Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Serial sections of normal and diseased pancreatic tissue were stained for CXCL12, CXCR4, CXCR7, and CK19. CXCL12 staining apparent in normal exocrine ducts was diminished in PDAC tissue. CXCR4 staining increased in PanIN and PDAC relative to normal ductal epithelium. CXCR7 expression was variable in normal epithelium, PanIN lesions, and PDAC. (A)Normal tissue from a single patient with healthy pancreas represents observations from 25 different normal tissues from 21 individual patients. (B)PDAC tissue from one patient represents observations from 82 different tissues from 29 different patients. 1000× magnification represents inset box at 200×. (C) Staining was quantified by blinded scoring of serial sections in relation to CK19 staining. (***) denotes P ≤0.001.

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Representative 200× images of the same (A) normal or (B) pancreatic ductal adenocarcinoma (PDAC) tissues shown at 1000× magnification in . Representative serial section images of a pancreatic intestinal neoplasm (PanIN) lesion at (C) 200× or (D) 1000× magnification. Serial tissue sections were immunostained with antibodies to CK19, CXCL12, CXCR4, and CXCR7, or the isotype controls. n = 25 different normal tissues from 21 individual patients, 82 different tissues from 29 different PDAC patients, or 19 pathologically confirmed PanIN lesions.

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques:

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: RT-PCR analysis revealed that (A) patient-derived pancreatic cancer cell lines (#1, #2, #3, #4) or (B) established cell lines lacked expression of CXCL12 and maintained expression of CXCR4. CXCR7 mRNA was present in 3 of 8 PDAC lines.Flow cytometric detection(C)of surface CXCR4 or CXCR7 protein expression. Cell lines treatedseven days with a concentration curve of (D) 5-aza-2-deoxycytidine (5-aza) restored expression of CXCL12.Linestreated 4 days with a concentration curve of (E) Trichostatin-A (TSA)restored CXCL12 mRNA expression in Capan2 cells.

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Expressing, Concentration Assay

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: (A) Panc1 and MiaPaCa2 cells migrated towards exogenous gradients of CXCL12 in a range from 1 nM to 1000 nM under serum-free conditions(*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001, respectively, in comparison to unstimulated cells (NS). Receptor-null HPAFII cells did not migrate in response to CXCL12. (B) CXCL12 directs Panc1 cell chemoinvasion into a three-dimensional Matrigel plug. The positive control (+) was 10% serum-containing medium. (*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001, respectively, in comparison to unstimulated cells (NS).

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques: Positive Control

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Luciferase levels in control GFP or three different (31, #2, #3) CXCL12-expressing MiaPaCa2 clones as measured by spectrophotometer (A) or IVIS-100 biophotonic imager (B). Levels of CXCL12 were measured by ELISA (C) in established PDAC cell lines as well as GFP- and CXCL12-expressing MiaPaCa2-luciferase clones. (D) CXCL12-secreted by transfected MiaPaCa2-luciferase clones #1 and #2 stimulated U937 chemotaxis. Cells treated with neutralizing antibody to CXCL12 (αL12) confirmed the specificity of U937 chemotaxis. Values in A, C, and D are mean±SEM, n = 2–3.

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques: Luciferase, Expressing, Clone Assay, Spectrophotometry, Enzyme-linked Immunosorbent Assay, Transfection, Chemotaxis Assay

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: (A) Transwell migration assays revealed significantly reduced chemotaxis of CXCL12-expressing clones (#1 and #2) compared to ligand null (GFP) cells. Attractants were serum-free media (--), 10% serum (+), or 10 nM CXCL12 (L12) in serum-free media. denote P ≤0.01 and P ≤0.001, respectively, compared to 10 nM CXCL12-stimulated GFP cells, n = 5. (B) CXCL12 re-expression diminished TGF-β (5 ng/mL)-induced chemotaxis relative to the CXCL12-null cells. (##) denotes P ≤0.01 compared to TGF-β-stimulated GFP cells, n = 4. (C) & (D) Representative images of experiments in (A) and (B) respectively. (E) CXCL12-expressing cells were significantly more adherent to tissue culture plastic compared to CXCL12-null cells. Untreated cells = (--), neutralizing antibody for CXCL12 activity = (αL12), and a positive control = 1 ng/mL of EGF (+). (##) denotes P ≤0.01 compared to 10% serum-stimulated control cells. (*), (**), and (***) denote P ≤0.05, P ≤0.01 and P ≤0.001, respectively, compared to unstimulated GFP cells, n = 7.

