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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 has no effect on tumor growth but increases percent tumor TEMs and augments angiogenesis.(A) After two weeks of treatment, tumors were removed, homogenized and immunostained with antibodies specific for F4/80 and Tie2 to identify total F4/80+ cells and F4/80+/Tie2+ cells (Tie2-expressing macrophages, TEMs). While there was a marked increase in total F4/80+ macrophages with CSF1 treatment, the percent of F4/80+/Tie2+ TEMs was significantly increased in response to CSF1 suggesting a regulatory role for CSF1 in expanding the TEM population. N = 5 mice per group and results represent the mean ± SEM of total F4/80+ and F4/80+/Tie2+ TEMs within the tumors. (B, top and bottom left) PyMT tumors without CSF1 treatment and (top and bottom right) with CSF1 treatment immunostained with CD31 for blood vessels, F4/80 for macrophages, Tie2 for F4/80+/Tie2+ TEMS, and DAPI. Confocal images (using 60× objective (top) and with 3× zoom (bottom) suggest an increase in both F4/80 macrophages and F4/80+/Tie2+ TEMS in the CSF1-treated tumors. Multiply overlap indicates those areas where F4/80 and Tie2 positivity overlap. Individual stains are in SupplementaryFigure 3. (C, top) Orthotopically implanted PyMT mammary tumors in wild type C57Bl/6 female mice were allowed to become palpable then intraperitoneally treated with PBS (PBS), CSF1 (100 ng in 100 µls) (CSF1), a neutralizing antibody for the CSF1R (50 mg/kg) 4 hours prior to CSF1 treatment (100 ng in 100 µls) (CSF1R NAb+CSF1), the CSF1R antibody alone (CSF1R NAb), an isotype antibody (50 mg/kg) 4 hours prior to CSF1 (100 ng in 100 µls) treatment (CSF1+IgG), or the isotype antibody alone (IgG) three times per week for two additional weeks. The tumors were immunostained with a CD31-Alexa Flour 546 antibody to recognize endothelial cells that comprise blood vessels. Qualitatively, CSF1 treatment increased the percent of CD31-postitive pixels per high powered field compared to PBS treated tumors, while the neutralizing antibody to CSF1R suppressed the CSF1 effect on angiogenesis. (B, bottom) Quantitatively, the percent of CD31+ pixels per high powered field were quantified as blood vessels (angiogenesis) using Adobe Photoshop histogram analysis. CSF1 treatment significantly increased CD31-positive pixels (angiogenesis) compared to PBS. The neutralizing antibody for CSF1R significantly reduced the ability of CSF1 to up-regulate angiogenesis. N = 5 mice per group and results represent the mean ± SEM of percent CD31-positive pixels per high powered field (HPF).
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pone-0098623-g004: CSF1 has no effect on tumor growth but increases percent tumor TEMs and augments angiogenesis.(A) After two weeks of treatment, tumors were removed, homogenized and immunostained with antibodies specific for F4/80 and Tie2 to identify total F4/80+ cells and F4/80+/Tie2+ cells (Tie2-expressing macrophages, TEMs). While there was a marked increase in total F4/80+ macrophages with CSF1 treatment, the percent of F4/80+/Tie2+ TEMs was significantly increased in response to CSF1 suggesting a regulatory role for CSF1 in expanding the TEM population. N = 5 mice per group and results represent the mean ± SEM of total F4/80+ and F4/80+/Tie2+ TEMs within the tumors. (B, top and bottom left) PyMT tumors without CSF1 treatment and (top and bottom right) with CSF1 treatment immunostained with CD31 for blood vessels, F4/80 for macrophages, Tie2 for F4/80+/Tie2+ TEMS, and DAPI. Confocal images (using 60× objective (top) and with 3× zoom (bottom) suggest an increase in both F4/80 macrophages and F4/80+/Tie2+ TEMS in the CSF1-treated tumors. Multiply overlap indicates those areas where F4/80 and Tie2 positivity overlap. Individual stains are in SupplementaryFigure 3. (C, top) Orthotopically implanted PyMT mammary tumors in wild type C57Bl/6 female mice were allowed to become palpable then intraperitoneally treated with PBS (PBS), CSF1 (100 ng in 100 µls) (CSF1), a neutralizing antibody for the CSF1R (50 mg/kg) 4 hours prior to CSF1 treatment (100 ng in 100 µls) (CSF1R NAb+CSF1), the CSF1R antibody alone (CSF1R NAb), an isotype antibody (50 mg/kg) 4 hours prior to CSF1 (100 ng in 100 µls) treatment (CSF1+IgG), or the isotype antibody alone (IgG) three times per week for two additional weeks. The tumors were immunostained with a CD31-Alexa Flour 546 antibody to recognize endothelial cells that comprise blood vessels. Qualitatively, CSF1 treatment increased the percent of CD31-postitive pixels per high powered field compared to PBS treated tumors, while the neutralizing antibody to CSF1R suppressed the CSF1 effect on angiogenesis. (B, bottom) Quantitatively, the percent of CD31+ pixels per high powered field were quantified as blood vessels (angiogenesis) using Adobe Photoshop histogram analysis. CSF1 treatment significantly increased CD31-positive pixels (angiogenesis) compared to PBS. The neutralizing antibody for CSF1R significantly reduced the ability of CSF1 to up-regulate angiogenesis. N = 5 mice per group and results represent the mean ± SEM of percent CD31-positive pixels per high powered field (HPF).

