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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 expands the TEM population in peripheral blood.To reach serum levels of CSF1 similar to breast cancer patients and determine that effect on TEM expansion, PBS or CSF1 (20 ng/ml) was intravenously injected into non-tumor bearing wild type C57Bl/6 female mice every other day for a total of three treatments. Bone marrow and atrial blood was collected and immunostained with CD45 and Tie2 antibodies (for bone marrow) or CD11b, CD31, Gr-1, and Tie2 antibodies (for blood). There was no difference in the percentage of TEMs in the bone marrow of the PBS- and CSF1-treated mice. In peripheral blood, CSF1 treatment significantly reduced the percent of CD11b+/CD31+/Gr-1lo/Tie2- cells while significantly increasing CD11b+/CD31+/Gr-1lo/Tie2+ TEMs. N = 5 mice per group and results represent the mean ± SEM of percent total CD45+ and CD45+/Tie2+ cells from bone marrow, and the percent of CD11b+/CD31-/Gr-1lo/Tie2- and CD11b+/CD31-/Gr-1lo/Tie2+ TEMs in peripheral blood.
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pone-0098623-g005: CSF1 expands the TEM population in peripheral blood.To reach serum levels of CSF1 similar to breast cancer patients and determine that effect on TEM expansion, PBS or CSF1 (20 ng/ml) was intravenously injected into non-tumor bearing wild type C57Bl/6 female mice every other day for a total of three treatments. Bone marrow and atrial blood was collected and immunostained with CD45 and Tie2 antibodies (for bone marrow) or CD11b, CD31, Gr-1, and Tie2 antibodies (for blood). There was no difference in the percentage of TEMs in the bone marrow of the PBS- and CSF1-treated mice. In peripheral blood, CSF1 treatment significantly reduced the percent of CD11b+/CD31+/Gr-1lo/Tie2- cells while significantly increasing CD11b+/CD31+/Gr-1lo/Tie2+ TEMs. N = 5 mice per group and results represent the mean ± SEM of percent total CD45+ and CD45+/Tie2+ cells from bone marrow, and the percent of CD11b+/CD31-/Gr-1lo/Tie2- and CD11b+/CD31-/Gr-1lo/Tie2+ TEMs in peripheral blood.

Mentions: Because we observed increased numbers of TEMs within the tumors of the CSF1-treated mice, we hypothesized that CSF1 may be inducing recruitment of TEM progenitors from bone marrow. Because breast cancer patients can have higher than normal levels of serum CSF1[39], [40], we emulated these serum levels of CSF1 in non-tumor bearing wild type mice to see if in the absence of a tumor that CSF1 could expand circulating levels of TEMs. We intravenously injected PBS or CSF1 at a concentration which emulates human breast cancer patients. Flow cytometry analysis revealed no difference between treatment groups in CD45+/Tie2+ cells from the bone marrow, described as TEM bone marrow precursors[24], [27] (Figure 5). Interestingly, we observed a significant reduction in circulating CD11b+/CD31-/Gr-1lo/Tie2- monocytes (p<0.0001) and a significant increase in circulating CD11b+/CD31-/Gr-1lo/Tie2+ cells (TEMs) (p<0.0001) with CSF1 treatment (Figure 5). This data suggests that serum CSF1 can regulate TEM expansion in circulation and not solely once monocytes enter the tumor proper.


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 expands the TEM population in peripheral blood.To reach serum levels of CSF1 similar to breast cancer patients and determine that effect on TEM expansion, PBS or CSF1 (20 ng/ml) was intravenously injected into non-tumor bearing wild type C57Bl/6 female mice every other day for a total of three treatments. Bone marrow and atrial blood was collected and immunostained with CD45 and Tie2 antibodies (for bone marrow) or CD11b, CD31, Gr-1, and Tie2 antibodies (for blood). There was no difference in the percentage of TEMs in the bone marrow of the PBS- and CSF1-treated mice. In peripheral blood, CSF1 treatment significantly reduced the percent of CD11b+/CD31+/Gr-1lo/Tie2- cells while significantly increasing CD11b+/CD31+/Gr-1lo/Tie2+ TEMs. N = 5 mice per group and results represent the mean ± SEM of percent total CD45+ and CD45+/Tie2+ cells from bone marrow, and the percent of CD11b+/CD31-/Gr-1lo/Tie2- and CD11b+/CD31-/Gr-1lo/Tie2+ TEMs in peripheral blood.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098623-g005: CSF1 expands the TEM population in peripheral blood.To reach serum levels of CSF1 similar to breast cancer patients and determine that effect on TEM expansion, PBS or CSF1 (20 ng/ml) was intravenously injected into non-tumor bearing wild type C57Bl/6 female mice every other day for a total of three treatments. Bone marrow and atrial blood was collected and immunostained with CD45 and Tie2 antibodies (for bone marrow) or CD11b, CD31, Gr-1, and Tie2 antibodies (for blood). There was no difference in the percentage of TEMs in the bone marrow of the PBS- and CSF1-treated mice. In peripheral blood, CSF1 treatment significantly reduced the percent of CD11b+/CD31+/Gr-1lo/Tie2- cells while significantly increasing CD11b+/CD31+/Gr-1lo/Tie2+ TEMs. N = 5 mice per group and results represent the mean ± SEM of percent total CD45+ and CD45+/Tie2+ cells from bone marrow, and the percent of CD11b+/CD31-/Gr-1lo/Tie2- and CD11b+/CD31-/Gr-1lo/Tie2+ TEMs in peripheral blood.
Mentions: Because we observed increased numbers of TEMs within the tumors of the CSF1-treated mice, we hypothesized that CSF1 may be inducing recruitment of TEM progenitors from bone marrow. Because breast cancer patients can have higher than normal levels of serum CSF1[39], [40], we emulated these serum levels of CSF1 in non-tumor bearing wild type mice to see if in the absence of a tumor that CSF1 could expand circulating levels of TEMs. We intravenously injected PBS or CSF1 at a concentration which emulates human breast cancer patients. Flow cytometry analysis revealed no difference between treatment groups in CD45+/Tie2+ cells from the bone marrow, described as TEM bone marrow precursors[24], [27] (Figure 5). Interestingly, we observed a significant reduction in circulating CD11b+/CD31-/Gr-1lo/Tie2- monocytes (p<0.0001) and a significant increase in circulating CD11b+/CD31-/Gr-1lo/Tie2+ cells (TEMs) (p<0.0001) with CSF1 treatment (Figure 5). This data suggests that serum CSF1 can regulate TEM expansion in circulation and not solely once monocytes enter the tumor proper.

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