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques: Migration, Chemotaxis Assay, Expressing, Clone Assay, Activity Assay, Positive Control

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: (A) Representative bioluminescence images of mice xenografted with GFP- or CXCL12-expressing cells at implantation (Day 0) or study endpoint (Day 28). (B) Whole-body in vivo radiance over time of GFP- and CXCL12-mice. (C) Ex vivo radiance of excised spleen (C), reflecting tumor cells at the site of injection, or the metastatic destination (D), reflecting decreased hepatic metastasis of CXCL12-expressing cells relative to GFP-cells. (**) denotes P ≤0.01, n = 4–5. (E) Representative H&E images showing pronounced tumor mass in the liver of control (GFP), relative to experimental (CXCL12) xenografted mice.

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques: Expressing, In Vivo, Ex Vivo, Injection

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Apoptosis of GFP and CXCL12-expressing MiaPaCa2 cells were assessed using the caspase 3/7 glo assay.Cells were starved for 24 hours and cultured in 0% serum (A, C) or 1% serum (B, D) containing medium. (A–B) Apoptosis in adherent CXCL12-expressing cells was elevated compared to either GFP-expressing clones in serum-free conditions with no change seen in 1% serum. GFP-cells werestimulated with 100 µM gemcitabine as a control. (C–D) To measure cell number and detachment based apoptosis of cells in suspension, MiaPaCa2-luciferase cells were cultured on poly-HEMA. Using the Viacount reagent and flow cytometric cell counting, there was no difference in live cell number observed in non-adherent CXCL12-null (GFP) or expressing cells when cultured either in 0% (C) or 1% serum (D). Apoptosis of poly-HEMA cultured cells revealed an in increase in active caspase-3/7 restricted to cells cultured in serum-free conditions only (C) with no change observed in 1% serum (D). Gemcitabine (GEM) was used as a control for decreased cell count and increased apoptosis.(*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001 respectively in comparison to control cells (GFP). Values are mean±SEM, n = 4–5.

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques: Expressing, Glo Assay, Cell Culture, Clone Assay, Luciferase, Cell Counting

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Population growth of adherent GFP and CXCL12-expressing MiaPaCa2 cells was assessed using the Viacount reagent and flow cytometric cell counting (A–B). CXCL12-expressing cells starved for 24 hours and cultured in both 0% serum (A) or 1% serum (B) containing medium were found to have decreased population growth.(B) Two CXCL12-expressing clones (#1, #2) were compared to GFP alone or GFP + gemcitabine (GEM) controls in 1% serum containing medium. Doubling time of adherent clones (T 2 ) was calculated using a linear regression of the data to determine slope and the intercept at y = 10 6 , with increased T 2 observed in both CXCL12-expressing clones. (C–D) Propidium Iodide cell cycle analysis revealed a decrease in percentage of cells in the G 2 phase in CXCL12-expressing cells compared to GFP controls. (*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001 respectively in comparison to control cells (GFP). Values are mean±SEM, n = 4–5.

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques: Expressing, Cell Counting, Cell Culture, Clone Assay, Cell Cycle Assay

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: (A) Kaplan-Meier survival curves for GFP- and CXCL12-expressing cell groups. Three experimental CXCL12 PDAC injected mice were removed for non-study reasons (tick marks). (B) Percent change in bioluminescence from baseline-level measured at day 7 for both GFP and CXCL12 engrafted mice. Dotted lines represent individual mice. Solid lines are quadratic regression fitted curves of each group. Statistical comparison was done between both groups independent of time. (C–D) Representative bioluminescence images of mice from each groupat days 7, 49, and endpoint for GFP (98) or CXCL12 (106). (E) Tumor wet weight was significantly reduced in CXCL12-expressing tumors relative GFP-tumors. Representative photomicrographs are shown in lower panels. (F) CXCL12-producing tumors had significantly fewer Ki-67 positive cells compared to GFP-expressing tumors, as counted in a cross-section of each tumor normalized to the total cross-sectional area of each tumor. (G) Representative images of Ki-67immunostaining and rabbit isotype control (inset, Rab IgG) are shown.n = 8–10. (**) and (***) denote P ≤0.01 and P ≤0.001 respectively, between CXCL12-expressing and control xenografted mice.

