Macrophage Colony-Stimulating Factor Augments Tie2-Expressing Monocyte Differentiation, Angiogenic Function, and Recruitment in a Mouse Model of Breast Cancer
Authors: Amy C Gross
Sara L Cole
Tim D Eubank
Leni Moldovan
Clay B Marsh
Jeffrey L Voorhees
Mary A Forget
Randall Evans
Ivory L Patterson
Xiaokui Mo
Duaa Dakhlallah
Journal: PLoS ONE (2014)
Discipline: Multidisciplinary
Keywords: M-CSF; tumor progression; macrophage numbers; breast cancer; CD14+ monocytes

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. 

Retracted: Yes

PubPeer comments:
 Concern about Figure 1B. Two flow cytometry panels look very similar to each other. The thick black ellipse is part of the original figure, but the colored ellipses highlight some (not all) areas that look unexpectedly similar to each other. Reported to the journal in June 2014, 4 days after it was published, with a friendly reminder in 2016, but no update yet. In the meantime, it was cited 60 times. ![file] , 
 Figure 1C. Two flow cytometry panels appear to share unexpected similarities. Boxes of the same color bring attention to some of those similar-looking areas. ![file] , 
 2020 retraction. https: journals.plos.org plosone article?id=10.1371 journal.pone.0235096 After publication of this article [1], concerns were raised about results reported in Figure 1: In Figure 1B, several areas of the 0.1 ng ml CSF1 panel appear similar to the corresponding areas in the 1 ng ml CSF1 panel. In Figure 1C, several areas of the ANG2 panel appear similar to areas in the CSF1 + CSF1R Nab panel. The authors noted that the original flow cytometry data (.fcs files) underlying the results in Figure 1 are no longer available. Quantitative data supporting the graphs in Figure 1 were provided, but without the original flow cytometry data we are unable to clarify the above concerns or confirm the reliability of the quantitative results. In light of the unresolved concerns about Figure 1, the PLOS ONE Editors retract this article. MAF, JLV, DD, LM, XM, CBM and TDE agreed with retraction but stand by the results and conclusions reported in this article. The other authors either did not reply directly or could not be reached. Reference 1.Forget MA, Voorhees JL, Cole SL, Dakhlallah D, Patterson IL, Gross AC, et al. (2014) Macrophage Colony-Stimulating Factor Augments Tie2-Expressing Monocyte Differentiation, Angiogenic Function, and Recruitment in a Mouse Model of Breast Cancer. PLoS ONE 9(6): e98623. https: doi.org 10.1371 journal.pone.0098623 , 
 As I mentioned above, I reported this paper to the journal in June 2014, 4 days after publication. The interval between reporting and retraction was six full years and 67 (per Google Scholar) citations later. ,