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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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Related in: MedlinePlus

Conditioned supernatants from CSF1-treated TEMs augments HUVEC branching.(A) Human monocytes were isolated from whole blood and differentiated to macrophages over five days using rhCSF1 (5 ng/ml). The cells were serum-starved in endotoxin-free RPMI containing polymyxin B (10 µg/ml) for 24 hours then treated with either a neutralizing antibody for CSF1R or isotype antibody (40 µg/ml) for 1 hour prior to stimulation with CSF1 (10 ng/ml) for 18 hours. The cells were then washed three times and treated with ANG2 (10 ng/ml) for 24 more hours. The conditioned supernatants from these cells (CSF1/ANG2; CSF1R NAb/CSF1/ANG2) or minimal media alone, (minimal), media containing ANG2 alone (10 ng/ml) (ANG2), media containing CSF1 alone (10 ng/ml) (CSF1), or media containing VEGF (10 ng/ml)(VEGF) were used to culture 1.5×104 human umbilical vein endothelial cells (HUVEC) on growth factor-reduced Matrigel for 8 hours. Digital images were captured to determine HUVEC branching. The number of branches was quantified in a blinded manner per field. CSF1, VEGF, and ANG2 each stimulated significantly more branch points from the HUVECs than minimal media. The conditioned media from the macrophages pre-treated with CSF1 induced significantly more branch points than CSF1, VEGF, and ANG2 treatment alone and pre-treatment of the macrophages with the CSF1R neutralizing antibody significantly reduced branch points comparable to media containing VEGF, CSF1, and ANG2 alone levels. N = 5 and results represent the mean ± SEM of HUVEC branch points. (B) In the same manner as above, human monocytes were differentiated to macrophages over five days and serum-starved for 24 hours. The cells were transfected with a siRNA targeting human Tie2 receptor (50 nM) or a scrambled siRNA (50 nM) for 24 hours. The macrophages were washed three times with PBS and then treated with CSF1 (10 ng/ml) for 18 more hours. These conditioned supernatants were collected and used to culture HUVEC cells grown on growth factor-reduced Matrigel for eight hours to detect branch points. Digital images were captured to determine HUVEC branching. The macrophages pre-treated with CSF1 then ANG2 (CSF1/ANG2) induced a significant number of branch points compared to HUVEC cultured in media alone (Media). The conditioned supernatants from the macrophages transfected with siTie2 then treated with CSF1 then ANG2 (Tie2 siRNA/CSF1/ANG2) had significantly less branch points than the CSF1-conditioned supernatants and similar to the minimal conditions. The macrophages transfected with the scrambled siRNA and pre-treated with CSF1 then ANG2 (scrmbsiRNA/CSF1/ANG2) induced significantly more branch points than the conditioned supernatants from the siTie2 samples but still significantly less than the CSF1-conditioned media. N = 5 and results represent the mean ± SEM of HUVEC branch points. (C) CD14+ monocytes were left untreated (UTX) or treated with CSF1 (100 ng/ml) (CSF1), ANG2 (100 ng/ml) (ANG2), or the combination CSF1 (100 ng/ml) and ANG2 (100 ng/ml) (CSF1+ANG2) for 48 hrs. RT-PCR analysis for mRNA expression of the following angiogenic factors was performed: VEGF, uPA, COX2, TNFα, MMP2, MMP7, and MMP9. Data is represented as relative expression for each target mRNA relative to GAPDH housekeeping mRNA. VEGF, uPA, and COX2 mRNA were all significantly increased in CD14+ monocytes treated with CSF1+ANG2 compared to untreated CD14+ monocytes. Each condition was repeated at least 3 times (N = at least 3 and results represent the mean ± SEM for relative mRNA expression).
