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Macrophage colony-stimulating factor augments Tie2-expressing monocyte differentiation, angiogenic function, and recruitment in a mouse model of breast cancer.

Forget MA, Voorhees JL, Cole SL, Dakhlallah D, Patterson IL, Gross AC, Moldovan L, Mo X, Evans R, Marsh CB, Eubank TD - PLoS ONE (2014)

Bottom Line: We found that CSF1 pre-treatment significantly augmented chemotaxis and that Tie2 receptor upregulation was responsible as siRNA targeting Tie2 receptor abrogated this effect.While supernatants from CSF1-pre-treated TEMs increased HUVEC branching, a neutralizing antibody against the CSF1R abrogated this activity, as did siRNA against the Tie2 receptor.To test our hypothesis in vivo, we treated PyMT tumor-bearing mice with CSF1 and observed an expansion in the TEM population relative to total F4/80+ cells, which resulted in increased angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, The Ohio State University, Columbus, Ohio, United States of America; Molecular Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
Reports demonstrate the role of M-CSF (CSF1) in tumor progression in mouse models as well as the prognostic value of macrophage numbers in breast cancer patients. Recently, a subset of CD14+ monocytes expressing the Tie2 receptor, once thought to be predominantly expressed on endothelial cells, has been characterized. We hypothesized that increased levels of CSF1 in breast tumors can regulate differentiation of Tie2- monocytes to a Tie2+ phenotype. We treated CD14+ human monocytes with CSF1 and found a significant increase in CD14+/Tie2+ positivity. To understand if CSF1-induced Tie2 expression on these cells improved their migratory ability, we pre-treated CD14+ monocytes with CSF1 and used Boyden chemotaxis chambers to observe enhanced response to angiopoietin-2 (ANG2), the chemotactic ligand for the Tie2 receptor. We found that CSF1 pre-treatment significantly augmented chemotaxis and that Tie2 receptor upregulation was responsible as siRNA targeting Tie2 receptor abrogated this effect. To understand any augmented angiogenic effect produced by treating these cells with CSF1, we cultured human umbilical vein endothelial cells (HUVECs) with conditioned supernatants from CSF1-pre-treated CD14+ monocytes for a tube formation assay. While supernatants from CSF1-pre-treated TEMs increased HUVEC branching, a neutralizing antibody against the CSF1R abrogated this activity, as did siRNA against the Tie2 receptor. To test our hypothesis in vivo, we treated PyMT tumor-bearing mice with CSF1 and observed an expansion in the TEM population relative to total F4/80+ cells, which resulted in increased angiogenesis. Investigation into the mechanism of Tie2 receptor upregulation on CD14+ monocytes by CSF1 revealed a synergistic contribution from the PI3 kinase and HIF pathways as the PI3 kinase inhibitor LY294002, as well as HIF-1α-deficient macrophages differentiated from the bone marrow of HIF-1αfl/fl/LysMcre mice, diminished CSF1-stimulated Tie2 receptor expression.

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CSF1 pre-treatment augments the migratory response to ANG2 by CD14+ monocytes.(A) CD14+ monocytes were isolated and cultured in Boyden chemotaxis chambers in minimal media alone (Media), in media containing 0.1, 1, 10, 100 or 300 ng/ml rhANG2, or with the same ANG2 doses but first pre-treated for 24 hours with media containing 10 ng/ml rhCSF1 and analyzed for their migratory ability. A significant synergistic effect of CSF1 pre-treatment was first observed at 1 ng/ml rhANG2 and peaked at 100 ng/ml rhANG2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber. (B) CD14+ monocytes were treated with rhANG2 (10 ng/ml) (ANG2) or rhCSF1 (10 ng/ml) (CSF1) alone, or pre-treated with rhCSF1 (10 ng/ml) for 24 hours, washed 3x, then treated with rhANG2 (10 ng/ml) for another 24 hours and transfected with a scrambled siRNA or an siRNA targeting the human Tie2 receptor. While ANG2 and CSF1 did not induce significant migration, the CSF1-pre-treated cells transfected with the scrambled siRNA migrated significantly more than those cells transfected with siRNA targeting Tie2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber.
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pone-0098623-g002: CSF1 pre-treatment augments the migratory response to ANG2 by CD14+ monocytes.(A) CD14+ monocytes were isolated and cultured in Boyden chemotaxis chambers in minimal media alone (Media), in media containing 0.1, 1, 10, 100 or 300 ng/ml rhANG2, or with the same ANG2 doses but first pre-treated for 24 hours with media containing 10 ng/ml rhCSF1 and analyzed for their migratory ability. A significant synergistic effect of CSF1 pre-treatment was first observed at 1 ng/ml rhANG2 and peaked at 100 ng/ml rhANG2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber. (B) CD14+ monocytes were treated with rhANG2 (10 ng/ml) (ANG2) or rhCSF1 (10 ng/ml) (CSF1) alone, or pre-treated with rhCSF1 (10 ng/ml) for 24 hours, washed 3x, then treated with rhANG2 (10 ng/ml) for another 24 hours and transfected with a scrambled siRNA or an siRNA targeting the human Tie2 receptor. While ANG2 and CSF1 did not induce significant migration, the CSF1-pre-treated cells transfected with the scrambled siRNA migrated significantly more than those cells transfected with siRNA targeting Tie2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber.

