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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 up-regulates Tie2 receptor on CD14+ human monocytes.(A) CD14+ monocytes were isolated from whole blood using CD14+ microbeads. Cells were fixed and immunostained using anti-human Tie2 receptor antibody or isotype control antibody immediately following isolation (Freshly isolated) or after treated without (-CSF1) or with rhCSF1 (100 ng/ml) (+CSF1) for 24 hours. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (B) CD14+ monocytes treated with rhANG1 (100 ng/ml), rhANG2 (100 ng/ml) or a dose-response of rhCSF1 (0, 0.1, 1, 10, 100 ng/ml). ANG2 up-regulated Tie2 expression compared to ANG1 and CSF1 induces a dose-escalation of Tie2 on CD14+ monocytes. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (C) CD14+ monocytes were left untreated (Utx) or treated with rhANG2 (100 ng/ml) (ANG2), rhCSF1 (100 ng/ml) (CSF1), CSF1R neutralizing antibody alone, or pre-treated with the CSF1R Nab for 30 minutes prior to stimulation with rhCSF1 (100 ng/ml) (CSF1R NAb+CSF1) for 24 hours. ANG2- and CSF1-treatment significantly increased Tie2 expression while the CSF1R NAb abrogated this effect. N = 8 per group and results represent the mean ± SEM of Tie2-positivity by flow cytometry. (D) CD14+ monocytes were left untreated (Untreated), pre-treated with CSF1R NAb (40 µg or 80 µg) for 30 minutes then treated with rhCSF1 (100 ng/ml) (CSF1R NAb+CSF1), or with rhCSF1 (100 ng/ml) alone (CSF1) for 10 minutes. Western blot analysis indicates that the CSF1R NAb was effective at reducing Akt1 phosphorylation.
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pone-0098623-g001: CSF1 up-regulates Tie2 receptor on CD14+ human monocytes.(A) CD14+ monocytes were isolated from whole blood using CD14+ microbeads. Cells were fixed and immunostained using anti-human Tie2 receptor antibody or isotype control antibody immediately following isolation (Freshly isolated) or after treated without (-CSF1) or with rhCSF1 (100 ng/ml) (+CSF1) for 24 hours. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (B) CD14+ monocytes treated with rhANG1 (100 ng/ml), rhANG2 (100 ng/ml) or a dose-response of rhCSF1 (0, 0.1, 1, 10, 100 ng/ml). ANG2 up-regulated Tie2 expression compared to ANG1 and CSF1 induces a dose-escalation of Tie2 on CD14+ monocytes. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (C) CD14+ monocytes were left untreated (Utx) or treated with rhANG2 (100 ng/ml) (ANG2), rhCSF1 (100 ng/ml) (CSF1), CSF1R neutralizing antibody alone, or pre-treated with the CSF1R Nab for 30 minutes prior to stimulation with rhCSF1 (100 ng/ml) (CSF1R NAb+CSF1) for 24 hours. ANG2- and CSF1-treatment significantly increased Tie2 expression while the CSF1R NAb abrogated this effect. N = 8 per group and results represent the mean ± SEM of Tie2-positivity by flow cytometry. (D) CD14+ monocytes were left untreated (Untreated), pre-treated with CSF1R NAb (40 µg or 80 µg) for 30 minutes then treated with rhCSF1 (100 ng/ml) (CSF1R NAb+CSF1), or with rhCSF1 (100 ng/ml) alone (CSF1) for 10 minutes. Western blot analysis indicates that the CSF1R NAb was effective at reducing Akt1 phosphorylation.

Mentions: CSF1 drives the differentiation of peripheral blood monocytes to macrophages resulting in a pro-angiogenic and immune regulatory phenotype [13], [32]. In breast tumors, CSF1 expression increases the number of macrophages which enhances tumor vessel density and metastasis [33]–[35]. Further, loss of CSF1 (op/op mice) hinders the development of tumor blood vessels and tumor progression [20]. Tie2-expressing monocytes/macrophages (TEMs) are a subset of myeloid cells that function similar to M2, tumor-associated macrophages (TAMs) as their activity increases tumor blood vessel area and metastasis in mouse models of breast cancer[24], [26], [27]. As stated above, CSF1 plays a role in normal mammary gland physiology and ductal development [21]. But, in breast cancer patients, CSF1 levels are augmented and predict poor outcome [15]. In 2003, we reported that CSF1 drove a pro-angiogenic phenotype in CD14+ monocytes via the production of VEGF [13]. However, in that study we did not consider that CSF1 may differentiation alternate subpopulations of monocytes. Thus, our current study examines a direct link between CSF1 and the expression of the Tie2 receptor on CD14+ human monocytes to become TEMs. To elucidate this effect, human monocytes were isolated from whole blood using CD14+ microbeads and incubated in non-adherent culture tubes in RPMI media containing fetal bovine serum and cultured for 24 hours with or without 100 ng/ml recombinant human CSF1. Even though lower doses can drive the differentiation, survival and function of macrophages [6], [36]in vitro, we elected to use 100 ng/ml to maximally-activate macrophage signaling pathways. After, monocytes were immunostained with an antibody specific for the human Tie2 receptor and analyzed by flow cytometry (Figure 1A). Our data suggests that CD14+ cells incubated in the absence of CSF1 for 24 hours express the Tie2 receptor similar to freshly-isolated CD14+ cells. In contrast, CD14+ monocytes cultured in the presence of CSF1 for 24 hours significantly increased Tie2 receptor expression (p<0.0001) (Figure 1A). Cell viability of all monocytes was confirmed by Trypan Blue staining (data not shown).