Mentions: Upon sacrifice, fresh tumors were collected, homogenized using collagenase, immunostained with F4/80 and Tie2 receptor antibodies and subjected to flow cytometry for the percent of total F4/80+ cells and percent of F4/80+/Tie2+ TEMs relative to tumor cells. CSF1 treatment induced a significantly higher percentage of F4/80+/Tie2+ TEMs (p = 0.0003) than the percentage of overall tumor F4/80+ cells (Figure 4A) while a neutralizing antibody to CSF1R abrogated this effect. These data suggest that CSF1 contributes more to the expansion of the population of tumor TEMs than overall F4/80+ cells. To corroborate the flow cytometry data, we immunostained +/- CSF1 treated tumors with CD31, F4/80, or Tie2 antibodies and analyzed by confocal microscopy and found differences in both overall F4/80+ infiltration as well as an increase in F4/80+/Tie2+ cells (TEMs) in the CSF1-treated tumors (Figure 4B).


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 has no effect on tumor growth but increases percent tumor TEMs and augments angiogenesis.(A) After two weeks of treatment, tumors were removed, homogenized and immunostained with antibodies specific for F4/80 and Tie2 to identify total F4/80+ cells and F4/80+/Tie2+ cells (Tie2-expressing macrophages, TEMs). While there was a marked increase in total F4/80+ macrophages with CSF1 treatment, the percent of F4/80+/Tie2+ TEMs was significantly increased in response to CSF1 suggesting a regulatory role for CSF1 in expanding the TEM population. N = 5 mice per group and results represent the mean ± SEM of total F4/80+ and F4/80+/Tie2+ TEMs within the tumors. (B, top and bottom left) PyMT tumors without CSF1 treatment and (top and bottom right) with CSF1 treatment immunostained with CD31 for blood vessels, F4/80 for macrophages, Tie2 for F4/80+/Tie2+ TEMS, and DAPI. Confocal images (using 60× objective (top) and with 3× zoom (bottom) suggest an increase in both F4/80 macrophages and F4/80+/Tie2+ TEMS in the CSF1-treated tumors. Multiply overlap indicates those areas where F4/80 and Tie2 positivity overlap. Individual stains are in SupplementaryFigure 3. (C, top) Orthotopically implanted PyMT mammary tumors in wild type C57Bl/6 female mice were allowed to become palpable then intraperitoneally treated with PBS (PBS), CSF1 (100 ng in 100 µls) (CSF1), a neutralizing antibody for the CSF1R (50 mg/kg) 4 hours prior to CSF1 treatment (100 ng in 100 µls) (CSF1R NAb+CSF1), the CSF1R antibody alone (CSF1R NAb), an isotype antibody (50 mg/kg) 4 hours prior to CSF1 (100 ng in 100 µls) treatment (CSF1+IgG), or the isotype antibody alone (IgG) three times per week for two additional weeks. The tumors were immunostained with a CD31-Alexa Flour 546 antibody to recognize endothelial cells that comprise blood vessels. Qualitatively, CSF1 treatment increased the percent of CD31-postitive pixels per high powered field compared to PBS treated tumors, while the neutralizing antibody to CSF1R suppressed the CSF1 effect on angiogenesis. (B, bottom) Quantitatively, the percent of CD31+ pixels per high powered field were quantified as blood vessels (angiogenesis) using Adobe Photoshop histogram analysis. CSF1 treatment significantly increased CD31-positive pixels (angiogenesis) compared to PBS. The neutralizing antibody for CSF1R significantly reduced the ability of CSF1 to up-regulate angiogenesis. N = 5 mice per group and results represent the mean ± SEM of percent CD31-positive pixels per high powered field (HPF).
