, machines, blades, etc are best left to adults or with close adult supervision."
"I would have to say any craft requiring the use of machinery (IE woodworking, sewing ) is specifically meant for adults only. Anything requiring the use of chemicals as well. However these can all be taught to children in a form of respect for safety by a supervising adult D"
Victorian Vintage Bottles
By: Mitzi Curi from Mitzi Made It
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Click here to view your Craft Projects.
Glam up some old bottles with colorful vintage paper and embellishments. These Victorian Vintage Bottles from Mitzi Curi are perfect for storing your favorite beads and buttons, and also make lovely flower vases. Mix and match sizes for a lovely vintage display that's sure to catch everyone's eye.
- Vintage or newer bottles of various shapes and sizes
- Interesting paper, including sheet music or old wallpaper
- Ribbon, lace, or seam binding
- Corks that fit the bottles if they don't have caps
- Pearls or beads to put inside the bottles (optional)
- Jewelry bits for embellishment
- E-6000 glue or hot glue
- Brayer (optional)
- Mod Podge (matte)
- Sponge brush
- Distress Ink to age the paper (optional)
Once your bottles are clean and dry, begin a layering process. I select the paper pieces first, and either cut or tear them to fit the front of the bottles. I had some great embossed vintage wallpaper that I used for this project, and I really wanted to highlight it.
Attach your paper pieces with Mod Podge by coating the back of the paper and pressing it or using the brayer to get a smooth attachment to the bottle. A topcoat of Mod Podge can be given and the edges wiped clean with a damp paper towel.
Additional embellishments can be added, including cameos, buttons, and vintage millinery. Use hot glue to tack the items down. My paper was so pretty, I decided not to glue anything on top of it.
The bottle caps or corks can be embellished too. I used Rub-n-Buff in Grecian Gold to color the corks and the screw on metal cap. I just use my finger, but I think a rag or paper towel is recommended.
- I added vintage gold plastic roses and a fancy button to the caps for a final bit of embellishment, along with some pearls and beads in the bottom of the jars.
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elvirawells 245915 7
Mar 19, 2013
love it: i have some many cute bottle that I have bought : now I know what to do
kraftykat16 002802 6
Nov 16, 2012
I absolutely adore these! Perfect idea for all the perfume bottles I've benn saving!
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Top Tips Pack A of 4
Written by Rebecca Rissman
Illustrated by
Part of the Read Me!: Top Tips Series
Lovereading4kids Price £27.33
£34.16 saving £6.83 (20%)
when in stock
As soon as this book is back in stock we will send you an email.Compare book price We'd love you to buy your books from Lovereading - especially if we helped you to choose them! But as our mantra is guidance, and that includes guidance on price, please do use our 'Compare Book Price' service to discover if the best value deal is from Lovereading or another specialist bookstore.
Top Tips Pack A of 4 by Rebecca RissmanLearn to outsmart your classmates with the tips in this exciting, top secret book! The secret tips included in this series use simple mnemonics, easy-to-remember tips, and core curricular content to help even the most reluctant students succeed!
About the Author
Other Formats
Book Info
Contains 4 Paperbacks
128 pages
Interest Age: From 8
Rebecca Rissman
More books by Rebecca Rissman
Raintree an imprint of Capstone Global Library Ltd
Publication date
18th July 2013
I love Lovereading as it provides an honest opinion and showcases a range of fiction. Suited to both parents & kids alike, it's a must-use.Georgie Rowe – age 16
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You give me age appropriate ideas of books I can read and buy for the children and find out what other children their age think of them too.Katie Lonsdale
I am so pleased to have signed my kids up as they are reading a much wider range of books and even choosing books out of their comfort zone.Angela East
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We love Lovereading4kids because they put books in front of us we wouldn't otherwise have read. They make us more adventurous readers!Emily Jacques


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The digital edition of the Fusion Product Guide has been updated to version 1.5. Download an updated copy today and easily find plastic piping systems parts and accessories along with so much more.
Latest News
To celebrate the end of year and recharge the batteries, Fusion will be closing for the holiday period. Find out more about our
Fusion implemented a strategy to deliver more globally recognised product ranges in our 2016 Product Guide. Adding Asahi, Charlotte, Flowline & Polypipe...
"We received one of your new product guides. Out of all we have seen from all over the world, yours is by far the most well thought out and best designed we have seen. Congrats on stepping away from the norm & taking a risk.
It's fantastic."
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Successfully Completed Projects
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English Country Inns
Welcome to English Country Inns, the largest online directory of inns in England. Famous around the world for their warm hospitality, the English inn offers great food, good company and comfortable accommodation. Whether you are looking for romantic breaks, weekend breaks in the countryside or a late availability deal, we have it covered!
There is no better time to visit the English countryside than in autumn, when the colours of the leaves on the trees in the hazy morning light are simply stunning. And after a brisk walk enjoying the last of the warm weather, what better than to relax over a drink and a delicious meal at a cosy country inn!
At this time of year our thoughts turn to romance! The most romantic day of the year is coming up: why not treat your loved one to a Valentine's break or weekend away at a cosy country inn?