Mentions: ANG2 binds the Tie2 receptor on endothelial cells allowing for the detachment of cellular adhesions and facilitating the migration of these cells during angiogenesis [37]. We next sought to determine if CSF1 up-regulation of the Tie2 receptor on CD14+ monocytes modulated migration towards ANG2. We pre-treated CD14+ human monocytes with or without 10 ng/ml CSF1 in the bottom well of Boyden chemotaxis chambers for 18 hours then washed the cells and replaced the media with FBS-containing media without or with a dose escalation of ANG2 in the top chamber. After 24 hours, the filters were inspected for migration of monocytes to the top chamber (chemotaxis) and cells blindly counted by microscopy. We observed that ANG2 stimulation alone induced significant migration at 100 ng/ml ANG2 compared to ANG2-free media (p<0.0001) (Figure 2A). Interestingly, pre-treating CD14+ monocytes with 10 ng/ml CSF1 significantly increased the number of migrating cells responsive to ANG2 compared to monocytes not pre-treated with CSF1 and reduced the significant migration response to ANG2 from 100 ng/ml without CSF1 to 1 ng/ml with CSF1 (p = NS without CSF1 pre-treatment and p = 0.0003 with CSF1 pre-treatment) (Figure 2A) suggesting that CSF1 pre-treatment increased the number of CD14+/Tie2+ cells during the 18 hour pre-incubation and resulted in a larger population cells able to respond to ANG2.


Macrophage colony-stimulating factor augments Tie2-expressing monocyte differentiation, angiogenic function, and recruitment in a mouse model of breast cancer.

Forget MA, Voorhees JL, Cole SL, Dakhlallah D, Patterson IL, Gross AC, Moldovan L, Mo X, Evans R, Marsh CB, Eubank TD - PLoS ONE (2014)

CSF1 pre-treatment augments the migratory response to ANG2 by CD14+ monocytes.(A) CD14+ monocytes were isolated and cultured in Boyden chemotaxis chambers in minimal media alone (Media), in media containing 0.1, 1, 10, 100 or 300 ng/ml rhANG2, or with the same ANG2 doses but first pre-treated for 24 hours with media containing 10 ng/ml rhCSF1 and analyzed for their migratory ability. A significant synergistic effect of CSF1 pre-treatment was first observed at 1 ng/ml rhANG2 and peaked at 100 ng/ml rhANG2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber. (B) CD14+ monocytes were treated with rhANG2 (10 ng/ml) (ANG2) or rhCSF1 (10 ng/ml) (CSF1) alone, or pre-treated with rhCSF1 (10 ng/ml) for 24 hours, washed 3x, then treated with rhANG2 (10 ng/ml) for another 24 hours and transfected with a scrambled siRNA or an siRNA targeting the human Tie2 receptor. While ANG2 and CSF1 did not induce significant migration, the CSF1-pre-treated cells transfected with the scrambled siRNA migrated significantly more than those cells transfected with siRNA targeting Tie2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043882&req=5