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pone-0098623-g003: Conditioned supernatants from CSF1-treated TEMs augments HUVEC branching.(A) Human monocytes were isolated from whole blood and differentiated to macrophages over five days using rhCSF1 (5 ng/ml). The cells were serum-starved in endotoxin-free RPMI containing polymyxin B (10 µg/ml) for 24 hours then treated with either a neutralizing antibody for CSF1R or isotype antibody (40 µg/ml) for 1 hour prior to stimulation with CSF1 (10 ng/ml) for 18 hours. The cells were then washed three times and treated with ANG2 (10 ng/ml) for 24 more hours. The conditioned supernatants from these cells (CSF1/ANG2; CSF1R NAb/CSF1/ANG2) or minimal media alone, (minimal), media containing ANG2 alone (10 ng/ml) (ANG2), media containing CSF1 alone (10 ng/ml) (CSF1), or media containing VEGF (10 ng/ml)(VEGF) were used to culture 1.5×104 human umbilical vein endothelial cells (HUVEC) on growth factor-reduced Matrigel for 8 hours. Digital images were captured to determine HUVEC branching. The number of branches was quantified in a blinded manner per field. CSF1, VEGF, and ANG2 each stimulated significantly more branch points from the HUVECs than minimal media. The conditioned media from the macrophages pre-treated with CSF1 induced significantly more branch points than CSF1, VEGF, and ANG2 treatment alone and pre-treatment of the macrophages with the CSF1R neutralizing antibody significantly reduced branch points comparable to media containing VEGF, CSF1, and ANG2 alone levels. N = 5 and results represent the mean ± SEM of HUVEC branch points. (B) In the same manner as above, human monocytes were differentiated to macrophages over five days and serum-starved for 24 hours. The cells were transfected with a siRNA targeting human Tie2 receptor (50 nM) or a scrambled siRNA (50 nM) for 24 hours. The macrophages were washed three times with PBS and then treated with CSF1 (10 ng/ml) for 18 more hours. These conditioned supernatants were collected and used to culture HUVEC cells grown on growth factor-reduced Matrigel for eight hours to detect branch points. Digital images were captured to determine HUVEC branching. The macrophages pre-treated with CSF1 then ANG2 (CSF1/ANG2) induced a significant number of branch points compared to HUVEC cultured in media alone (Media). The conditioned supernatants from the macrophages transfected with siTie2 then treated with CSF1 then ANG2 (Tie2 siRNA/CSF1/ANG2) had significantly less branch points than the CSF1-conditioned supernatants and similar to the minimal conditions. The macrophages transfected with the scrambled siRNA and pre-treated with CSF1 then ANG2 (scrmbsiRNA/CSF1/ANG2) induced significantly more branch points than the conditioned supernatants from the siTie2 samples but still significantly less than the CSF1-conditioned media. N = 5 and results represent the mean ± SEM of HUVEC branch points. (C) CD14+ monocytes were left untreated (UTX) or treated with CSF1 (100 ng/ml) (CSF1), ANG2 (100 ng/ml) (ANG2), or the combination CSF1 (100 ng/ml) and ANG2 (100 ng/ml) (CSF1+ANG2) for 48 hrs. RT-PCR analysis for mRNA expression of the following angiogenic factors was performed: VEGF, uPA, COX2, TNFα, MMP2, MMP7, and MMP9. Data is represented as relative expression for each target mRNA relative to GAPDH housekeeping mRNA. VEGF, uPA, and COX2 mRNA were all significantly increased in CD14+ monocytes treated with CSF1+ANG2 compared to untreated CD14+ monocytes. Each condition was repeated at least 3 times (N = at least 3 and results represent the mean ± SEM for relative mRNA expression).

Mentions: Tumor angiogenesis requires endothelial cell proliferation, mobilization, and sprouting blood vessels. The human umbilical vein endothelial cell (HUVEC) tube branching assay is a method to analyze the angiogenic potential of endothelial cells in vitro. Conditioned supernatants from CSF1-treated human monocytes contribute to endothelial cell tube formation [13]. More specifically, conditioned supernatants from TEMs increase endothelial tube branching over that observed by conditioned supernatants from a TEM-depleted monocyte population [28]. Given our observation that CSF1 increases Tie2 receptor expression on CD14+ monocytes, we hypothesized that pre-treatment with CSF1 would induce macrophages to release factors which increase HUVEC tube branching and that blocking Tie2 receptor expression with an siRNA targeting monocyte Tie2 receptor would abrogate this effect. To determine the angiogenic potential of CSF1 up-regulation of the Tie2 receptor on monocytes, we isolated CD14+ monocytes from whole blood and differentiated to macrophages over five days using low-dose (5 ng/ml) rhCSF1. After starvation in minimal media, the cells were treated with either a neutralizing antibody for CSF1R or isotype antibody (40 µg/ml) for 1 hour prior to stimulation with CSF1 for 18 hours. The cells were then washed three times and treated with ANG2 for 24 more hours. The cell-free conditioned supernatants from these cells or minimal media alone, media containing ANG2 alone, media containing CSF1 alone, or media containing VEGF alone were used to culture 1.5×104 human umbilical vein endothelial cells on growth factor-reduced Matrigel for 8 hours. The conditioned supernatants from the macrophages pre-treated with CSF1 induced significantly more HUVEC branch points than media containing CSF1, VEGF, and ANG2 alone (p<0.0001 for each condition). More importantly, pre-treatment of the macrophages with the CSF1R neutralizing antibody before CSF1 and ANG2 stimulation significantly reduced HUVEC branch points comparable to HUVECs cultured in media containing VEGF, media containing CSF1, and media containing ANG2 alone (p<0.0001) (Figure 3A).


