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. 2014 Jun 3;9(6):e98623. doi: 10.1371/journal.pone.0098623

Figure 4. CSF1 has no effect on tumor growth but increases percent tumor TEMs and augments angiogenesis.

Figure 4

(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 Supplementary Figure 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).