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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 and HIF pathways independently and synergistically regulate Tie2 receptor expression on monocytes.(A) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1αfl/fl/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were treated with PBS or CSF1 (100 ng/ml) for 24 hours followed by immunostaining with antibodies specific for F4/80 and Tie2 receptor. CSF1 induced an increase in F4/80+/Tie2+ cells over PBS-treated cells from the macrophages derived from the bone marrow of wild type LysMcre mice. The macrophages derived from the HIF-1αfl/fl/LysMcre bone marrow and treated with CSF1 had a significantly smaller percentage of F4/80+ Tie2+ cells than those from the wild type mice. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (B) Macrophages were derived from the bone marrow of wild type female mice over five days in non-adherent tubes. The cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were pre-treated with DMSO (vehicle), the PI3 kinase/Akt inhibitor LY294002 (50 µM) (LY294002), the MEK inhibitor U0126 (10 µM) (U0126), or the NF-Kb inhibitor PDTC (100 µM) (PDTC) for 30 minutes. After, the cells were treated with CSF1 (100 ng/ml) (Vehicle+CSF1), LY294002+CSF1 (100 ng/ml) (LY294002+CSF1), U0126+CSF1 (100 ng/ml) (U0126+CSF1), or PDTC+CSF1 (100 ng/ml) (PDTC+CSF1) or left untreated for 24 hours followed by immunstaining with antibodies specific for F4/80 and Tie2. CSF1 induced an increase in the percent of F4/80+/Tie2+ cells compared to vehicle alone. The inhibitors LY294002, U0126, and PDTC alone had no effect on TEM levels. LY294002 pre-treatment significantly reduced the percent of TEMs regulated by CSF1 while U0126 and PDTC had no effect on CSF1 expansion of TEMs. Results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (C) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1αfl/fl/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were then pre-treated with DMSO or LY294002 (50 µM) for 30 minutes followed by CSF1 (100 ng/ml) (LY294002+CSF1) or not (vehicle) for 24 hours and then immunostained with antibodies specific for F4/80 and Tie2. The macrophages derived from HIF-1αfl/fl/LysMcre mice in combination with the PI3 kinase inhibitor LY294002 significantly reduced the percent TEMs to that similar to untreated levels. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells.
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pone-0098623-g006: CSF1 and HIF pathways independently and synergistically regulate Tie2 receptor expression on monocytes.(A) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1αfl/fl/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were treated with PBS or CSF1 (100 ng/ml) for 24 hours followed by immunostaining with antibodies specific for F4/80 and Tie2 receptor. CSF1 induced an increase in F4/80+/Tie2+ cells over PBS-treated cells from the macrophages derived from the bone marrow of wild type LysMcre mice. The macrophages derived from the HIF-1αfl/fl/LysMcre bone marrow and treated with CSF1 had a significantly smaller percentage of F4/80+ Tie2+ cells than those from the wild type mice. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (B) Macrophages were derived from the bone marrow of wild type female mice over five days in non-adherent tubes. The cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were pre-treated with DMSO (vehicle), the PI3 kinase/Akt inhibitor LY294002 (50 µM) (LY294002), the MEK inhibitor U0126 (10 µM) (U0126), or the NF-Kb inhibitor PDTC (100 µM) (PDTC) for 30 minutes. After, the cells were treated with CSF1 (100 ng/ml) (Vehicle+CSF1), LY294002+CSF1 (100 ng/ml) (LY294002+CSF1), U0126+CSF1 (100 ng/ml) (U0126+CSF1), or PDTC+CSF1 (100 ng/ml) (PDTC+CSF1) or left untreated for 24 hours followed by immunstaining with antibodies specific for F4/80 and Tie2. CSF1 induced an increase in the percent of F4/80+/Tie2+ cells compared to vehicle alone. The inhibitors LY294002, U0126, and PDTC alone had no effect on TEM levels. LY294002 pre-treatment significantly reduced the percent of TEMs regulated by CSF1 while U0126 and PDTC had no effect on CSF1 expansion of TEMs. Results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (C) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1αfl/fl/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were then pre-treated with DMSO or LY294002 (50 µM) for 30 minutes followed by CSF1 (100 ng/ml) (LY294002+CSF1) or not (vehicle) for 24 hours and then immunostained with antibodies specific for F4/80 and Tie2. The macrophages derived from HIF-1αfl/fl/LysMcre mice in combination with the PI3 kinase inhibitor LY294002 significantly reduced the percent TEMs to that similar to untreated levels. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells.

Mentions: The hypoxia inducible factor, HIF-1α, has been linked to CSF1 and PI3 kinase/AKT signaling pathways [41]–. Further, macrophages reside in hypoxic patches in solid tumors. We asked if HIF-1α was involved in the CSF1-induced increase in Tie2 receptor expression on TEMs. We differentiated bone marrow-derived macrophages from HIF-1αfl/fl/LysMcre and LysMcre (wild type) mice and stimulated these cells with PBS or CSF1. After 24 hours, we analyzed the macrophages for expression of Tie2 receptor by flow cytometry. We found no difference in the percent of F4/80+ macrophages expressing Tie2 receptor in the PBS-treated wild type LysMcre cells compared to HIF-1α-deficient cells (Figure 6A). In the CSF1-treated cells, we found that the loss of HIF-1α significantly inhibited the ability of these F4/80+ macrophages to express Tie2 receptor (p<0.0001) (Figure 6A). These data suggest an oxygen-independent role for HIF-1α in CSF1 augmentation of TEMs.