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)

Conditioned supernatants from CSF1-treated TEMs augments HUVEC branching.(A) Human monocytes were isolated from whole blood and differentiated to macrophages over five days using rhCSF1 (5 ng/ml). The cells were serum-starved in endotoxin-free RPMI containing polymyxin B (10 µg/ml) for 24 hours then treated with either a neutralizing antibody for CSF1R or isotype antibody (40 µg/ml) for 1 hour prior to stimulation with CSF1 (10 ng/ml) for 18 hours. The cells were then washed three times and treated with ANG2 (10 ng/ml) for 24 more hours. The conditioned supernatants from these cells (CSF1/ANG2; CSF1R NAb/CSF1/ANG2) or minimal media alone, (minimal), media containing ANG2 alone (10 ng/ml) (ANG2), media containing CSF1 alone (10 ng/ml) (CSF1), or media containing VEGF (10 ng/ml)(VEGF) were used to culture 1.5×104 human umbilical vein endothelial cells (HUVEC) on growth factor-reduced Matrigel for 8 hours. Digital images were captured to determine HUVEC branching. The number of branches was quantified in a blinded manner per field. CSF1, VEGF, and ANG2 each stimulated significantly more branch points from the HUVECs than minimal media. The conditioned media from the macrophages pre-treated with CSF1 induced significantly more branch points than CSF1, VEGF, and ANG2 treatment alone and pre-treatment of the macrophages with the CSF1R neutralizing antibody significantly reduced branch points comparable to media containing VEGF, CSF1, and ANG2 alone levels. N = 5 and results represent the mean ± SEM of HUVEC branch points. (B) In the same manner as above, human monocytes were differentiated to macrophages over five days and serum-starved for 24 hours. The cells were transfected with a siRNA targeting human Tie2 receptor (50 nM) or a scrambled siRNA (50 nM) for 24 hours. The macrophages were washed three times with PBS and then treated with CSF1 (10 ng/ml) for 18 more hours. These conditioned supernatants were collected and used to culture HUVEC cells grown on growth factor-reduced Matrigel for eight hours to detect branch points. Digital images were captured to determine HUVEC branching. The macrophages pre-treated with CSF1 then ANG2 (CSF1/ANG2) induced a significant number of branch points compared to HUVEC cultured in media alone (Media). The conditioned supernatants from the macrophages transfected with siTie2 then treated with CSF1 then ANG2 (Tie2 siRNA/CSF1/ANG2) had significantly less branch points than the CSF1-conditioned supernatants and similar to the minimal conditions. The macrophages transfected with the scrambled siRNA and pre-treated with CSF1 then ANG2 (scrmbsiRNA/CSF1/ANG2) induced significantly more branch points than the conditioned supernatants from the siTie2 samples but still significantly less than the CSF1-conditioned media. N = 5 and results represent the mean ± SEM of HUVEC branch points. (C) CD14+ monocytes were left untreated (UTX) or treated with CSF1 (100 ng/ml) (CSF1), ANG2 (100 ng/ml) (ANG2), or the combination CSF1 (100 ng/ml) and ANG2 (100 ng/ml) (CSF1+ANG2) for 48 hrs. RT-PCR analysis for mRNA expression of the following angiogenic factors was performed: VEGF, uPA, COX2, TNFα, MMP2, MMP7, and MMP9. Data is represented as relative expression for each target mRNA relative to GAPDH housekeeping mRNA. VEGF, uPA, and COX2 mRNA were all significantly increased in CD14+ monocytes treated with CSF1+ANG2 compared to untreated CD14+ monocytes. Each condition was repeated at least 3 times (N = at least 3 and results represent the mean ± SEM for relative mRNA expression).