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques: Expressing, Injection

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Ex vivo bioluminescence analysis revealed significantly decreased metastasis to the liver (A), lung (C), and mesenteric lymph nodes (D) of CXCL12-expressing cells compared to GFP-controls. Representative biophotonic images of hepatic metastases are shown in panel B. (**) and (***) denote statistically significant P ≤0.01 and P ≤0.001, respectively, differences between CXCL12-expressing and control tumor engrafted mice. n = 8–10 mice in each group.

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques: Ex Vivo, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Heparin-induced Leukocytosis Requires 6- O -Sulfation and Is Caused by Blockade of Selectin- and CXCL12 Protein-mediated Leukocyte Trafficking in Mice *

doi: 10.1074/jbc.M111.314716

Figure Lengend Snippet: Heparin-induced leukocytosis depends on blockade of CXCL12 function. The kinetics of leukocytosis (A) and the levels of CXCL12α (B) and CXCL12β (C) in peripheral blood and in BM after heparin administration. The data were summarized from three experiments with 10 mice per group. D, blocking CXCL12 function with neutralizing antibody attenuated HIL response. The mice were preinjected i.v. with neutralizing anti-CXCL12 antibody prior to heparin or saline injection and then analyzed for HIL. The data were summarized from three experiments with 7–9 mice per group. Each bar represents the average value ± S.E. The statistical analysis was carried out by paired Student's t test. WBC, white blood cells. Ab, antibody.

Article Snippet: CXCL12α and CXCL12β levels in mouse plasma and BM were determined by ELISA using monoclonal antibodies specific for CXCL12β (BAF351) or CXCL12α (BAF310) with capture antibody MAB350 and recommended protocols from R&D Systems ( 12 ).

Techniques: Blocking Assay, Saline, Injection

Journal: The Journal of Biological Chemistry

Article Title: Heparin-induced Leukocytosis Requires 6- O -Sulfation and Is Caused by Blockade of Selectin- and CXCL12 Protein-mediated Leukocyte Trafficking in Mice *

doi: 10.1074/jbc.M111.314716

Figure Lengend Snippet: Heparin disrupted CXCL12 binding to heparan sulfate in a competitive and 6-O-sulfation-dependent manner and abolished the CXCL12 gradient-directed bone marrow cell migration. A, effects of heparinoids on the binding of CXCL12 to endothelial cell surface. CXCL12α was preincubated with heparan sulfate (HS), Hep, N-des-hep, 2/3-des-hep, 6-des-hep, or CR-hep. CXCL12α alone or the preincubated CXCL12a solution was incubated with untreated endothelial cells (control) or cells pretreated with heparinases. CXCL12α bound to the cell surface was detected by an ELISA. The data were summarized from three independent experiments. Bars represent mean ± S.E. The p values were calculated using the paired Student's t test in comparison with saline treatment. B, heparin but not 6-desulfated-heparin competitively inhibited CXCL12 binding to immobilized heparin in the SPR assay. CXCL12α was premixed with Hep or 6-des-hep and then the mixture was injected over heparin-immobilized CM5 chip surface. The SPR sensorgram shown is representative of three experiments. C, CXCL12 induced transendothelial cell migration of BM cells via both chemotaxis and chemokinesis, and heparin inhibited the CXCL12-induced chemotaxis of BM cells. The transendothelial cell migration assay was carried out with BM cells placed in the upper chamber and CXCL12 supplemented in the lower chamber (to determine chemotaxis) or immobilized to both abluminal and luminal surfaces (to determine chemokinesis). The BM cells that migrated into the lower chamber were counted. To determine the effect of heparin on CXCL12-induced chemotaxis of BM cells, heparin was added in both upper and lower chamber prior to initiation of cell migration. The data were summarized from three experiments. Each bar represents the average value ± S.E. The statistical analysis was carried by paired Student's t test.

Article Snippet: CXCL12α and CXCL12β levels in mouse plasma and BM were determined by ELISA using monoclonal antibodies specific for CXCL12β (BAF351) or CXCL12α (BAF310) with capture antibody MAB350 and recommended protocols from R&D Systems ( 12 ).

Techniques: Binding Assay, Migration, Incubation, Control, Enzyme-linked Immunosorbent Assay, Comparison, Saline, SPR Assay, Injection, Chemotaxis Assay, Cell Migration Assay