Gene sets enrichment analysis of genes enriched in wild type compared to <i>Ccr2</i>^<i>-/-</i> monocytes.

<p>(A) Enrichment plots of wound healing, inflammation and locomotory behavior pathways. (B) Common genes enriched in similar pathways.</p

<i>Csfr1</i> mRNA is reduced in monocytes from <i>Ccr2</i>^<i>-/-</i> mice.

<p>Monocytes were purified from the blood of wild type, <i>Ccl2</i>^<i>-/-</i>, and <i>Ccr2</i>^<i>-/-</i> mice (5 mice in each group). mRNA was isolated and mRNA encoding CSF-1R was quantified by PCR. * p > 0.05 by t-test; ** p < 0.05 by t-test.</p

Measurement of tumor-associated leukocytes and endothelial cells.

<p>Tissue microarrays were prepared from MMTV-<i>neu</i>-driven tumors in wild type, <i>Ccl2</i>^<i>-/-</i>, and <i>Ccr2</i>^<i>-/-</i> mice at 60, 80, 100, 120 and 140 days of age. Four or five mice were included per data point. Tissue microarray slides treated with fluorescent antibodies against (<b>A</b>) Ki-67, (<b>B</b>) vWF, (<b>C</b>) CD31, and (<b>D</b>) Mac2 and imaged by fluorescence or immunohistochemistry. Quantified stains were compared using one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

CCL2’s effects on mammary carcinoma cells are non-cell autonomous.

<p>(<b>A</b>) <i>In vitro</i> growth of mammary carcinoma cell lines derived from MMTV-<i>neu</i>-induced tumors in wild type, <i>Ccl2</i>^<i>-/-</i>, and <i>Ccr2</i>^<i>-/-</i> mice. Two representative cell lines were tested from each genotype. (<b>B</b>) Neutralization of CCL2 in cultures of wild type murine tumor cells or addition of exogenous CCL2 to <i>Ccl2</i>^<i>-/-</i> tumor cells did not affect cell proliferation <i>in vitro</i> as measured by ³H-thymidine incorporation. Etoposide was used as positive control for growth inhibition. (<b>C</b>) Concentration of CCL2 protein in medium or cell lysates of cultured MCF7, SK-BR-3 or MDA-MB-231 human breast cancer cell lines determined by ELISA. (<b>D</b>) Immunoblot detection of full-length and cleaved poly(ADP-ribose) polymerase (PARP) in lysates from MCF7, SK-BR-3 or MDA-MB-231 cells treated with CCL2-neutralizing antibody. Etoposide was used as a positive control for PARP cleavage. Blots were stripped and reblotted for tubulin as a loading control. (<b>E</b>) MDA-MB-231 cells were treated with two different CCL2-neutralizing antibodies, and cell proliferation was measured by ³H-thymidine incorporation. (<b>F</b>) Luciferase-expressing MDA-MB-231 cells were injected into the mammary fat pads of SCID mice. Mice were treated with ABN912, a neutralizing anti-human CCL2 antibody, or isotype control. Arrows indicate time of antibody treatment. Tumor growth was followed by bioluminescence imaging.</p

Effect of mammary tumors, <i>Ccl2</i> disruption, and <i>Ccr2</i> disruption on monocyte accumulation in spleen, bone marrow, and tumors.

<p>(<b>A</b> and <b>B</b>) Proportions of monocytes in the spleen (<b>A</b>) and bone marrow (<b>B</b>) of tumor-free and tumor-bearing wild type, <i>Ccl2</i>^<i>-/-</i> and <i>Ccr2</i>^<i>-/-</i> mice were measured by flow cytometry. Comparisons were made by one-way ANOVA and Bonferroni’s multiple comparison test. #, p < 0.05; ##, p < 0.01 wild type <i>versus Ccl2</i>^<i>-/-</i> or <i>Ccr2</i>^<i>-/-</i> mice. (<b>C</b>) Total leukocyte content of tumors was determined as the percentage of CD45⁺ cells. (<b>D</b>) Proportions of tumor-infiltrating T cells, B cells, and monocytes/macrophages were determined by flow cytometry using antibodies against CD90⁺, B220⁺, and CD11b⁺, respectively.</p

Effect of pharmacologic inhibition of CCR2 on mouse mammary carcinoma development.

<p>(<b>A</b> and <b>B</b>) Overall survival and tumor-free survival of <i>neu</i>^<i>+</i> mice treated with the CCR2 antagonist CCX872 (n = 26) or vehicle (n = 25). Median survivals were compared using Log-rank (Mantel-Cox) test. Mice were sacrificed when the diameter of any single tumor reached 2 cm, if a tumor became necrotic, or if mice were unable to reach food or water (<b>C</b> and <b>D</b>) Tumor growth in mice treated with CCX872 or vehicle measured as total tumor volume in a mouse <b>(C)</b> or as the volume of the single largest tumor mass in a mouse <b>(D)</b>. Tumors were measured twice weekly and averaged. Comparisons were made by two-way ANOVA.</p

CCL2 production associated with MMTV-neu-driven tumors.

<p>(<b>A</b>) CCL2 concentrations were measured by ELISA in sera from 4 wild type and 6 <i>neu</i>^<i>+</i> mice at the indicated ages. The difference between wild type and tumor bearing mice was significant by two-way ANOVA (p < 0.05). (<b>B</b>) Cell lines were developed from <i>neu</i>^<i>+</i>, <i>neu</i>^<i>+</i>/<i>CCL2</i>^<i>-/-</i>, and <i>neu</i>^<i>+</i>/<i>CCR2</i>^<i>-/-</i> mice. CCL2 released into the culture medium by 10⁶ cells over a 72 hr period was measured by ELISA.</p