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 and HIF pathways independently and synergistically regulate Tie2 receptor expression on monocytes.(A) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1αfl/fl/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were treated with PBS or CSF1 (100 ng/ml) for 24 hours followed by immunostaining with antibodies specific for F4/80 and Tie2 receptor. CSF1 induced an increase in F4/80+/Tie2+ cells over PBS-treated cells from the macrophages derived from the bone marrow of wild type LysMcre mice. The macrophages derived from the HIF-1αfl/fl/LysMcre bone marrow and treated with CSF1 had a significantly smaller percentage of F4/80+ Tie2+ cells than those from the wild type mice. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (B) Macrophages were derived from the bone marrow of wild type female mice over five days in non-adherent tubes. The cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were pre-treated with DMSO (vehicle), the PI3 kinase/Akt inhibitor LY294002 (50 µM) (LY294002), the MEK inhibitor U0126 (10 µM) (U0126), or the NF-Kb inhibitor PDTC (100 µM) (PDTC) for 30 minutes. After, the cells were treated with CSF1 (100 ng/ml) (Vehicle+CSF1), LY294002+CSF1 (100 ng/ml) (LY294002+CSF1), U0126+CSF1 (100 ng/ml) (U0126+CSF1), or PDTC+CSF1 (100 ng/ml) (PDTC+CSF1) or left untreated for 24 hours followed by immunstaining with antibodies specific for F4/80 and Tie2. CSF1 induced an increase in the percent of F4/80+/Tie2+ cells compared to vehicle alone. The inhibitors LY294002, U0126, and PDTC alone had no effect on TEM levels. LY294002 pre-treatment significantly reduced the percent of TEMs regulated by CSF1 while U0126 and PDTC had no effect on CSF1 expansion of TEMs. Results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (C) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1αfl/fl/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were then pre-treated with DMSO or LY294002 (50 µM) for 30 minutes followed by CSF1 (100 ng/ml) (LY294002+CSF1) or not (vehicle) for 24 hours and then immunostained with antibodies specific for F4/80 and Tie2. The macrophages derived from HIF-1αfl/fl/LysMcre mice in combination with the PI3 kinase inhibitor LY294002 significantly reduced the percent TEMs to that similar to untreated levels. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells.
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pone-0098623-g006: CSF1 and HIF pathways independently and synergistically regulate Tie2 receptor expression on monocytes.(A) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1αfl/fl/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were treated with PBS or CSF1 (100 ng/ml) for 24 hours followed by immunostaining with antibodies specific for F4/80 and Tie2 receptor. CSF1 induced an increase in F4/80+/Tie2+ cells over PBS-treated cells from the macrophages derived from the bone marrow of wild type LysMcre mice. The macrophages derived from the HIF-1αfl/fl/LysMcre bone marrow and treated with CSF1 had a significantly smaller percentage of F4/80+ Tie2+ cells than those from the wild type mice. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (B) Macrophages were derived from the bone marrow of wild type female mice over five days in non-adherent tubes. The cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were pre-treated with DMSO (vehicle), the PI3 kinase/Akt inhibitor LY294002 (50 µM) (LY294002), the MEK inhibitor U0126 (10 µM) (U0126), or the NF-Kb inhibitor PDTC (100 µM) (PDTC) for 30 minutes. After, the cells were treated with CSF1 (100 ng/ml) (Vehicle+CSF1), LY294002+CSF1 (100 ng/ml) (LY294002+CSF1), U0126+CSF1 (100 ng/ml) (U0126+CSF1), or PDTC+CSF1 (100 ng/ml) (PDTC+CSF1) or left untreated for 24 hours followed by immunstaining with antibodies specific for F4/80 and Tie2. CSF1 induced an increase in the percent of F4/80+/Tie2+ cells compared to vehicle alone. The inhibitors LY294002, U0126, and PDTC alone had no effect on TEM levels. LY294002 pre-treatment significantly reduced the percent of TEMs regulated by CSF1 while U0126 and PDTC had no effect on CSF1 expansion of TEMs. Results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (C) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1αfl/fl/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were then pre-treated with DMSO or LY294002 (50 µM) for 30 minutes followed by CSF1 (100 ng/ml) (LY294002+CSF1) or not (vehicle) for 24 hours and then immunostained with antibodies specific for F4/80 and Tie2. The macrophages derived from HIF-1αfl/fl/LysMcre mice in combination with the PI3 kinase inhibitor LY294002 significantly reduced the percent TEMs to that similar to untreated levels. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells.
Mentions: The hypoxia inducible factor, HIF-1α, has been linked to CSF1 and PI3 kinase/AKT signaling pathways [41]–. Further, macrophages reside in hypoxic patches in solid tumors. We asked if HIF-1α was involved in the CSF1-induced increase in Tie2 receptor expression on TEMs. We differentiated bone marrow-derived macrophages from HIF-1αfl/fl/LysMcre and LysMcre (wild type) mice and stimulated these cells with PBS or CSF1. After 24 hours, we analyzed the macrophages for expression of Tie2 receptor by flow cytometry. We found no difference in the percent of F4/80+ macrophages expressing Tie2 receptor in the PBS-treated wild type LysMcre cells compared to HIF-1α-deficient cells (Figure 6A). In the CSF1-treated cells, we found that the loss of HIF-1α significantly inhibited the ability of these F4/80+ macrophages to express Tie2 receptor (p<0.0001) (Figure 6A). These data suggest an oxygen-independent role for HIF-1α in CSF1 augmentation of TEMs.

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