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 up-regulates Tie2 receptor on CD14+ human monocytes.(A) CD14+ monocytes were isolated from whole blood using CD14+ microbeads. Cells were fixed and immunostained using anti-human Tie2 receptor antibody or isotype control antibody immediately following isolation (Freshly isolated) or after treated without (-CSF1) or with rhCSF1 (100 ng/ml) (+CSF1) for 24 hours. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (B) CD14+ monocytes treated with rhANG1 (100 ng/ml), rhANG2 (100 ng/ml) or a dose-response of rhCSF1 (0, 0.1, 1, 10, 100 ng/ml). ANG2 up-regulated Tie2 expression compared to ANG1 and CSF1 induces a dose-escalation of Tie2 on CD14+ monocytes. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (C) CD14+ monocytes were left untreated (Utx) or treated with rhANG2 (100 ng/ml) (ANG2), rhCSF1 (100 ng/ml) (CSF1), CSF1R neutralizing antibody alone, or pre-treated with the CSF1R Nab for 30 minutes prior to stimulation with rhCSF1 (100 ng/ml) (CSF1R NAb+CSF1) for 24 hours. ANG2- and CSF1-treatment significantly increased Tie2 expression while the CSF1R NAb abrogated this effect. N = 8 per group and results represent the mean ± SEM of Tie2-positivity by flow cytometry. (D) CD14+ monocytes were left untreated (Untreated), pre-treated with CSF1R NAb (40 µg or 80 µg) for 30 minutes then treated with rhCSF1 (100 ng/ml) (CSF1R NAb+CSF1), or with rhCSF1 (100 ng/ml) alone (CSF1) for 10 minutes. Western blot analysis indicates that the CSF1R NAb was effective at reducing Akt1 phosphorylation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098623-g001: CSF1 up-regulates Tie2 receptor on CD14+ human monocytes.(A) CD14+ monocytes were isolated from whole blood using CD14+ microbeads. Cells were fixed and immunostained using anti-human Tie2 receptor antibody or isotype control antibody immediately following isolation (Freshly isolated) or after treated without (-CSF1) or with rhCSF1 (100 ng/ml) (+CSF1) for 24 hours. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (B) CD14+ monocytes treated with rhANG1 (100 ng/ml), rhANG2 (100 ng/ml) or a dose-response of rhCSF1 (0, 0.1, 1, 10, 100 ng/ml). ANG2 up-regulated Tie2 expression compared to ANG1 and CSF1 induces a dose-escalation of Tie2 on CD14+ monocytes. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (C) CD14+ monocytes were left untreated (Utx) or treated with rhANG2 (100 ng/ml) (ANG2), rhCSF1 (100 ng/ml) (CSF1), CSF1R neutralizing antibody alone, or pre-treated with the CSF1R Nab for 30 minutes prior to stimulation with rhCSF1 (100 ng/ml) (CSF1R NAb+CSF1) for 24 hours. ANG2- and CSF1-treatment significantly increased Tie2 expression while the CSF1R NAb abrogated this effect. N = 8 per group and results represent the mean ± SEM of Tie2-positivity by flow cytometry. (D) CD14+ monocytes were left untreated (Untreated), pre-treated with CSF1R NAb (40 µg or 80 µg) for 30 minutes then treated with rhCSF1 (100 ng/ml) (CSF1R NAb+CSF1), or with rhCSF1 (100 ng/ml) alone (CSF1) for 10 minutes. Western blot analysis indicates that the CSF1R NAb was effective at reducing Akt1 phosphorylation.
Mentions: CSF1 drives the differentiation of peripheral blood monocytes to macrophages resulting in a pro-angiogenic and immune regulatory phenotype [13], [32]. In breast tumors, CSF1 expression increases the number of macrophages which enhances tumor vessel density and metastasis [33]–[35]. Further, loss of CSF1 (op/op mice) hinders the development of tumor blood vessels and tumor progression [20]. Tie2-expressing monocytes/macrophages (TEMs) are a subset of myeloid cells that function similar to M2, tumor-associated macrophages (TAMs) as their activity increases tumor blood vessel area and metastasis in mouse models of breast cancer[24], [26], [27]. As stated above, CSF1 plays a role in normal mammary gland physiology and ductal development [21]. But, in breast cancer patients, CSF1 levels are augmented and predict poor outcome [15]. In 2003, we reported that CSF1 drove a pro-angiogenic phenotype in CD14+ monocytes via the production of VEGF [13]. However, in that study we did not consider that CSF1 may differentiation alternate subpopulations of monocytes. Thus, our current study examines a direct link between CSF1 and the expression of the Tie2 receptor on CD14+ human monocytes to become TEMs. To elucidate this effect, human monocytes were isolated from whole blood using CD14+ microbeads and incubated in non-adherent culture tubes in RPMI media containing fetal bovine serum and cultured for 24 hours with or without 100 ng/ml recombinant human CSF1. Even though lower doses can drive the differentiation, survival and function of macrophages [6], [36]in vitro, we elected to use 100 ng/ml to maximally-activate macrophage signaling pathways. After, monocytes were immunostained with an antibody specific for the human Tie2 receptor and analyzed by flow cytometry (Figure 1A). Our data suggests that CD14+ cells incubated in the absence of CSF1 for 24 hours express the Tie2 receptor similar to freshly-isolated CD14+ cells. In contrast, CD14+ monocytes cultured in the presence of CSF1 for 24 hours significantly increased Tie2 receptor expression (p<0.0001) (Figure 1A). Cell viability of all monocytes was confirmed by Trypan Blue staining (data not shown).

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