* indicates 0.05 versus siCon group. of MDSCs in bone marrow, spleens and main tumors. Mechanistically, we found that KLF4 knockdown resulted in a significant decrease of circulating GM-CSF, an important cytokine for MDSC biology. Consistently, recombinant GM-CSF restored the frequency of MDSCs in purified bone marrow cells incubated with conditioned medium from KLF4 deficient cells. In addition, we recognized CXCL5 as a critical mediator to enhance the expression and function of GM-CSF. Reduced CXCL5 expression by KLF4 knockdown in main tumors and breast malignancy cells was correlated with a decreased GM-CSF expression in our mouse models. Finally, we found that CXCL5/CXCR2 axis facilitated MDSC migration and that anti-GM-CSF antibodies neutralized CXCL5-induced accumulation of MDSCs. Taken together, our data suggest that KLF4 modulates maintenance of MDSCs in bone marrow by inducing GM-CSF production via CXCL5 and regulates recruitment of MDSCs into the main tumors through the CXCL5/CXCR2 axis, both of which contribute to KLF4-mediated mammary tumor development. culture of bone marrow cells Bone marrow cells from siCon cell- and siKLF4 cell-inoculated mice were extracted. 1 108 bone marrow cells were sequentially incubated and purified with 25 l Biotin-conjugated Gr-1 Ab and 200 l anti-Biotin microbeads (Miltenyi Biotech). MDSCs were cultured in 10-cm plates (5 106 cells/plate) for a total of 6 days. Recombinant mouse GM-CSF (100 ng/ml), IL-4 (50 ng/ml), or IL-6 (50 ng/ml) (Sigma-Aldrich) were added directly to the culture medium on day 0. For MDSC maintenance, 1 107 bone marrow cells were cultured with CXCL5 (25 ng/ml) and mouse anti-GM-CSF monoclonal antibody (250 ng/ml, ab9471) or mouse IgG (250 ng/ml) (eBioscience). 6 days later, bone marrow cells were collected and MDSC populace was detected by FACS. To examine GM-CSF expression in bone marrow, mammary tumor tissues (50 mg) from siCon and siKLF4 cell-inoculated mice were cut into 1 mm 1 mm pieces and incubated with 1 106 bone marrow cells. 24 h later, bone marrow cells were collected and RT-PCR was performed. T cell suppression assay Splenocytes were isolated from wild type BALB/c mice and CD4+ T or CD8+ cells were sorted using Miltenyi Biotech magnetic beads as explained in Rabbit Polyclonal to UBA5 Materials and Methods of the main text. Different numbers of gamma-irradiated (9 Gy) MDSCs derived from siCon cell- and siKLF4 cell-inoculated mouse spleens or tumors, as indicated in the figures, were cocultured with purified CD4+ T cells (5 105) or CD8+ cells (1 106) stimulated with Con A (5 g/mL) in 24-well plates. T-cell SCH28080 proliferation was decided after 72 h culture by pulsing with 1 Ci/well [3H] thymidine (PerkinElmer Life Sciences, Boston, MA) during the final 12 h of culture. Cultures were harvested and thymidine incorporation was measured by scintillation counting (Perkin Elmer). Data are expressed as cpm (mean SE) of triplicate cultures. Three independent experiments were performed. Arginase activity 1 106 gamma-irradiated MDSCs derived from siCon cell- and siKLF4 cell-inoculated mouse spleens were cultured in 24-well plates for 24 h. Cells were collected and lysed with 200 l of lysis buffer (0.1% Triton X-100 plus 1 tablet of protease inhibitor mixture). 10 l of 10 mM SCH28080 MnCl2 was added. Arginase was activated by heating the solution for 10 min at 55C. The lysate was incubated with 100 l of 0.5 M L-arginine (pH 9.7) for 1 h at 37C. The reaction was halted with 800 l quit answer (96% H2SO4:85% H3PO4:H2O ratio, 1:3:7). The urea concentration was measured at 540 nm after addition of 40 l of a-isonitrosopropiophenone, followed by heating at 100C for 30 min. A standard SCH28080 curve consisting of serial dilutions of urea was run in parallel. Data are offered as mean SE of triplicate cultures. Three independent experiments were performed. Immunohistochemistry (IHC) and Western blotting Paraffin-embedded tumor sections were fixed in 4% paraformaldehyde and incubated with a Biotin-conjugated Gr-1 Ab (BD PharMingen). A streptavidin-conjugated HRP was applied and peroxidase activity was localized with diaminobenzidine (Vectastain ABC kit, Vector Laboratories). CXCL5 and GM-CSF expression in tumor and lung tissues of siCon and siKLF4 cell-inoculated mice was examined by Western blotting. The antibodies used were as follows: rabbit polyclonal anti-CXCL5.