pone-0098623-g002: CSF1 pre-treatment augments the migratory response to ANG2 by CD14+ monocytes.(A) CD14+ monocytes were isolated and cultured in Boyden chemotaxis chambers in minimal media alone (Media), in media containing 0.1, 1, 10, 100 or 300 ng/ml rhANG2, or with the same ANG2 doses but first pre-treated for 24 hours with media containing 10 ng/ml rhCSF1 and analyzed for their migratory ability. A significant synergistic effect of CSF1 pre-treatment was first observed at 1 ng/ml rhANG2 and peaked at 100 ng/ml rhANG2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber. (B) CD14+ monocytes were treated with rhANG2 (10 ng/ml) (ANG2) or rhCSF1 (10 ng/ml) (CSF1) alone, or pre-treated with rhCSF1 (10 ng/ml) for 24 hours, washed 3x, then treated with rhANG2 (10 ng/ml) for another 24 hours and transfected with a scrambled siRNA or an siRNA targeting the human Tie2 receptor. While ANG2 and CSF1 did not induce significant migration, the CSF1-pre-treated cells transfected with the scrambled siRNA migrated significantly more than those cells transfected with siRNA targeting Tie2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber.
Mentions: ANG2 binds the Tie2 receptor on endothelial cells allowing for the detachment of cellular adhesions and facilitating the migration of these cells during angiogenesis [37]. We next sought to determine if CSF1 up-regulation of the Tie2 receptor on CD14+ monocytes modulated migration towards ANG2. We pre-treated CD14+ human monocytes with or without 10 ng/ml CSF1 in the bottom well of Boyden chemotaxis chambers for 18 hours then washed the cells and replaced the media with FBS-containing media without or with a dose escalation of ANG2 in the top chamber. After 24 hours, the filters were inspected for migration of monocytes to the top chamber (chemotaxis) and cells blindly counted by microscopy. We observed that ANG2 stimulation alone induced significant migration at 100 ng/ml ANG2 compared to ANG2-free media (p<0.0001) (Figure 2A). Interestingly, pre-treating CD14+ monocytes with 10 ng/ml CSF1 significantly increased the number of migrating cells responsive to ANG2 compared to monocytes not pre-treated with CSF1 and reduced the significant migration response to ANG2 from 100 ng/ml without CSF1 to 1 ng/ml with CSF1 (p = NS without CSF1 pre-treatment and p = 0.0003 with CSF1 pre-treatment) (Figure 2A) suggesting that CSF1 pre-treatment increased the number of CD14+/Tie2+ cells during the 18 hour pre-incubation and resulted in a larger population cells able to respond to ANG2.

Bottom Line: We found that CSF1 pre-treatment significantly augmented chemotaxis and that Tie2 receptor upregulation was responsible as siRNA targeting Tie2 receptor abrogated this effect.While supernatants from CSF1-pre-treated TEMs increased HUVEC branching, a neutralizing antibody against the CSF1R abrogated this activity, as did siRNA against the Tie2 receptor.To test our hypothesis in vivo, we treated PyMT tumor-bearing mice with CSF1 and observed an expansion in the TEM population relative to total F4/80+ cells, which resulted in increased angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, The Ohio State University, Columbus, Ohio, United States of America; Molecular Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
Reports demonstrate the role of M-CSF (CSF1) in tumor progression in mouse models as well as the prognostic value of macrophage numbers in breast cancer patients. Recently, a subset of CD14+ monocytes expressing the Tie2 receptor, once thought to be predominantly expressed on endothelial cells, has been characterized. We hypothesized that increased levels of CSF1 in breast tumors can regulate differentiation of Tie2- monocytes to a Tie2+ phenotype. We treated CD14+ human monocytes with CSF1 and found a significant increase in CD14+/Tie2+ positivity. To understand if CSF1-induced Tie2 expression on these cells improved their migratory ability, we pre-treated CD14+ monocytes with CSF1 and used Boyden chemotaxis chambers to observe enhanced response to angiopoietin-2 (ANG2), the chemotactic ligand for the Tie2 receptor. We found that CSF1 pre-treatment significantly augmented chemotaxis and that Tie2 receptor upregulation was responsible as siRNA targeting Tie2 receptor abrogated this effect. To understand any augmented angiogenic effect produced by treating these cells with CSF1, we cultured human umbilical vein endothelial cells (HUVECs) with conditioned supernatants from CSF1-pre-treated CD14+ monocytes for a tube formation assay. While supernatants from CSF1-pre-treated TEMs increased HUVEC branching, a neutralizing antibody against the CSF1R abrogated this activity, as did siRNA against the Tie2 receptor. To test our hypothesis in vivo, we treated PyMT tumor-bearing mice with CSF1 and observed an expansion in the TEM population relative to total F4/80+ cells, which resulted in increased angiogenesis. Investigation into the mechanism of Tie2 receptor upregulation on CD14+ monocytes by CSF1 revealed a synergistic contribution from the PI3 kinase and HIF pathways as the PI3 kinase inhibitor LY294002, as well as HIF-1α-deficient macrophages differentiated from the bone marrow of HIF-1αfl/fl/LysMcre mice, diminished CSF1-stimulated Tie2 receptor expression.

Show MeSH
Related in: MedlinePlus