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Related In: Results  -  Collection

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Show All Figures
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pone-0098623-g004: CSF1 has no effect on tumor growth but increases percent tumor TEMs and augments angiogenesis.(A) After two weeks of treatment, tumors were removed, homogenized and immunostained with antibodies specific for F4/80 and Tie2 to identify total F4/80+ cells and F4/80+/Tie2+ cells (Tie2-expressing macrophages, TEMs). While there was a marked increase in total F4/80+ macrophages with CSF1 treatment, the percent of F4/80+/Tie2+ TEMs was significantly increased in response to CSF1 suggesting a regulatory role for CSF1 in expanding the TEM population. N = 5 mice per group and results represent the mean ± SEM of total F4/80+ and F4/80+/Tie2+ TEMs within the tumors. (B, top and bottom left) PyMT tumors without CSF1 treatment and (top and bottom right) with CSF1 treatment immunostained with CD31 for blood vessels, F4/80 for macrophages, Tie2 for F4/80+/Tie2+ TEMS, and DAPI. Confocal images (using 60× objective (top) and with 3× zoom (bottom) suggest an increase in both F4/80 macrophages and F4/80+/Tie2+ TEMS in the CSF1-treated tumors. Multiply overlap indicates those areas where F4/80 and Tie2 positivity overlap. Individual stains are in SupplementaryFigure 3. (C, top) Orthotopically implanted PyMT mammary tumors in wild type C57Bl/6 female mice were allowed to become palpable then intraperitoneally treated with PBS (PBS), CSF1 (100 ng in 100 µls) (CSF1), a neutralizing antibody for the CSF1R (50 mg/kg) 4 hours prior to CSF1 treatment (100 ng in 100 µls) (CSF1R NAb+CSF1), the CSF1R antibody alone (CSF1R NAb), an isotype antibody (50 mg/kg) 4 hours prior to CSF1 (100 ng in 100 µls) treatment (CSF1+IgG), or the isotype antibody alone (IgG) three times per week for two additional weeks. The tumors were immunostained with a CD31-Alexa Flour 546 antibody to recognize endothelial cells that comprise blood vessels. Qualitatively, CSF1 treatment increased the percent of CD31-postitive pixels per high powered field compared to PBS treated tumors, while the neutralizing antibody to CSF1R suppressed the CSF1 effect on angiogenesis. (B, bottom) Quantitatively, the percent of CD31+ pixels per high powered field were quantified as blood vessels (angiogenesis) using Adobe Photoshop histogram analysis. CSF1 treatment significantly increased CD31-positive pixels (angiogenesis) compared to PBS. The neutralizing antibody for CSF1R significantly reduced the ability of CSF1 to up-regulate angiogenesis. N = 5 mice per group and results represent the mean ± SEM of percent CD31-positive pixels per high powered field (HPF).
Mentions: Upon sacrifice, fresh tumors were collected, homogenized using collagenase, immunostained with F4/80 and Tie2 receptor antibodies and subjected to flow cytometry for the percent of total F4/80+ cells and percent of F4/80+/Tie2+ TEMs relative to tumor cells. CSF1 treatment induced a significantly higher percentage of F4/80+/Tie2+ TEMs (p = 0.0003) than the percentage of overall tumor F4/80+ cells (Figure 4A) while a neutralizing antibody to CSF1R abrogated this effect. These data suggest that CSF1 contributes more to the expansion of the population of tumor TEMs than overall F4/80+ cells. To corroborate the flow cytometry data, we immunostained +/- CSF1 treated tumors with CD31, F4/80, or Tie2 antibodies and analyzed by confocal microscopy and found differences in both overall F4/80+ infiltration as well as an increase in F4/80+/Tie2+ cells (TEMs) in the CSF1-treated tumors (Figure 4B).

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