EDA-Fibronectin Originating from Osteoblasts Inhibits the Immune Response against Cancer - Fig 9

<p><b>Myeloid cells are intrinsically different based on exposure to EDA-FN.</b> (A) Adoptive transfer using similar numbers of isolated CD11b⁺-cells from cKO and CT animals mixed with B16 melanoma cells and injected subcutaneously showed a significant decrease in growth in the tumors that contained cKO CD11b⁺-cells, <i>n</i> = 16/10/18/18. Isolated CD11b⁺-cells were pooled for each experiment from at least three mice, and the experiment was repeated four times. ANOVA followed by <i>t</i> tests was performed. <b>EDA-containing FN, by acting on α5β1, modifies CD11b</b>⁺<b>-cell behavior and arginase-1 expression.</b> (B) Newly formed CD11b⁺-cells generated in the presence of EDA and then mixed with B16 melanoma cells and injected subcutaneously resulted in larger tumors compared to CD11b⁺-cells generated in the presence of pFN or EDB <i>n</i> = 7/7/5 in three experiments. (C) Inhibition of EDA effects using an antibody directed against α5β1 prevented growth stimulation by EDA, <i>n</i> = 6/6/6/6 in two experiments. ANOVA followed by <i>t</i> tests was used for comparisons. (D) Expression of arginase-1 in CD11b⁺-cells isolated from the bone marrow of CT and cKO animals, from the tumors in CT and cKO animals, (E) from CD11b⁺-cells generated in the presence of pFN, EDA, or EDB, or (F) from CD11b⁺-cells in which EDA effects were prevented by inhibiting integrin α5β1. EDA resulted in opposite effects to cKO or use of the inhibitory antibody. (G) Even though inducible nitric oxide synthase (iNOS) was elevated in the absence of EDA (cKO bone marrow and tumor) and (H) diminished in the presence of EDA (+EDA columns), (I) the inhibitor of α5β1 failed to prevent the effect of EDA on the mRNA expression of iNOS, suggesting that it is not involved in mediating EDA effects through α5β1 integrin, <i>n</i> = 8/10, 5/7, 13/12/8, 8/8/8/8. The numbers represent biological replicates. The samples were collected over four separate experiments for bone marrow, three experiments for tumor, three experiments for EDA treatment, and three experiments for use of the inhibitory antibody. ANOVA followed by <i>t</i> tests was performed. Underlying data for A–I are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002562#pbio.1002562.s001" target="_blank">S1 Data</a>.