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pone-0098623-g003: Conditioned supernatants from CSF1-treated TEMs augments HUVEC branching.(A) Human monocytes were isolated from whole blood and differentiated to macrophages over five days using rhCSF1 (5 ng/ml). The cells were serum-starved in endotoxin-free RPMI containing polymyxin B (10 µg/ml) for 24 hours then treated with either a neutralizing antibody for CSF1R or isotype antibody (40 µg/ml) for 1 hour prior to stimulation with CSF1 (10 ng/ml) for 18 hours. The cells were then washed three times and treated with ANG2 (10 ng/ml) for 24 more hours. The conditioned supernatants from these cells (CSF1/ANG2; CSF1R NAb/CSF1/ANG2) or minimal media alone, (minimal), media containing ANG2 alone (10 ng/ml) (ANG2), media containing CSF1 alone (10 ng/ml) (CSF1), or media containing VEGF (10 ng/ml)(VEGF) were used to culture 1.5×104 human umbilical vein endothelial cells (HUVEC) on growth factor-reduced Matrigel for 8 hours. Digital images were captured to determine HUVEC branching. The number of branches was quantified in a blinded manner per field. CSF1, VEGF, and ANG2 each stimulated significantly more branch points from the HUVECs than minimal media. The conditioned media from the macrophages pre-treated with CSF1 induced significantly more branch points than CSF1, VEGF, and ANG2 treatment alone and pre-treatment of the macrophages with the CSF1R neutralizing antibody significantly reduced branch points comparable to media containing VEGF, CSF1, and ANG2 alone levels. N = 5 and results represent the mean ± SEM of HUVEC branch points. (B) In the same manner as above, human monocytes were differentiated to macrophages over five days and serum-starved for 24 hours. The cells were transfected with a siRNA targeting human Tie2 receptor (50 nM) or a scrambled siRNA (50 nM) for 24 hours. The macrophages were washed three times with PBS and then treated with CSF1 (10 ng/ml) for 18 more hours. These conditioned supernatants were collected and used to culture HUVEC cells grown on growth factor-reduced Matrigel for eight hours to detect branch points. Digital images were captured to determine HUVEC branching. The macrophages pre-treated with CSF1 then ANG2 (CSF1/ANG2) induced a significant number of branch points compared to HUVEC cultured in media alone (Media). The conditioned supernatants from the macrophages transfected with siTie2 then treated with CSF1 then ANG2 (Tie2 siRNA/CSF1/ANG2) had significantly less branch points than the CSF1-conditioned supernatants and similar to the minimal conditions. The macrophages transfected with the scrambled siRNA and pre-treated with CSF1 then ANG2 (scrmbsiRNA/CSF1/ANG2) induced significantly more branch points than the conditioned supernatants from the siTie2 samples but still significantly less than the CSF1-conditioned media. N = 5 and results represent the mean ± SEM of HUVEC branch points. (C) CD14+ monocytes were left untreated (UTX) or treated with CSF1 (100 ng/ml) (CSF1), ANG2 (100 ng/ml) (ANG2), or the combination CSF1 (100 ng/ml) and ANG2 (100 ng/ml) (CSF1+ANG2) for 48 hrs. RT-PCR analysis for mRNA expression of the following angiogenic factors was performed: VEGF, uPA, COX2, TNFα, MMP2, MMP7, and MMP9. Data is represented as relative expression for each target mRNA relative to GAPDH housekeeping mRNA. VEGF, uPA, and COX2 mRNA were all significantly increased in CD14+ monocytes treated with CSF1+ANG2 compared to untreated CD14+ monocytes. Each condition was repeated at least 3 times (N = at least 3 and results represent the mean ± SEM for relative mRNA expression).
Mentions: Tumor angiogenesis requires endothelial cell proliferation, mobilization, and sprouting blood vessels. The human umbilical vein endothelial cell (HUVEC) tube branching assay is a method to analyze the angiogenic potential of endothelial cells in vitro. Conditioned supernatants from CSF1-treated human monocytes contribute to endothelial cell tube formation [13]. More specifically, conditioned supernatants from TEMs increase endothelial tube branching over that observed by conditioned supernatants from a TEM-depleted monocyte population [28]. Given our observation that CSF1 increases Tie2 receptor expression on CD14+ monocytes, we hypothesized that pre-treatment with CSF1 would induce macrophages to release factors which increase HUVEC tube branching and that blocking Tie2 receptor expression with an siRNA targeting monocyte Tie2 receptor would abrogate this effect. To determine the angiogenic potential of CSF1 up-regulation of the Tie2 receptor on monocytes, we isolated CD14+ monocytes from whole blood and differentiated to macrophages over five days using low-dose (5 ng/ml) rhCSF1. After starvation in minimal media, the cells were treated with either a neutralizing antibody for CSF1R or isotype antibody (40 µg/ml) for 1 hour prior to stimulation with CSF1 for 18 hours. The cells were then washed three times and treated with ANG2 for 24 more hours. The cell-free conditioned supernatants from these cells or minimal media alone, media containing ANG2 alone, media containing CSF1 alone, or media containing VEGF alone were used to culture 1.5×104 human umbilical vein endothelial cells on growth factor-reduced Matrigel for 8 hours. The conditioned supernatants from the macrophages pre-treated with CSF1 induced significantly more HUVEC branch points than media containing CSF1, VEGF, and ANG2 alone (p<0.0001 for each condition). More importantly, pre-treatment of the macrophages with the CSF1R neutralizing antibody before CSF1 and ANG2 stimulation significantly reduced HUVEC branch points comparable to HUVECs cultured in media containing VEGF, media containing CSF1, and media containing ANG2 alone (p<0.0001) (Figure 3A).

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