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Event: Mika Singh, Nana Patekar, Rahul Vaidya and other Bollywood celebs attended renowned singer Suresh Wadkar�s birthday bash.
Guest List: Mika Singh, Nana Patekar, Rahul Vaidya
Bollywood celebs Kangana Ranaut, Vishal Bhardwaj, Randeep Hooda, Aanand L Rai, Vikas Bahl, Vidhu Vinod Chopra, Anupama Chopra, Prasoon Joshi among others were snapped at the special screening of RANGO...
Bollywood celeb Adah Sharma promoted her upcoming film COMMANDO 2.
Bollywood celebs Riya Sen, Kyra Dutt, Gulshan Nain, Vivek Vaswani among others were snapped at the special screening of LONELY GIRL.
Bollywood celebs Ranveer Singh, John Abraham, Kiran Rao, Manyata Dutt, Poonam Dhillon, Baba Siddiqui, Shekhar Suman comes out to vote.
Bollywood celebs Varun Dhawan and Alia Bhatt were snapped at Mehboob studio.


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Can i replace air shocks with gas shocks?
Guys i have a cadillac 2006 sts v8 base model, so
the front shocks are leaking, and the rear shocks
are completely gone, and i need to replace them,
but i found out that they are air shocks, and i can't
afford getting new air shocks because they are too
expensive, so i was thinking of replacing them with
gas shocks because they are cheap, it says that
they fit my vehicle, but i need some advices and i
want to know if they would fit or not
1 Answer
If the STS is in good condition, not beat.. repair the original system with the correct air suspension parts its (slightly) more in cost! Unless you don't buy right.. than a "conversion" which destroys the ride and the vehicles value. Every high line vehicle whether performance or luxury uses air suspension or electronic magnetic ride in 2016 a good reason not to change a '06 Cadillac. When you want to sell it you'll find out. Clean original Cadillac STS models bring $ one of last American V8 sedans
Your Answer
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All corporations, including S corporations, must make certain calculations for accounting and tax purposes. Though there are several distinct differences between S corporations and other corporate entities, the way in which they determine shareholder equity is the same. However, the shareholder equity of most corporate entities is necessary for tax purposes, while the shareholder equity of an S corporation isn't.
About S Corporations
S corporations do not file their own separate tax returns. Instead, their income is divided among their shareholders according to their stakes in the company. Each shareholder reports his portion of the income on his tax return and pays the appropriate amount of income tax. Shareholders also divide the corporation's losses and use them to offset the income they earn during the year.
Determining Shareholder Equity
To determine the shareholder equity of an S corporation, subtract the value of the corporation's treasury shares from its paid-in capital. The corporation's treasury shares are those shares that it issued to shareholders and then repurchased. Its paid-in capital is the money it received from the sale of stock or other investments from shareholders. On a balance sheet, paid-in capital typically appears as "common stock" and "additional paid-in capital." If any shareholders paid a price higher than par value for their shares, some paid-in capital may also appear as "paid-in capital in excess of par value."
If an S Corporation's balance sheet shows common stock issued in the amount of $500,000, additional paid-in capital in the amount of $300,000 and treasury shares in the amount of $40,000, then its shareholder equity is $760,000. To determine this value, combine the values of the issued stock and the additional paid-in capital for a sum of $800,000 and subtract the value of the treasury shares.
Tax Issues
While most other types of corporations must report their shareholder equity to the Internal Revenue Service for tax purposes, S corporations do not pay income taxes. Those with more than one shareholder, however, still report shareholder equity and other balance sheet entries to the IRS for informational purposes.


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Here's a "red" iris for Martin Weber who asked on
"I saw some very
red SDB, are there also TBs that are also so red?"
(Barry Blyth, R. 2003), Sdlg.
L209-2. TB, 42" (107 cm), EM "Bright burnt brick rose, F. with small
lavender chevrons by tangerine red beard; sweet musky fragrance.
Safari Sunset X Wearing Rubies.
Tempo Two 2005/06 "
Will try to make a link from Iris-talk to


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Greetings Guest!!Dismiss Notice
In order to combat SPAM on the forums, all users are required to have a minimum of 2 posts before they can submit links in any post or thread.
How to reforge 50+ DI onto a sword?
Discussion in 'UO Craftsman' started by DJAd, Sep 21, 2013.


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New Era Rubberized Belt
Made of 90% Polyester, Woven Canvas, 10% Metal
Canvas belt with an rubberized buckle from New Era. Length of the belt is 50". Belts can be cut to fit. One size fits most.
- 20540164
- Color:
- Black/Gold
- Availability:
- In Stock
International shipping available to Canada - info